Mitochondrial and immunoallergic injury increase risk of positive drug rechallenge after drug-induced liver injury: A systematic review †
Potential conflict of interest: C. M. Hunt is an employee of GlaxoSmithKline.
In prospective studies, drug rechallenge following drug-induced liver injury (DILI) is associated with up to 13% mortality,1 whereas retrospective case series report a 2% mortality rate across a broad range of drugs2 and a 51% mortality rate with halothane rechallenge.3 However, risk factors for severe liver injury with rechallenge are poorly characterized.1-4 Clinical outcomes following drug rechallenge appear to vary markedly by drug,2, 3, 5-7 suggesting that rechallenge risk may be related to drug-specific mechanisms of injury.
Drug rechallenge is rarely deliberately performed due to potential fatalities.4 However, drug rechallenge may be considered in life-threatening disease, when no other treatments are available with informed consent and close follow-up. Because limited data on drug rechallenge are available, additional data are needed to enhance clinical decision making. The aim of this systematic analysis is to examine clinical outcomes and mechanisms of liver injury that may influence a drug's potential for serious or fatal injury following rechallenge.
When compared with the initial liver injury, rechallenge injury occurs more rapidly (generally after days or weeks, but sometimes within hours of rechallenge).2 Rechallenge events commonly result in jaundice (64%), hospitalization (52%), or allergic/hypersensitivity features (39%).4 Most rechallenge events are inadvertent and hence preventable if initial DILI is accurately identified and clearly communicated to the patient.
DILI results from the cumulative effects of oxidative stress, reactive metabolites, immune injury from protein adducts, inhibition or disruption of transporters or drug metabolism, mitochondrial impairment,8 loss of ion gradients and adenosine triphosphate (ATP), and activation of programmed cell death or necrosis.9 Intraindividual and environmental factors, inflammatory mediators, and glutathione stores further modulate the outcome of DILI. For example, diabetes increases the risk of acute liver failure,10 which may be related to diminished mitochondrial function.8, 10, 11 In addition, approximately 1 in 8,000 adults harbor inherited pathogenic mitochondrial DNA mutations.12
Most drugs withdrawn from the market are mitochondrial toxicants, as are more than half of marketed drugs with black box warnings for hepatotoxicity or cardiotoxicity.13 Mitochondria are the key cellular energy source, supplying more than 90% of cellular ATP.13 Therefore, drug-induced mitochondrial impairment directly affects hepatocyte viability.8 Mitochondrial energy is largely generated by fatty acid oxidation; mitochondrial respiration also generates reactive oxygen species.13 Drugs or metabolites that inhibit the mitochondrial electron transport chain increase reactive oxygen species and can trigger the mitochondrial permeability transition (depleting the mitochondrial membrane potential difference), resulting in cell death.8, 13 Nucleoside analogues for treatment of human immunodeficiency virus inhibit mitochondrial DNA synthesis in a concentration-dependent fashion, resulting in decreased mitochondrial respiration and ATP and increased lactate.13 Drugs that are lipophilic and cationic (such as tacrine) accumulate in mitochondria and increase the risk of mitochondrial toxicity.13 Cell death occurs when a critical number of a hepatocyte's mitochondria are disabled.8 Upon withdrawal of mitochondrial toxicants, mitochondria can successfully regenerate over one to several weeks.14, 15
The likelihood of DILI is significantly increased in patients exhibiting polymorphisms of mitochondrial DNA polymerase gamma,16, 17 superoxide dismutase 2 (which scavenges mitochondrial superoxide),18 or specific human leukocyte antigen (HLA) markers associated with immunoallergic injury.19-23 Immunoallergic injury or hypersensitivity is associated with fever, rash, eosinophilia, and antidrug antibodies and occurs rapidly on rechallenge (sometimes within hours). Immunoallergic injury is initiated when a reactive drug or metabolite binds to intracellular proteins and generates a hapten, which is taken up by antigen-presenting cells and presented with major histocompatibility complex to helper T cells, and further activated by a danger signal of cytokines, such as high mobility group box 1 or other markers of necrosis or inflammation, to stimulate an immune response.24
Among the potential mechanisms of hepatotoxicity, concomitant mitochondrial impairment and immunoallergic injury appear most likely associated with fatal or positive rechallenge. Drug-specific rechallenge outcomes were systematically reviewed to examine the hypothesis that drug-related mitochondrial impairment and/or immunoallergic injury may particularly increase the risk of positive rechallenge or fatality and to further assess other rechallenge risk factors. With mitochondrial injury, rechallenge within several weeks of the primary DILI would be expected to greatly increase the risk of positive or fatal rechallenge. This results from residual mitochondrial injury persisting from the primary liver injury and incomplete mitochondrial regeneration lowering the threshold for incapacitating cellular injury. Therefore, clinically important rechallenge injury occurs more rapidly. Immunoallergic injury should also occur more quickly on rechallenge than observed with the primary injury. Combined mitochondrial and immunoallergic injury likely negatively impacts rechallenge clinical outcomes.
