3. Factors affecting Drug Metabolism Flashcards
(114 cards)
1
Q
What is induction of xenobiotic metabolising enzymes?
A
The process whereby the expression and activity of enzymes that metabolise xenobiotics are unregulated.
2
Q
Why is drug induction significant?
A
-Inducers modulate the duration of the action of drugs
-Either reducing toxicity or the duration of drug actions
-May contribute to polypharmacy interactions
-May contribute to drug tolerance
3
Q
Name the transcriptional mechanisms of xenobiotic metabolising enzyme induction
A
-Activation of Nuclear receptors
-Oxidative stress pathways
-Mitogen-Activated Protein Kinase (MAPK) Pathway
-Epigenetic Modifications
4
Q
Describe the induction of xenobiotic metabolising enzymes through activation of nuclear receptors
A
-Xenobiotics bind to nuclear receptors, which form complexes that bind to specific DNA response elements
-Leading to transcriptional activation of XME genes
4
Q
Describe the induction of xenobiotic metabolising enzymes through oxidative stress pathways
A
-Xenobiotics that generate ROS can activate pathways such as the Nrf2-Keap1 pathway
-Nrf2 when dissociated from Keap1 translocates to the nucleus and induces antioxidant response elements
-Which up regulate Phase II enzymes
5
Q
Describe the induction of xenobiotic metabolising enzymes through mitogen activated protein kinase (MAPK) pathway
A
-Xenobiotic exposure can activate MAPK signalling
-Affecting transcription factors
6
Q
Describe the induction of xenobiotic metabolising enzymes through epigenetic modifications
A
-Xenobiotics can induce changes in DNA methylation, histone acetylation or miRNA expression
-Leading to increased transcription of these enzymes
7
Q
Name some in vitro methods to studying drug induction
A
-Cell culture models, eg reporter gene assays
-mRNA expression analysis, eg qRT-PCR
-Enzyme activity assays
-Northern blotting
8
Q
Name some molecular and biochemical approaches to studying drug induction
A
-Chromatin Immunoprecipitation: Focusing on binding of transcription factors to the promoters of PME genes, confirming whether nuclear receptors or TFs are directly interacting with DNA response elements
-Electrophoretic Mobility Shift Assay (EMSA): Focusing on binding of nuclear receptors or TFs to DNA response elements, with shifts in electrophoretic mobility indicating binding
9
Q
Name an in vivo method to studying drug induction
A
-Using knockout models to study the role of a specific gene (eg receptors) in XME induction, if its presence or activity is involved
-Using knock-in models to study the
10
Q
Give types of nuclear receptors
A
-Steroid binders
-Ligand binders
-Bile acid binders
11
Q
Give the receptor responsible for inducing CYP1A enzymes, and their inducers
A
-AhR (Aryl hydrocarbon receptor)
-Polyaromatic hydrocarbons, tryptophan derived products
12
Q
Give the receptor responsible for inducing CYP2B enzymes
A
-CAR (Constitutive androstane receptor)
-Phenobarbitone drugs, bile acids
13
Q
Give the receptor responsible for inducing CYP3A enzymes
A
-PXR (Pregnane X receptor)
-Many drugs, bile acids
14
Q
Give the receptor responsible for inducing CYP4A enzymes
A
-PPAR⍺ (Peroxisome proliferator-activated receptor alpha)
-Fibrate drugs, fatty acids
15
Q
Give the receptor responsible for inducing CYP7A enzymes
A
-LXR (Liver X receptor) and FXR (Farnesoid X receptor)
-Cholesterol, bile acids
16
Q
Give examples of steroid binding nuclear receptors
A
-Glucocorticoid receptors
-Mineralocorticoid receptors
-Androgen receptors
-Estrogen receptors
16
Q
Give examples of ligand binding nuclear receptors
A
-Retinoid X receptors
-Retinoic acid receptor
-Thyroid hormone receptor
-Vitamin D receptor
17
Q
Give examples of bile acid binding nuclear receptors
A
-Pregnane X receptors
-Constitutive activated receptor
18
Q
Give examples of drug induction
A
-Rifampicin and contraceptive pill: rifampicin induces expression of progesterone and oestrogen clearance, reducing their effect on inhibition of ovulation
-Barbiturates induce the CYPs that metabolise them, resulting in increased clearance
-Smokers/grilled meat eaters may experience an increased metabolism of theophylline as PhIP will induce enzymes that break down theophylline
19
Q
Give the consequences of Xenobiotic metabolising enzyme inhibition
A
-Increased Drug Half-Life: Reduced metabolism prolongs drug action.
