Genetic Polymorphism Governing the CYP2D6 Cytrochrome P450 Enzyme Subfamily in Drug Metabolism

I. Abstract

The decoding of the human genome has opened up an immense opportunity for further research in designing treatment plans that can be more personalized. The composition of a person's genome varies amongst individuals and also within populations. Individual responses to drug are inherited. The clinical implication of inter-individual variations is implicit in Cytochrome P450 enzymes that are prominent in drug metabolism. Polymorphism of over 20 enzymes involved in drug metabolism has been characterized and most of these involve the Cytochrome P450 enzymes. The Cytochrome P450 enzymes have been subjected to numerous evolutionary pressures over time, consequently producing various isoforms. The frequency of variant alleles can alter the pharmacokinetic parameters of the drug, especially of a drug with a narrow therapeutic index. These alleles can either have heightened responses to certain drugs causing toxicity or show very low compliance leading to therapeutic failure. Specifically, CYP2D6 is known to vary tremendously amongst different ethnic groups. Polymorphism of drug metabolizing enzymes such as CYP2D6 can severely affect the clinical outcome in regards to drug response. CYP2D6 gene is shown to have 74 variant alleles, when expressed in homozygous or heterozygous manners give rise to four distinct phenotypes. In this new era of genomic advancements, there is much hope to decipher variations pertaining to drug metabolism and gear the focus towards individualized medicine. Patient selection can be drastically improved by the employment of genotyping. Innovative technologies have made genotyping prevalent and we have come a long way since the advent of pharmacogenetic in the early 19th century.

Sir William Osler (1849-1919) documented that "variability is the law of life, and as no two faces are the same, no two bodies are alike, and no two individuals react alike, and behave alike under the abnormal conditions we know as disease."

II. Personalized Medicine and Pharmacogenomics

A. Pharmacogenomics

The human genome project has it made possible for researchers to comprehend the complexity of biological pathways involved in disease states and focus on variations amongst individuals in regards to drug regimens (Ginsburg and Willard, 2009). The pharmacokinetic aspect of the body's way of dealing with the drug such as adsorption, distribution, metabolism and elimination of the substrate factors into the variability of individual drug response (Kroemer and Meyer zu Schwabedissen, 2010). The pharmacogenetic variation in absorption and elimination are quite rare compared to the variation seen in drug elimination (Nebert, 1999). According to Nebert et al. (2004) "Clinical pharmacology is any particular response seen after a drug is administered". However, this phenotypical drug response is rather ambiguous and has various biological and environmental influences as illustrated in Fig.1, which can lead to a gradient in drug efficacy and toxicity (D. R. Nelson et al., 2004). The phenomenon of genetic variability causing different reactions to drugs has been recognized for awhile as seen in Fig 2 but only recently has the idea become prevalent (March, 2000). In 1902, Sir Archibold Garrard regarded enzymes as vital endogenous biochemical substances required for detoxification in alkaptonuria (Hood, 2003). Sir Archibold Garrard later exemplified the enzyme deficit leading to adverse drug reactions as "in born errors of metabolism" (Hood, 2003). An inherited difference in tasting ability of phenylthiocarbamide was first discovered by a chemist, Arthur Fox in 1931. Arthur Fox's finding in 1931 on genetic variability was considered a breakthrough finding in the field of pharmacogenetic (Hood, 2003). During World War II, the antimalarial drug such as primaquine showed differing results in Caucasian soldiers compared to the African American soldiers; African American soldiers showed greater occurrences of hemolytic anemia when administered drug (March, 2000). Metabolism as a concept became prevalent in mid 19th century when scientists began to decipher the excretory metabolites of consumed substances (Nebert and Vesell, 2004).

Pharmacogenomics, the term coined in 1995, focuses on a person's genetic composition, gene and respective gene products, and illustrates how this variability affects drug metabolism (Nebert and Vesell, 2004)(Maria Almira Correia, 2009). The two major aspects of pharmacogenomics are a) To recognize the genes that are affected in a disease state; and b) To focus on the variant alleles that alter our response to the drugs (Wolf, Smith, and Smith, 2000).

Figure 1 Factors influencing individual drug response.

Reprinted from "Pharmacology, pharmacogenetics, and pharmacoepidemiology: three Ps of individualized therapy" By S. Dawood , 2009, Cancer Investigation, 27, 809-815

Figure 2 Favism is implicit in certain population that consume fava beans

A Greek philosopher Pythagoras first noted this phenomenon that was later found to be associated with acute hemolytic anemia in people who consume the legumes. These people have deficiency in glucose-6-phospahte dehydrogenase and can show altered response to antimalarial drug Reprinted from "Pharmacogenomics: the promise of personalized medicine" by Hood Emily, 2003, Environ Health Perspect.; Aug;111(11):A581-9.

Pharmacogenomics encompasses the whole human genome, DNA, RNA and the associated gene products involved in the study of drug metabolism, drug transport, "target proteins (receptor, ion channels, enzymes)" and links these gene products to their affects on xenobiotics (Mini and Nobili, 2009). A drug that exhibits reduced efficacy does not always correlate with reduced levels of toxicity since remedial values and noxious side effects of a drug are often exerted via diverse biochemical pathways (Mini and Nobili, 2009). The study of pharmacogenomics, therefore, has vital therapeutic value because most disease states entail some sort of drug treatment (Kroemer and Meyer zu Schwabedissen, 2010). The study of genomics is now made it possible to predict safety, toxicity and efficacy of drugs and opt for a personalized treatment plan by targeting variant alleles (Dawood, 2009). The empirical notion of patients with a certain disease state reacting to drugs homogenously is flawed (Dawood, 2009). This conviction, however, does not account for genetic variation, which unfortunately leads to over 40% of patients either getting the incorrect drug or wrong dosage of the drug (Bordet, Gautier, Le Louet, Dupuis, and Caron, 2001). A Meta analysis study done in 1994, estimated that more than 2 million patients hospitalized in the US had fatalities related to adverse drug reactions (Lazarou, Pomeranz, and Corey, 1998). These results concluded that in 1994, the 106,000 fatalities associated with adverse drug effects ranked between fourth to sixth leading causes of death in the US(Lazarou et al., 1998). Regardless of strict and regulated standards for drug efficacy and prevention of toxicity, adverse drug reactions are prominent and a drug is never equivalently effective on a general population (Roses, 2000). Financially, neither the patients and/or the health insurance companies find it feasible to pay for drugs that are either ineffective or cause adverse effects (Roses, 2000). If a patient has blunted ability to metabolize a drug that is administered to them in normal doses this could easily lead to mortality due to toxic levels of the exogenous substance left in the system (Hood, 2003). Patients react to drugs in a heterogeneous manner compared to the notion of homogenous efficacy, which is particularly imminent in chemotherapeutic drugs (Dawood, 2009). Most chemotherapeutic drugs have narrow therapeutic index and any variability in metabolism of this drug can lead to adverse drug reaction (Dawood, 2009). The approach employed currently often leads to therapeutic failure and waste of time leading to expensive health care costs and valuable time (Hood, 2003). Therapeutic failure related to drug metabolism in diseases such as cancer, psychiatric disorders, and hypertension can be severely detrimental if the drugs do not take effect due to the presence of variantions in enzymes leading to high and low metabolizers (Hood, 2003). Although, genetic variability alone does not account for all the adverse effects of drugs seen in a patient, pinpointing the altered gene can be beneficial in tailoring a more precise therapy that involves less adverse effects (Hood, 2003). Therefore, understanding the complex interaction of individuals with their environment and underlying genetic variation will allow for a gradual shift from "one drug fits all" perception to an embodiment of individualized medicine (Dawood, 2009).

B. Individualized Medicine

Individualized medicine encompasses many attributes such as clinical, genetic, and environmental factors all intertwined in a complex meshwork affecting a disease state (Ginsburg and Willard, 2009). Thorough understanding of these various attributes can aid in development of personalized treatment plans and medication types/dosages leading to an effective patient care, reduction in drug toxicity and increase in drug efficacy (Ginsburg and Willard, 2009).

The ultimate goal of the drug is to have the most efficacious and least toxic effect on the patient (Dawood, 2009). However, clinical variables such as drug-drug interaction and metabolism of drug and drug transport show pronounced differences accounting for toxicity (Dawood, 2009). The statistics reveal that a certain drug is known to produce therapeutic effect only in 30% of the patients, whereas 30% of the patient show little or no advantageous effect to the drug, 10% are shown to have only deleterious effects (Maitland-van der Zee, de Boer, and Leufkens, 2000). For example if a patient is on an antidepressant, which usually take two weeks to take effect, predicting drug response for patients with a variant allele is advantageous in regards to predicting efficacy (Kirchheiner and Seeringer, 2007). Predicting drug response poses just as many challenges as do the study of inherited diseases related to genes (McCarthy and Hilfiker, 2000). The variant gene products involved in drug metabolism are related to regulation at the level of gene expression, post translational modification and drug-drug interaction, all of which affects individual responses to xenobiotics (McCarthy and Hilfiker, 2000).Typically, drug doses are determined by body surface area and for certain group of individuals the systemic exposure is presumed to be homogenous if the surface area is similar The surface area is mainly determined based on height and weight (Dawood, 2009). The variation however stems not necessarily from differences in physical factors but rather from discrepancy in drug metabolism and drug clearance (Galpin and Evans, 1993). Although, systemic monitoring for drugs with low therapeutics indicies has been employed, it still is not efficient enough to prevent therapeutic failure (Nebert and Vesell, 2004).

