Biochemical Modification

The actual breakdown process, which usually takes place in the liver, varies with different drugs. Some are oxidized completely to carbon dioxide, which is exhaled through the lungs. Others are excreted in modified forms either via the kidneys into the urine, or by the liver into the bile and thence the feces. Many drugs undergo biochemical modifications that increase their solubility, resulting in their being more readily excreted.

One important biochemical modification of many drugs is conjugation, which involves union with the carbohydrate glucuronic acid. Glucuronide conjugation occurs primarily in the liver. The elimination of morphine and its derivatives, such as codeine, is dependent almost entirely on this process. Isoniazid, used in the treatment of tuberculosis, and a number of other drugs, including the sulfonamides, are modified by the introduction of an acetyl group into the molecule, a process known as acetylation (Figure 12.2).

FIGURE 12.2 Acetylation of the antituberculosis drug isoniazid.

Kinetics of Drug Metabolism

The study of the metabolism and effects of a particular drug usually involves giving a standard dose of the drug and then, after a suitable time interval, determining the response, measuring the amount of the drug circulating in the blood or determining the rate at which it is metabolized. Such studies show that there is considerable variation in the way different individuals respond to certain drugs. This variability in response can be continuous or discontinuous.

If a dose–response test is carried out on a large number of subjects, their results can be plotted. A number of different possible responses can be seen (Figure 12.3). In continuous variation, the results form a bell-shaped or unimodal distribution. With discontinuous variation the curve is bimodal or sometimes even trimodal. A discontinuous response suggests that the metabolism of the drug is under monogenic control. For example, if the normal metabolism of a drug is controlled by a dominant gene, R, and if some people are unable to metabolize the drug because they are homozygous for a recessive gene, r, there will be three classes of individual: RR, Rr, and rr. If the responses of RR and Rr are indistinguishable, a bimodal distribution will result. If RR and Rr are distinguishable, a trimodal distribution will result, each peak or mode representing a different genotype. A unimodal distribution implies that the metabolism of the drug in question is under the control of many genes—i.e., is polygenic (p. 143).

FIGURE 12.3 Various types of response to different drugs consistent with polygenic and monogenic control of drug metabolism. A, Continuous variation, multifactorial control of drug metabolism. B, Discontinuous bimodal variation. C, Discontinuous trimodal variation.

N-Acetyltransferase Activity

Isoniazid is one of the drugs used in the treatment of tuberculosis. It is rapidly absorbed from the gut, resulting in an initial high blood level that is slowly reduced as the drug is inactivated and excreted. The metabolism of isoniazid allows two groups to be distinguished: rapid and slow inactivators. In the former, blood levels of the drug fall rapidly after an oral dose; in the latter, blood levels remain high for some time. Family studies have shown that slow inactivators of isoniazid are homozygous for an autosomal recessive allele of the liver enzyme N-acetyltransferase, with lower activity levels. N-acetyltransferase activity varies in different populations. In the United States and Western Europe, about 50% of the population are slow inactivators, in contrast to the Japanese, who are predominately rapid inactivators.

In some individuals, isoniazid can cause side effects such as polyneuritis, a systemic lupus erythematosus–like disorder, or liver damage. Blood levels of isoniazid remain higher for longer periods in slow inactivators than in rapid inactivators on equivalent doses. Slow inactivators have a significantly greater risk of developing side effects on doses that rapid inactivators require to ensure adequate blood levels for successful treatment of tuberculosis. Conversely, rapid inactivators have an increased risk of liver damage from isoniazid. Several other drugs are also metabolized by N-acetyltransferase, and therefore slow inactivators of isoniazid are also more likely to exhibit side effects. These drugs include hydralazine, which is an antihypertensive, and sulfasalazine, which is a sulfonamide derivative used to treat Crohn disease.