-Drug Accumulation & Toxicity: High plasma levels can lead to adverse effects.
-Drug Interactions: Concurrent drugs metabolised by the same enzyme may be affected.
20
Q
Describe how type II substrates may inhibit CYP450s
A
-Contain electron-donating groups (e.g., amines, imidazoles) that directly coordinate Fe³⁺, -stabilizing the low-spin hexacoordinate state.
-This inhibits enzyme activity by preventing the Fe³⁺ to Fe²⁺ transition, blocking oxygen binding and catalysis.
21
Q
What is the action of the CYP inhibitor: SKF525a
A
Binds haem prosthetic group
22
Q
Give an example of a specific inhibitor of the CYP1A2 isoform
A
Furafylline
23
Give an example of a specific inhibitor of the CYP2C9 isoform
Sulfaphenazole
24
Give an example of a specific inhibitor of the CYP2D6 isoform
Quinidine
25
Give an example of a specific inhibitor of the CYP3A4 isoform
Troleandomycin
26
Describe changes to Km and Vmax in competitive reversible inhibition
Km: Increases
Vmax: Same
27
Describe changes to Km and Vmax in mixed reversible inhibition
Km: Increases
Vmax: Decreases
28
Describe changes to Km and Vmax in irreversible inhibition
Km: Same
Vmax: Same
29
Describe types of reversible competitive inhibition of CYP450s, and examples
-Competitive inhibition by a non-substrate (ie a pure competitive inhibitor)
-Competitive inhibition by another substrate (eg omeprazole and diazepam in CYP2C19)
30
Describe reversible mixed inhibition of CYP450s, and examples
-Mix of competitive (binding site) and uncompetitive (ES complex) via nitrogen groups covalently binding to Fe in haem
-eg Metyrapone, Ketoconazole
31
Describe irreversible inhibition of CYP450s, and examples
-Suicide substrates. Often covalent link with inhibitor and enzyme-substrate complex
-eg troleandomycin on CYP3A4
32
How may we compare inhibition to determine if it is reversible or irreversible?
-Preincubating the CYP enzyme with either a competitive inhibitor or a suicide (mechanism-based) inhibitor.
-Then measuring residual enzymatic activity after the preincubation step to determine the inhibitor's effect.
-With returning enzymatic activity suggesting reversible inhibition
33
Give a clinical example of complications surrounding CYP inhibition
-CYP3A4 may be inhibited by azole (anti fungal agent) or macrolide antibiotics or grapefruit juice
-These may lead to cardiac arrhythmia in those taking terfenadine, as the K+ channel blocker is not metabolised
34
Give an example of an enzyme inhibitor used in a clinical setting
-Disulphiram (Antabuse) inhibits alcohol dehydrogenase, causing buildup of acetaldehyde if individual drinks alcohol
-Leading to flushing, nausea, vomiting, contributing towards an "aversion therapy" which is used in some treatment regimes for alcoholism
35
When may inhibition of drug metabolisers be a problem?
-Two drugs are metabolised by the same enzyme eg a P450 isoform
-The therapeutic range of one or both drugs is narrow eg warfarin
-One drug binds more strongly to the metabolising enzyme than the other eg terfenadine and erythromycin
36
What things may you measure to investigate drug X's metabolism?
-Typical monooxygenase reaction? If so P450
-NADPH dependence
-Production of metabolite inhibited by CO
-Other general CYP inhibitors
-Specific isoform inhibitors
-Inhibitory antibodies
37
Why are microsomal fractions better for measuring inhibition compared to whole cells?
