Intro to Pharmacology and Toxicology
Topics
A xenobiotic is a chemical compound foreign to a living organism, may be a drug, a toxicant, etc. Metabolism expedites the removal of xenobiotics from the body by enzymatically transforming the chemical. Almost all transformations are irreversible. Nearly all metabolic transformations result in products more polar (more water soluble) than the parent chemical, facilitating xenobiotic removal from the organism (easier to excrete in urine and other fluids).
Xenobiotic metabolism may deactivate (terminate action) of a xenobiotic (example: ibuprophen) or activate (increase action) of the xenobiotic (example: codeine). Some substrates may interact with the enzyme forming stable complexes that inhibit individual enzyme molecules and remove them from the "active pool" (example: nerve gases) .
The main site of xenobiotic metabolism is the liver, although many other tissues may participate (gut, lungs, kidneys, brain, skin, etc.).
An isoenzyme (or isozyme) is one of a group of enzymes that are very similar in catalytic properties but may be differentiated by variations in physical properties (e.g. isoelectric point, electrophoretic mobility, etc.).
Drug
metabolism may be divided into two types of reactions: Phase 1 or functionalization
reactions, and Phase 2 or conjugation reactions. Phase 1 include oxidative,
and reductive reactions that alter and create new functional groups, and hydrolytic
reactions that cleave esters and amides to release "masked" functional
groups, usually resulting in increased polarity. An example is the conversion
of diazepam to desmethyldiazepam:
In
Phase 2 (conjugation) reactions the compound is coupled to an endogenous
substrate such as glucoronic acid, acetic acid or sulfuric acid. An example
is the conversion of oxazepam to oxazepam glucoronide:
Metabolic enzymes may be induced by some chemicals, increasing protein levels and total activity. In general, induction affects pharmacokinetics by:
Common enzyme inducers include ethanol and aryl hydrocarbons. Other xenobiotics are known for inducing important proteins like the peroxisome proliferator activated receptor (PPAR), the constitutive androstane receptor (CAR), and the pregnane X receptor (PXR).
Ethanol targets CYP2E1. The ligand binds and prolongs the destabilization of the enzyme (?). The effects are minor mostly because ethanol is metabolized by different pathways.
Aryl hydrocarbons (for example, as in tobacco smoke) induce an increase in the synthesis of CYP1A1 and CYP1A2. This is initiated by the binding of the inducer to the intracellular aryl hydrocarbon (Ah) receptor. An example of an interaction related to this induction is the increase metabolism of caffeine (by CYP1A2) by smoking.
Two classes of drugs bind to peroxisome proliferator activated receptor (PPAR): glitazones and fibrates. Peroxisome proliferation has not been observed in humans and targets are unknown (?).
Phenobarbital induction of several cytochrome P450 enzymes occurs by the constitutive androstane receptor (CAR) and may affect up to 50 genes (CYP2B6, CYP2C8, CYP2C9, CYP3A4).
Rifampin and glucocorticoids like dexamethasone act on the pregnane X receptor (PXR). PXR binds to the rifampin/dexamethasone response element in the CYP3A4 promoter region. All inducers of PXR are CYP3A4 substrates.
The human enzymes responsible for metabolic clearance of a xenobiotic can be determined. The results of drug metabolism studies may be able to predict drug-drug interactions. Metabolic studies can result in identification of toxic metabolites and the identification of species differences in metabolite formation.
In vivo metabolic studies use a dynamic system. Drugs are administered and samples of blood, urine, saliva or feces are collected to quantify drugs and/or metabolites. In vitro systems need energy (usually NADPH) and oxygen for reactions to occur. Commonly used in vitro systems include microsomes, individual enzymes, hepatocytes and liver slices.
Microsomes are subcellular fractions comprised of mostly liver smooth endoplasmic reticulum (~ 85% SER). The contain Phase 1 and Phase 2 enzymes, and are stable and convenient to use. Human tissue microsomes are hard to obtain.
Individual drug metabolizing enzymes are usually genetically engineered. They are stable and selective, very useful in characterizing new compounds. But they may exhibit nonspecific activity.
Hepatocytes are used to study integrated hepatic metabolism (Phase 1 and Phase 2). They may be maintained in monolayer cultures, however CYP activity becomes less stable over time. Collagenase, a connective tissue degradation enzyme, is used to dissolve the tissue.
Liver slides have properties similar to hepatocytes but may be more robust since the structural integrity of the tissue is preserved. They are not subject to treatment with collagenase, which may damage hepatocyte membranes. The tissue and enzyme stability in liver slides may still be questionable.
The quantification of metabolites is usually indirect: the sample must be treated with an enzyme that degrades conjugates prior to assay. Drugs and metabolites are usually measured by High Pressure Liquid Chromatography (HPLC), Gas Chromatography (GC), Liquid Chromatography / Mass Spectrometry (LC-MS) and other methods. An example of chromatographs to determine codeine/dextromethorphan metabolism is shown below.
Continue to "Phase
1 Reactions" or take a quiz: [Q1] [Q2]
[Q3] [Q4].
Need more practice? Answer the review questions below (after sponsor).