II. Genetic Polymorphism

A. Introduction

Genetic polymorphism is the variation in allele that is present at a locus and occurs in more than 1% of the population (Phillips, Veenstra, Oren, Lee, and Sadee, 2001). The allele is considered a mutation when it occurs in less than 1% of the population (Mini and Nobili, 2009). The human genome is 3 billion base pair long and the variation in one nucleotide sequence in the DNA occurs in every 100-300 bases (Hood, 2003). Single nucleotide polymorphism (SNP) is the most extensively studied genetic polymorphism, which accounts for most of the variation in drug metabolism (Schmith et al., 2003). The human genome has over 1.4 million single nucleotide polymorphisms 60, 000-100,000 is associated with drug effects ((Dawood, 2009)(Schmith et al., 2003). These SNP can gives rise to polygenic gene variants that can alter the pharmacokinetic and the pharmacodynamic portfolio of a drug leading to innate deviation in metabolism (W. E. Evans and McLeod, 2003). The gene loci that encodes for proteins involved in drug metabolism are inherently shown to have about 47-61% polymorphism, which in turn correlates to the immense differences in substrate breakdown (Nebert, 1999). Genes that have SNPs in the coding region usually change the amino acid sequence of the protein whereas the SNP in the regulatory region are known to control the concentration of the proteins (McCarthy and Hilfiker, 2000). An exogenous substance relays its effect by interacting either on the cell membrane, cytoplasm or in the plasma (Mini and Nobili, 2009). However, a substance that is known to be efficacious in most individuals can cause detrimental effects in some if they are homozygous for the variant alleles as seen in Fig 3. This variation can affect any of the compartment of interaction a drug asserts its effects (Mini and Nobili, 2009). These alterations can manifest into phenotypes that can cause adverse effects by enhancing or inhibiting normal physiological activity (Mini and Nobili, 2009). The human genome project has simplified the identification of roughly 100,000 SNP's in the human genome, which can be employed to acquire accurate information on individual drug responses (Schmith et al., 2003). A haplotype is regarded as a blueprint in which not one but many SNP occur on the same chromosome (Hood, 2003). Although a single SNP may cause altered response to drugs, it is more likely the combination of SNPs on a single chromosome that may play a role in drug metabolism leading to polygenic phenotype (Hood, 2003). In the near future, clinical trials might be required to incorporate genotyping for potential drugs. The cost of genotyping for clinical trials has been predicted to cost approximately 1 million dollars (McCarthy and Hilfiker, 2000). Even though the additional cost to the trial is of concern, the overall end results might provide valuable information on drug metabolism amongst different ethnic groups, which would be beneficial in the long run.

Characterization of genes of enzymes involved in drug metabolism are shown to have considerable variations; about 3 to 10 variant alleles are considered to be of the common type and over 12 to 100 variant alleles that are uncommon and occur rarely (Nebert and Vesell, 2004). Initially, when the Human Genome Project was undertaken, there was little concern about the difference in sequencing of chromosome amongst different ethnic groups (Nebert, 1999). Most scientists at the time believed there would be no substantial discrepancy between chromosomes of an individual who is of an Asian descent compared to an individual of European descent (Nebert, 1999). Graham and Smith in the 1999 study showed that there is significant variation in drug metabolism amongst individuals of different ethnic backgrounds, which effects the pharmacokinetic variability of the enzyme that are involved in drug metabolism (Graham and Peterson, 1999)(Maitland-van der Zee et al., 2000). Recent study on Asian, Whites and Blacks showed that different ethnic populations differ in the frequency of alleles of a gene and this variant can result in altered drug responses (Limdi et al., 2010). The functional consequence on drug metabolism of the variant allele depends on the extension of mutation and frequency of occurrence in an individual subgroup (Maitland-van der Zee et al., 2000). To establish an accurate overall picture of variant alleles in different ethnic subgroups, an extensive SNP genotyping is needed, with an average group size of 1000 individuals in each subgroup (Nebert, 1999). The information derived from this can then be utilized for an extensive genotype-phenotype linkage study (Nebert, 1999).

Figure 3 Polymorphism affecting the concentration of a drug leading to toxic doses and low efficacy in individuals who are homozygous for the variant gene.

Reprinted from "Pharmacogenetics: implementing personalized medicine" By Enrico Mini; Stefania Nobili, Clinical Cases in Mineral and Bone Metabolism 2009; 6(1): 17-24

B. Adverse Drug Reaction

Drug-drug interactions are common when numerous drugs are ingested simultaneously (Wolf et al., 2000). These drug-drug interactions can induce or inhibit enzymes in the common pathway of metabolism causing adverse effects (Oesch, 2009). An individual who has reduced ability to metabolize a substrate can easily accumulate the drug if an alternative route is not accessible (Oesch, 2009). The pharmacokinetic differences in individuals can cause poor metabolizers to have increased amounts of systemic exposure to the drug and fast metabolizers having less than normal amounts resulting in therapeutic failure or even toxicity. (Bailey, Bondar, and Furness, 1998). Comprehending this inherited genetic variability in drug metabolism can herald a new era in individualized therapy (Dawood, 2009)(Oesch, 2009)(Wolf et al., 2000). Study of pharmacogenomics allows for ways to reduce adverse drug reactions by identifying the nature of the drug, reaction to the drug and metabolic targets of the drug (Phillips et al., 2001). All of the above can be utilized to create an extensive biomarker, which can then be employed by physicians to make appropriate dosing changes for individuals with variant alleles (Ginsburg, Konstance, Allsbrook, and Schulman, 2005). Alternatively, if reducing the dose is not a viable option, physicians can alter the treatment to include drugs that can by pass the deficient biochemical pathway (Ginsburg et al., 2005; Phillips et al., 2001). In order to utilize genotyping as a beneficial tool, physicians need to quantify variant drug responses to the specific gene unambiguously (Nebert, 1999). It is imperative that the candidate locus that is affected by the drug is identified and positive tests are employed for the variant alleles (Holmes et al., 2009). The

Genetic polymorphism plays a major role in drug efficacy and also in adverse drug reactions (Dawood, 2009). Pharmacogenomic studies are hard to conduct because the variation in drug metabolism is only known after the administration of the exogenous substance, as compared to inherited diseases which have clear family linkage (McCarthy and Hilfiker, 2000). It is highly unlikely that an entire family would be prescribed a certain drug at the same time so the variation in the allele is only known under clinical trials (McCarthy and Hilfiker, 2000). SNP profiling can be beneficial if it can predict the drug response in patients and the demographics of people affected (McCarthy and Hilfiker, 2000). For example, a study by Drazen in 1999 showed that variation in ALOX5 was correlated 100% of the time with patients being non-receptive to an antiasthmatic drug (Drazen et. al, 1999). However, the prevalence of the non-variant gene in ALOX5 occurs in only 6-10 % of the patients; therefore, for a drug to be efficacious, the percent frequency of variant allele needs to be determined (Drazen et. al, 1999;McCarthy and Hilfiker, 2000). The major questions to be addressed then is how prevalent is the variant gene? How often are patients in a certain demographic group prescribed a drug that can cause adverse effects (Maitland-van der Zee et al., 2000)?

A potential drug is marketed and distributed worldwide, however, most of the clinical trials are never encompass a broad range of population and most polymorphisms go undetected (McCarthy and Hilfiker, 2000). The clinical trials mainly consist of the Caucasian population in America and Europe, but a wider range of population is needed to pinpoint major variation amongst different ethnic groups (McCarthy and Hilfiker, 2000). Consequently, polymorphisms that are relevant in certain populations need to be studied and the target must be to address variant genes that are prevalent in drug metabolism (Maitland-van der Zee et al., 2000). Currently, there is little to no information on most of the drugs that are already in the market regarding genetic variability in drug metabolism (Maitland-van der Zee et al., 2000). In the future, potential drugs should include such "population based studies" in their clinical trials so fewer drugs would conform to one drug fits all motto (Maitland-van der Zee et al., 2000). Polymorphism profiling can have major implication in drug safety because a drug that poses adverse effects on a large subgroup could be restricted from being launched into the market (Ginsburg et al., 2005). Genotyping can permit physicians to detect different polymorphism in individuals and allow them to create drug regimens that are not only efficacious but pose least toxic effects (Oesch, 2009). Preferential genotyping by clinicians for variant alleles could drastically reduce drug related adverse effects and in turn will be economically feasible and productive in the long run (March, 2000; Nebert and Vesell, 2004). Patient selection could be drastically improved by employment of genotyping.

C. When is Genotyping Appropriate?

Most drug targets are not key candidates for genotyping (Kirchheiner and Seeringer, 2007). The blood sample is collected from the patient after a day or two of administration of the drug. Therefore, drugs that require an immediate attention to dose adjustment or drugs that have a high therapeutic index may not be feasible for genotyping (Kirchheiner and Seeringer, 2007). In addition drugs that are metabolized via more than one overlying biochemical pathway pose extreme difficulties in pinpointing the variant allele and do not benefit from genotyping. However there are enzymes that have variant alleles such as the Cytochrome P450 enzymes which metabolize drugs such as warfarin, morphine, tamoxifen etc. and this polymorphism can lead to altered response to a drug (Kirchheiner and Seeringer, 2007). Adjusting the dose based on plasma level concentration of the drug is not always adequate for these patients (Dawood, 2009). Genotyping in these cases can lead to increased efficacy by identification of polymorphism, which can reduce the costly and time-consuming dose adjustment period. For example, CYP2D6 is a major enzyme involved in the breakdown of antidepressants. The therapeutic effects of antidepressants are only seen after a few weeks of treatment (Kirchheiner and Seeringer, 2007). Therefore, if a patient is a poor metabolizer they will accumulate the drug vs. a person who is an ultra rapid metabolizer, who will show no therapeutic value. In the case of antidepressants, genotyping for the CYP2D6 polymorphism may be beneficial prior to the start of therapy.

Innovative technologies have made genotyping prevalent and we have come a long way since the advent of pharmacogenetic in the early 19th century. Pharmacogenetic disciplines if employed in pharmaceutical industry can aid in development of drugs that cater to the individual; this will allow for prospective drugs to be well suited for fewer people in comparison to drugs that assert mediocre efficacy in a vast group of individual. Food and Drug administration in 2004 permitted the employment of Chip technology known as AmpliChip by Rosche for identification of variant genes in the Cytochrome P450 pathway (http://www.roche-diagnostics.us/press_room/2005/011105.htm); (Ginsburg et al., 2005) Companies like Genelex Corporation of Seattle, Washington and Gentris are now enabling pharmaceutical companies and patients respectively to utilize Cytochrome P450 genotype profiling for CYP 2D6, CYP 2C9 and CYP2C19 enzymes (Hood, 2003). The marriage of genetics and medicine is going to become prominent in the years to come and by the year 2020 pharmacogenomics will become a vital tool utilized to market drugs. The information derived from these test will allow patients to be on customized "designer drugs"(Collins and McKusick, 2001), allow physicians to set appropriate prescription amount for initial dosing and establish monitoring system for individuals with variant alleles (Tweardy and Belmont, 2009).