-Microsomes do not contain plasma membranes
-Removing extraneous factors that may skew results
38
Describe the common trends in xenobiotic metabolism across age
Neonatal stage - Minimal phase I and II metabolism, with reduced excretion
Adult - Peak metabolic state as enzymes are fully developed
Elderly - Phase I and Renal excretion is reduced
39
Describe how neonates metabolise bilirubin
-Have severe depression of glucuronidation enzymes in neonates
-Lack of bilirubin glucuronidation leads to build up
40
Describe how neonates clear chloramphenicol, and its results.
-Unable to clear chloramphenicol, suppressing respiration in cardiac myocytes
-Leads to "grey baby syndrome" caused by cyanosis
-Leads to abdominal distention, vomiting, cyanosis, cardiovascular collapse, irregular respiration and death
41
Give an example of a phase I metabolising enzyme not found in neonates, and this impact?
-CYP1A2 is barely detectable at neonatal ages
-Caffeine is metabolised mostly instead by 8-hydroxylation
41
How much CYP450 is found in foetal livers?
30% of adult liver amounts
42
Why is species differences in drug metabolising important?
Important because most drug metabolism studies initially involve animals and toxicology studies always do
43
What is coumarin?
-Naturally occurring compounds, present in a wide variety of plants, microorganisms and in some animal species
-Used in perfumes, soap, detergents, toothpaste, tobacco, alcoholic beverages, etc
-Discontinued as a food flavouring due to hepatotoxic effects in rats and dogs fed coumarin in diet.
44
Describe the routes of metabolism of coumarin
-Either by 7 hydroxylation (healthy) and formation of epoxides (hepatotoxic)
-Epoxide is conjugated with GSH meaning unable to cause damage
-In rats, mouse, hamster, guinea pig, only a small fraction is metabolised via 7 hydroxylation, meaning rest is metabolised to epoxide form
45
Describe the species differences in drug metabolism of coumarin between humans and other mammals
-Humans: Most have functioning CYP2A6, meaning they can metabolise safely
-Rats, mouse, hamster, guinea pig: Most is metabolised to epoxide, making it dangerous
46
Is coumarin intake always safe in humans?
-Some lack CYP2A6 activity due to an amino acid change, meaning it "could" have toxic effects
-Coumarin however is demonstrated to be a non-genotoxin carcinogen, meaning unless hitting a threshold concentration, there will be no carcinogenesis
47
Give an example of marked differences in phase II metabolism between strains of species
-AhR metabolism in strains of mouse
-The B6 strain has a high-affinity AhR allele, leading to robust transcriptional activation of CYP1A1 in response to AhR ligands (e.g., TCDD, benzo[a]pyrene).
-The D2 strain has a low-affinity AhR allele, which results in weaker CYP1A1 induction, leading to slower detoxification of these xenobiotics.
48
Give hormones affecting P450 levels (in rats)
-GH
-Oestrogen
-Progesterone
-Testosterone
-Insulin
-Thyroid hormone
-Glucocorticoids
49
Describe what causes sex differences in xenobiotic metabolism
Differences in drug metabolism between male and female rats arise primarily from variations in the expression of cytochrome P450 (CYP) enzymes, particularly CYP2A, CYP2B, CYP2C, and CYP3A isoforms. These differences are regulated by sex hormones (testosterone, estrogen) and sex-specific growth hormone (GH) secretion patterns.
50
Describe how alcoholic liver disease affects drug metabolism
-Alcohol metabolism eats up NAD+ supply
-Resulting in inability to produce UDP glucuronic acid, meaning an inability for glucuronidation
-Alcohol may also compete with certain enzymes eg CYP2E1, and induction
51
Describe how Porphyria mediated liver disease affects drug metabolism
-Impaired synthesis of harm and accumulation of toxic precursors
-Levels of cytochrome P450 enzymes may be lower due to limitation in supply of haem
-Drugs such as barbiturates which induce P450s will trigger increased synthesis of harm precursors triggering a clinical attack
52
Describe how liver infection may affect drug metabolism
-Inflammation has a general repressive effect on CYP450 expression
-Limiting drug metabolism.
53
What is pharmacogenetics?
The study of variations in drug response between individuals that have a hereditary basis
54
Describe genetic polymorphisms in pharmacogenetics
-Genetic polymorphisms refer to variations in DNA sequences that occur in at least 1% of the population and can influence drug metabolism, efficacy, and toxicity.
