Lecture 11 - Hepatotoxicity 2 Flashcards
(36 cards)
(I?)DILI: why is it so hard to account for?
Hard to detect, relatively low incidence (5-20/100,000)
IDILI: what is it, what is it mostly caused by, why, and what are the suggested mechanisms that result in IDILI?
Idiosyncratic DILI
Antibiotic drugs - not exactly clear whether these drugs specifically result in it or whether they are the most frequently taken type of drugs
Basis of these reactions are poorly understood - understood that there is the production of active metabolites that activate the immune system - act as haptens and cause cell damage/production of anti-drug antibodies
Several hypothesis exist to explain them and the most common is “The Adaptive Immunity Hypothesis of IDILI”:
* T-cell reactions
* Cytokines produced (TNFα, IFNγ, etc) - prevent new cells forming to compensate damage
The Adaptive Immunity Hypothesis of IDILI: what clinical evidence is there for it?
- Fever, skin rash, eosinophilia - associated with immune reactions, these are not seen in intrinsic DILI
- HLA (class II) polymorphisms
- Production of active metabolites that activate the immune system - act as haptens and cause cell damage/production of anti-drug antibodies
IDILI: what is the proposed mechanism for its formation?
Hepatotoxic drug results in cell injury and activation of innate and adaptive immunity
This results in an individual response which takes into account susceptibility factors
In cases with no IDILI adaptation occurs and immune tolerance and cell repair happens
In those with failed adaptation (FA), IDILI occurs (what causes FA is poorly understood):
* APCs present drug antigens/drug-modified self antigens
* CD8+ cytotoxic T-cell activation and CD4+ helper T-cell activation promotes an immune response
* Hepatic cell injury, apoptosis, necrosis, steaatosis, etc, all occur
Flucloxacillin-induced IDILI: what is flucloxacillin, how may it cause liver injury, why is this an important medical problem, how long do flucloxacillin reactions occur, and how may any liver injuries present?
flucloxacillin - a beta-lactam semi-synthetic antibiotic is used widely in the treatment of gram-positive bacterial infections
It causes liver injury, which is predominantly cholestatic in nature and may be irreversible
The risk of cholestasis amongst first-time users of flucloxacillin is thought to be in the region of 8.5 in 100,000 patients- there are ~2 million prescriptions per year in the UK for this drug, and given that the injury caused may be irreversible, flucloxacillin-induced liver injury is a significant medical problem
In the case of flucloxacillin reactions, this delay is typically between 1 and 45 days (drug administration likely occurs between 4-5 days, can’t often stop using it before IDILI occurs)
All immune-mediated:
* Mitochondrial stress (minor)
* Apoptosis
* Bile duct injury (cholestasis)
Cholestasis
Reduced or stopped bile flow
Flucloxacillin: what is the main enzyme that metabolises it, what is its main metabolite, and how is it formed?
CYP3A4
The biologically active 5′-hydroxymethylflucloxacillin
The hydroxylation of the 5-methyl group of the isoxazole ring by CYP3A4
Metabolite
a substance formed in or necessary for metabolism
Proposed mechanism of immune-mediated bile duct injury with flucloxacillin
5′-hydroxymethylflucloxacillin is produced following flucloxacillin metabolism
* 5′-hydroxymethylflucloxacillin causes inhibited bile acid secretion
* Bile acid builds up within cells, causing cell stress and the release of DAMPS
* These result in immune stimulation and, if tolerance doesn’t occur, an immune reaction occurs (resulting in IDILI)
* Immune response causes hepatocytes to undergo apoptosis, and cytokines prevent cell proliferation
Flucloxacillin: how may it induce apoptotic cell death?
Immune cells, such as T-cells and macrophages, support programmed cell death through:
* Pro-inflammatory cytokines (e.g., TNF-α, IFN-γ)
* Fas/FasL interactions
* caspase activation
Flucloxacillin: what are the genetic links involved with increased risk, how may these genetic links enable the predictions of susceptibility to other antibiotics, and what is its similarity to amoxicillin?
Association with HLA-B57:01 and B57:03 - not perfect, there is a correlation but not 100% confirmation that someone will/won’t have an IDILI reaction with flucloxacillin
Does not enable the prediction of susceptibility to idiosyncratic reaction to other (or newly developed) antibiotics
Amoxicillin & flucloxacillin have a lot of structural similarity, but hepatotoxicity is associated with different HLA genes
Why is the prediction/detection of hepatotoxicity important?
