Pharmacogenetics: Hidden Phenotype and Medication

1 Pharmacogenetics: Hidden Phenotype and Medication Maria Elena Jolly University of Lisbon 2012  2 Pharmacogenetics: Hidden Phenotype and Medication Introduction The purpose of this presentation is to discuss current developments in pharmacogenetic research and its implications for clinical practice using the example of antidepressant response. The sequencing of the human genome completed in 2003, a major breakthrough in modern science, has significantly advanced understanding of genetic influences on therapeutic response to medication and gave rise to a new discipline – pharmacogenetics. Analyses of the results of the Human Genome Project are still continuing and have yielded new data leading to new approaches in pharmacology, one of which is identification of genetic differences among patients regarding their response to various pharmaceuticals. Further study of interindividual variability in DNA sequence related to drug response is expected to eventually make truly personalized medicine a reality. Why Study Pharmacogenetics? At the beginning of the 20th century Archibal Garrod was the first to recognize the connection between genes and biochemical reactions. In his research on rare metabolic disorders, he discovered that faulty metabolism of some compounds could be caused by deficiencies in appropriate enzymes, an inherited condition he called the “inborn errors of metabolism”1 (Cummings, 2003, p. 249). Even though the relationship between proteins and phenotype has been known ever since, rigorous pharmacogenetics studies did not begin until almost a hundred years later. The delay can be explained by the lack of interdisciplinary approach that would combine research in genetics, medicine, and pharmacology. The main goal of the current genetic approach in pharmacology is to determine the hereditary foundations for a wide range of reactions to therapeutic drugs observed in clinical practice and use this knowledge for optimization of treatment according to the unique genetic makeup of each patient. In practical terms this means control of the dosage of the drug in view of genetic peculiarities of the organism to ensure the drug efficacy, to minimize toxicity, and to prevent unexpected side effects. 1 Garrod presented his conclusions in his famous book Inborn Errors of Metabolism in 1908.  3 The need to approach medical treatment from a more individualized perspective is also justified by safety concerns as adverse drug reactions (ADRs) constitute a serious clinical issue. For example, in 1994 in the US hospitals over 100 thousand patients had fatal ADRs and 2.2 million had serious ADRs (Lazarou, Pomeranz, & Corey, 1998). In view of such grave outcomes, the US Food and Drug Administration (FDA) has taken into consideration accomplishments in pharmacogenetics and introduced pharmacogenomic labeling information for medications (Perlis, 2007). In the past decade, a paradigm shift from the concept “one drug fits all” to a more personalized perspective has been observed in drug treatment approaches; however, there has been insufficient clinical benefit so far. Even though there is a general agreement in the medical community with Garrod’s view of every patient as a biochemically unique individual due to their genotypes, slow progress in pharmacogenetic research and economic considerations have held back the implementation of personalized approach to drug treatment. Pharmacogenetics and Pharmacogenomics Pharmacogenetics and pharmacogenomics refer to the study of the role of heritable factors in individual variation in patients’ response to medication - “a phenotype that varies from potentially life-threatening adverse drug reactions to equally serious lack of therapeutic efficacy” (Wang, 2010). There is no consensus among researchers on the exact definition of pharmacogenetics and pharmacogenomics. For example, Pinto (2011) defined pharmacogenetics as a “study of genetic factors that influence the functioning of pharmaceuticals” and pharmacogenomics as “apparently new science that makes a systematic identification of all human genes, their products, and individual variability with the goal to predict an appropriate treatment of a certain individual and to design new pharmaceuticals”.2 Porcelli et al. (2011) offered the following definition of pharmacogenetics: “the study of how an individual’s genetics affects his or her response to drugs, combining traditional pharmaceutical sciences, such as biochemistry, with annotated knowledge of genes, proteins and single-nucleotide polymorphisms (SNPs)”. Rosania (2005) proposed a straightforward definition of pharmacogenomics as “using genetic information to predict whether