III Cytochrome P450 Enzyme

A. Background

Variant alleles that lead to functional changes of gene product can have therapeutic consequences. These alleles can either have heightened responses to certain drugs causing toxicity or show none to very low compliance (Wolf et al., 2000). Polymorphism of over 20 enzymes involved in drug metabolism has been characterized and most of these involve the Cytochrome P450 enzymes (CYP) (Wolf et al., 2000). Cytochrome P450 enzymes are involved in metabolism of over 60% of drugs currently in the market today (Hood, 2003). Polymorphisms in the CYP enzymes are known to alter the pharmacokinetic aspects of exogenous substances affecting mainly the biotransformation of the substance (Kirchheiner and Seeringer, 2007). Polymorphism of the Cytochrome P450 enzyme was first discovered in relation to debrisoquine, a hypertension-correcting drug (March, 2000). Bob Smith, of Imperial College in London ingested debrisoquine and experienced severe hypotension after administration. In addition, his blood levels showed 20 fold decreased levels of drug metabolite compared to his colleagues (March, 2000; Nebert 1997). In 1988, Gonzalez and his group characterized and showed that the gene product that was causing the altered response to debrisoquine as CYP2D6; it was also found to be a liver microsomal enzyme. The cloning of this microsomal enzyme was the first look at genetic polymorphism at the molecular level (Gonzalez et al., 1988; Mini and Nobili, 2009). The study by Gonzales et al. and his group paved way for further studies geared to identify genetic polymorphism in a population that linked variant genes to alteration in drug metabolism and drug response (Mini and Nobili, 2009). Cytochrome P450s are mainly found in endoplasmic reticulum and in the mitochondria of a cell, and are copious in the liver (Porter and Coon, 1991). The CYP enzymes consist of about 49 genes that function primarily in drug metabolism (Maitland-van der Zee et al., 2000; Porter and Coon, 1991). In humans the CYP enzymes are major constituents in metabolism of fatty acids, prostoglandins, steroids and xenobiotics (Graham and Peterson, 1999). Daily diet intake of mammals consists of many natural products such as "terpenes, steroids, and alkoloids" and the CYP enzymes are major catalysts in the biotransformation and breakdown of these exogenous substances (Guengerich, 1991). Cytochrome P450 enzymes comprise of a super family of gene that encompass proteins predominantly involved in metabolizing of xenobiotics as well as endogenous substrates such as steroids, fatty acids, prostaglandins and arachidonate metabolites as shown in Table 1, therefore genetic polymorphism in the CYP enzymes can lead to many health related risks such as hypertension and cancer (Graham and Peterson, 1999; Jiang et al., 2005; Mayer et al., 2005). CYP enzymes are monooxygenases that catalyze non-specific oxidations of many substrates (Guengerich, 1991), (Porter and Coon, 1991). The synthetic exogenous substrates of the cytochrome enzymes range to approximately 200,000 entities, which can all have complex interplay amongst each other in inducing or inhibiting the various isoforms of the CYP enzymes (Porter and Coon, 1991). These enzymes however are capable of catalyzing novel substrates as well and therefore one cannot cap an upper limit on the number of possible potential substrates (Porter and Coon, 1991). Therefore, the evolutionary advantage in the immensity of the CYP isoform is a crucial survival tool for our cultivating environment as well as our dynamically changing physiological system.

Table 1. Exogenous and endogenous substrates of Cytochrome P450 enzymes

The substrate for the CYP enzymes are just as diverse for endogenous substance as they are for exogenous substances. The CYP enzymes are prominent catalytic enzymes involved in biotransformation of various substances.

Reprinted from "Miniereview: Cytochrome P450" By Todd D. Porter and Minor J. Coon, The Journal of Biological Chemistry, 1991; 266(21): 13649-13472

The rates of catalyzation of the CYP enzymes are relatively slow and this can provide further explanation into their pivotal role in drug disposition (Guengerich, 1991). In addition, most of the CYP enzymes are involved in rate-limiting steps of drug metabolism and this is a key determinant of the in vivo kinetics of the drug (Pelkonen, 2002). CYP enzymes are key players in the systemic exposure of a drug and the time period a drug can assert its action (Brockmoller, Kirchheiner, Meisel, and Roots, 2000). The CYP enzymes are involved in either forming the active metabolite of the drug from a prodrug or in metabolizing the drug into inactive by-products,both of which can influence the functional temporal aspect of a drug (Brockmoller et al., 2000). Metabolites created by the CYP enzymes can also be toxic; exerting their own mutagenic and allergenic effects (Brockmoller et al., 2000). The FDA requires pharmaceutical companies to identify on the product brochure one of twenty CYP enzymes that are involved in the biotransformation of the drug (Brockmoller et al., 2000). Interactions of different drugs concerning CYP enzymes are good predictor of drug-drug interaction, therefore marketed drugs are required to indicate the CYP enzyme involved in biotransformation of the drug on the product information (Andersson, 1991)(Brockmoller et al., 2000). However, this information does not include the polymorphism prominent within these CYP enzymes. The need for such information is crucial since these enzymes are notorious for genetic polymorphism (Brockmoller et al., 2000). Functional variations in the CYP enzymes are known to show a gradient in efficacy and variation in the quantity of the substrate present in the subject (Maitland-van der Zee et al., 2000; Wolf et al., 2000). Allelic variants causing poor, fast and ultrarapid metabolizing enzymes have been identified in most of the CYP enzymes. Most of the CYP enzymes in the liver show some degree of polymorphism (Anzenbacherova et al., 2000).

B. Cytochrome Gene Family Evolution

CYP enzymes are ubiquitous as they are found in every domain of living organism from Bacteria, Archaea and Eukarya and known to have originated from an ancestral gene approximately three and half billion years ago. The modern cytochrome probably originated with the Prokaryotes 1.5 billion years before the prevalence of atmospheric oxygen (Graham and Peterson, 1999; Nebert and Gonzalez, 1987; Werck-Reichhart and Feyereisen, 2000). In early eukaryotes, these enzymes not only maintained membrane veracity but also were primarily involved in the biosynthesis of endogenous hydrophobic substances such as fatty acids, cholesterol (Nebert and Gonzalez, 1987). The CYP mutilgene family diverged again 900 hundred million years later giving rise to enzymes predominantly involved in biosynthesis of steroids (Nebert and Gonzalez, 1987). This expansion lead to the another divergence of the two most important mammalian CYP families implicit in drug and carcinogen metabolizing enzymes currently known as CYP1 and CYP2 gene family (Nebert and Gonzalez, 1987). Finally, 400 million years ago dramatic expansion ensued primarily in CYP2, CYP3 and CYP4 families (Nebert and Gonzalez, 1987). This current expansion correlates to the time frame when aquatic animals merged onto the terrestrial land and were exposed to many hydrocarbon-based combustion material in the environment along with toxic plant products in their diet (Gonzalez and Nebert, 1990; D. R. Nelson and Strobel, 1987)

The generation of this multigene family is due to the multiple mechanistic changes over time that reflect the complexity and diversity of the CYP enzymes. Most of the changes are related to lack of intron conservation (Werck-Reichhart and Feyereisen, 2000), exon shuffling (Doolittle, 1985; Patthy, 1985), expression of redundant genes (Anderson et al., 1981; Barrell, Air, and Hutchison, 1976), alternative splicing, frame shit mutations and RNA editing (Andreadis, Gallego, and Nadal-Ginard, 1987; Atkins, Weiss, and Gesteland, 1990; Blinkowa and Walker, 1990; Leff, Rosenfeld, and Evans, 1986). The gene duplications that lead to the expansion of the CYP enzymes gene was not devoid of mutations. Once the gene is duplicated it will inherently begin to acquire new mutation independent of each other due to the imposed pressure of natural selection (Gonzalez and Nebert, 1990). This process can create various isoforms and can do so as long as one copy of the duplicated gene retains its original function, the other copy can drift and acquire functional mutations that can be beneficial for the organism (Gonzalez and Nebert, 1990). The dynamic nature of our environment creates many potential xenobiotics over time, therefore a phylogenetic analysis shows that these enzymes are prone to acquire mutations and evolve functional isoforms that equips them to deal with possible xenobiotics (D. R. Nelson et al., 2004). In comparison to the conserved sequence of the histone proteins which take 400 million years to show 1% change in their amino acid sequence, the CYP enzymes only require two to four million years to show unit evolutionary changes (Gonzalez and Nebert, 1990)

CYP enzymes evolutionarily evolved for biosynthesis of endogenous substrates and most likely were important in maintenance of cellular functions (Gonzalez and Nebert, 1990). The mark of modern pharmaceuticals most likely dates back to the past billion years with the advent of herbivorous animals ingesting plants that produced allelochemicals as a defensive machinery (Gonzalez and Nebert, 1990). The ability of these herbivorous animals to detoxify natural toxic plant products was essential for their survival. This unquestionably connects the modern CYP enzymes to their ability to metabolize xenobiotics, which arose due to the nature imposed selection pressures (Gonzalez and Nebert, 1990)

CYP enzymes were classified as hemeproteins in 1961 when they were shown to bind carbon monoxide in their active site to a ferrous iron and elucidate maximum absorption spectra at 450 nm (Nebert and Russell, 2002). This major Soret spectral band at 450 nm is due to the thiolate sulfur of the cysteine residue of the core enzyme binding to the heme iron, which happens to be conserved in 80% of the CYP enzyme. This conserved region appears at the N terminus (Guengerich, 1991). The cytochrome enzyme in the early to mid 1960 was perceived to be a single microsomal enzyme involved in metabolism of steroids and drugs (Nebert and Russell, 2002). In 1970, six CYP enzymes were identified and it wasn't until 1988 when Gonzales and his group utilized advances in mRNA purification to fully categorize the first CYP enzyme (Nebert and Russell, 2002). Currently there 11, 500 CYP enzymes and an updated list can be accessed on the following website (http://drnelson.uthsc.edu/CytochromeP450.html)(D. Nelson, 2010).

Figure 4 History of the Cytochrome P450 enzymes and its place in the molecular biology research

Reprinted from "Pharmacogenetic diagnostics of cytochrome P45 polymorphisms in clinical drug development and in drug treatment" By Jurgen Brockmoller ; Julia Kirchheiner, Christian Meisel & Ivar Root , Pharmacogenomics, 2000; 1(2): 125-151

The CYP enzyme isoforms are just as selective as they are evolutionary diverse in their specificity for the exogenous substrates and are known to be the most prominent group of enzyme catalysts in the mammalian system (Porter and Coon, 1991). A structural homology schematic nomenclature is now employed to name the evolutionarily diverse CYP enzymes because the genetic redundancy and deficiency in preservation of the primary amino acid sequence makes it imminent to have an ordered naming system (Danielson, 2002). Arabic numeral is employed for distinct gene families, capital letters for subfamilies, and roman numerals for isoforms within each subfamily (Nebert et al., 1987). The amino acid sequences are the key determinant in how the CYP enzymes are sub grouped into families and subfamilies (Nebert et al., 1987). The CYP enzymes that show greater than 40% homology are pooled together into a family and those that share grater than 55% homology are grouped into subfamilies and 97% homology is regarded as alleles (Nebert et al., 1987). The advent of genomics and advancements in molecular biology has enabled researches to identify and characterize the CYP enzymes once known to solely localize in the microsomal membranes of the liver (Porter and Coon, 1991). The CYP enzymes are not only vital aspects in metabolizing xenobiotics, they are also key players in many endogenous reactions such as initiating oxidative, peroxidative and reducing changes into small molecules of diverse substrates (Porter and Coon, 1991). The cytochrome enzymes are component of catalytic reaction for saturated and unsaturated fatty acids, eicasonoids, retinoids, and uropophyrinogens (Porter and Coon, 1991). In addition, cloning and crystallography have given us unprecedented insight into these biologically diverse sets of enzymes and their role in cholesterol, steroid hormones, bile synthesis and xenobiotic metabolism (Porter and Coon, 1991). CYP enzymes can metabolize xenobiotics such as pollutants and irritants and reduce the toxicity of these exogenous substances. However, at times the metabolic by-products of some of these reactions can create reactive toxins and mutagens, which can cause cancer, toxicity and birth defects (Nebert and Russell, 2002; Williams, Cosme, Sridhar, Johnson, and McRee, 2000). The carcinogenic and mutagenic potential of CYP enzymes stems from the fact that most inert substance need to be activated into their active form before they can form adducts within DNA (Nebert and Gonzalez, 1987). In the absence of these CYP enzymes most mutagens cannot cause DNA damage since they need to be metabolized by the monooxygenases to form adducts (Nebert and Gonzalez, 1987).

