Drug induced liver injury

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New drugs are required to undergo pre-clinical trials before approval for market sale. Failure in these trials is mainly due to adverse drug reactions (ADRs) (1). Even with preclinical and clinical testing, 20% of new drugs will cause ADRs that only become apparent after a drug is on the market (1) when it is used by a large number of people (2). The occurrence of ADRs has risen greatly since the baby boomer generation due to their increased use of prescription medications (3).
ADRs can be classified into two broad groups: dose-dependent and predicable; and idiosyncratic (4). Predictable ADRs generally occur during a set time frame and all patients are susceptible to them (4). Idiosyncratic ADRs are more difficult to predict because toxicity is a result of a combination of factors such as drug properties, environmental factors, and genetic variation (4). A drug’s toxicity can also be the result of unintended off-target activity or non-specific reactions (1). Studying potential toxicity of a drug is complex because there is no ‘one size fits all’ method (1).
When a new drug causes an ADR that was not observed during testing, it can either be withdrawn from the market or its sales restricted (4). Drug withdrawal is not an ideal situation for pharmaceutical companies because of the high costs of removing a drug from the market and loss of buyer trust and reputation (1). This is also a concern for patients because taking the drug can result in a life-threatening or fatal outcome (5). It is estimated that 6.7% of all hospitalisations are the result of ADRs (6). In Western countries, ADRs are ranked on as the fifth most common cause of inpatient death (6).
ADRs can affect a majority of organs but most commonly manifest in blood cells, liver, and skin (7). The liver is the primary site for drug metabolism, making it a frequent off-target site for drug-induced toxicity (8). A majority of drugs on the market do not cause DILIs (9). However, DILIs are still the leading cause of drug withdrawal (10).
The reported incidence is believed to be one in every 10,000 – 100,000 patients (9, 11). Clinical trials normally have a sample size of 2,000 – 5,000 patients; the incidence of DILIs in clinical trials is therefore too small to detect (11). It is also suspected that the incidence of DILIs is underestimated (4) due to underreporting, incomplete observations, or detection/diagnosis difficulties (9).
Hepatotoxicity can result in clinical consequences such as cholestasis, hepatocellular damage, and steatosis (12). In the United States, the leading cause of liver transplant referral in acute liver failure patients is druginduced hepatotoxicity (2, 9). 10% of acute liver failure is the result of idiosyncratic DILI (4). If a patient with DILI also has jaundice, there is a 10% chance of fatality (2). Only a quarter of DILI patients will recover spontaneously (2). In the case of drug-induced hepatotoxicity, there are two suggested responses: discontinuation of the suspected drug (9) and/or therapeutic intervention to prevent or relieve toxicity (13).
It is often difficult to determine DILI because its symptoms overlap with those of all known liver diseases (14). In order to diagnose DILI, several criteria must be met. First, the causative medication needs to be identified and the onset of symptoms has to correlate with the suspected medication addition (2). The symptoms of the patient need to correspond with published reports and other potential competing causes need to be ruled out (2). When the drug is removed, the patient’s symptoms should improve (9). If the toxicity is immunological in nature, a rechallenge with the treatment should result in a more severe and rapid toxicity (9). A drug re-challenge with the suspected causative agent is helpful for further confirmation (2). However, this rarely occurs and does not always guarantee a reproducible response (2).
The tests for DILI include liver function tests and histological results from liver biopsies (15). However, liver function tests are limited by the lack of reliable and accurate biomarkers (2). Alanine transaminase (ALT) is a highly sensitive marker; however it is not able to distinguish between DILI or non-hepatic causes (10). For example, ALT activity can be elevated due to metabolic or skeletal muscle disorders (10). Even with elevations in liver enzymes, this may not affect liver function due to its ability to heal from injury (9). A liver biopsy is considered the gold standard for liver diagnosis, but is limited by its invasiveness (16).
Several factors are believed to influence idiosyncratic DILI such as age, cytochrome P450 polymorphism, drugdrug interactions, gender, genetics, immune-mediated responses, lack of adaptation, and nutritional status (10).
It is difficult to identify who is at risk due to the low incidence of DILI and limited knowledge on the specific biochemical mechanism of the toxicity (2).

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1. INTRODUCTION
1.1. Drug induced liver injury
1.2. Toxicological endpoints
1.3. Drugs of interest
1.4. Hepatitis B virus
1.5. Potential co-diseases
1.6. Summary
2. METHODS
2.1. Materials and equipment
2.2. Cell Culture
2.3. Cell viability assay
2.4. Determination of human nuclear genes, B2M and GAPDH, and human mitochondrial genes, ND1 and ND4, levels in HepG2 cell lysates by qPCR analysis
2.5. Determination of AMP, ADP, and ATP levels in HepG2 cell lysates by HPLC analysis
2.6. Determination of reduced glutathione levels in HepG2 cell lysates by HPLC analysis
2.7. Statistical methods
3. RESULTS
3.1. Optimisation of literature methods
3.3. The effect of short-term exposure of HepG2 cells to drugs at ten-fold maximal drug plasma concentration
3.4. The effect of 20-day exposure of HepG2 cells to drugs at twice maximal drug plasma concentration