The report by Metushi et al. addresses the role of immune response in isoniazid (INH)-mediated liver injury. Isoniazid is an essential drug in the treatment of tuberculosis. It is used as one component of combination therapy for the treatment of tuberculosis and as prophylaxis for patients deemed to have been exposed to tuberculosis. Whereas INH is generally considered to be safe and cost-effective, liver-related adverse drug reactions may occur and involve two forms of liver injury. Mild hepatitis is typically asymptomatic and self-limiting, occurs in the early stages of a course of treatment, and is thought to occur in approximately 10% of patients receiving monotherapy. Alanine aminotransferase (ALT) elevations are generally less than 3-fold the upper limit of normal. Overt hepatitis occurs in 0.5-1.0% of patients receiving INH as monotherapy, generally occurs early in the course of therapy, and is associated with gastrointestinal complaints, jaundice, and progression to liver failure. Identified risk factors for INH acute liver failure (ALF) include female gender, age >35 years, concomitant treatment with other antituberculous drugs, slow acetylator N-acetyltransferase 2 (NAT2) genotype (NAT2*7), cytochrome P450 2E1 (CYP2E1 c1/c1) genotype, daily dose of INH >50 mg/kg, concomitant treatment with acetaminophen (APAP), and daily ethanol use.
Although the metabolism of INH (i.e., oxidation of INH to a reactive metabolite) is known to contribute to the development of toxicity,[4, 5] the potential role of the immune system in the development of liver injury is unclear. Previous clinical and laboratory data have been inconclusive in this area, because INH liver injury is not universally associated with fever or rash, does not necessarily recur with rechallenge, and anti-INH antibodies (Abs) have not been detected in previous studies. However, new data, including Metushi et al.'s report in this issue of Hepatology, contribute to an evolving understanding regarding the contribution of the immune response to the etiology of INH hepatotoxicity.
Utilizing various immunoassay approaches, Metushi et al. measured Abs to INH and native proteins in clinical samples from two patient groups. Patient samples (n = 19) obtained from the Acute Liver Failure Study Group (ALFSG) registry deemed to have at least probable or higher (>50%) likelihood of toxicity resulting from INH represented the first group. Comparison samples were obtained from patients receiving INH prophylaxis (n = 20), of whom 15 had no liver injury response, whereas 5 developed mild hepatitis (ALT, 47-147 IU/L). Baseline demographic characteristics differed between the two groups by gender (73.7% female, ALFSG group vs. 40% female, prophylaxis group), whereas mean age was comparable between groups. An antigen for detection of anti-INH Ab in the patient's serum was prepared by modifying lysozyme with the N-hydroxysuccinimide ester of isonicotinic acid (INA). To test the specificity of the patient's Ab to INH, free INH was mixed with patient serum. Western blotting assays showed inhibition of binding, confirming the assay's specificity for INH. Samples from the two patient groups were subsequently tested with the synthetic Lys-INH antigen. Eight of the nineteen samples from patients with INH ALF were positive (42%), whereas the samples from the INH prophylaxis group were all negative for Abs to LYS-INH.
Additional assays were performed in the ALFSG samples to examine the immune response to native proteins involved in the metabolism of INH. These assays showed that a significant fraction (14 of 19) of ALFSG samples contained Abs that reacted with one or more of the CYPs, specifically CYP2E1, CYP3A4, or CYP2C9. These CYP P450 isoforms activate INH, as confirmed in this report and previously supported by clinical reports showing that INH inhibits the metabolism of known pharmacologic substrates of CYP2E1, CYP3A4, and CYP2C9.7 Though some of the patient samples contained Abs to INH and several of the CYP P450s, the pattern of detection of the various antigens was variable among the 8 patients with positivity to INH. In addition, the ALFSG samples were tested with a composite antigen comprised of CYP2E1 modified with INA to produce CYP2E1-INH. Thus, the composite antigen contained antigenic determinants that would be recognized by the same Abs that recognize LYS or LYS-INH and/or CYP2E1 protein. Some patient samples (e.g., ALF-3 and ALF-10) were negative for anti-CYP-2E1, but were positive for anti-INH (LYS-INH) and CYP2E1-INH. In this context, the positive reaction of select patients (ALF-1, −6, −13, −15, −16, and −17) to the CYP2E1-INH composite antigen appears to be the result of reactivity with the 2E1 protein antigenic determinants; the positive reaction of patient ALF-7 appears to be the result of reactivity with the INH antigenic determinant; and the positive reaction of other patients (ALF-3, −10, −14, −18, and −19) could be the result of either or both antigenic determinants. This concept could be ultimately confirmed in the future by competitive inhibition studies. For example, one could predict that the anti-CYP2E1-INH reactivity of ALF-1, −6, −13, −15, −16, and −17 could be inhibited by incubating the Ab with free INH.
The overall conclusion of the Metushi et al. study is consistent with the concept that in some patients with INH hepatotoxicity, CYPs bioactivate INH to a reactive metabolite that covalently modifies the CYP and/or other native proteins, and that in some (as yet undefined) circumstances, this reaction constitutes an immunogenic experience, and that the resultant immune response may have specificity for the INH hapten, CYPs, or both. Although the Metushi et al. data add support for an immune mechanism of injury, there is no proof that the detected anti-INH Abs are associated with injury. The anti-INH Abs and autoAbs may be the result of a nonpathogenic immune response associated with INH metabolism, but not associated with injury. Additional, larger studies are needed to more fully examine the relationship of detectable immune responses to LYS-INH and relevant CYP P450s in relationship to the onset and degree of liver injury, with appropriate comparison to non-INH-exposed human samples and INH prophylaxis samples. In addition, additional work is needed to examine whether or not the immune response to the CYP P450s examined in this report (CYP2E1, CYP3A4, and CYP2C9) with activity to INH is limited to these particular CYP P450 isoforms. For example, INH also interacts with CYP2C19, a highly polymorphic enzyme involved in the metabolism of a large number of drugs used clinically, such as phenytoin. The drug-drug interaction of INH and phenytoin is well documented and represents a clinically important issue for patients receiving INH because of the potential for phenytoin toxicity secondary to the inhibitory effects of INH on the metabolism of phenytoin. In vitro studies have shown that INH potently inhibits the catalytic activity of CYP2C19 and CYP3A in a concentration-dependent manner, whereas it is a noncompetitive inhibitor of CYP2E1.7 Other drug interactions with potential relevance in the clinical setting of INH hepatotoxicity include the concurrent use of INH with CYP2C19 substrates (e.g., omeprazole, diazepam, citralopram, and nelfenavir), CYP3A4 substrates (e.g., carbamazepine, ethosuximide, and vincristine), and/or CYP2E1 substrates (APAP and ethanol).[7, 8]
Clinical studies of drug-induced liver injury are very challenging because of the rare and the multifactorial nature of the condition and the lack of mechanism-based analytical assays. Future strategies involving the collaborative use of international clinical sample banks—representing broad genetic diversity—would broaden and facilitate ongoing research in this area. An analysis of INH Ab positivity by ethnicity, drug metabolism genotype status (e.g., N-acetylation and CYP P450), concomitant use of other medications, and the inclusion of appropriate negative controls would help to advance our growing understanding of the role of the immune system and its interaction with drug metabolism in the pathogenesis of INH hepatotoxicity. Ultimately, a strategy that incorporates new analytical approaches—addressing both the immune response and pharmacogenetic vulnerability—can be envisioned.
Laura James, M.D.
Dean Roberts, Ph.D.
Department of Pediatrics University of Arkansas for Medical Sciences Section of Clinical Pharmacology and Toxicology Arkansas Children's Hospital Little Rock, AR