B. Cytochrome P450 Structure

The cytochrome enzymes are localized in three different regions of the cell. Their sub-cellular locations are as follows: soluble cytoplasmic, mitochondrial membrane bound or bound to the microsomal membrane (D. R. Nelson et al., 1996). Most of the prokaryotic CYP enzymes are soluble cytoplasmic proteins whereas the eukaryotic are mainly membrane bound (D. R. Nelson et al., 1996). The primary sequences of the CYP enzyme differ drastically amongst prokaryotes and eukaryotes, however, the enzymes show remarkable similarity in their structural folds (Hasemann, Kurumbail, Boddupalli, Peterson, and Deisenhofer, 1995) .Surprisingly, the CYP enzymes have very similar tertiary structure and the heme-binding domain, where transport of electrons and protons occurs, seems to be conserved ubiquitously (Hasemann et al., 1995; Jean, Pothier, Dansette, Mansuy, and Viari, 1997; Werck-Reichhart and Feyereisen, 2000). Sandra Graham and Julia Peterson showed in six crystallized soluble CYP enzymes that there was remarkable similarity in the structural fold of the proteins in spite of having less than 20% homology amongst the amino acid sequences (Graham and Peterson, 1999). Although the tertiary structures are very similar, the primary and secondary structures confer enough variability amongst the CYP families that a diverse group of substrates can be catalyzed with immense specificity (Graham and Peterson, 1999). The Protein Data Bank now consists of a growing number of crystal structures of the CYP enzymes (Berman et al., 2002). The crystal structure comparisona show that these enzymes only have 10-30% homology in their amino acid sequence across the gene family. (Hasemann et al., 1995; D. R. Nelson et al., 1993)

Comparative analyses illustrate that prokaryotic and eukaryotic CYP enzymes have a well conserved globular structure that comprises a carboxyl terminal half prominent with alpha helices and the amino terminal half which contains the beta sheet (Danielson, 2002). All the cytochrome P450s have a heme protoporphyrin IX molecule in its active site as shown in Fig 5, which confers the CYP enzymes with a strong absorption spectral band at 450 nm (Mestres, 2005). The iron complexed with the heme is bound to the protoporphyrin ring by hydrophobic interactions and hydrogen bonding (Porter and Coon, 1991). The heme ring is surrounded by the catalytic active site of the CYP enzymes, which can have different amino acids, and this variation in the amino acid sequences allows for catalysis of assortment of substrates (Peterson and Graham, 1998). A comparative analysis done by Peterson and Graham showed that conservative core of the CYP enzymes is localized around the heme and consists of helices D, E, I, L; each of which is composed of a collection of 4 helix. In addition, the structural core also contains the J and K helices, two β pleated sheets and a coil known as the "meander" as seen in Fig. 6 (Peterson and Graham, 1998; Werck-Reichhart and Feyereisen, 2000). The β sheets consist of β 1 and β 2 comprised of 5 and 1 strands respectively. (Graham and Peterson, 1999). The sheets together form a channel that is highly hydrophobic and allow the substrate to access the conserved core. The substrates of CYP enzymes are mainly hydrophobic and the access channel is thermodynamically favorable for the entrance of the substrate (Graham and Peterson, 1999). The preserved sequence markers present in the conserved region allow for precise identification of the structural core unmistakably (Mestres, 2005). The consensus amino acid sequence of Phe-X—X-Gly-X-Arg-X-Cys-X-Gly in the heme-binding loop of the conserved region core is highly distinctive of the CYP enzymes (Peterson and Graham, 1998)(Danielson, 2002). In addition, the conserved cysteine residue that binds to heme in the protoporphyrin ring serves as the "fifth ligand" and is located below the L helices on the proximal face of the heme group (Peterson and Graham, 1998). The heme molecule is hidden within the heart of the CYP enzyme and is edged by helix I on the distal side and helix L on the proximal side (Danielson, 2002). The microsomal CYP enzymes differ in comparison to the mitochondrial CYP enzymes at the L helix. All the mitochondrial CYP enzymes have two positive charged arginines flanking the beginning of the L helix (Peterson and Graham, 1998).

The core structure is stabilized by the presence of a conserved amino sequence Glu-X-X-Arg located in the K helices facing the proximal portion of the heme (Werck-Reichhart and Feyereisen, 2000). This sequence in the K helix interacts with the "meander" which is a 14 amino acid unit located in the amino terminal of the heme binding loop. Finally, the distal portion of heme domain is composed of the proton transfer groove, which is composed of Ala-Gly-Gly-X-Asp/Glu-Thr-Thr-Ser and is located on the central part of the I helix (Graham and Peterson, 1999; Werck-Reichhart and Feyereisen, 2000). The threonine residue forms a pocket allowing for the oxygen molecule to bind. This pocket is critical for the monooxygenation reaction to occur and introduction of the activated oxygen into the sunbstrate (Poulos, Finzel, and Howard, 1987).

Figure 5. The protoporphyrin ring of a heme-conjugated CYP enzyme.

Reprinted from "Mechanism of Oxidation Reactions Catalyzed by Cytochrome P40 Enzymes" By Bernard Meunier, Samuel P de Visser and Sason Shaik, Chem Rev, 2004, 104: 3947-3980

Figure 6 Ribbon drawing of the P450 BM.

The drawing is shown from the distal face. This bacterial CYP enzyme has many similarities with the eukaryotic CYP enzymes.

Reprinted from "How Similar are P450s and What can Their Differences Teach Us?" By Sandra E. Graham and Julia A. Peterson, Archives of Biochemistry and Biophysics, 1999; 369(1): 24-29

The variant or the non-conserved regions of the CYP enzymes are mainly in the amino terminal-anchoring domain, in the substrate recognition domain, or in the carrier transport-associating domain (Graham and Peterson, 1999). The structure specificity is in the variable helices "A, B, B', F, G, H, K', β sheets 3 and 4 and their adjacent loops" (Graham and Peterson, 1999). Gotah in 1991, categorized 6 presumed sites that occur mostly in the variable region of the CYP enzymes and regarded them as substrate recognition cites or (SRS), which comprise 16% of the total CYP enzyme residue (Gotoh, 1992). The substrate recognition sites are flexible and can repose upon the binding of the substrate (Gotoh, 1992)(Werck-Reichhart and Feyereisen, 2000). The binding of the substrate occurs in the variable substrate binding helices A, B, B', F, G and their associated loops. The SRS-1 is comprised of loops B-B', B'-C (Gotoh, 1992). The SRS-2 and SRS-3, which are components of the access channel and the ceiling of the active site, contain the F and G helices and their coupled loops (Gotoh, 1992). SRS-4 is usually in the central region of helix I and this region is mostly conserved in all the CYP enzymes. The N terminus of the β sheets 1-4 projects into the active site and this region of extension into the active site comprise the SRS-5. Finally the overhang of the β turn portion of the β sheet 4 into the active site comprises the SRS-6 (Gotoh, 1992; Graham and Peterson, 1999). The differences amongst the CYP enzymes ultimately relate to their corresponding substrates, which apparently exert an evolutionary pressure on the variability of the active site. Difference in the amino acid sequence in the SRS region of the various isoforms of the CYP enzymes has been correlated with substrate specificity (Danielson, 2002; Graham and Peterson, 1999). The conservation of SRS or lack of conservation of SRS can both be identified by performing a homology based amino acid substitution study. This study employs recognition of specific conserved amino acid region as markers to comprehend evolutionary divergence within the various CYP families of enzymes (Danielson, 2002). In addition to substrate specificity, the stability of the active site also varies within the different isoforms of the enzyme and has shown to have fluctuating degrees of flexibility (Anzenbacherova et al., 2000). Spectroscopic study executed under pressure and at varying temperatures on CYP3A4 and CYPBM3 enzymes show diverging results for the active site flexibility and protein denaturation. CYPBM3 (CYP102), whose crystal structure has been well characterized shows an immense flexibility in the active site upon substrate binding, nevertheless it is a very stable protein and not easily degraded (Anzenbacherova et al., 2000). In contrast, the CYP 3A4 shows relative flexibility upon substrate binding, but it is readily degraded to its inactive form under conditions of increasing temperature (Anzenbacherova et al., 2000). Consequently, the active site flexibility does not always correlate with the resistance to denaturation.

C. Eukaryotic CYP enzyme properties

The eukaryotic CYP enzymes are mainly found linked to a membrane (Williams et al., 2000). In the membrane anchoring range, these CYP enzymes have a sequence of proline residues that form a hinge. A bundle of basic amino acids flank the membrane-anchoring region between the hydrophobic N terminus and the region between the globular portions of the protein as shown in fig. 7 (Williams et al., 2000). The globular section contains the SRS variable region of the CYP enzymes (Werck-Reichhart and Feyereisen, 2000). The structural similarities between the soluble CYP enzymes and the membrane bound enzymes are remarkable due to the fact that the transmembrane N terminal region is the only defined membrane-anchoring region that sets the two major classes of CYP enzymes apart (Williams et al., 2000). The microosmal CYP enzymes have a signal recognition peptide at the N terminus of about 20-25 amino acids long (Williams et al., 2000). The eukaryotic CYP enzymes are co-translationally translocated to the endoplasmic reticulum by the signal recognition peptide (SRP) after they bind to the leader sequence on the N terminus of the nascent peptide (Sakaguchi, Mihara, and Sato, 1984). This N terminal leader sequence not only functions as a signal for the SRP but it is also a stop transfer signal (Sakaguchi, Mihara, and Sato, 1987). The N terminus signal is highly hydrophobic and halts the transfer of the nascent protein in the ER lumen, thereby anchoring the protein in the membrane and exposing most of the mature protein to the cytoplasmic portion of the cell. In addition, the mammalian microsomal enzymes form a hydrophobic surface that allows for "monofacial" interaction of the enzymes with the membrane (Williams et al., 2000). The enzymes interact with the microsomal membrane unilaterally, close to the N-terminus anchoring region, and this conformation allows for most of the enzymes to be buried in the lipid bilayer (Williams et al., 2000). The hydrophobic surfaces that are created by "non-contiguous" portions of the enzymes assemble in a way to form a stable catalytic domain that is more firm compared to the stability generated if the enzyme was only anchored at the N-terminus domain (Williams et al., 2000).

