Download 3. Metabolism - Professor Monzir Abdel

3. Metabolism
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Many xenobiotics undergo chemical transformation
(biotransformation; metabolism) when introduced
into biologic systems like the human body.
Biotransformation is often mediated by enzymes
End result of biotransformation is either alteration of
the parent molecule, or conjugation of the parent
molecule (or its metabolites) with endogenous
substances in the body.
Enzymes involved in biotransformation can act on
either endogenous or xenobiotic compounds,
especially if the xenobiotics are structurally similar to
endogenous compounds
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Example: Monoamine oxidase (MAO) is an
enzyme that normally metabolizes biologic
amines like epinephrine. MAO can also
oxidize a variety of drugs. If a person is taking
a drug that inhibits MAO activity (like many
blood pressure medications), it can be
dangerous for that person to take other drugs
that can be metabolized by MAO.
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The products of biotransformation can be either less
toxic, more toxic, or about as toxic as the parent
molecules.
Enzymes involved in biotransformation are
sometimes called “drug metabolizing enzymes”.
Although strictly speaking this is a misnomer because
many of the substrates are not drugs, the term is still
commonly used.
Location of metabolic enzymes
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Species – found in virtually every species, although the
type and amount vary tremendously.
Organs – present in many tissues. Many enzymes are
particularly abundant in the liver.
Subcellular drug-metabolizing enzymes are located in the
smooth endoplasmic reticulum (SER).
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Types of Biotransformation Reactions
Basically, two types of reactions, nonsynthetic (Phase
I) and synthetic (Phase II)
Phase I reactions
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Involve modification of the basic structure of the substrate
Do not involve covalent binding of the substrate to an
endogenous compound
Phase I enzymes are often membrane-bound (e.g.,
microsomal). This is because they generally act on
more lipid-soluble (nonpolar) substrates, and their
purpose is to make the compounds MORE POLAR
and therefore, MORE EASILY EXCRETABLE by
the kidney and biliary tract.
Major Biotransformation Reactions
Phase I
Phase II
Oxidation
Sulfation
Reduction
Glucuronidation
Hydrolysis
Glutathione conjugation
Hydration
Acetylation
Dehalogenation
Amino acid conjugation
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Oxidation
Uses molecular oxygen (O2). One atom of oxygen is
combined with hydrogen to form water, and the other
atom of oxygen is introduced into the substrate
molecule.
 Involves several enzymatic steps.
 The oxidative system is often known as the “mixed
function oxidase” system”. These enzymes are some of
the most thoroughly researched enzymes in biological
systems.
One subfamily of the mixed function oxidase system is the
group of enzymes known as Cytochrome P-450 enzymes.
They are so called because of their absorbance characteristics
at wavelengths of 448-450 nm.
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Anything that affects the activity of any oxidative
enzyme can affect the way the body reacts to a given
drug or other xenobiotic.
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Deamination – replacement of an amine group (NH2) with an
oxygen (O) atom
N-, O-, or S-Dealkylation – replacement of an alkyl group
(e.g., CH3) with a hydrogen atom. Typically, the alkyl group in
the parent molecule is bonded to a N, O, or S atom.
Aliphatic or aromatic hydroxylation – addition of a hydroxyl
group (OH) to a molecule
N-oxidation – replacement of a hydrogen atom on an amine
with an oxygen
S-oxidation – addition of an oxygen atom to a sulfur atom
Conversion of a hydroxyl group (alcohol) to a carboxyl
group (acid)
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Reduction
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Azo reduction – reduction of an azo bond (N=N) to two
amines (NH2)
Nitro reduction – reduction of a nitro group (NO2) to an
amine
Hydrolysis
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Addition of water (H2O) to an ester bond (CO-O-C) to
form an alcohol (C-OH) and a carboxylic acid (COOH)
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Phase II reactions
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Involve addition of a cofactor to a substrate to form a
new product. Therefore, the rate of these reactions can be
limited by the availability of the cofactor.
Phase II enzymes may be either microsomal or cytosolic.
This is because the primary purpose of the Phase II
reactions is not so much to increase the polarity of the
parent compound (although that is part of what they
accomplish). The primary purpose is to increase the
molecular weight of the parent compound to make it a
better substrate for active transport mechanisms in the
biliary tract.
Various factors can affect the availability of cofactors. For
example, fasting markedly reduces the amount of
glutathione available in the liver.
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Sulfation
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Replacement of a hydrogen atom (H) with a sulfonate
(SO3-)
Uses the enzyme sulfotransferase
Uses the cofactor called PAPS (phosphoadenosine
phosphosulfate)
Produces a highly water-soluble sulfuric acid ester
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Glucuronidation
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Replacement of a hydrogen atom with a glucuronic acid
Uses the enzyme UDP-glucuronosyl transferase (UDPGT)
Uses the cofactor called UDPGA (uridine diphosphate
glucuronic acid)
One of the major Phase II enzymatic pathways
Example: Conjugation of a phenol and a carboxylic acid
with glucuronic acid
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Acetylation
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Replacement of a hydrogen atom with an acetyl group
Uses the enzyme acetyltransferase
Uses the cofactor called acetyl CoA (acetyl coenzyme
A)
Sometimes results in a less water-soluble product
Methylation
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Replacement of a hydrogen atom with a methyl group
Uses the enzyme methyltransferase
Uses the cofactor called SAM (S-adenosyl methionine)
Common but relatively minor pathway
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Glutathione conjugation
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Adds a glutathione molecule to the parent compound,
either by direct addition or by replacement of an
electrophilic substituent (e.g., a halogen atom)
Uses the enzyme glutathione transferase (GST)
Uses the cofactor called glutathione (a tripeptide made
up of glycine, cysteine, and glutamic acid
One of the major Phase II enzymatic pathways
Example: Metabolism of naphthalene
showing the conjugation of naphthalene
epoxide with glutathione and the subsequent
formation of a N-acetylcysteine conjugate
(mercapturic acid)
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Amino acid conjugation
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Adds an amino acid to the parent compound.