a drug will help make a patient well or ill”, adding that this field of study combines genetics, genomics, 2 In the original Portuguese text: “Farmacogenética estuda factores genéticos que influenciam o funcionamento dos fármacos” and “Farmacogenómica - aparentemente uma nova ciência que: faz uma identificação sistemática de todos os genes humanos, dos seus produtos e variabilidade interindividual com o objectivo de prever o tratamento certo para um determinado individuo e de desenhar novos fármacos.” (Luís Abegão Pinto, 2011)  4 molecular biology, pharmacology, pharmaceutics, toxicology, population biology, and statistics. For the purpose of this paper pharmacogenetics is defined as the study of interindividual genetic variations in DNA sequence that underlie drug response and pharmacogenomics as the individualized drug therapy in clinical practice based on the unique genetic makeup of the patient (Cummings, 2003). Genetic individuality regarding metabolism of various chemical compounds is a theoretical truism, but practical evaluation of the metabolic idiosyncrasies can be made only if they are demonstrated in real situations when people come in contact with specific substances. In the words of Cummings, “Differences in reactions to therapeutic drugs represents a hidden set of phenotypes that are not revealed until exposure occurs” (2003, p.266). The Role of Genetic Variability in Drug Response Genetic variations can produce a range of phenotypic responses – drug resistance, toxic sensitivity to low doses, atypical reaction to a combination of drugs, or development of diseases after prolonged exposure to the drug. Some of the reactions can be harmless while others can be life threatening or even lethal. These changes are thought to occur because of the presence of mutations – “heritable changes in the base sequence within DNA strand” that persist through generations and may alter the amino acid sequence of the protein for which the gene codes (Rang et al., 2012, p. 132). Mutations are the source of genetic differences between people, for altered genes are inherited as a recessive trait in the Mendelian way. Mutations can give rise to polymorphisms which are “alternative sequences at a locus within DNA that persist in a population” (p.133). The presence of polymorphisms explains unusual genotypic tendencies in populations of common ethnic ancestry. In pharmacogenetic terms, mutations and polymorphisms are factors that cause lack or insufficient production of certain enzymes and thus can block some biochemical reactions, change their metabolic pathways, and ultimately affect drug response. Three types of interindividual differences in drug response have been recognized: pharmacokinetic, pharmacodynamic, and idiosyncratic. Pharmacokinetic effect refers to the variations in amounts of the drug in the body that reach the site of action – insufficient or excessive; pharmacodynamic effect reflects drug interaction with the site of action/receptor, meaning levels of concentration that produce effect; and idiosyncratic response represents an atypical, usually rare, reaction to the drug (Rang et al., 2012).  5 Genetic variations may cause alterations in drug absorption, distribution, metabolism, elimination (ADME); additionally, genetic influence extends to signal transduction, production of enzyme co-factors, and transcription factors (Pinto, 2011). Specifically, genetic factors can change the expression of proteins involved in ADME (pharmacokinetics), can affect drug targets (pharmacodynamics), and can provoke changes in enzymes or immune mechanisms (idiosyncratic reactions). (Rang et al., 2012) Interestingly, even one nucleotide alteration in alleles can unleash a series of events that lead to individual genetic differences. Such alterations are known as single nucleotide polymorphism (SNP). Selected SNPs may have provided an evolutionary advantage to certain populations in a distant human past but represent a health risk in modern times. For example, thrombophilia may have saved early humans from bleeding to death but today blood clots are a grater risk than hemorrhage. In addition to being determinants of some diseases, SNPs can also cause diverse individual food preferences and reactions to chemical substances in general and drugs in particular (Cummings, 2003). For example, a peculiar reaction to fava beans that consisted in life-threatening anemia was recorded in ancient Greece. A similar reaction was observed in certain populations in mid-20th century, but this time the causing substance was anti-malaria medication (primaquine and pamaquine). It was established that both, beans and drugs, caused massive destruction of erythrocytes (hemolytic anemia) by