Patients and Methods
A comprehensive search of PubMed was completed using the terms “liver injury and drug rechallenge,” “liver injury and rechallenge,” and “hepatotoxicity and rechallenge” with a secondary search of selected English-language references. Drugs with at least 10 well-documented rechallenge events25 were systematically summarized by clinical outcome (fatality, liver transplantation, or other), demographics, predominant liver injury type, drug dosage,26 timing of drug readministration relative to the initial liver injury event, percent positive rechallenge, evidence of potential hypersensitivity (defined as fever, rash, peripheral eosinophilia, or eosinophilic infiltrate on liver histopathology), putative risk factors, and mechanisms of liver injury.
Liver injury was categorized as hepatocellular, mixed, or cholestatic.27 Drug rechallenge was defined by Council for International Organizations of Medical Sciences criteria27 with hepatocellular injury as a doubling of alanine aminotransferase (ALT) on rechallenge with ALT >2× upper limit of normal (ULN) and ALT (ULN)/alkaline phosphatase (ULN) >5 (or R > 5) and cholestatic or mixed injury as a doubling of alkaline phosphatase (or bilirubin) on rechallenge with alkaline phosphatase >2× ULN and ALT (ULN)/alkaline phosphatase (ULN) <5 (or R < 5). When the data permitted, positive rechallenge was confirmed following the initial drug injury to prevent inadvertently including chronic liver injury as a positive rechallenge event.
The systematic review yielded drug-specific rechallenge data for seven drugs, of which three were derived from controlled clinical trials and four from case series. In drug rechallenge series, most subjects exhibited hepatocellular injury, jaundice, and/or hepatitis symptoms.1, 2, 4 Antibiotics, antiretrovirals, azathioprine, H2 antagonists, and 5-HT3 antagonists were the most frequently implicated medications in rechallenge injury. Most drug rechallenges were inadvertent. Most rechallenge events occur more rapidly than primary injury: 40 days to rechallenge injury versus 93 days for primary injury in prospective studies,4 and liver injury appearing within 1 week of rechallenge in nearly half of patients in a retrospective series (and within hours in 2 of 88 patients, exhibiting predominantly immunoallergic injury).2 Although most patients had jaundice or hepatitis symptoms with the initial or rechallenge liver injury, asymptomatic liver chemistry elevations were reported in >50% of patients upon rechallenge, and jaundice or hepatitis symptoms were reported less commonly on rechallenge than the initial liver event in a retrospective series.2 Positive rechallenge events were observed over a broad age range (6 months to 83 years) and at generally similar rates in both sexes.2, 4
Drug-Specific Rechallenge Results.
Many drugs with positive or fatal rechallenge are associated with mitochondrial impairment, hypersensitivity, or immunoallergic injury, hepatocellular injury, reactive metabolites, and high dose. Rechallenge data for seven drugs are outlined in Table 1.