The mitochondrial CYP enzymes also have a leader sequence at their N-terminus. This motif serves as a signal for delay so that the catalytic domain is not improperly folded before the heme subunit is attached. This lag time is essential for maintaining the integrity of the catalytic domain of the mitochondrial CYP enzymes (Kusano, Sakaguchi, Kagawa, Waterman, and Omura, 2001). The hydrophobic region in the N-terminus is utilized to anchor the enzyme to the inner mitochondrial membrane once the protein has been translocated to the mitochondrion. The signal sequence is cleaved by a membrane protease once the enzyme has been translocated into the mitochondria (Kusano et al., 2001). The presence of this unique leader sequence in the N-terminal confers the eukaryotic CYP enzymes to either the microsomal or the mitochondrial membrane (Williams et al., 2000). Soluble CYP enzymes do not contain the N-terminus signal and therefore are not found associated to a membrane (P.B Danielson 2002)

Figure 7 Structure of the Eukaryotic CYP enzyme

(a) A ribbon drawing of the eukaryotic CYP2C5. The eukaryotic CYP enzyme shows similarities to the P450BM (bacterial CYP enzyme). The ER binding domain is shown in purple at the N terminus. The drawing is shown from the distal face. The heme is in orange and the substrate is in yellow. The I helix is on top of the heme, close to the substrate binding site. The back of the enzyme associates with the redox partner and transfers the electrons to the active site. (b) Primary structure of the P450 protein. Class (II) ER membrane bound CYP enzymes form a hinge with a cluster of basic amino acid residues followed b a hudle of Proline amino acid residues. The SRS are in the globular domain. The heme-binding domain has the conserved cysteine residue.

Reprinted from "Cytochrome P45: A Success Story?" By Daniele Werck-Reichhart and Rene Feyereisen, Genome Biology, 2000; 1(6): 3003.1-3003.9

(a) (b)

D. Classes of CYP Enzymes

The "redox partner" associated with the CYP enzymes that donates the electron from NADPH (reduced substrate) are commonly used to classify the CYP enzymes into four different classes (Mestres, 2005; Werck-Reichhart and Feyereisen, 2000). Class I consists of soluble CYP enzymes that are prominent in bacteria and are also found in the inner mitochondrial membrane of eukaryotes (Mestres, 2005). The redox partners in the Class I enzymes consist of flavin adenine dinucleotide (FAD) reductase and an iron sulfur protein (Mestres, 2005). Steroids and Vitamin D3 biotransformation and synthesis utilize predominantly Class I of CYP enzymes (Werck-Reichhart and Feyereisen, 2000). Class II enzymes are the most abundant in eukaryotes and are mainly associated with the membrane-bound microsomes. The redox partners for these enzymes are FAD containing reductase and flavin mononucleotides (FMN). Class I and Class II are major groups involved in xenobiotic transformation (Werck-Reichhart and Feyereisen, 2000). Class III enzymes are special in a sense they do not require a redox partner for electron transfer or molecular oxygen because they catalyze substrates that utilize electrons from integral oxygen (Mestres, 2005). Class IV of CYP enzymes utilize pyridine nucleotides for their electrons and also do not require electron carriers, as some of the classes of CYP enzyme (Mestres, 2005). Almost all classes of CYP enzymes are found in eukaryotes and there is no preferential distribution within the tissue (Anzenbacherova et al., 2000). The liver has the most abundant expression of the CYP enzymes; however, since their function is so diverse, they are found in almost all types of tissue throughout the body. This expression is regulated at development and can be influenced via induction, inhibition (Anzenbacherova et al., 2000; Werck-Reichhart and Feyereisen, 2000).

D. Cytochrome Families in Humans

There are 57 genes, which encode for the CYP family of enzymes in humans, are present throughout the body and are broken down into 18 families and 43 subfamilies (Meunier, de Visser, and Shaik, 2004). Most drugs are chemicals and have structural similarities to some if not many plant products or plant derived metabolites (Nebert et al., 1987). The most prominent CYP enzymes in drug metabolism fall under the category of CYP1, CYP2 or the CYP 3 and CYP4. The divergence of the P450 gene families (I, II, III, IV) of the cytochrome enzymes now prominent in animals link back to the exposure to many plants products dating to the past billion years (Nebert and Gonzalez, 1987). Humans, on a daily basis, consume and/or are exposed to many foreign substances such as pollutants, plant byproducts, halogenated hydrocarbons, arylamines, pesticides, herbicides, acid metabolites. They are also involved in endogenous reactions such as eicosanoid biosynthesis, cholesterol synthesis, steroid metabolism and synthesis, neuroamine and biogenic amine metabolism all use one of the four CYP gene families (Nebert and Russell, 2002). These CYP families consist of variant alleles and this polymorphism creates heterogeneity amongst individuals, which can alter drug metabolism and drug response. The heterogeneity is mainly due to the differential expression of these enzymes in the liver causing modified functional activity (Danielson, 2002).

  1. CYP1 Gene Family

The CYP1 gene family products are involved in metabolism of foreign chemicals, eicosonoids, and arachidonic acids and comprise CYP1A1, CYP1A2, and CYP1B1 (Brockmoller et al., 2000). The CYP1 family of genes is induced via the aromatic hydrocarbon receptor, a transcription factor that is activated in the presence of aromatic hydrocarbons and heterocyclic amines. The polycyclic aromatics are prominent in cigarette smoke, charcoal grilled foods and industrial incineration products (Brockmoller et al., 2000; Nebert and Gonzalez, 1987). CYP1A1 and CYP1A2 have 70% amino acid homology, however their tissue distribution is very diverse (Danielson, 2002). CYP1A1 enzyme is known as aryl hydrocarbon hydroxylase and is expressed in hepatic and extrahepatic tissues in varying amounts. This enzyme's major substrate is polycyclic aromatic hydrocarbons . CYP1A2, a hepatically expressed enzyme, is known to be important in metabolizing 10-20 drugs mainly aromatic hydrocarbon drugs (Brockmoller et al., 2000)(Nebert and Gonzalez, 1987). CYP1B1 is not as localized as CYP1A1 and CYP1A2 and is constitutively expressed in various different tissue types (Nebert and Gonzalez, 1987).

2. CYP2 Gene Family

This prevalent gene family originally called the "Phenobarbital inducible family" consists of the most extensively studied polymorphic enzymes (Nebert and Gonzalez, 1987). This functional classes of microsomal enzymes are comprised of 16 genes (Danielson, 2002). The CYP2 gene family is composed of subfamilies A, B, C, D, and E and is the largest of all the CYP families. The CYP2C subfamily of enzymes plays a vital role in metabolizing drugs that are prescribed on a regular basis, most importantly CYP2C8, CYP2C9, CYPC18 and CYP2C19 (Brockmoller et al., 2000; Nebert and Gonzalez, 1987; Nebert and Russell, 2002). CYP2D6 is known to metabolize well over 75 drugs on its own (Nebert and Russell, 2002).

3. CYP3 Gene Family

This family comprises four members: CYP3A4, CYP3A5, CYP3A7, and CYP3A43 (Nebert and Russell, 2002). These enzymes metabolize more than 120 drugs and mostly large amphiphilic substrates. They are found in hepatic cells as well as in the gastrointestinal tract. These groups of enzymes are easily induced by a transcription factor known as pregnane X factor or xenobiotic receptor. Pregnane binds to small ligands and becomes activated. This activated transcription factor can then bind to a corresponding response element on the gene and increase transcription of CYP3A family of genes (Nebert and Russell, 2002).

4. CYP4 Gene Family

This family consists of 12 members and is mainly involved in biotransformation of endogenous biochemical susbstances such as "fatty acid, arachidonic acid, leukotrienes, prostaglandins, epoxyeicosatrienoic acids, and hydroxyeicosatrienoic". CYP4 family of enzymes is also found to play a role in salt and water metabolism and consequently blood pressure (Nebert and Russell, 2002).

D. Cytochrome Biochemistry

The CYP proteins were once regarded as heme-thiolate proteins, however this distinctions to identify the CYP enzymes is no longer used (Anzenbacherova et al., 2000). This is due to the fact that many other physiological enzymes such as nitric oxide synthase and chloroperoxidases, also contain a heme-thiolate residue (Anzenbacherova et al., 2000). The heme group located in the inner core of the enzyme is a chromophore and is responsible for the enzymes red pigmentation (Danielson, 2002). The Iron (III) in the heme moiety can co exist between the low spin (S=1/2) and the high spin state (S=5/2). In the low spin state, a water molecule usually occupies the sixth ligand position on the heme- iron molecule forming a partial hexapeptide bond (Meunier et al., 2004). Binding of a substrate to the heme iron can causes a shift in the spin state of the iron from "penta-coordinated" species to a "hexa-coordinated species" (Meunier et al., 2004). The binding of the substrate causes the water molecule to be kicked out from the hydrophobic pocket of the enzyme, and subsequently the iron also undergoes a redox potential change from -300 to -170 mV (Meunier et al., 2004). This change is important because the associated reductase can now efficiently transfer the electrons to the CYP enzyme facilitating the overall mono-oxgenation reaction (Meunier et al., 2004). This change in the spin can be easily assayed by spectroscopy in the presence of carbon monoxide, which binds to the reduced iron (Fe2+). The CO bound CYP enzymes have a Soret absorbance, which peaks at 450nm, therefore giving the CYP enzymes their name as Cytochrome P450s (Danielson, 2002).