-Often affect genes encoding drug-metabolising enzymes (e.g., CYP450 enzymes), drug transporters, or drug targets.
55
Give some common types of genetic polymorphisms in pharmacogenetics
-Single nucleotide polymorphisms (SNPs): A single base change in a gene, which can alter enzyme activity (e.g., CYP2C19 polymorphisms affecting clopidogrel metabolism).
-Insertions/deletions (Indels): Addition or loss of DNA bases, potentially leading to loss of function.
56
How may functional polymorphisms have an effect on biological activity of XMEs?
Often due to:
-Amino acid substitution
-Splice site change
-Affecting transcription factor binding
57
What are some methods of analysing XME polymorphisms?
Phenotyping – Measure enzyme activity (phenotype) directly by giving a probe drug and measuring metabolites or direct enzyme assay (eg on red or white blood cells)
Genotyping – Look directly for presence of mutation in individuals DNA. Need to know gene responsible for the defect and what mutation to screen for
58
Why may pharmacogenetics be important?
-Impact on drug kinetics and dynamics
-Safety
-Efficacy
-Economic considerations
59
Describe debrisoquine phenotyping
-Classifies CYP2D6 activity
-Essentially measuring the ratio of debrisoquin(prodrug):4-Hydroxydebrisoquin(active metabolite)
-Utilised this to quantify the rate of metabolism, eg 0.01 ratio was ultrarapid, 100 ratio was poor metabolisers
60
Give some loss of activity CYP2D6 polymorphisms
-CYP2D6*3
-CYP2D6*4
-CYP2D6*5
61
What causes the loss of activity in the CYP2D6*3 polymorphism?
A is deleted in exon five leading to frameshift and loss of function
62
What causes the loss of activity in the CYP2D6*4 polymorphism?
G->A in intron 3 exon 4, leading to alternative splicing and introduction of early stop codon
63
What causes the loss of activity in the CYP2D6*5 polymorphism?
Entire CYP2D6 gene is deleted
64
What are the two classes of loss of activity XMEs
Low-function vs null-function
65
What are the types of metabolisers, based on CYP2D6 polymorphisms, and what causes this?
-Poor metabolisers (homozygous loss of function)
-Intermediate metabolisers (heterozygous loss and normal function)
-Extensive/Normal metabolisers (homozygous normal function)
-Ultrarapid metabolisers (More than two functional alleles)
66
What causes ultra rapid metabolisers?
-Some individuals inherit multiple copies (≥2) of a functional allele, resulting in excessive enzyme production.
-Due to structural variations in the genome, specifically copy number variations (CNVs). These variations result from unequal crossover events during meiosis, leading to extra copies of the gene
67
Give the consequences of each type of xenobiotic metaboliser
-Poor metabolisers (Increased drug levels, reduced efficacy of prodrugs)
-Intermediate metabolisers (less severe than poor metabolisers)
-Extensive/Normal metabolisers (typical drug response)
-Ultrarapid metabolisers (decreased drug efficacy, increased toxicity for prodrugs)
68
Describe how CPIC allocates different genotypes to different phenotypes of drug metabolisers
Activity scores are assigned to certain alleles, of which classes of metabolisers (eg poor, ultra rapid) are assigned ranges, of which different combinations of alleles fall into
69
Describe how CYP2D6 interacts with morphine, and how ultrametabolisers may react
-Opiate prodrug, converted to morphine
-Metabolism of codeine to morphine dependent on CYP2D6
-Severe respiratory depression in ultra metabolising children
-Increased morphine in breast milk of Ultra-fast metabolisers breast feeding mothers
70
Describe NAT2 polymorphisms
-Arylamine N-acetyltransferase 2 (NAT2), expressed in 8p22
-Individuals with normal activity are fast acetylators
-50% of Europeans lack NAT2 activity, slow acetylators
-Slow acetylators have 2 mutated copies of the gene
-Phenotyping with eg caffeine and/or genotyping
71
Give some alleles of NAT2 associated with variable activity
-NAT2*4 normal activity
-NAT2*5B decreased activity
-NAT2*6A decreased activity
-NAT2*7B decreased activity
72
Is NAT2 subject to functional polymorphisms?