Table 1. Rechallenge Events with Marketed Drugs
|Halothane (n = 141)3||51%3||Inhibits mitochondrial complex 1 and 2; inhibits mitochondrial fatty acid and pyruvate oxidation30, 31||HLA A-1129; hypersensitivity28, 29; reactive metabolite28, 29||Inhalation|
|Tacrine (n = 145)5||33%5||Impairs mitochondrial DNA polymerase gamma and topoisomerase I and II; depletes mitochondrial DNA32||Reactive metabolite32||20-160 mg26|
|Atorvastatin, simvastatin, lovastatin (n = 10)34||30%34||Decreases mitochondrial membrane potential; inhibits fatty acid beta-oxidation36; uncouples electron transport from phosphorylation37||Atorvastatin electrophilic acyl glucuronide metabolite35||20-80 mg26|
|Isoniazid, rifampin, pyrazinamide (n = 220)6, 7||11%6-24%7||Decreases mitochondrial membrane potential; releases cytochrome c40||HLA DQB1*020139; reactive metabolite||300-1,500 mg26|
|Azathioprine (n = 14)2||—||Oxidative stress; depletes mitochondrial glutathione and decreases ATP42||14% hypersensitivity2||100 mg26|
|Lamivudine, stavudine, didanosine, zidovudine (n = 11)2||—||Inhibits mitochondrial DNA synthesis45||Stavudine epoxide intermediate reactive metabolite44||80-600 mg26|
|Amoxicillin/clavulanate (n = 10)2||—||—||HLA DQB1*060223; HLA DRB1*150119; 38% hypersensitivity41||500-3,000 mg26|
Halothane rechallenge is associated with the highest fatality rate, approaching 50% in two case series when rechallenge occurred within 1 month of anesthesia complicated by halothane-associated jaundice.3 Females and obese subjects exhibit an increased susceptibility to injury.28 Postulated mechanisms of halothane liver injury include both immunoallergic injury/hypersensitivity and mitochondrial impairment. Halothane is oxidized to a trifluoroacetyl halide, which forms protein adducts,24, 28 and it forms a free radical in hypoxic conditions.29 Fatal halothane rechallenge is widely attributed to immunomediated liver injury with rapid injury with rechallenge, associated fever, eosinophilia, anti-CYP2E1, anti–liver-kidney-microsomal and adduct antibodies,24, 28 and association with HLA A-11.29 Halothane also causes mitochondrial impairment, due to inhibition of complex I and II,30 as well as fatty acid and pyruvate oxidation in nonclinical studies.31 Therefore, halothane's high fatality rate on rechallenge appears related to its combined mitochondrial impairment and immunoallergic injury, most frequently observed with rechallenge occurring within 1 month of initial injury.
Tacrine, a cholinesterase inhibitor for Alzheimer's disease, is associated with a 33% positive rechallenge rate. Asymptomatic liver injury is commonly observed with initial tacrine treatment, with 6% of subjects exhibiting ALT exceeding 10× ULN and 25% of subjects with ALT exceeding 3× ULN in controlled clinical trials.5 One hundred forty-five subjects with ALT exceeding 3× ULN during initial tacrine treatment underwent tacrine rechallenge after a mean of 6 weeks (2-24 weeks). One-third (48/145) exhibited positive rechallenge, and ALT elevations occurred earlier with rechallenge than initial treatment (22 versus 48 days), although ALT normalized in 62% despite continuing treatment, suggesting adaptation. ALT elevations were generally lower with rechallenge than the initial liver event, and no patients exhibited bilirubin exceeding 3 mg/dL or 52 μmol/L.5
Possible mechanisms of tacrine-related predominantly hepatocellular injury include generation of a reactive tacrine metabolite and depletion of mitochondrial DNA.32 With 12-28 day tacrine administration in mice, tacrine accumulates in mitochondria, impairs DNA polymerase gamma and topoisomerase I and II, and depletes mitochondrial DNA.32 Tacrine increases p53, Bax, affecting mitochondrial permeability transition, cytosolic cytochrome c, and caspase-3 activity, resulting in hepatocyte apoptosis or necrosis.32 Therefore, tacrine markedly impairs mitochondria, and its reactive metabolite could potentially trigger immunoallergic injury in susceptible individuals.