E. Reactions Catalyzed by the CYP Enzymes

The reaction catalyzed by the CYP enzymes involves substrates that are mainly hydrophobic. These enzymes can utilize oxygen and carry out multiple reactions such as hydroxylation of saturated bonds, epoxidation, reduction and oxidation reactions (Goeptar, Scheerens, and Vermeulen, 1995). Organic molecules cannot utilize oxygen at low temperatures due to spin barriers of oxygen when it is in its lowest singlet state, therefore living systems require enzymes that can activate dioxygen for certain oxidation reactions (Filatov et al. 2000; Meunier et al. 2004. CYP enzymes can regiospecifically and stereospecifically catalyze reaction that would otherwise require extremely high temperatures (Werck-Reichhart and Feyereisen, 2000). The CYP enzymes are capable of binding an oxygen molecule and activating it (Mestres, 2005). The activated oxygen then forms a reactive oxygen species that can attack stable substances, such as hydrocarbons and aromatic rings. The monoxygenation reaction by CYP enzymes utilizes an electron carrier or "redox partner" to mediate the flow of electrons from a reduced substrate such as NADPH to the oxygen molecule (Anzenbacherova et al., 2000). This flow of electron from the electron carrier to the oxygen results in the activation of the oxygen molecule (Anzenbacherova et al., 2000; Mestres, 2005). The CYP enzyme then introduces one of the activated oxygen atoms into the substrate, which is bound to the heme-iron as the sixth ligand on the protoporphyrin ring (Sheweita, 2000), (Meunier et al., 2004). The other oxygen moiety is then released as a water molecule (Sheweita, 2000).

In this way the CYP enzymes can modify exogenous substance to make them hydrophilic and easily extractable (Meunier et al., 2004). This biotransformation consists of two distinctive phases, Phase I, which is conducted primarily by the CYP enzymes and Phase II, which is executed by various transferases (Meunier et al., 2004). Phase I reactions mainly consists of reactions such as hydroxylation, epoxidation, oxidation of aromatic rings and oxidation of heteroatoms, which make the substrates hydrophilic and Phase II includes conjugation reactions (Sheweita, 2000):

F. Catalytic Cycle of CYP enzymes

Most of the catalytic mechanism has been studied on soluble CYP proteins, which have several well-defined crystal structures (Werck-Reichhart and Feyereisen, 2000). The catalytic cycle comprises four distinct steps starting with the binding of the substrate to the active site of the CYP enzyme as shown in fig. 8 (Werck-Reichhart and Feyereisen, 2000):

(1) The binding of the substrate has a relatively slow rate of reaction (Meunier). The binding of the substrate initiates the catalytic cycle, subsequently causing the bound water molecules at the sixth position of the heme-iron to be dislodged. This dehydration reaction causes an increase in entropy and is also thermodynamically favorable (Griffin and Peterson, 1972). The thermodynamic favorability arises from the fact that water molecules bound to the heme-iron are distributed over a large surface area and are highly mobile (Meunier et al., 2004). The substrate binding to the active site also pushes the iron out of the plane of the porphyrin ring creating a shift from 0.30 Å to 0.44 Å. The shift in the position of the heme-iron generates a suitable environment for the electron transfer to transpire from the redox partner to the enzyme (Meunier et al., 2004). The electrons are then transferred from the redox partner to the iron center, changing the oxidation state of the heme iron from to (+) 3 to (+) 2. The reduced heme-iron makes the CYP a potent reducing agent. The sulfur-iron bond between the heme-iron and the cysteinyl sulfur changes as the iron goes from the ferric state to the ferrous state. (2) Dioxygen then covalently binds to the distal axial position of the heme-iron. The iron (II) then binds an electron of dioxygen molecule forming an iron (III)-O complex intermediate. This bond can split forming an oxygen reactive species (superoxide anion) (Meunier et al., 2004). This constitutes what is known as a decoupling reaction and can halt the catalytic cycle of the CYP enzyme. The oxygen reactive species formed are detrimental to the system as most can be carcinogenic (Loida and Sligar, 1993). The rate-determining step of most CYP reactions is the transfer of the second electron to the ferrous-dioxygen bond (Meunier et al., 2004). This step generates negative charged anion known as the peroxo group bound to iron (III) moiety (Meunier et al., 2004). This intermediate is fairly short lived and readily gets protonated to form iron (III)-hydroperoxo complex. The peroxo and the hydroperoxo complex are excellent nucleophiles. Subsequently, the hydroperoxo terminal oxygen accepts another proton forming an irono-peroxocomplex (Meunier et al., 2004). Consequently the O-O bond cleavage results in the formation of a water molecule with the aid of two electrons and two protons. One of the cleaved oxygen atom stays bound to the iron moiety, forming a ferryl oxygen species, which is eventually transferred to the substrate (Meunier et al., 2004).

Figure 8 Catalytic Mechanism of P450

The substrate binding initiates the catalytic cycle, the dixogen molecule is split with one molecule of oxygen being inserted into the substrate and the other being released as water.

Reprinted from "Cytochrome P45: A Success Story?" By Daniele Werck-Reichhart and Rene Feyereisen, Genome Biology, 2000; 1(6): 3003.1-3003.9

The CYP catalyzed reaction is broadly divided into four major categories depending on the microenvironment where the oxygen residue is introduced (Goeptar et al., 1995). Hydroxylation is when a hydroxyl group is substituted by a hydrogen atom, epoxidation consists of insertion of an oxygen atom between two carbon residues leading to formation of an epoxide molecule, heteroatom oxidation reaction is when an oxygen is added to a nitrogen or a sulfur molecule and finally reduction reactions, which occur in the oxygen depleted environment (Goeptar et al., 1995).

Most CYP catalyzed reactions involve hydroxylation reactions. The carbon atoms in aliphatic and polycyclic hydrocarbons are prone to get hydroxlated by the CYP enzymes. This reaction is most prevalent in the metabolism of xenobiotics. The hydroxylation reaction requires an insertion of an oxygen atom in between a carbon and a hydrogen bond of a hydrocarbon. The position where the oxygen gets inserted in the hydrocarbon chain is determined predominantly by thermodynamic factors (P.B Danielson 2002). The carbon hydrogen bond is highly reactive in its tertiary state compared to when it is in primary bond formation (Frommer, Ullrich, and Staudinger, 1970; White, McCarthy, Egeberg, and Sligar, 1984). Epoxidation carried out by the CYP enzymes involves olefins, which get converted to their respective epoxides (P. R. Ortiz de Montellano, Stearns, and Langry, 1984). In rare occasions, the toxicological property of the substrate can be affected if epoxidation is not concerted and the catalysis leads to "suicide inactivation" of the enzyme (P. R. Ortiz de Montellano et al., 1984). In suicide inactivation, the pyrrole nitrogen of the heme group assoociated with the CYP enzyme can be N-alkylated by the terminal carbon of the olefin (P. R. Ortiz de Montellano et al., 1984). The CYP enzymes are also capable of hydroxylation of aromatic hydrocarbons such as benzene rings. The biotransformation of aromatic rings is critical in understanding the metabolite profile generated via the CYP monooxygenases in relation to drug detoxification (Danielson, 2002). Therefore, this area of research has been prominent in many laboratories and continues to be so in regards to toxicological significance of some of these aromatic drugs (Danielson, 2002).

Finally the CYP enzymes easily catalyze oxidation of heteroatoms such as nitrogen and sulfur (Danielson, 2002). Nitrogen and sulfur are readily oxidized forming their corresponding oxides, N-oxide and sulfoxide metabolites. Nitrogen and sulfur have free electrons and are easier targets to be oxidized by the CYP enzymes in comparison to oxygen, which is highly electronegative (Danielson, 2002). For example, Tylenol-Acetaminophen (4-hydroxy acetanilide) can be biotransformed by the CYP enzymes via the heteroatom oxidation pathway (Dong, Haining, Thummel, Rettie, and Nelson, 2000). Acetaminophen is safe at therapeutic doses, however in case of an overdose-CYP mediated N-hydroxylation reaction becomes a prominent pathway for detoxification of Tyelonol. This pathway generates a reactive metabolite N-acetylbenzoiminoquinone, which in the presence of glutathione (GSH), is readily conjugated. However in the absence of hepatic glutathione, the reactive metabolite can accumulate (Maria Almira Correia, 2009). This nucleophillic metabolite has the ability to alkylate proteins in the liver consequently leading to liver necrosis (Dong et al., 2000).

G. Regulation/ Modification of CYP enzymes

The CYP enzyme levels can be regulated and or/modified by induction, inhibition and activation. These modifications can be detrimental in subjects with variant alleles as they can affect the levels of the enzymes, consequently affecting the metabolism of the substrate (Hollenberg, 2002). Simultaneous administration of a drug that utilizes the same CYP enzyme can lead increased competition of the specific enzyme. This competition can lead to increased levels of one or both the drugs, leading to adverse drug reactions, even death (Ferslew, Hagardorn, Harlan, and McCormick, 1998; Kudo et al., 1997). This affect can be elevated if the person has polymorphism for the targeted CYP enzyme (P. R. Ortiz de Montellano and Correia, 1995). The inhibitors of the CYP enzyme tend to be specific; however, some inhibitors can block a wide range of CYP enzymes. The basic type of inhibitors include i) "compounds that bind the reversibly ii) compounds that form a "quasi irreversible" complex with the iron of the heme prosthetic group iii) compounds that bind irreversibly to the prosthetic heme or to the protein, or that cause covalent binding of the heme prosthetic group or its degradation product to the apoprotein" (Hollenberg, 2002). The CYP enzymes induction was first documented in relation to an environmental toxicant 2,3,7,8-tetrachlorodibenzo-p-dioxin (Whitlock, 1999). The CYP enzymes are capable of being induced when they are exposed to distinct substrates. These substrates could surprisingly upregulate one or more isoforms of CYP enzymes and increase their own capacity (Conney, 1967). Induction may have evolved so that, physiological system can efficiently deal with the xenobiotics (Whitlock, 1999). Although most of the CYP enzymes are regulated/induced by the xenobiotics, there are certain isoforms that are developmentally regulated and are tissue/sex specific (Hollenberg, 2002). Regardless of the mode of induction, the baseline regulation is always at the level of transcription (Whitlock, 1999). Induction of an enzyme causes in an increase in the levels of the enzyme and frequently due to increased transcription activity for the gene encoding the enzyme (Hollenberg, 2002). Since induction mainly occurs at the level of transcription, the increased level of the enzyme is not seen instantaneously. The CYP enzymes can also be activated by the presence of certain substances and this can result in increased activation of the catalytic activity of the enzyme. The overall effect is more or less instantaneous and the outcome resembles of what one might find in the inductory pathway (Hollenberg, 2002).

Genetic variation, in addition to induction, inhibition and activation of the CYP enzymes can have profound effect on biotransformation of drugs. Polymorphisms in certain CYP subfamilies are documented and are shown to contribute to the variability in the levels of CYP enzymes.