producing peroxides in the blood. Normally peroxides would be inactivated in the blood by an enzyme; however, some populations show deficiency of the specific enzyme G6PD (glucose-6-phosphate dehydrogenase), so peroxides cannot be broken down in the blood and their high concentration destroys red blood cells. The G6PD enzyme deficiency is a wide spread genetic disorder affecting over 400 million people in the world, among who the majority are of Mediterranean ancestry (Cummings, 2003). The G6PD deficiency allele shows a selective advantage because malarial parasites do not reproduce well in the absence of the enzymes it codes for, which explains the fact that the distribution of the G6PD deficiency allele follows traditional distribution of malaria in the world. This example represents a case of balanced polymorphism, which means that the advantage of this genetic variant (X-linked gene for G6PD) is compensatory, at least in certain times and places. Current research in pharmacogenetics aims to detect polymorphisms that influence drug response. This is a daunting task because there are estimated 11 million of SNPs in the human population (Pinto, 2011) and the identification of key variations is extremely difficult.  6 Nevertheless, with increase in pharmacogenetic research, the number of known genes that are responsible for variations in drug metabolism has grown in recent years. For example, CYP450 enzymes are associated with the metabolism of such medications as fluoxetine (brand names: Prozac, Sarafem, Fontex), paroxetine (Aropax, Paxil, Seroxat), and citalopram (Celexa, Cipramil). (Porcelli et al., 2011). In another instance, findings suggest that the 5- HTTLPR polymorphism (serotonin-transporter promoter region) is associated with antidepressant-induced mania in patients with bipolar disorder (Daray, Thommi, & Ghaemi, 2010). Most pharmacogenetic studies have, so far, focused on pharmacokinetic aspects of drug response. The SNP pharmacokinetic disorders that are significant in pharmacogenetic terms include such single-gene disorders as plasma cholinesterase deficiency that alters human response to anesthetics, porphyria which is implicated in various responses to sedatives, drug acetylation deficiency which interferes with detoxification, and amino glycoside ototoxicity which involves a toxic reaction to some antibiotics and may cause deafness (Rang et al., 2012). Research has also advanced in the identification of single-gene variations implicated in illness. For instance, 75 variations in CYP2D6 have been established; some of these alleles are associated with arterial hypertension, leukemia, apnea, thyroid and breast cancers, Alzheimer disease and Parkinson disease, hepatitis, pulmonary conditions, as well as with antidepressants metabolism (Allele Nomenclature Database, 2012). Pharmacogenetics and Antidepressants Recently, there has been an increased usage of antidepressant drugs; however, the efficacy of drug treatment remains ambiguous. For example, about 40% of patients do not respond to antidepressants and 70% do not achieve depression remission (Kato & Serretti, 2010). Moreover, adverse drug reactions and overdoses have been reported. Empirical evidence of the variability in antidepressant response has led to the hypothesis that vulnerability to unexpected drug effects – dynamic, kinetic, or idiosyncratic – can be genetically determined and it has been established that, indeed, several known pharmacogenetic disorders influence drug response. Pharmacogenetic research has provided some explanations for this diversity in the treatment response: variations of DNA within coding genes have been found to affect the rates of metabolism of certain drugs. For example, the metaanalysis by Kato and Serretti (2010) showed that genetic variants in 5-HTT (5-HTTLPR and STin2), STin2, HTR1A,  7 HTR2A, TPH1 and BDNF may modulate antidepressant response. Another metaanalysis (Porcelli et al., 2011) indicated that variations in CYP2D6 have been associated with people’s ability to metabolize antidepressants. According to Porcelli et al. (2011), current pharmacogenetic studies have focused on genes associated with metabolism, those that code for receptors and transporters and those related to second-messenger systems (p. 87). In the area of pharmacokinetics, variations of genes coding for CYP2D6 and P-glycoprotein have been implicated in the differences in drug response. The following example of such differences in drug metabolism refers to bupropion (brand names: Wellbutrin, Wellbutrin SR, Wellbutrin XL, Zyban). Bupropion is an atypical antidepressant medication that affects neurotransmitters; it works through an increased noradrenergic activity, weak dopaminerguc