1- What is a xenobiotic?
2- What is the overall effect of metabolism on xenobiotics?
3- List 3 possible fates of xenobiotics when they undergo metabolism and give an example of each.
4- List the main metabolic organ and other 5 organs where metabolism occur.
5- What is an isozyme?
6- List 2 types of metabolic reactions.
7- What are Phase 1 reactions? Give one example.
7- List 3 types of Phase 1 reactions.
8- What are Phase 2 reactions? Give one example.
9- List 3 endogenous substrates used in conjugation reactions.
10- What is enzyme induction?
11- List 3 ways enzyme induction affects pharmacokinetics.
12- List 7 enzyme or receptor inducers and their targets.
13- List 3 applications of metabolic studies.
14- List 3 characteristics of in vivo metabolic studies.
15- List 2 characteristics of
in vitro metabolic studies.
16- List 4 in vitro systems.
17- What are microsomes?
18- What are the general characteristics of individual enzymes as experimental systems?
19- What are the general characteristics of hepatocytes as experimental systems?
20- What is collagenase?
21- What are the general characteristics of liver slides as experimental systems?
22- In general, how are metabolites quantified?
Continue scrolling to answers below (after sponsor).
Hey! DON'T PEEK!!! Finish the questions fist!
1- What is a xenobiotic?
Chemical compound foreign to a living organism.
2- What is the overall effect
of metabolism on xenobiotics?
Expedites their removal by enzymatic transformation, almost always irreversible
and resulting in polar products easier to excrete in urine and other fluids.
3- List 3 possible fates of xenobiotics
when they undergo metabolism and give an example of each.
deactivation (ibuprophen)
activation (codeine)
complex with and inhibit the enzyme (nerve gases)
4- List the main metabolic organ
and other 5 organs where metabolism occur.
liver (main)
gut
lungs
kidneys
brain
skin
5- What is an isozyme?
One of a group of enzymes that are very similar in catalytic properties but
may be differentiated by variations in physical properties like isoelectric
point or electrophoretic mobility.
6- List 2 types of metabolic reactions.
Phase 1 or functionalization reactions
Phase 2 or conjugation reactions
7- What are Phase 1 reactions?
Give one example.
Functionalization reactions that alter existing functional groups, create new
functional groups, or cleave esters and amides to release "masked"
functional groups, usually increasing polarity. Example: conversion of diazepam
to desmethyldiazepam.
7- List 3 types of Phase 1 reactions.
oxidation
reduction
hydrolysis
8- What are Phase 2 reactions?
Give one example.
Conjugation reactions where the substrate is coupled to and endogenous molecule.
Example: oxazepam to oxazepam glucuronide.
9- List 3 endogenous substrates
used in conjugation reactions.
glucuronic acid
acetic acid
sulfurc acid
10- What is enzyme induction?
Metabolic enzymes may be induced by some chemicals, increasing protein levels
and total activity.
11- List 3 ways enzyme induction
affects pharmacokinetics.
decreasing bioavailability of orraly administered drugs
increasing hepatic clearance
increasing formation of active or unwanted metabolites
12- List 7 enzyme or receptor
inducers and their targets.
ethanol - CYP2E1
aryl hydrocarbons - CYP1A1 and CYP1A2
glitazones and fibrates - PPAR
phenobarbital - several CYP450 enzymes
rifampin and glucocorticoids - CYP3A4
13- List 3 applications of metabolic
studies.
predict drug-drug interactions
identify toxic metabolites
identify species differences in metabolite formation
14- List 4 characteristics of
in vivo metabolic studies.
use a dynamic system
drugs are administered
blood, urine. saliva or fecens samples are collected
xenobiotic or metabolites are quantify in samples
15- List 2 characteristics of
in vitro metabolic studies.
need energy (usually NADPH)
need oxygen
16- List 4 in vitro systems.
microsomes
individual enzymes
hepatocytes
liver slices
17- What are microsomes?
Subcellular fractions of mostly liver smooth endoplasmic reticulum, containing
Phase 1 and Phase 2 enzymes, stable and convenient to use.
18- What are the general characteristics
of individual enzymes as experimental systems?
Usually obtained by genetic engineering, are stable and selective but may be
nonspecific.
19- What are the general characteristics
of hepatocytes as experimental systems?
Used to study integrated hepatic metabolism (Phase 1 and Phase 2), may be maintained
in monolayer cultures. CYP activity becomes less stable over time. Collagenase
is used to dissolve tissue.
20- What is collagenase?
A connective tissue degradation enzyme used to dissolve tissue in culture. May
damage hepatocyte membranes.
21- What are the general characteristics
of liver slides as experimental systems?
Have properties similar to hepatocytes but may be more robust since the structural
integrity of the tissue is preserved. Are not subject to collagenase treatment.
Tissue and enzyme stability may still be questionable.
22- In general, how are metabolites
quantified?
Indirectly by treating the sample with an enzyme that degrades conjugates prior
to assay by High Pressure Liquid Chromatography (HPLC), Gas Chromatography (GC),
Liquid Chromatography / Mass Spectrometry (LAGS), and other methods.
Sponsor (again...):