PHARMACOKINETICS AND BIOPHARMACEUTICS(46:138)

LECTURE TUTORIAL

Pharmacogenetics

 

Key Terms:

Pharmacogenetics: The study of genetically controlled variations in drug response. Historically, this term has been used to refer to variations that arise from variants in a single gene that result in variants in a single protein, such that the activity or content of that protein is modified. For example, variants in pseudocholinestrase produce proteins that have reduced activity compared to the protein produced by the wild-type gene. This focus on ‘single gene-single protein’ has been expanded with advances arising from the Human Genome Project. Now, the term pharmacogenomics is supplanting the term pharmacogenetics. Theses two terms are sometimes used as synonyms, but the term pharmacogenomics takes a much more global approach to the impact of variation in genetic information on drug response. In addition, much of the work in the area of pharmacogenomics is focused on identifying new molecular targets for therapeutic intervention. Considered another way, pharmacogenetics is an area of study within the broad field of pharmacogenomics in much the same way that synthetic chemistry is an area of study within the broad field of chemistry. The understanding that drug response may be multi-factorial (meaning it may be dependent upon the response of many genes) helps us to recognize the importance of examining more than the classical ‘single gene-single protein’ concept. Moreover, there is growing body of evidence that modifications besides outright mutation of genes (e.g., methylation of promoter regions) may have a profound impact on gene expression and, by implication, drug response.

 

Phenotype: Derives from two Greek words that mean ‘to show forth’ and ‘model’. It is the assignment of an individual to a group or category based on observable (e.g., eye color) or measurable (e.g., enzyme activity) characteristics. The phrase deduced phenotype refers to the phenotype an individual would be presumed to display based upon their identified genotype. Importantly, genotype is not the only determinant of phenotype. For example, an individual whose genotype is that of an extensive metabolizer via CYP2D6 can display a phenotype that would characterize them as a poor metabolize if they are co-administered low doses of quinidine, which is a potent inhibitor of CYP2D6.

 

Genotype: Derives from two Greek words that mean ‘birth/decent’ and ‘model’. The genotype is determined by the specific nucleotide sequence of the gene of interest. Different genotypes may give rise to the same phenotype. For example, there are a variety of mutations in NAT2 that give rise to a slow acetylator phenotype. In addition, some genotypic variations fail to give rise to phenotypic variation. For example, some nucleotide substitutions do not result in differences in the amino acid sequence (often referred to as silent mutations). Moreover, some mutations give rise to amino acid changes that do not result in changes in the fundamental activity or structure of the protein. Thus, observation of genotypic variation alone is not adequate to tell us whether or not a genetic polymorphism is of clinical significance. Historically, the terms wild-type and mutant alleles were used to characterize genotypes. However, what one identified as wild-type could vary based upon what population was studied. Should the wild-type (sometimes even called ‘normal’) allele be that which is found most commonly in a Caucasian population or that which is found most commonly in an Aboriginal population? These and other questions, which can be linked with emotionally charged implications, has caused the field to migrate to the use of the terms reference and mutant alleles.

 

Polymorphic Variation:  The term polymorphic literally means ‘many forms’. When used in the field of pharmacogenetics, it refers to a characteristic by which, when examined on a frequency distribution plot, one is able to designate the population into various sub-groups. For example, if a drug metabolizing enzyme displays an activity that exhibits a normal distribution on a frequency distribution plot, the sub-division of the population would, of necessity, be arbitrary. On the other hand, when the frequency distribution plot exhibits a distinct non-normal distribution with some magnitude of demarcation between sub-groups of the population, one can readily characterize the population by division into those distinct sub-groups. The magnitude of difference between the sub-groups may vary between characteristics. For example, while the phenotypic differentiation between the two major phenotypes resulting from variation in CYP2D6 is substantial, the difference between phenotypes resulting from variation in NAT2 is much less. This results in greater difficulty in the accurate assignments of NAT phenotypes than CYP2D6 phenotypes. Moreover, the potential for environmental factors altering enzyme activity and resulting in a phenotype that differs from that which would be predicted from the genotype is far greater for those traits where distinction between phenotypic groups is small in magnitude (i.e., an environmental factor is more likely to ‘change’ the phenotype from that determined by genetics alone).