activity, and augmenting SSRIs effects (Preston, O’Neal, & Talaga, 2005); the expected treatment outcome is an increased presence of neurotransmitters in the synapse and thus relief from the symptoms of depressed mood. The enzyme that metabolizes bupropion is CYP2B6; it belongs to the Cytochrome P450 group of enzymes responsible for the oxidation and reduction of endogenous substrates and drugs; and in humans is encoded by the CYP2B6 gene. There is a great variation in cytochromes - more than 400 individual forms of cytochromes have been found in humans – which potentially can explain the diversity of individual drug response (Allele Nomenclature Database, 2012). Gene variants are associated with different rates of metabolizing drugs. Depending on the drug metabolism rate, patients are distinguished as poor, intermediate, extensive and rapid metabolizers on the basis of their genetic makeup. The analysis of experimental data indicates that differences between patients who are poor metabolizers and those who are rapid metabolizers depends on the number of CYP2B6 copies as rapid metabolizer phenotype have multiple copies of CYP2B6 with a direct influence on plasma drug concentration. On the other hand, the study found that the majority of poor metabolizer phenotypes had four deficient-activity CYP2D6 alleles (Porcelli et al., 2011). Kirchheiner et al. (2004) explored the relationship between polymorphic variations CYP2D6 or CYP2C19 and response to large number of antidepressants and found that, in order for the treatment to be effective, for 14 of these drugs patients with this genetic variation who were extensive metabolizers would need twice as large a dose as poor metabolizers.  8 Another example of patient response variations with antidepressants concerns the medication task to penetrate the brain in order to have an effect and refers to P-glycoprotein, one of the factors that regulate the blood–brain barrier. This protein controls drug intake in the brain and is known to remove from the brain such antidepressant drugs as amitriptyline, nortryptyline, citalopram, venlafaxine, sertraline and trimipramine. Thus, changes in P- glycoprotein function are thought to affect drug response and patterns of side effects. Altered P-glycoprotein expression and function are associated with mutations at positions 2677 (rs2032582) and 3435 (rs1045642); P-glycoprotein is coded by the ABCB1 gene (Porcelli et al., 2011). In the field of pharmacodynamics, the following genes have been found to affect antidepressant drug response: the genes coding for tryptophan hydroxylase, catechol-O- methyltransferase (COMT), monoamine oxidase A (MAOA), serotonin transporter (5-HTT), norepinephrine transporter (NET), dopamine transporter (DAT), monoamine receptors (5- HT1A, 5-HT2A, 5-HT6, 5-HT3A, 5-HT3B, β1 adrenoceptor), dopamine (DA) receptors, G protein β3 subunit, corticotropin-releasing hormone (CRH) receptor I (CRHR1), glucocorticoid receptor, angiotensin-converting enzyme, circadian locomotor output cycles kaput (CLOCK), nitric oxide synthase, interleukin (IL)-1β and brain-derived neurotrophic factor (BDNF). (Porcelli et al., 2011). For example, monoamine oxidase A (MAOA) genetic variations are thought to affect the action of SSRIs through an interaction with 5-HT transporters. MAOA is an enzyme that degrades monoamine neurotransmitters (NE, DA, 5-HT); its absence in humans has been associated with psychiatric disorders. In another example, the authors mention the role of the dopamine transporter. The gene for this transporter (DAT1) has over 500 known variants and one of the polymorphisms (40bp VNTR in exon 15) is thought to affect DAT expression; its allele related to an enhanced expression is associated with a faster onset of response to a wide range of antidepressants (Porcelli et al., 2011). Even though clinical practice has evidence that antidepressant medication has brought symptom relief for many patients, science has not found a clear explanation of the exact functioning of these pharmaceuticals. “Despite our knowledge of some of the important mechanisms of action of these medications, we still do not really know how they relieve depression” (Preston, O’Neal & Talaga, 2005). For instance, one of the hypotheses about the operation of antidepressants in the organism concerns the dopamine system which is associated with depressive symptomatology. It is hypothesized that depressed patients may have decreased dopaminergic neurotransmission and that its cause may be hypersensitivity of  9 inhibitory 5-HT2 receptors located on dopaminergic neurons (Porcelli et al., 2011). Antidepressants have a down-regulating effect on the inhibition which, in turn, may provoke dopaminergic