Mercapturic acid formation
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Formed by cleavage of the glycine and glutamic acid
substituents from a glutathione conjugate, followed by
N-acetylation of the resulting product
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Significance of Biotransformation Reactions
in Toxicology
Biotransformation is a major part of the
pathway for elimination of many xenobiotic
compounds.
Biotransformation can result in either a
decrease or an increase (or no change) in
toxicity.
Biotransformation can result in the formation
of reactive metabolites.
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Example – metabolism of acetaminophen
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Acetaminophen is ordinarily metabolized in the liver by
sulfation and glucuronidation to form non-toxic conjugates
These are low capacity pathways, in that the cofactors are
available in only limited concentrations, so these are ratelimiting.
As long as the amount of acetaminophen in the liver is
relatively low, the Phase II pathways can handle the
compound, and there is no toxicity.
If the concentration of acetaminophen becomes high
enough to overwhelm the capacity of the Phase II
pathways, an alternate metabolic pathway, involving Phase
I enzymes, becomes active.
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The product of the Phase I reaction is a highly reactive
quinoneimine, which can form adducts with (bind
covalently to) cellular macromolecules, especially proteins.
The binding of the reactive intermediate to cellular
macromolecules destroys the activity of those molecules,
and can lead to compromised cell function and, ultimately,
cell death.
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Another good example – metabolism of carbon
tetrachloride
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Carbon tetrachloride is metabolized by the
cytochrome P-450 system in the liver by
abstraction of one of the four chlorine atoms.
This results in formation of a highly reactive
trichloromethane radical, which initiates a cascade
of lipid peroxidation by removing a hydrogen atom
from membrane phospholipids.
Damage to the cell membrane causes loss of
osmotic integrity, cell swelling and death.
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The activity of drug metabolizing enzymes is
dependent on numerous factors
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Species
Age (activity is generally lower in very young and aged
animals)
Sex (activity is generally higher in males than in females)
Genetics (remember slow versus fast acetylators)
Organ (activity of many enzymes is highest in the liver)
General health status (e.g., hepatic injury decreases
metabolic activity in the liver)
Diet (remember how fasting decreases the amount of
glutathione available for GST)
Previous exposure to other compounds
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Induction – an increase in the activity of one or more
enzymes as a result of previous exposure of the
organism to compounds that serve as substrates for
the enzyme(s)
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Classic example of an inducer is phenobarbital, which
induces the activity of cytochrome P-450 enzymes
Induction may involve either increases in the synthesis of
enzymatic protein, or increases in activation of
proenzymes.
One effect of induction of microsomal enzymes is an
increase in the amount of smooth endoplasmic reticulum in
a cell.
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Induction is usually temporary, and enzyme activity levels
return to normal after several weeks.
Induction can result in tolerance to drugs, if the
metabolism of the drugs results in a product with lower (or
no) pharmacologic activity. This is why, for example,
patients can develop tolerance to Phenobarbital anesthesia
after repeated administration.
Induction may result in increases or decreases in toxicity,
depending on whether the metabolite is more or less toxic
than the parent compound. This is why, for example,
alcoholics are more susceptible to acetaminophen toxicity,
since alcohol induces the enzyme that is responsible for
production of the reactive metabolite from acetaminophen.
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Inhibition – a decrease in the activity of one or more
enzymes
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Classic example of an inhibitor is SKF-525A, which
inhibits microsomal enzymes
Inhibition may be either competitive or non-competitive.
Competitive inhibition
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Occurs when an inhibitor binds to the same active site on the
enzyme as the substrate. The higher the concentration of the
inhibitor, the less likely it is that the substrate molecule will be able
to find and bind to an available enzyme molecule.
Reversible, since the binding of the inhibitor to the active site is not
covalent
Example: Omeprazole and diazepam are both metabolized by
cytochrome P-450 2C19 (CYP2C19). Co-administration of these
two drugs results in prolonged plasma half-life for diazepam.
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Non-competitive inhibition
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May occur when an inhibitor binds to the same active
site on the enzyme as the substrate, but binds so tightly
that it is effectively not released. Thus, the binding site
is permanently blocked.
May also occur when an inhibitor binds tightly
(sometimes covalently) to a different site on the enzyme
than the active site. This can result in conformational or
affinity changes that effectively inactive the enzyme.
Non-competitive inhibition is generally not reversible.
Therefore, recovery takes much longer because it
requires the synthesis of new enzyme.
Factors affecting metabolism
Age
The metabolizing enzymes in neonates are not fully
developed, therefore those cannot efficiently
metabolize drugs. Also in the elderly, enzymatic
systems may not function well leading to same
conclusion.
2. Sex
Males who are deficient in glucose -6-phosphate
dehydrogenase are more prone to hemolysis when
subjected to some drugs like sulfonamides
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3. Pharmacogenetic factors
Some individuals may be deficient in some
enzymes, regardless of sex
4. Pregnancy
Hepatic metabolism of drugs is decreased in
pregnancy
5. Nutritional status/ liver dysfunction
Malnutrition can cause a decreased level of some
enzyme system and liver dysfunction can lead
to decreased metabolism
6. Bioactivation
Some drugs may be transformed to more toxic
metabolites
7. Enzyme induction / inhibition
A result of this is either an increase in the
metabolism or a decrease in the drug
metabolism
8. Changes in the kinetic mechanism: depending
on whether the concentration of drug is in the
therapeutic or overdose range