Mild to modest ALT elevations are common with statin initiation, affecting 0%-3%.33 In a retrospective analysis of a large insured population in the United States, 23,000 patients received statins.34 Of these patients, 2% exhibited ALT exceeding 3× ULN and 0.1% (17/23,000) exhibited symptomatic hepatitis and statin-related ALT exceeding 10× ULN (with most occurring within 4 weeks of therapy initiation). Among those patients with ALT exceeding 10× ULN with initial statin treatment, 10 were rechallenged with the same statin. Three of the 10 (30%) patients exhibited a positive rechallenge, with one event reported as being severe.34 With statin cessation, liver chemistry elevations resolved within 2-8 weeks.
Statin injury is predominantly hepatocellular.33 The formation of electrophilic metabolites covalently binding to proteins is frequently implicated in immune-based hepatotoxicity; an electrophilic acyl glucuronide metabolite has been reported for atorvastatin.35 When tested in vitro, lipophilic statins (e.g., atorvastatin, simvastatin, cerivastatin, fluvastatin) decrease mitochondrial membrane potential and beta-oxidation and increase mitochondrial swelling, cytochrome c release, and DNA disruption.36 In isolated rat hepatic mitochondria, simvastatin uncouples electron transport from phosphorylation.37 Statin-induced mitochondrial impairment and/or reactive metabolite formation contributes to rechallenge injury.
Due to the high rate of liver injury associated with tuberculosis medications and their critical public health role in global tuberculosis control, two prospective controlled clinical trials have examined the clinical outcomes of rechallenging patients with active disease with tuberculosis medications.6, 7 One hundred seventy-five Indian patients, 93% of whom exhibited symptomatic hepatitis or jaundice on initial treatment with isoniazid, rifampin, and pyrazinamide, were observed until liver injury resolved and then randomized to rechallenge with tuberculosis medications, all restarted simultaneously or with each drug restarted sequentially.6 Both treatment groups exhibited similar positive rechallenge rates with an overall 11% positive rechallenge rate and no fatalities. Positive rechallenge was associated with a lower pretreatment albumin value (3.4 g/dL versus 3.9 g/dL in patients with negative rechallenge; P < 0.01).6 However, the risk of positive rechallenge was unaffected by the severity of the initial DILI.6
Forty-five Turkish patients with liver injury on initial tuberculosis treatment were followed until liver injury resolved and then rechallenged with isoniazid, rifampin, ethambutol, and pyrazinamide in two different ways: simultaneous rechallenge of all four TB medications resulted in a 24% positive rechallenge rate (n = 25), whereas exclusion of pyrazinamide and dose escalation of the remaining three medications resulted in a 0% positive rechallenge rate (n = 20).7 Pyrazinamide DILI is reportedly more severe/fatal than isoniazid or rifampin,38 and its exclusion favorably affected rechallenge.7 A higher risk of positive rechallenge was associated with hypoalbuminemia, extensive tuberculosis, and female sex.7
Possible mechanisms of the predominantly hepatocellular injury observed with tuberculosis medications include the generation of a reactive metabolite of isoniazid, high daily dose of 300-1,500 mg,26 immunoallergic injury, with an HLA DQB1*0201 marker associated with liver injury (odds ratio, 1.9),39 and mitochondrial impairment. In cell cultures, isoniazid decreases mitochondrial membrane potential, releasing cytochrome C.40 Risk factors for liver injury include advanced age, female sex, alcohol use, and hypoalbuminemia.39 Therefore, tuberculosis medications induce both mitochondrial impairment and immunoallergic injury.