III. CYP2D6

A. Background

Variation in the CYP enzymes can affect how an individual responds to a drug in vivo. The variation can stem from induction, inhibition and most commonly, from mutation. This is prevalent in the CY2D6 isoforms, which carry out about 30% of drug oxidation (Phase I) reactions in vivo (Ingelman-Sundberg, 2005; Rendic, 2002). CYP2D6 comprises 2-4% of the hepatic content of all the CYP enzymes and is known to metabolize 20-25% of drugs currently in market (Cascorbi, 2003; Ingelman-Sundberg, 2005). The major substrates of CYP2D6 enzyme are distinct as they all contain basic nitrogen and an aromatic ring, which binds to the active site of the enzyme (Marechal et al., 2008). This basic nitrogen is shown to interact with the acidic amino acid (Glu 216) in the active site of the enzyme located on the F helix as show in Fig 9. Most cardiovascular and central nervous system drugs contain the basic nitrogen or the aromatic ring and are mainly metabolized by the CYP2D6 enzyme (Marechal et al., 2008). The substrates for the CYP2D6 are mainly "lipophillic bases and include some antidepressants, antipsychotic, antiarrythmics, antiemetics, b blockers and opioids" (Zhou, 2009). The enzyme has a relatively low Kd and gets maximally saturated at low concentrations due to it high affinity for its substrate (Zhou, 2009). In addition, CYP2D6 show varying degrees of first-pass elimination to all its substrates after oral administration (Zhou, 2009). CYP2D6 is not inducible by steroids, cigarette smoking and over the counter contraceptives like most of the other CYP enzymes; however, other drugs are capable of inhibiting it, causing severe drug-drug interaction (Bock et al., 1994; Zhou 2009). CYP2D6 gene is known to have 74 variant allelic forms, which confer to a wide range of enzyme activity (Human Cytochrome P450 (CYP) Allele Nomenclature Committee, 2008). Mahgoub and his group first discovered the polymorphic characteristic of CYP2D6 in 1977 when they found deviations amongst different ethnic groups in their ability to metabolize debrisoquine (Mahgoub et al., 1977). The alleles possess a gradient of functional ability; they can be fully functional and metabolize drugs normally, show a decrease in enzyme function, or possess no enzymatic activity (Zhou, 2008). This variation in alleles can consequently generate phenotypes ranging from poor metabolizers to ultra rapid metabolizers and show a range in inter-individual drug clearance (Zanger, Raimundo, and Eichelbaum, 2004; Zhou et al., 2008). This variance in allele can then alter drug responses and a patient can receive no therapeutic effect or, on the flip side, experience adverse effects (Zhou, 2009). In a study done on a Caucasian population of 589 unrelated German volunteers, the percentages of variant phenotypes measured with debrisoquine or dextromethorphan revealed the following results: ultra-rapid metabolizers comprised 3-5% of the study population, extensive metabolizers made up 70-80% of the population, 10-17% were intermediate metabolizers and 5-10% were poor metabolizers (Sachse, Brockmoller, Bauer, and Roots, 1997). Poor metabolizers are very uncommon in the Chinese population, comprising of only 1% of the polymorphic population according to a study done on 124 Chinese residents living in Taiwan (Wang, Huang, Lai, Liu, and Lai, 1993). Among African Americans poor metabolizers consist of approximately 5% of the population (Nebert, 1997). Ultra rapid metabolizers arise due to an extra functional gene, which is prevalent in 1-2% of the Swedish population with a MR of 0.1 and 16% of Black Ethiopians with a MR of (0.9-5) (Aklillu et al., 1996; Dahl, Johansson, Bertilsson, Ingelman-Sundberg, and Sjoqvist, 1995). The presence of two non-functional alleles results in a poor metabolizing gene product, whereas extensive metabolizing enzymes result from the presence of one or two additional copies of the normal gene (Zanger et al., 2004; Zhou et al., 2008). An intermediate metabolizing enzyme results from a non-functional gene and an extra copy of a gene with decreased function. This variability in gene frequency, although prevalent in varying degrees in different ethnic groups, does not pinpoint the exact outcome of the genotypic variation with the observed phenotype (Zanger et al., 2004; Zhou et al., 2008)

Figure 9 Crystal Structure of CYP2D6

Reprinted from "Insights into drug metabolism by cytochrome P450 from modeling studies of CYP2D6-drug interactions"By J.D Marechal et. al, British Journal of Pharmacology, 2008; (153): S82-S89

B. CYP2D6 Locus

The CYP2D6 in vivo is charted on the long arm of chromosome 22 in the region of q13.1 (Eichelbaum et al., 1987)(Gough et al., 1993). The CYP2D6 gene is 4378 base pairs long coding region that contains 9 exons with an open reading frame of 1491 base pairs (Kimura, Umeno, Skoda, Meyer, and Gonzalez, 1989). This reading frame accounts for a 497 amino acid polypeptide chain (Heim and Meyer, 1990). The CYP2D6 gene is nestled in a cluster of homologous pseudogenes CYP2D8 and CYP2D7P (Kimura et al., 1989). The CYP2D genes have shown differential evolution pressures between rodents and humans. In rodents there are 9 active CYP2D genes, however, in the humans, there is only one active gene, which has been proposed to undergo detrimental mutations over time resulting from gene conversion (D. R. Nelson et al., 2004; Kimura et al., 1989). The differential selection in rodents is due to the immense amount of dietary detoxification required on a daily basis in comparison to humans, who have altered their diets over generations to avoid toxic substances (Zhou, 2009). The evolution of CYP2D6 in humans is most likely due to gene duplication that also gave rise to CYP2D7P pseudogene (Kimura et al., 1989).

C. Prototypical Substrate for CYP2D6

The archetypal substrates for CYP2D6 are sparteine and debrisoquine as these drugs have been used quite often to determine the phenotypical variability in patients (Zanger et al., 2004). Sparteine is broken down to N-oxide via the N-oxidation reaction (Eichelbaum, Spannbrucker, and Dengler, 1975). The break down product, N-oxide, can form 2-dehydrosparteine or 5-dehydrosparteine via a dehydration reaction (Osikowska-Evers, Dayer, Meyer, Robertz, and Eichelbaum, 1987). The rearrangement and formation of 5-dehyrosparteine showed a 32-fold difference between poor metabolizers and extensive metabolizers in monophasic Michealis-Menton kinetics (Osikowska-Evers et al., 1987). Since poor metabolizers or (non-metabolizers) cannot undergo the N-oxidation reaction for sparteine effectively; most of the clearance is dependent on the kidney, which accounts for 99.9% excretion of the drug (Eichelbaum, Spannbrucker, and Dengler, 1979).

Debrisoquine is an antihypertensive agent and it is metabolized by the CYP2D6 via a hydroxylation reaction, which was discovered by administering 20 healthy Swedish Caucasians and oral dose 20mg of the drug (Eiermann et al., 1998). The metabolites of the hydroxylation reaction are as follows: 4-hydroxy-debrisoquine, 3- hydroxy-debrisoquine and 1- hydroxy-debrisoquine (Eiermann et al., 1998). The CYP2D6 phenotype is determined by calculating the metabolic ratio of 4-hydroxydebrisoquine in urine sampled of fasting subjects over a period of 8 hours. The metabolic ratio was determined by dividing the percent dose debrisoquine over percent dose as 4-hydroxydebrisoquine (D. A. Evans, Mahgoub, Sloan, Idle, and Smith, 1980). The CYP2D6 has a bimodal distribution with an antimode metabolic ratio of 12.6 in the Caucasian population to distinguish between poor metabolizers and extensive metabolizers (Bertilsson et al., 1992). In a study conducted on 695 Chinese subjects, less than 1% qualified as poor metabolizers based on the 12.6 antimode distribution normally employed for Caucasians. The metabolic ratio for extensive metabolizers in the Chinese population is higher, consequently lowering the mean value of the CYP2D6 activity (Bertilsson et al., 1992). Although the use of sparteine and debrisoquine is inexpensive and fairly effective in testing for CYP2D6 variant, these drugs are no longer available in the market for phenotype testing (Zhou, 2009).

Dextromethorphan, used as an over-the counter antitussive agent, is a synthetic analogue of a narcotic agent. Dextrorphan, a metabolite of dextromethorphan, is a pharmacologically active agent and is formed by O-methylation reaction by the CYP2D6 (Dayer 1989). The human liver microsomes assayed in vitro showed that dextromethorphan associates with debrisoquine like phenotypic variations (Dayer, Leemann, and Striberni, 1989). Dextromethorphan is a probe drug frequently used to assay the CYP2D6 activity in vivo and in vitro (Zhou, 2009). Dextromethorphan has a bimodal distribution and the antimode of 0.3 is commonly used in Caucasians to distinguish between poor metabolizers and extensive metabolizers (Schmid, Bircher, Preisig, and Kupfer, 1985). A serum assay conducted by employing an antimode value of 0.126 is far efficient in determining the distinction between IM and PM. The MR of 0.126 decreases the overlap between PM and IM (Kohler et al., 1997)

Bufuralol is also frequently used as a probe drug for in vitro study phenotype assay for CYP2D6 (Zanger et al., 2004). Bufuralol is metabolized in the hepatic microsomes by the CYP2D6 into three metablites: 1-hydroxybufuralol, 1-oxobufuralol, and 1'2'-ethenylbufuralo. The hydroxylation reaction was inhibited when antibodies against the CYP2D isoforms were applied (Hiroi, Chow, Imaoka, and Funae, 2002). The levels of 1-hydroxybufuralol are employed to assay the CYP2D6 activity in vitro and low levels of this metabolite relates to poor metabolizer phenotype (Carcillo et al., 2003). Bufuralol and dextromethorphan are both commonly used in vitro probe drugs to assay CYP2D6 (Yuan, Madani, Wei, Reynolds, and Huang, 2002). The pharmaceutical industry investigators preferentially use bufuralol over dextromethorphan to assay for CYP2D6 phenotypes (Yuan et al., 2002).

D. Genetic Variation of the CYP2D6 and Effects on the Enzyme Activity

There are four known distinctive categories of the CYP2D6 enzymes ranging from PM (poor metabolizer), IM (intermediate metabolizer), EM (extensive metabolizer) and Ultra-rapid metabolizers (UM) (Zhou, 2009). The PM and UM show a 5 to 15 fold difference in rate of metabolism (substrate to metabolite) ratio in comparison to the EM. However, the comparison between IM and EM is not as distinctive due to complexity and overlay of the alleles. The clinical significance in targeting the PM and UM phenotypes are pivotal in regards to drug dosage; the UM phenotype, however, requires further research in order to draw meaningful conclusion for genotyping (Zhou, 2009).