 

Co-segregation: When it is stated that the metabolism of Compound B co-segregates with the metabolism of Compound A, a patient’s phenotype for one compound can be predicted by the other. In other words, if an individual is a poor metabolizer of dextromethorphan, they will also be poor metabolizers of agents whose metabolism co-segregates with dextromethorphan. This is one indication that the compounds are metabolized by the same enzyme.

 

Epigenetic: A factor that alters gene expression without directly effect the DNA sequence. The most prevalent example of this in the area of pharmacogenomics is methylation of the promoter region of a gene. This area of investigation is relatively new and should yield important information on non-sequence factors that control gene expression.

 

Single nucleotide polymorphisms (SNPs): A SNP (pronounced ‘snip’) is a change in a single base pair in a particular gene that occurs in the population with a frequency of at least 1%. SNPs are the most common form of variation in DNA sequence. Currently, 1.4 million SNPs have been identified in the human genome (Nature 409:928, 2001). A SNP Consortium has been formed (http://snp.cshl.org/) with a mission “to develop up to 300,000 SNPs distributed evenly throughout the human genome and to make the information related to these SNPs available to the public without intellectual property restrictions.” It is anticipated that this database will be extremely useful in identifying genetic markers with predictive capacity for drug response and disease progression. For a good review of SNPs, how they are detected, and their potential application, see http://www.ncbi.nlm.nih.gov/About/primer/snps.html.

 

Key Principles:

 

Phenotype is influenced by factors other than nucleotide sequence on a gene: It is important to recognize that genotype is not the only determinant of phenotype. For example, an individual whose genotype is that of an extensive metabolizer via CYP2D6 can display a phenotype that would characterize them as a poor metabolize if they are co-administered low doses of quinidine, which is a potent inhibitor of CYP2D6.

 

The consequence of phenotypic variation will depend upon the drug and which species is pharmacologically active: Differences in phenotype do not necessarily translate into differences in pharmacologic response between subjects. For some drugs, the parent compound and metabolite display similar pharmacologic activity. As a consequence, though parent drug and metabolite levels differ substantially between phenotypes, dosing adjustments are not necessary.

 

Adverse effects to drugs may correlate with genetic polymorphisms: As genetic polymorphisms can result in significant differences in drug exposure among subjects, individuals with certain phenotypes may be predisposed to the development of adverse reactions. For example, subjects who possess mutant alleles of CYP2D6 are predisposed to developing hypotension after administration of debrisoquine. Similarly, individuals who exhibit the slow acetylator phenotype develop symptoms of drug-induced lupus after procainamide more rapidly than subjects who are rapid acetylators.

           

Practice Problems:

 

1.     Propafenone is an antiarrhythmic agent with beta-blocking activity. The drug is metabolized to two primary metabolites, 5-hydroxypropafenone and N-desalkylpropafenone. The metabolism of propafenone to 5-hydroxypropafenone is catalyzed by CYP2D6. The impact of polymorphism in CYP2D6 on propafenone disposition can be seen in the data tabulated below, which shows the steady-state concentration of propafenone and its metabolites in a group of extensive and poor metabolizers of debrisoquine. Studies have demonstrated that the QRS, PR and QTc intervals do not differ between extensive metabolizers given propafenone alone and those that are given propafenone + an inhibitor of CYP2D6. In contrast, reduction in CYP2D6 capacity in subjects receiving propafenone results in a significant increase in beta-blocking. Propose an explanation for the lack of impact of CYP2D6 phenotype on the antiarrhythmic activity of propafenone, while at the same time seeing differences in beta-blockade that correlate with phenotype.  Answer

 

Table 1. Mean plasma concentrations (micromoles per mL) of propafenone and its metabolites after dosing subjects 225 mg orally every 8 hrs for 5 days. Data from: Lee JT, et al. N Engl J Med 322:1764, 1990.

                       BLD – below limit of detection for the assay

 

 

2.     The NAT2 genotype and phenotype was determined in a group of volunteers. The phenotype observed for each genotype is given in the table below. NAT2*4 is the reference allele, while the others listed are mutant alleles. Provide a plausible explanation as to why an individual homozygous for NAT2*13 (a mutant allele) displays the rapid acetylator phenotype. Answer

 

 

 

 

Last revised 07/19/04

 

ã 2004 - Craig K. Svensson, Pharm.D., Ph.D.

 

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