Whereas liver injury is frequently delayed 1-4 weeks posttreatment in initial amoxicillin/clavulanate-associated liver injury, injury appeared in only 4 days of positive rechallenge in 10 patients in a retrospective series.2 In comparison with the initial event, the severity of liver chemistry elevations was generally similar in most rechallenge events, although it was increased in one subject.2 This 41-year-old man developed hepatocellular hepatitis with initial treatment with amoxicillin/clavulanate and developed cirrhosis on subsequent rechallenge with amoxicillin/clavulanate, requiring liver transplantation.2, 4
Administered at a high daily dose of 500-3,000 mg,26 amoxicillin/clavulanate is frequently associated with cholestatic or mixed disease; only 36% of patients exhibit hepatocellular injury.41 Hypersensitivity was observed in 38% of amoxicillin/clavulanate-associated DILI in the prospective Spanish DILI Registry.41 In further support of immunoallergic injury, two HLA markers are associated with amoxicillin/clavulanate-associated liver injury: HLA DQB1*060223 and HLA DRB1*1501.19 Therefore, amoxicillin/clavulanate's rechallenge injury is primarily immunoallergic.
In a retrospective series, azathioprine-positive rechallenge was associated with hepatocellular injury in 71% of patients, jaundice in 43% of patients, and hypersensitivity in 14% of patients (n = 14).2 Whereas the initial liver injury occurred following 3 months of treatment, liver injury with positive rechallenge was observed after only 10 days.2
The key mechanisms of azathioprine liver injury include hypersensitivity,2 glutathione depletion, oxidative stress with resultant mitochondrial impairment, and depletion of mitochondrial glutathione,42 ATP, and lipid peroxidation.43
Nucleoside Reverse-Transcriptase Inhibitors.
Positive rechallenge events with nucleoside reverse-transcriptase inhibitors generally resulted in asymptomatic hepatocellular injury in a retrospective series.2 Graded liver chemistry elevations were similar in severity in most patients undergoing rechallenge in comparison with the initial liver injury.2
The mechanisms involved in nucleoside reverse-transcriptase inhibitor-associated hepatotoxicity include the formation of a stavudine epoxide intermediate reactive metabolite44 and progressive mitochondrial impairment resulting from depletion of mitochondrial DNA. Among nucleoside reverse-transcriptase inhibitors, the rates of hepatotoxicity are highest in those drugs most potently inhibiting mitochondrial DNA synthesis in vitro: zalcitabine-didanosine, stavudine, and zidovudine-lamivudine.45 Therefore, mitochondrial impairment is a key mechanism of rechallenge injury, with a possible contribution from reactive metabolites.
Drug-specific mechanisms of liver injury appear to influence the incidence, severity, and temporal onset of positive rechallenge events. Mitochondrial impairment and immunoallergic injury are associated with the highest rate of positive rechallenge (ranging from 11% to 51%) and fatality. Rechallenge within 1 month of halothane-induced jaundice results in a mortality rate of nearly 50%.3 However, for other drugs, rechallenge mortality rates range from 2% to 13%, and the severity of the primary liver injury does not clearly affect the severity of the rechallenge injury.2, 4 Hepatocellular injury and high daily drug dose (>50 mg) are also commonly observed in positive rechallenge.
Drug rechallenge is generally avoided due to associated severe liver injury and fatalities.1, 2, 4 However, because drug rechallenge may be considered for life-saving therapies (e.g., oncology), evaluation of the mechanisms of liver injury may elucidate a drug's potential for serious or fatal injury.
