**2. Problem statement**

TB is an important global public health problem but has cure in almost 100% of the new cases if correct quimiotherapy is applied. The American Thoracic Society (ATS) treatment guidelines recommend an initial phase for TB treatment which consists of rifampicin 10 mg/kg (maximum 600 mg), isoniazid 5 mg/kg (maximum 300 mg), pyrazinamide 15–30 mg/kg (maximum 2 g), and ethambutol 15–20 mg/kg (maximum 1.6 g) given daily for 8 weeks, followed by a contin‐ uous phase of isoniazid 15 mg/kg (maximum 900 mg) and rifampicin 10 mg/kg (maximum 600 mg) administered 2–3 times/week for 18 weeks [6]. The use of fixed-dose combination (FDC) tablets containing anti-TB drugs has been recommended by the World Health Organization (WHO) as an additional measure to improve treatment adherence by reducing the number of tablets to be taken. The principal disadvantages of combining three or more drugs in one tablet include (a) the possibility of overdosage or underdosage resulting from a prescription error, (b) changes in the bioavailability of rifampicin and (c) difficulties in determining which drug is responsible for adverse effects [7].

Isoniazid (INH) is an important drug in the TB treatment and was introduced in chemo‐ therapic scheme since 1952. It is the hidrazine of isonicotinic acid and shows cytotoxic ac‐ tivity for *Mycobacterium tuberculosis* both in rest (during latency) and proliferation phases. This drug enters easily in macrophague cells to kill bacilli in multiplication and is specif‐ ic for mycobacteria [1].

INH-induced adverse reactions include fever, nausea, vomiting, hepatotoxicity, skin reactions, gastrointestinal and neurological disorders. Only in the early 1970s, the occurrence of severe liver injury as a side effect of this drug was recognized, resulting in the death of some patients [8]. Among the first-line anti-TB drugs, INH is the main associated with drug-induced hepatotocixity with a frequency ranging from 1 to 30% in different populations [9]. Other drugs causing liver injury are mainly reported in combination with INH [10, 11]. Drug-induced hepatotoxicity is defined as a serum alanine aminotransferase (ALT) level three times greater than the upper limit of normal (ULN) with clinical symptoms or five times the ULN without symptoms. In both cases treatment should be interrupted and, generally, a modified or alternative regimen is introduced [9]. Because these adverse reactions do not only affect morbidity and mortality rate but also lead to treatment interruptions, failure and relapse, adverse reactions contribute to the spread of the disease and the emergence of multidrug resistence (MDR).

slow and rapid drug absorption, poor, efficient or ultrarapid drug metabolism and poor or efficient receptor interactions [3]. The consequences of such variations can lead to ad‐

In this context, pharmacogenetics is the study of genetic variations associated with individual variability in drug response, including differences in efficacy, drug-drug interactions, and the relative risk of an adverse response to drugs. It includes the study of genetic polymorphisms that could affect the expression or activity of drug transporters, drug metabolizing enzymes

It's estimated that 99.9% of the human genome sequence between individuals is identical and genetic differences in polulations are called mutations if they are present in less than 1% and polymorphisms when present in at least 1% of a population. A single-nucleotide polymor‐ phism (SNP) involves a replacement of one nucleotide base with any one of the other three and occuring at approximately one out of every 1,000 bases in the human genome [5].

A mutation or polymorphism in genes that encode metabolic enzymes, carriers or receptors can affect the drug pharmacokinetics and pharmacodinamics leading to undesired therapeutic effects. The identification of these genetic markers which predicted if a person responds well or not to a specific drug could help to select the right medication in right dosage, maximizing

TB is an important global public health problem but has cure in almost 100% of the new cases if correct quimiotherapy is applied. The American Thoracic Society (ATS) treatment guidelines recommend an initial phase for TB treatment which consists of rifampicin 10 mg/kg (maximum 600 mg), isoniazid 5 mg/kg (maximum 300 mg), pyrazinamide 15–30 mg/kg (maximum 2 g), and ethambutol 15–20 mg/kg (maximum 1.6 g) given daily for 8 weeks, followed by a contin‐ uous phase of isoniazid 15 mg/kg (maximum 900 mg) and rifampicin 10 mg/kg (maximum 600 mg) administered 2–3 times/week for 18 weeks [6]. The use of fixed-dose combination (FDC) tablets containing anti-TB drugs has been recommended by the World Health Organization (WHO) as an additional measure to improve treatment adherence by reducing the number of tablets to be taken. The principal disadvantages of combining three or more drugs in one tablet include (a) the possibility of overdosage or underdosage resulting from a prescription error, (b) changes in the bioavailability of rifampicin and (c) difficulties in determining which drug