Yes, however NAT2 is encoded by a single open reading frame meaning no alternative splicing
73
Give examples of how NAT2 variations may affect therapies
Isoniazid
-Slow acetylators more likely to have increased risk of hepatotoxicity
-Fast acetylators more likely to show poor response to intermittent dosing
Hydralazine
-Slow acetylators and autoimmune systemic lupus erythematosus (SLE)
74
Give examples of different NAT2 allele distributions across the world
-NAT2*5 50% of Europeans, but <5% east Asians
-NAT2*7 20% of East Asians, but <5% European or African
75
What may inadequate assessment of metabolism and toxicity lead to?
-Adverse reactions in trial participants
-Drug attrition rates
-Drug recall even after approval
76
What is reaction phenotyping in drug metabolism studies?
The process of identifying which drug-metabolizing enzymes (mainly cytochrome P450s, UGTs, etc.) are responsible for the metabolism of a drug
77
Why is reaction phenotyping important?
-Preducts drug drug interactions
-Explains interindividual variability
-Guides drug development and dosing
78
What should be identified when investigating a new drug's metabolism?
-Primary metabolic pathways (determines how the drug is cleared, and major vs minor metabolic routes, predicting variability)
-Metabolising enzymes (predicts drug-drug interactions, predicting genetic variability)
-Metabolite identification and activity (assess efficacy, safety and side effects)
-Drug drug identifications
79
Give methods of reaction phenotyping to study drug metabolism
-Recombinant enzyme systems
-Human liver microsomes and S9 fractions
-Selective chemical inhibitors and antibodies
-Hepatocyte based studies
-Animal models
-Hepatic cell lines
-In silicon computational approaches
-Inhibition and induction studies
80
Describe using hepatocytes to study reaction phenotyping
-Uses fresh, cryopreserved, or immortalized human hepatocytes (liver cells) to study metabolism in a fully intact system.
-Parent drug/metabolite levels are measured.
Enzyme contributions can be inferred using chemical inhibitors or gene knockdown techniques.
81
Give the pros and cons of using hepatocytes to study drug metabolism
+Most physiologically relevant system (includes transporters, cofactors, and enzyme interactions).
+Covers both phase I and phase II metabolism.
+Allows time-dependent metabolism studies (e.g., prodrug activation).
-Expensive and requires fresh or high-quality cryopreserved hepatocytes.
-Hard to separate individual enzyme contributions.
82
Describe using human liver microsomes and S9 fractions to study drug metabolism
-Liver microsomes are vesicles containing membrane-bound phase I and II enzymes (e.g., CYPs, UGTs).
-S9 fractions contain both microsomal (CYPs, UGTs) and cytosolic (GSTs, NATs) enzymes, covering more metabolic pathways.
-The drug is incubated with either, and metabolite formation is measured
-Contribution of specific enzymes can be determined using selective inhibitors
83
Give the pros and cons of using liver microsomes and S9 fractions to study drug metabolism
+More physiologically relevant than recombinant enzymes.
+Allows study of multiple enzymes simultaneously.
+Useful for both phase I and II metabolism (S9 fractions).
-Cannot fully separate individual enzyme contributions.
-Lacks transporters, so may not reflect in vivo metabolism accurately.
84
What must we consider when reaction phenotyping, depending on the type of metabolising reaction?
Which experimental systems must be chosen, eg Alcohol dehydrogenase is present in cytosol so use S9 or Hepatocytes
85
What must we consider when using a human liver microsome bank?
-Genetic variations (SNPs)
-Lifestyle differences
-Different personal lifetime exposure to inducers
86
Describe using recombinant enzyme systems to study drug metabolism
-Uses individually expressed human enzymes (e.g., CYP450s, UGTs, FMOs, etc.) to assess drug metabolism.
-Generated using various host cells, eg E coli, yeast, insect, mammalian cells
-Helps identify which enzymes metabolise the drug by incubating it with single enzyme isoforms
-Drug is incubated with a panel of recombinant enzymes
-Metabolite formation rate is measured using techniques such as LC-MS
87
Give the pros and cons of using E coli in recombinant enzyme systems to study drug metabolism
+High yield: E. coli can produce large amounts of recombinant protein quickly and inexpensively.