Drug-induced hepatotoxicity remains one of the most frequent reasons for drug withdrawal:
* During clinical phases of development
* During postmarketing pharmacovigilance
Predictability from in vivo and in vitro assays is not perfect:
* False positives
* Species differences – some toxicities are animal-specific; animals dosed at high levels
* False negatives
* Absence of predisposing factors; species differences; insufficient exposure
* Idiosyncratic toxicity - while intrinsic DILI can be predicted, IDILI cannot
Modelling DILI - In vitro testing: what is it, what is one of the easiest and efficient methods of testing, and why is it ideal but not perfect?
In test-tube testing - the first-line screening for any developing drugs
Cell lines - cells are easily available, relatively cheap and easy to grow:
* Suitable for high-throughput screening (HTS) assays
* Small amounts of the compound can be used
* Use of a single translatable endpoint
Detection of intrinsic DILI is easy to do (monitoring effect of dosage on cells), but idiosyncratic DILI is hard to detect
Modelling DILI - In vitro testing - immortalised cell lines: what are the types used, which is the current gold standard, and what is the new gold standard??
- Fa2N-4
- HepG2 (current gold standard)
- HepaRG (new gold standard?)
Modelling DILI - In vitro testing - immortalised cell lines: why is HepG2 the current gold standard?
- Lack nuclear receptors and efflux transporters
- CYP enzymes may be of lower expression (but all present)
- Metabolic and secretory functions similar to primary hepatocytes
Modelling DILI - In vitro testing - immortalised cell lines: why is HepaRG seen as the new gold standard, but why is it not adopted as the new gold standard yet?
- Greater similarity to primary hepatocytes
- Greater CYP and transporter activities; functional bile canaliculi
These cells are much more expensive
Modelling DILI - In vitro testing - primary cell cultures: what is the gold standard of hepatocytes to use, why, how are cells pooled, and why is it difficult to obtain these hepatocytes?
Primary human or rat hepatocytes:
* Greatest metabolic activity;
* Can be cultured in 3D
Pooled according to gender, BMI, ethnicity, genetics, etc
Cells (especially human cells) are an exhaustible supply & expensive, and lose metabolic activity quickly in culture
Microsomes: what are they, why may they be used, what can they be used to detect, and what limitations are there?
Homogenise tissue and centrifuge them to obtain cellular fractions (ER)
Quicker and easier to use, especially to do initial testing
Phase 1 metabolism only
Cannot detect phase 2 metabolism well, there may be phase 2 enzymes present in fractions but they are limited
Modelling DILI - In vitro testing - primary cell cultures: what are the types of testing and what are the advantages and disadvantages to each?
- Primary cell cultures (PCC) - Cheap and easy to do, worst at showing hepatotoxicity
- Immortalised cell lines (ICL) - more expensive and harder to obtain than PCC, better at showing hepatotoxicity
- Liver slices - more expensive and harder to obtain than PCC/ICL, better at showing hepatotoxicity
- Whole perfused livers - more expensive and harder to obtain than PCC/ICL/liver slices, better at showing hepatotoxicity
Modelling DILI - In vitro testing: what are the things that can be tested, what controls are often used, and what results are found?
- Enzyme induction
- Enzyme inhibition
- Hepatocellular necrosis
- Loss of mitochondrial function (using IC50)
- Impaired tetrazolium salt reduction (MTT assay) (using IC50)
- Decreased ATP levels (using IC50)
EC50 determination of enzyme activity or mRNA levels
Determination of IC50 values using dose-response curves
EC50
The concentration of a drug that gives half-maximal response
IC50
The concentration of an inhibitor where the response (or binding) is reduced by half
Modelling DILI - In vitro testing: what are some other in vitro approaches and what are their advantages/disadvantages?
Embryonic stem cells - advantages:
* Better displays hepatotoxicity compared to traditional cell lines
Embryonic stem cells - disadvantages:
* Ethical Issues
* Maturation issues
* Variability
Induced Pluripotent cells - advantages:
* Better displays hepatotoxicity compared to traditional cell lines
* iPSC-derived human hepatic stellate cells have recently become commercially available for use
Induced Pluripotent cells - disadvantages:
* Maturation
* Functional duration in culture
Liver slices - advantages:
* Retain liver structure
* Zonal cytochrome activity
* All enzymes present
Liver slices - disadvantages:
* Necrosis occurs within 48-72hrs
* Kinetics are slightly different compared to isolated hepatocytes
Modelling DILI - In vivo testing: why is it more ideal than in vitro testing from a safety perspective but more inconvenient to do, and what similarities does it have to in vitro testing?
More relevant information and multiple endpoints can be screened
Expensive, longer to conduct, ethical issues, species differences
Similar to in vitro testing - good for the detection of intrinsic DILI, but struggles to detect idiosyncratic DILI