Studies in other animal species led to the cloning of the genes responsible for N-acetyltransferase activity in humans. This has revealed that there are three genes, one of which is not expressed and represents a pseudogene (NATP), one that does not exhibit differences in activity between individuals (NAT1), and a third (NAT2), mutations in which are responsible for the inherited polymorphic variation. These inherited variations in NAT2 have been reported to modify the risk of developing a number of cancers, including bladder, colorectal, breast, and lung cancer. This is thought to be through differences in acetylation of aromatic and heterocyclic amine carcinogens.

Glucose 6-Phosphate Dehydrogenase Variants

For many years, quinine was the drug of choice in the treatment of malaria. Although it has been very effective in acute attacks, it is not effective in preventing relapses. In 1926 primaquine was introduced and proved to be much better than quinine in preventing relapses. However, it was not long after primaquine was introduced that some people were found to be sensitive to the drug. The drug could be taken for a few days with no apparent ill effects, and then suddenly some individuals would begin to pass very dark, often black, urine. Jaundice developed and the red cell count and hemoglobin concentration gradually fell as a consequence of hemolysis of the red blood cells. Affected individuals usually recovered from such a hemolytic episode, but occasionally the destruction of the red cells was extensive enough to be fatal. The cause of such cases of primaquine sensitivity was subsequently shown to be a deficiency in the red cell enzyme glucose 6-phosphate dehydrogenase (G6PD).

G6PD deficiency is inherited as an X-linked recessive trait (p. 114), rare in Caucasians but affecting about 10% of Afro-Caribbean males and relatively common in the Mediterranean. It is thought to be relatively common in these populations as a result of conferring increased resistance to the malarial parasite. These individuals are sensitive not only to primaquine, but also to many other compounds, including phenacetin, nitrofurantoin, and certain sulfonamides. G6PD deficiency is thought to be the first recognized pharmacogenetic disorder, having been described by Pythagoras around 500 bc.

Coumarin Metabolism

Coumarin anticoagulant drugs, such as warfarin, are used in the treatment of a number of different disorders to prevent the blood from clotting (e.g., after a deep venous thrombosis). Warfarin is metabolized by the cytochrome P450 enzyme encoded by the CYP2C9 gene, and two variants (CYP2C9*2 and CYP2C9*3) result in decreased metabolism. Consequently, these patients require a lower warfarin dose to maintain their target international normalized ratio range and may be at increased risk of bleeding.

Debrisoquine Metabolism

Debrisoquine is a drug that was used frequently in the past for the treatment of hypertension. There is a bimodal distribution in the response to the drug in the general population. Approximately 5% to 10% of persons of European origin are poor metabolizers, being homozygotes for an autosomal recessive gene with reduced hydroxylation activity.

Molecular studies revealed that the gene involved in debrisoquine metabolism is one of the P450 family of genes on chromosome 22, known as CYP2D6. The mutations responsible for the poor metabolizer phenotype are heterogeneous; 18 different variants have been described.

CYP2D6 variation is important because this enzyme is involved in the metabolism of more than 20% of prescribed drugs, including the β-blockers metoprolol and carvedilol, the antidepressants fluoxetine and imipramine, the antipsychotics thioridazine and haloperidol, the painkiller codeine, and the anti-cancer drug tamoxifen.

Malignant Hyperthermia

Malignant hyperthermia (MH) is a rare complication of anesthesia. Susceptible individuals develop muscle rigidity as well as an increased temperature (hyperthermia), often as high as 42.3°C (108°F) during anesthesia. This usually occurs when halothane is used as the anesthetic agent, particularly when succinylcholine is used as the muscle relaxant for intubation. If it is not recognized rapidly and treated with vigorous cooling, the affected individual will die.

MH susceptibility is inherited as an autosomal dominant trait affecting approximately 1 in 10,000 people. The most reliable prediction of an individual’s susceptibility status requires a muscle biopsy with in vitro muscle contracture testing in response to exposure to halothane and caffeine.

MH is genetically heterogeneous, but the most common cause is a mutation in the ryanodine receptor (RYR1) gene. Seven other candidate genes have been identified and variants in these genes may influence susceptibility within individual families. This observation may explain the discordant results of the in-vitro contracture test and genotype in members of some families that segregate RYR1 mutations.