Isoniazid (INH) is an important drug in the TB treatment and was introduced in chemo‐ therapic scheme since 1952. It is the hidrazine of isonicotinic acid and shows cytotoxic ac‐ tivity for *Mycobacterium tuberculosis* both in rest (during latency) and proliferation phases. This drug enters easily in macrophague cells to kill bacilli in multiplication and is specif‐

the eficacy and preventing or reducing the adverse drug reactions.

verse drug reaction and/or terapeutic failure.

106 Tuberculosis - Current Issues in Diagnosis and Management

and drug receptors [2-4].

**2. Problem statement**

is responsible for adverse effects [7].

ic for mycobacteria [1].

Adverse Drug Reactions (ADRs) are common causes of hospitalization and lead to large costs to society. There are two main financial burdens due to illnesses caused by ADRs: that of treating and that of avoiding them [12]. The occurrence of serious and fatal ADRs has been extensively studied in hospitalized patients and a meta-analysis of prospective studies in approximately forty hospitals in the United States of America (USA) suggests that 6-7% of hospitalized patients suffer from serious ADRs and 0.32% of patients develop fatal ADRs [13]. This results in approximately 100,000 deaths annually in the U.S. and an annual cost of over a hundred billion dollars to the society due to prolonged hospitalization and reduced produc‐ tivity [3, 13]. Furthermore, it has been estimated that ADRs are responsible for up to 7% of all admissions in hospitals in the United Kindown (UK) and 13% in medical clinics in Sweden [3], which shows the magnitude of this problem in the context of chemotherapy and drug development. Additionally, in France, a 10-year study in the Liver Unit of Hôpital Beaujon in Paris showed that among all patients hospitalized with acute hepatitis, 10% were due to adverse reaction to drugs and the prevalence of drug hepatotoxicity in patients older than fifty years exceeded 40%. In Japan and other Eastern countries, drugs are responsible for about 10-20% of cases of fulminant hepatitis [14].

Liver injury is the most common ADR and the main complication during chemotherapy since liver is the central organ for the biotransformation and excretion of most drugs and xenobiotics [14-17]. There are basically six mechanisms involving primarily the hepatocyte injury. The reactions of mono-oxygenase cytochrome P450 (CYP450) with certain drugs generate toxic metabolites that bind to intracellular proteins, leading to calcium homeostasis pump dysfunc‐ tion with consequent disruption of actin fibers and cell lysis. Some drugs affect transport proteins in the cell membrane interrupting the flow of bile and then causing cholestasis. Several reactions involving CYP P450 can promote binding of the drug to the enzyme, with consequent exposure of this complex on the cell surface for recognition by T cells and antibody production as part of the autoimmune response. Finally, certain drugs may promote hepatic injury mediated by programmed cell death (apoptosis) or being capable of inhibiting respiration and/ or mitochondrial beta-oxidation [17].

Xenobiotics are usually lipophilic and this facilitates their transport in association with lipoproteins in the blood stream and their penetration of lipid membranes and entrance into organs. However, physicochemical properties of drug molecules difficult their removal from the organism by biliary or renal excretion and therefore, these substances require enzymatic conversion to water soluble compounds [1]. The xenobiotics metabolization, often through multiple pathways, can generate metabolites that are more toxic than the substrate and through their interaction with target macromolecules such as DNA, RNA, proteins and receptors, generate the toxic effects. The organ affected is generally that reponsible for drug metaboli‐ zation or excretion of metabolites [1].

from CYP2E1 [22]. Sulphydryl conjugation facilitates the elimination of metabolites from the

Tuberculosis Pharmacogenetics: State of The Art

http://dx.doi.org/10.5772/54984

109

**Figure 1.** Schematic representation of the INH metabolism. The major enzymes involved in this pathway are indicated

In the last few years, an increasing number of studies have suggested that genetic polymor‐ phisms in *NAT2*, *CYP2E1* and *GST* genes would be associated with susceptibility to druginduced hepatotoxicity during TB treatment. The present work focused in an overview of the role of such polymorphisms in occurence of liver injury induced by anti-TB drugs, and by INH