In a liver microsomal sample of 20 subjects of EM, there was more than 25 folds divergence in the breakdown of bufuralol into 1-hydroxybufuralol and correspondingly the concentration of mRNA coding for CYP2D6 showed similar fold increase. The levels of mRNA were measured by employing an oligonucleotide probe. This allows for accurate measurement of the CYP2D6 mRNA alone and excludes the surrounding pseudogenes (Carcillo et al., 2003). Peripheral blood mononuclear cells of 78 healthy subjects showed similar fold differences in the catalytic activity of the CYP2D6 in the breakdown of debrisoquine and also in the levels of the CYP2D6 mRNA transcript (Carcillo et al., 2003).

Drugs who get primarily cleared by the CYP2D6 enzyme show enormous discrepancies in the area under the plasma-concentration curve (AUC). This discrepancy was primarily eminent in comparative analysis of PM to EM (Zhou, 2009; Gibbs, Hyland, and Youdim, 2006). Gibbs and his team showed that "for drugs having <60% CYP2D6 involvement in vivo, a modest difference between EM and PM exposure was observed (<2.5-fold). For major CYP2D6 substrates (>60%), more dramatic exposure differences were observed (3.5- to 53-fold)"(Gibbs et al., 2006). Despite the fact that CYP2D6 is responsible for a wide range of variability in drug metabolism, there are other factors such as "co-medication, disease status, liver and renal failure" which can also have a profound effect on the overall enzyme activity (Zhou, 2009). These factors have to be accounted for before initiating a drug dosage (Zhou et al., 2008). In a study done by Ebner, subjects who were administered a 50 mg dose of quinidine showed increased inhibition for the CYP2D6 enzymes. This competitive inhibition also resulted in a phenomenon knows as "phenocopying" where an individual with an extensive metabolizing phenotype showed characteristics of a poor metabolizer (Ebner and Eichelbaum, 1993). Certain epidemiology studies have also shown that cigarette smoking in EM is associated with increased risk of bladder, pharynx, liver and stomach cancer due to accumulation of carcinogenic metabolites (Nebert D. W, 1997).

E. CYP2D6 Gene Alleles

The Human Cytochrome P450 Allele Nomenclature Committee has specified over 74 variant alleles (*2 to *75) for the CYP2D6 gene. *1A is the wild type or the normal allele (Human Cytochrome P450 (CYP) Allele Nomenclature Committee, 2008; Zhou, 2009). The National Center of Biotechnology consists of 196 SNP for humans on file, of which atleast 52 are non-synonymous SNP that have shown to alter the protein function (http://www.ncbi.nlm.nih.gov/sites/entrez; Wang et al., 1993). The non-synonymous SNP can functionally alter the proteins and these can broken down into three broad categories: alleles that cause an increase in protein function, alleles that cause a decrease in protein function or loss of function, alleles that have cause proteins normal function (Zhou, 2009). "These variants result from point mutations, deletion or additions, gene rearrangements an deletion or duplication/multiduplication of the entire gene" (Zhou, 2009).

A) Non-functional Alleles

CYP2D6 isoform arise from a set of alleles that are known as non-functional alleles and encode for a protein with no activity. The following alleles are known to have no enzymatic activity in 589 unrelated German volunteers by using dextromethorphan or debsrioquine as a probe drug: *3-*8, *11-*16, *18-*21, *38, *40, *42, *44, *56 and 62 (Sachse et al., 1997; Zhou, 2009). The alleles when present in collections of homozygous or heterozygous forms are responsible for the PM phenotype (Zhou, 2009). The PM in the Caucasian population of included 41 out of the 589 German volunteers, of which about 97% of the PM phenotype caused by alleles 3 to 6 (Sachse et al., 1997). There are several ways in which null allele can lead to non-functional enzyme: a point mutation or insertion or deletion of a base can shift the reading frame and/or the incorrect splicing, leading to an improperly folded protein (Kagimoto, Heim, Kagimoto, Zeugin, and Meyer, 1990; Zhou, 2009), a fully functional allele with non-functional protein activity (Evert, Eichelbaum, Haubruck, and Zanger, 1997), or "large sequence deletion" which can lead to formation of a hybrid gene such as a blend of CYP2D6 with the pseudogene CYP2D7 (Gaedigk, Blum, Gaedigk, Eichelbaum, and Meyer, 1991)(Zhou, 2009). An example of single base mutation was exemplified in a study done on French Caucasians, which showed that a point mutation in exon 1 resulted in complete loss of function of the protein and caused a PM phenotype. This mutation is now characterized as being the allelic variant CYP2D6 * 12 (Marez et al., 1996; Zhou, 2009). The CYP2D6 enzyme is subjected to various types and degree of mutations that can create a host of proteins with diverse activity ranging from no enzymatic activity to increased activity.

B. Alleles with Partial or Reduced Function

The reduced function of the CYP2D6 enzyme can occur due to "decreased protein stability, disrupted substrate recognition and reduced substrate-enzyme affinity" (Zhou, 2009). In some cases, the activity of the enzyme is co-dependent on the specific substrate and therefore an individual with any of the above can be categorized as IM. Since the distinction between IM and PM phenotypes is not clear, most individuals can be regarded as having borderline PM and IM phenotypes (Zhou, 2009). CYP2D6 *10 is a frequently occurring allelic isoform in the Asian population causing them to be IM (Ji et al., 2002). This CYP2D6 is prevalent in the Japanese, Korean, Chinese and Pacific Islanders and occurs at a frequency of about 33-43% (Bradford, 2002; Ishiguro, Kubota, Sasaki, and Iga, 2004; Ji et al., 2002; Johansson et al., 1994). This IM phenotype is not as prevalent in Caucasians and African Americans and occurs at a frequency of 2-5% (Bradford, 2002; Leathart et al., 1998). The SNP that gives rise to the *10 variant is due to 100 CàT causing a P34S substitution in the proline rich region (Zhou, 2009). This proline rich region is highly conserved in the CYP enzymes, consequently individuals with this allelic form are known to have reduced ability to clear sparteine and have a metabolic rate of > 1.5 (Yokota et al., 1993; Zhou, 2009). In comparison, individuals who have the allelic variant *17 also are known to be IM. However, this allele is very rare in Caucasians but quite prevalent in the African American community (Bradford, 2002). This explains why the metabolic rate (MR) median for this allele is higher in Africans in comparison to Caucasians (Leathart et al., 1998).

C. Alleles with Multigene Duplication

If a subject has duplicated gene, this could lead to ultra-rapid metabolizers (Zhou, 2009). A study done on a Caucassian family with two siblings and the father showed that 12 extra copies of the CYP2D6 gene was inherited in a dominant autosomal fashion. The extra copies result in UM phenotype with a MR of <0.02 for debrisoquine (Johansson et al., 1993). This variant 12 fold amplitude gene is known as CYP2D6L and results in two mutations, one in exon 6 causing Arg 296 to be replaced for a Cys and another mutation in exon 9 causes Ser 486 to replaced for Thr (Johansson et al., 1993). Individuals with an increased number of this variant gene are shown to have increased activity of the enzyme and decreased metabolic ratio for the substrate (Johansson et al., 1993). High levels of enzyme activity result from gene duplication of the alleles *1 and *2 fused in head to tail orientation (Gaedigk et al., 2007). The UM phenotype results primarily in North East Africa with a gene duplication of the *2 X N (N=2, 3, 4, 5, or 13) and Oceania with the gene duplication of the *1 X N variant allele (Sistonen et al., 2007; Sistonen et al., 2009). The PM can be easily distinguished by employing the 12.6 antimode, however the distinction for UM like IM is ambiguous because of the overlap (Zhou, 2009). Individuals with the UM phenotype can accumulate metabolites readily and can result in therapeutic failure due to lack of response (Zhou, 2009). In individuals with UM phenotype, there can be a 10-30 fold increase in the metabolite concentration leading to potential toxicity and adverse drug reaction (Dalen, Frengell, Dahl, and Sjoqvist, 1997). For example, odiene is a commonly used analgesic and is shown to have enhanced effects in subjects who are UM. The AUCs for morphine differs significantly for EM and UM. The UM are sedated in a shorter duration of time in comparison to the EM and PM (Kirchheiner J et al., 2007). Physicians need to be aware of such variant gene before prescribing codeine to UM and PM, since the dose requirements may vary depending on the phenotype.

E. Ethnic Phenotypic Variation in CYP2D6

CYP2D6 polymorphism is prevalent around the world and differs drastically in the frequency of the phenotypes as shown in Fig 10. For example, Caucasians have the highest occurrence of PM phenotype mainly the caucasians of Britain and Switzerland, with 8.9% and 10% respectively (Alvan, Bechtel, Iselius, and Gundert-Remy, 1990). The Caucasian population in Europe is well characterized and has the highest PM frequency of (8%) with an equivalent bimodal distribution (Zanger et al., 2004). Overall, 5-10% of the Caucasian population are regarded as poor metabolizers with CYP2D6*4 as the most common variant allele occurring at a frequency of 20% (Bradford, 2002). The incidence of PM is low in the Asian population (0-1.2%) in comparison to the Indian populations with a frequency of about (3%) and has a slight bimodal distribution (Lamba, Lamba, Dilawari, and Kohli, 1998). The second most widespread phenotype is the UM which occurs in the North African, Oceania, Middle Eastern & American (Caucasian & Black) population in 40, 26, 12 & 8% frequency respectively (Sistonen et al., 2007). For example: the Black Ethiopians and Saudi Arabians have the UM frequency of 29 and 20% (21 out of 101) respectively making it one of the most prevalent UM frequency observed. The UM in the Black Ethiopians was due to duplicated gene with three or four copies of the CYP2D6*2L allele. The UM in the Saudi Arabians is due to gene duplication caused by dietary pressures over time (Aklillu et al., 1996; McLellan, Oscarson, Seidegard, Evans, and Ingelman-Sundberg, 1997). The American and Oceania subgroup lack the PM phenotype and belong mainly to the UM and the EM phenotypes (Sistonen et al., 2007). The IM phenotype is caused mainly due to the variant alleles *10, *17 and *41 (Sistonen et al., 2007). The Chinese, Japanese and Koreans contain the *10 variant, which is categorized as the intermediate phenotype. This allele occurs with a frequency of 35-55% (Wang et at., 1993). The *17 allele is prevalent in the African American and Black population and results in the higher median MR (Leathart et al., 1998). The allele *41 is prevalent in the Middle Eastern community (Sistonen et al., 2007).

Figure 10. Phenotype Distribution of CYP2D6 in various geographical areas.

Reprinted from "CYP2D6 worldwide genetic variation shows high frequency of altered activity variants and no continental structure"By Sistonen, Johanna et al., Pharmacogenetics and Genomics, 2007; (17)2:93-101

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