Thiopurine Methyltransferase

A group of potentially toxic substances known as the thiopurines, which include 6-mercaptopurine, 6-thioguanine, and azathioprine, are used extensively in the treatment of leukemia to suppress the immune response in patients with autoimmune disorders such as systemic lupus erythematosus and to prevent rejection of organ transplants. They are effective drugs clinically but have serious side effects, such as leukopenia and severe liver damage. Azathioprine is reported to cause toxicity in 10% to 15% of patients and it may be possible to predict those patients susceptible to side effects by analyzing genetic variation within the thiopurine methyltransferase (TPMT) gene. This gene encodes an enzyme responsible for methylation of thiopurines, and approximately two-thirds of patients who experience toxicity have one or more variant alleles.

Dihydropyrimidine Dehydrogenase

Dihydropyrimidine dehydrogenase (DPYD) is the initial and rate-limiting enzyme in the catabolism of the chemotherapeutic drug 5-fluorouracil (5FU). Deficiency of DPYD is recognized as an important pharmacogenetic factor in the etiology of severe 5FU-associated toxicity. Measurement of DPYD activity in peripheral blood mononuclear cells or genetic testing for the most common DPYD gene mutation (a splice site mutation, IVS14 + 1G > A, which results in the deletion of exon 14) may be warranted in cancer patients before the administration of 5FU.

Maturity-Onset Diabetes of the Young

Maturity-onset diabetes of the young is a monogenic form of diabetes characterized by young age of onset (often before the age of 25 years), dominant inheritance and β-cell dysfunction (p. 236). Patients with mutations in the HNF1A or HNF4A genes are sensitive to sulfonylureas (Figure 12.4) and may experience episodes of hypoglycemia on standard doses. However, this sensitivity is advantageous at lower doses, and sulfonylureas are the recommended oral treatment in this genetic subgroup.

FIGURE 12.4 Response to the sulphonylurea gliclazide and the type 2 diabetes drug metformin in patients with HNF1A Maturity-onset diabetes of the young (MODY) and type 2 diabetes. FPG is fasting plasma glucose. Patients (n = 18 in each group) were treated with each drug for 6 weeks in a randomized trial.

(Modified from Pearson ER, Starkey BJ, Powell RJ, et al 2003 Genetic cause of hyperglycaemia and response to treatment in diabetes. Lancet 362[9392]:1275–1281.)

Neonatal Diabetes

The most frequent cause of permanent neonatal diabetes is an activating mutation in the KCNJ11 or ABCC8 genes, which encode the Kir6.2 and SUR1 subunits of the ATP-sensitive potassium (K-ATP) channel in the pancreatic β cell (p. 236). The effect of such mutations is to prevent K-ATP channel closure by reducing the response to ATP. Because channel closure is the trigger for insulin secretion, these mutations result in diabetes. Defining the genetic etiology for this rare subtype of diabetes has led to improved treatment, because the majority of patients can be treated successfully with sulfonylurea tablets instead of insulin. These drugs bind to the sulfonylurea receptor subunits of the K-ATP channel to cause closure independently of ATP, thereby triggering insulin secretion (Figure 12.5). High-dose sulfonylurea therapy results in improved glycemic control with fewer hypoglycemic episodes and, for some patients, an Hb A1c level (this is a measure of glycemic control) within the normal range.

FIGURE 12.5 Insulin secretion in the pancreatic beta cell. Activating mutations in the genes encoding the K_ATP channel subunits Kir6.2 and SUR1 prevent closure of the channel in the presence of glucose. Sulphonylureas bind to the SUR1 subunit to close the channel and restore insulin secretion.

(Courtesy Professor A.T. Hattersley, Peninsula Medical School, Exeter, UK.)