NAT2, the main enzyme responsible for the metabolism and inactivation of INH in humans, is a Phase II enzyme that catalyzes the transfer of the acetyl group from the cofactor acetyl coenzyme A (acetyl-CoA) to the nitrogen terminal of the drug. Variations in activity of NAT2 were discovered over 50 years ago when observing interindividual differences in the metab‐ olism of INH and the level of drug-induced toxicity in TB patients. NAT2 is encoded by the *NAT2* gene and according family genetic studies, variability of *NAT2* was directly related to

The molecular study of human N-acetyltransferases revealed the presence of three genetic loci, two very homologous encoding the enzymes NAT1 and NAT2, and a third including the

body and reduces the toxic effect [23] (Figure 1).

in boxes [20, 24].

in particular.

**3. State of the art**

**3.1. N-acetyltransferase 2**

the emergence of different phenotypes of acetylation [25].

The enzyme systems responsible for the biotransformation of many drugs are located in the endoplasmic reticulum of the liver (microsomal fraction). Such enzymes are also present in the kidneys, lungs and gastrointestinal epithelium, although at a lower concentration [1]. The metabolic modification in biotransformation usually takes place in two consecutive steps and results in the loss of biological activity. Phase I reactions convert the xenobiotic into a metab‐ olite with higher polarity by oxidation, reduction or hydrolysis and generates a pharmaco‐ logically inactive or less active, or in the case of a pro-drug, more active molecule. This metabolite is than either eliminated or go through Phase II reactions (so-called synthesis or conjugation reactions), involving binding to a primary metabolite or endogenous substrate such as glucuronate, sulfate, acetate, amino acids or glutathione (tripeptide). Such enzymatic reactions include glucuronidation, methylation, sulfation, acetylation, conjugation with glutathione and conjugation with glycine [1].

The risk for developing hepatotoxicity is associated both with genetic and acquired factors. The acquired factors include: age, gender, nutritional habits, drug abuse, pregnancy and extrahepatic disease. Genetic variations in isoenzymes involved in drug biotransformation can result in abnormal reactions leading to toxic effects [14,17]. In the case of INH in particular, advanced age is a risk factor for hepatotoxicity whereas deficiency in the ability of N-acetyla‐ tion represent a genetic risk factor for liver injury.

INH is administered orally and rapidly absorbed through the gastrointestinal tract passing through the liver by the portal venous system before reaching the general circulation where is metabolized by a process known as the first pass effect with reduction of its biodiponibility. About 75% to 95% of the INH is excreted by the kidneys during the first 24 hours, mainly as the metabolic forms acetyl-isoniazid and isonicotinic acid [1].

In the liver, INH is metabolized to acetylisoniazid by N-acetyltransferase 2 (NAT2), followed by hydrolysis to acetylhydrazine and then oxidized by cytochrome P4502E1 (CYP2E1) to hepatotoxic intermediates [18, 19]. These metabolites can destroy hepatocytes either by interfering with cell homeostasis or by triggering immunologic reactions in which reactive metabolites that are bound to hepatocyte plasma proteins may act as haptens [17]. The other metabolic pathway to generate toxic metabolites is direct hydrolysis of INH to hydrazine, a potent hepatotoxin. NAT2 is also responsible for converting acetylhydrazine to diacetylhy‐ drazine, a nontoxic component [18, 20, 21] (Figure 1). Glutathione S-transferase (GST), an important phase II detoxification enzyme, is thought to play a protective role as an intracellular free radical scavenger, which conjugates glutathione with toxic metabolites that are generated from CYP2E1 [22]. Sulphydryl conjugation facilitates the elimination of metabolites from the body and reduces the toxic effect [23] (Figure 1).

**Figure 1.** Schematic representation of the INH metabolism. The major enzymes involved in this pathway are indicated in boxes [20, 24].

In the last few years, an increasing number of studies have suggested that genetic polymor‐ phisms in *NAT2*, *CYP2E1* and *GST* genes would be associated with susceptibility to druginduced hepatotoxicity during TB treatment. The present work focused in an overview of the role of such polymorphisms in occurence of liver injury induced by anti-TB drugs, and by INH in particular.