Mitochondria supply most of a hepatocyte's ATP and are thus central to hepatocyte survival.37 Mitochondrial dysfunction arises from multiple mechanisms: oxidative stress; opening of the mitochondrial transition pore, which depletes the mitochondrial membrane potential; inhibiting or uncoupling electron transport, which diminishes ATP production; inhibiting fatty acid oxidation; and inhibiting synthesis of mitochondrial DNA or proteins, which prevents the replacement of damaged mitochondria with newly formed functional mitochondria.37 Approximately one-third of over 500 pharmaceuticals inhibit mitochondrial respiration or impair electron transport.46 Mitochondrial dysfunction can be caused by a diverse array of drugs, including antibiotics, antiretrovirals, antidepressants, antianginals, nonsteroidal anti-inflammatory agents, anticonvulsants, anesthetics, antiarrhythmics, and oncology agents.37, 47 In drugs initiating mitochondrial dysfunction, liver injury develops gradually over weeks, because cumulative mitochondrial impairment reaches a critical threshold with clinically evident liver injury (apoptosis or necrosis).8 Regeneration of individual mitochondria and full cellular repopulation of mitochondria takes several weeks. Therefore, if drug rechallenge occurs within days to weeks of an initial liver injury, impaired mitochondria have not been replaced, resulting in a more rapid and lower threshold for critical cell injury. Cumulative mitochondrial dysfunction likely explains the nearly 50% mortality rate for individuals receiving halothane within 1 month of prior liver injury, and <12% mortality rate overall with rechallenge after 1 month of initial liver injury, when hepatocyte mitochondria have been repopulated.3 This supports delaying rechallenge of critical medications resulting in mitochondrial dysfunction to allow mitochondrial repopulation, if possible.
Immunoallergic injury or hypersensitivity is a prominent factor in DILI in select drugs (particularly antibiotics, antiretrovirals, and anticonvulsants). Multiple HLA markers have recently been identified which are highly associated with liver injury.19-23 Hypersensitivity reactions result in rapid onset of rechallenge injury (within hours for some drugs) with accompanying fever, rash, or eosinophilia.
Most positive rechallenge events yield hepatocellular injury. A prospective series reports an overall rechallenge mortality rate of 13%,1 which is somewhat higher than the 7%-12.7% mortality rates reported for the initial or primary hepatocellular DILI in several series.1, 48-51 Most drugs resulting in positive rechallenge are administered at a high daily drug dose (>50 mg), which has been associated with a higher risk of DILI overall.52
Typically, fewer than 1 in 1,000 exposed patients develop severe DILI,53 suggesting a heightened vulnerability in those affected, which may be due to a concurrent, potentially transient, inflammation54, 55 and resultant oxidative stress, with concomitant medications contributing to defective liver regeneration/repair,56 high drug dose or hepatic metabolism,52 female sex or obesity,28 inherited pathogenic mitochondrial DNA mutations,12 other genetic susceptibility,57 or other factors. Therefore, in addition to drug-specific mechanisms of injury, interindividual factors greatly influence the reaction to DILI.57 These additional important factors are generally outside the scope of this drug-specific analysis. This review is also limited by the incomplete retrospective case data, which impedes the conclusive identification of DILI25 or rechallenge (which may affect the percentage with positive rechallenge); sparse drug rechallenge literature, with its likely bias to report the more severe events; and the possibility that the search terms or strategy did not identify some relevant literature.
In conclusion, a systematic review of published case series reveals that drug rechallenge should generally be avoided and considered only if the benefit exceeds the risk (e.g., in the case of a life-saving drug). Drug-specific rechallenge analyses suggest that drugs causing mitochondrial and immunoallergic injury are associated with a higher rate of clinically important and even fatal positive rechallenge reactions. Cumulative mitochondrial dysfunction may particularly increase the risk of positive rechallenge when a suspect drug is restarted within weeks of the primary injury, prior to repopulation of the hepatocyte's mitochondria. Publication of drug-specific rechallenge information from controlled clinical trials and liver injury registries will further elucidate risk factors for positive rechallenge. Understanding risk factors and mechanisms of primary and rechallenge liver injury, as well as clinical outcomes, through an expanded evidence base, will advance drug safety.
Acknowledgment: The author wishes to thank Julie Papay, Nancy Yuen, Dawn Clines, Rezvan Rafi, Roger Brown, John S. Walsh, and the GlaxoSmithKline Hepatotoxicity Board for expert input and Cynthia Traynham for administrative assistance.