Pharmacogenomics

Pharmacogenomics is defined as the study of the interaction of an individual’s genetic makeup and response to a drug. The key distinction between pharmacogenetics and pharmacogenomics is that the former describes the study of variability in drug responses attributed to individual genes and the latter describes the study of the entire genome related to drug response. The expectation is that inherited variation at the DNA level results in functional variation in the gene products that play an essential role in determining the variability in responses, both therapeutic and adverse, to a drug. If polymorphic DNA sequence variation occurs in the coding portion or regulatory regions of genes, it is likely to result in variation in the gene product through alteration of function, activity, or level of expression. Automated analysis of genome-wide single nucleotide polymorphisms (SNPs) (p. 67) allows the possibility of identifying genes involved in drug metabolism, transport and receptors that are likely to play a role in determining the variability in efficacy, side effects and toxicity of a drug.

The availability of whole-genome SNP maps will enable an SNP profile to be created for patients who experience adverse events or who respond clinically to the drug (efficacy). An individual’s whole-genome SNP type has been described as an ‘SNP print’. However, this raises issues pertaining to the disclosure of information of uncertain significance that is later shown to be associated with an adverse outcome unrelated to the reason for the original test. An example is apolipoprotein E (ApoE) genotyping, where ApoE ε4 was first reported to be associated with variation in cholesterol levels but later with age of onset of Alzheimer disease.

Adverse Events

It is estimated that around 15% of hospital inpatients will be affected by an adverse drug reaction. The objective of adverse-event pharmacogenetics is to identify a genetic profile that characterizes patients who are more likely to suffer such an adverse event. The best known example is abacavir, a reverse transcriptase inhibitor used to treat human immunodeficiency virus (HIV) infection. Approximately 5% of patients show potentially fatal hypersensitivity to abacavir and this limited its use. A strong association with the human leukocyte antigen allele B*5701 was proven in 2002. Today testing for B*5701 is routine practice before abacavir is prescribed.

At least 10% of Africans, North Americans, and Europeans are homozygous for a variant in the promoter of the UGT1A1 gene (UGT1A1*28). This results in reduced glucuronidation of irinotecan, a drug used to treat colorectal cancer, and increases the risk of severe neutropenia if exposed to the standard dose. A simple polymerase chain reaction–based test for UGT1A1*28 can be used to determine the appropriate treatment dose.

Efficacy

There is no doubt that the cost-effectiveness of drugs is improved if they are prescribed only to those patients likely to respond to them. Several drugs developed for the treatment of various cancers have different efficacy depending on the molecular biology of the tumour (see Table 12.1). For example, herceptin (trastuzumab) is an antibody that targets overexpression of HER2/neu protein observed in approximately one-third of patients with breast cancer. Consequently, patients are prescribed herceptin only if their tumor has been shown to overexpress HER2/neu.

Table 12.1 Examples of Drugs Effective for the Treatment of Specific Cancers

Gleevec (imatinib) is a protein tyrosine kinase inhibitor that has been used to treat chronic myeloid leukemia since 2001. It is a very effective treatment that works by binding the BCR-ABL fusion protein resulting from the t(9;22) translocation. This is an example of effective drug design resulting from knowledge of the molecular etiology. More recently it has been also shown to be effective in the treatment of gastrointestinal stromal tumours that harbour KIT mutations.

Approximately 13% of patients with non–small-cell lung cancer have an activating EGFR mutation. These mutations increase the activity of the epidermal growth factor receptor tyrosine kinase domain so that the receptor is constitutionally active in the absence of epidermal growth factor. This leads to increased proliferation, angiogenesis and metastasis. Drugs designed to block the EGFR tyrosine kinase domain and inhibit these effects have been developed. Patients with lung tumours harbouring an activating EGFR mutation can show a dramatic response to treatment with these drugs (gefitinib and erlotinib) as shown in Figure 12.6.

FIGURE 12.6 Example of the response to gefitinib in a patient with non–small-cell lung cancer and an activating EGFR mutation. A computed tomographic scan of the chest shows a large mass in the right lung before treatment (A) and marked improvement 6 weeks after gefitinib was initiated (B).

(Reproduced with permission from Lynch TJ, Bell DW, Sordella R, et al 2004 Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. N Engl J Med 350(21):2129–2139.)

Genetic profiling is a step toward personalized medicine. This information can be used to select the appropriate treatment at the correct dosage and to avoid adverse drug reactions.