+Fast growth: E. coli grows rapidly, which is ideal for scaling up the production of enzymes.
+Cost-effective: It is relatively inexpensive to culture and maintain E. coli
-Lack of co-factors: CYP450 enzymes require cofactors like NADPH to function properly. In E. coli, these cofactors are often not naturally present, so they must be supplied externally (e.g., by adding NADPH to the incubation).
-Membrane-bound enzymes: CYP450s are membrane-bound enzymes, and E. coli lacks the complex membrane structure found in eukaryotic cells, making it difficult to express these enzymes in their native, functiona
87
Give the pros and cons of using Yeast in recombinant enzyme systems to study drug metabolism
+Eukaryotic system: Yeast cells are better suited to express membrane-bound enzymes, such as CYP450s, and can process cofactors like NADPH more efficiently than E. coli.
+Yeast can perform some post-translational modifications (e.g., glycosylation, phosphorylation) that are sometimes required for the enzyme to function as it would in humans.
+Yeast cells provide a more mammalian-like environment for the enzymes, which can make them more functional and help them fold correctly
-Although yeast cells can produce large quantities of enzymes, they may not be as efficient at large-scale fermentation as E. coli.
-Yeast lacks some of the post-translational modifications that human liver cells perform, so it may not fully replicate all the human metabolic processes
88
Give the pros and cons of using insect cells in recombinant enzyme systems to study drug metabolism
+Insect cells are eukaryotic and can carry out post-translational modifications (like glycosylation and phosphorylation), which can make the expressed enzymes more functional than in E. coli.
+Insect cells can often express higher levels of recombinant proteins, especially membrane-bound enzymes like CYP450s, which are difficult to express in E. coli or yeast.
+Insect cells can handle the complex folding and functional structure of CYP450s, allowing them to express enzymes in their native, active form.
-Insect cells tend to grow slower than E. coli or yeast, and the viral infection process can be time-consuming.
-The baculovirus expression system can be more expensive to maintain, especially when compared to bacterial systems like E. coli.
89
How does using insect cells in recombinant enzyme systems differ from other types, when studying drug metabolism
The gene encoding the enzyme (e.g., CYP3A4) is inserted into a baculovirus vector, which is used to infect insect cells.
90
Describe the methodology of transient expression of recombinant proteins
-The gene of interest is transfected (injected) directly into the host cells using techniques like lipofection, electroporation, or viral infection.
-The cells transcribe and translate the gene into a protein in a relatively short amount of time (typically 2–7 days, depending on the host).
-After expression, the protein is either harvested from the cell culture media or the cell lysate.
91
Give the pros and cons of transient expression of recombinant proteins
+Fast, making it suitable for short term studies or rapid production
+High yield
+No selection required, no selection markers required
+Flexible
-Short term, stop producing after a few days
-Lower stability, since gene is not integrated into host genome
-Lower overall yield compared to stable systems
92
Describe the methodology of stable expression of recombinant proteins
-The gene of interest is inserted into the host genome using techniques like viral transfection, lipofection, or electroporation.
-Cells that successfully integrate the gene into their genome are selected using selection markers (e.g., antibiotic resistance).
-The selected cells are expanded into clonal cell lines.
-The recombinant protein is produced continuously as the cells divide and proliferate, often at large scale
93
Give the pros and cons of stable expression of recombinant proteins
+Continues to express the protein over a long period, months to years
+Produce consistent protein yields over time, making them ideal for large scale production
+Essential for the commercial production of therapeutic proteins
+Cell lines can be cryopreserved for future use
-Process is slow, weeks to months
-Selection is required
-Lower initial yield
-Potential for silencing
94
Give common mammalian cells used in recombinant enzyme systems to study drug metabolism
-HepG2 cells: Human liver-derived cancer cell line, widely used for drug metabolism studies because they express liver-specific enzymes (like CYP450).
-Huh7 cells: Human hepatoma cell line often used in drug metabolism and toxicology studies.
-HEK293 cells: Human embryonic kidney cells, commonly used for recombinant protein production but can also be used to study metabolism by expressing specific CYP450 enzymes.
