Timed Essay Topic Flashcards
What may inadequate assessment of metabolism and toxicity lead to?
-Adverse reactions in trial participants
-Drug attrition rates
-Drug recall even after approval
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
Why is reaction phenotyping important?
-Preducts drug drug interactions
-Explains interindividual variability
-Guides drug development and dosing
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 interactions
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 silico computational approaches
-Inhibition and induction studies
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.
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.
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
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.
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
What must we consider when using a human liver microsome bank?
-Genetic variations (SNPs)
-Lifestyle differences
-Different personal lifetime exposure to inducers
-Cofactor requirements
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
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
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
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.
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.
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.
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
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
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
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.
Which type of recombinant enzyme system may we use to study Sulfotransferases, GSTs in drug metabolism?
E Coli
Which type of recombinant enzyme system may we use to study UGT and FMOs in drug metabolism?
Insect cells
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.