firing resulting in therapeutic effect. Interesting as this and many other hypotheses are, they need to be tested and supported by empirical evidence. Therefore, in addition to its drug response predictive potential, pharmacogenetic research may elucidate the way antidepressants actually work. Pharmacogenetic studies take into consideration patients’ ethnic origin to control for polymorphisms with a geographical gradient. For example, the above mentioned CYP2D6 poor metabolizer phenotype has been reported in 5%–10% of the US white population but is rarer in black and Asian populations (Porcelli et al., 2011). Another example of ethnicity significance is a much studied marker of antidepressant response - the 5-HTTLPR – whose allele frequencies differ in Caucasian and Asian populations and are present in 42% and 79% of these groups respectively (Kato & Serretti, 2010). Genetic variation related to ethnicity in human populations is an important issue in pharmacogenetics because modern globalization processes have contributed to people’s mobility and, thus, geographical polymorphisms may appear anywhere in the world, which may present unexpected problems with adverse drug reaction for the patients in treatment. Pharmacogenomic Testing One of the goals of pharmacogenetic research is to develop reliable genetic tests that would predict the patients’ response to medication. Among the first tests developed was the TPMT (thiopurine methyltransferase) enzyme test. A deficiency in this enzyme, caused by non-functional alleles TPMT2, TPMT3A, and TPMT3C, renders several immunosuppressive drugs toxic to the bone marrow. This has led the US FDA to require a warning on such drugs (e.g.: azathioprine, brand names: Imuran, Azasan) and a recommendation of phenotype/genotype testing for TPMT deficiency (US FDA, 2010). Other existing in clinical practice pharmacogenetic tests include human leukocyte antigen variation, genes controlling aspects of drug metabolism, gene encoding drug targets, CYP2C9 and VKORC1 genotyping (for anticoagulant Warfarin metabolism), and human interferon gene (for Hepatitis-C interferon treatment) (Rang et al., 2012; US FDA, 2010). So far, the expected wide application of pharmacogenetic tests has not materialized for various reasons ranging from insufficient empirical evidence to support clinical use of such tests to political and commercial considerations. Garrison & Finley Austin (2006) noted that genetic hypotheses needed to be tested to determine the conditions of clinical usefulness  10 of pharmacogenetic testing; however, the authors are hopeful that, once scientific requirements are satisfied, this kind of testing will become routine because "…from an economic perspective, pharmacogenetics-based tests do not differ from other tests" (p. 1287). Ethical considerations Clinical implementation of pharmacogenetic testing presents ethical considerations as it produces highly personal information which, if accessed by unintended users, can have dire consequences. Ideally, only the patient and the physician should be privy to these data but, in the context of managed health care, other entities may have a claim to it. Use of obtained information by insurance companies, health care entities, and all other potential users can lead to discrimination against people with high risk of certain diseases. Applications of Pharmacogenetics Pharmacogenetic research has important implications for clinical practice. Pharmacogenetic methods have been used or are expected to be used in the near future for drug treatment in such areas as oncology, cardiovascular disease, psychological disorders (major depression, bipolar, and ADD), HIV, TB, asthma, diabetes, companion diagnostics, pharmacogenetics-based diagnostics (Pinto, 2011). Conclusion Pharmacogenetics can provide powerful diagnostic and therapeutic tools and contribute to the development of personalized medicine. Recent pharmacogenetic research has considerably advanced the understanding of the relationship between genotype and drug response and has provided empirical data on the pharmacokinetic, pharmacodynamic, and idiosyncratic effects of many drugs on genetically various populations. Even though pharmacogenetics as a discipline is still in its initial phase, it has been evolving rapidly and its research is promising. Pharmacogenetic approach has the potential to optimize pharmaceutical treatment, create safer medication, and develop more appropriate methods to determine drug dosage. The transition of scientific discoveries into clinical reality is being slowed down by some practical realities, including ethical, political, and commercial considerations. 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