95
Which type of recombinant enzyme system may we use to study Sulfotransferases, GSTs in drug metabolism?
E Coli
96
Which type of recombinant enzyme system may we use to study UGT and FMOs in drug metabolism?
Insect cells
97
Why use inhibitors in reaction phenotyping studies?
The use of inhibitors helps to deconvolute complex metabolic pathways and provide insight into enzyme kinetics, drug interactions, and species differences in drug metabolism.
98
Give examples of chemical inhibitors used to study CYP drug metabolism
-Furafylline (CYP1A2 inhibitor)
-Sulfaphenazole (CYP2C9 inhibitor)
-Quinidine (CYP2D6 inhibitor)
-Troleandomycin (CYP3A4 inhibitor)
99
Give types of inhibitors used in reaction phenotyping studies
-Chemical inhibitors
-Antibodies (polyclonal or monoclonal)
100
What may we use to identify specific P450 isoforms responsible for drug metabolism
-Recombinant P450 studies
-Inhibition studies
-Correlation analysis using human microsome bank
-Probe substrate marker reaction experiments
101
What may we use to study CYP induction potential?
-High throughput screening
-qRT-PCR
-Reporter gene assays
102
Describe how high throughput screening can be used to study CYP induction
-Cell based assays (test compounds are incubated with CYP enzyme isoforms)
-Monitoring CYP activity (measuring changes in metabolic activity)
-RNA or protein assays (measuring CYP gene expression)
-Automated assays
103
Describe how qRT-PCR can be used to study CYP induction
-Measure gene expression of CYP enzymes
-Assessing induction potential at the mRNA level
-Quantifying the transcriptional expression of CYP genes
104
Describe how reporter gene assays can be used to study CYP induction
-Reporter gene assays use a genetically modified cell line that contains a CYP promoter linked to a reporter gene (e.g., luciferase, green fluorescent protein (GFP), or β-galactosidase).
-These assays allow researchers to monitor CYP induction by detecting the activity of the reporter gene, which correlates with the expression of the CYP enzyme.
105
Give sources of human liver cell lines
-Primary human hepatocytes
-Isolation of hepatoma cells
-Isolation of progenitor cells using adult stem cells
-hESCs
-IPSCs
106
Describe hepatic cell lines
-Hepatic cell lines are either derived directly from liver tumour tissue (hepatoma) or generated from primary
hepatocytes in vitro
-Immortalised (non-tumour) cell lines generated with viral oncogenes or telomerase, may lead to mutation
-Easier to maintain and culture for experiments
-Human hepatoma cell line HepG2 is the liver cell line most commonly studied
-Cancer cell lines altered metabolism due to greater energy demand
-Metabolic activity of tumour lines is significantly lower than that of primary hepatocytes
107
Describe using in silico computational methods to study drug metabolism, and give examples
-In silico approaches use computational models and simulations to predict drug metabolism, enzyme interactions, and pharmacokinetics
-These methods are essential in early drug discovery to reduce experimental costs, refine candidate selection, and minimize failures in later stages.
-eg Molecular docking, Pharmacophore modelling, Quantitative Structure Activity Relationship, Machine learning and AI based models
108
Describe Molecular docking as an in silico method to study drug metabolism
-Simulates how a drug binds to metabolic enzymes (e.g., CYP450 enzymes)
-Predicts potential substrates, inhibitors, or inducers
-Identifies binding affinity of a drug to CYP enzymes
-Predicts competitive vs non competitive inhibition, and helps in understanding drug drug interactions
109
Describe pharmacophore modelling as an in silico method to study drug metabolism
-Defines the structural features (e.g., hydrogen bonds, hydrophobic areas) that are critical for drug binding to metabolic enzymes
-Identifies which molecular structures influence CYP metabolism
-Helps design new drugs to avoid rapid metabolism or toxic metabolites
110
Describe quantitative structure-activity relationship as an in silico method to study drug metabolism
-Uses mathematical models to relate a drug’s chemical structure to its metabolic behavior
-Predicts if a drug is a CYP substrate, inhibitor, or inducer
-Estimates metabolic stability based on structural features
-Identifies toxic metabolites
