Extent of Absorption

Extent of Absorption
The extent of absorption is important in determining the total
body exposure or internal dose, and therefore is an important
variable during chronic toxicity studies and/or chronic human
exposure. The extent of absorption depends on the extent to
which the chemical is transferred from the site of
administration into the local tissue, and the extent to which it
is metabolized or broken down by local tissues prior to
reaching the general circulation. An additional variable
affecting the extent of absorption is the rate of removal from
the site of administration by other processes compared with the
rate of absorption
١٨

Chemicals given via the gastrointestinal tract may be
subject to a wide range of pH values and
metabolizing enzymes in the gut lumen, gut wall, and
liver before they reach the general circulation. The
initial loss of chemical prior to it ever entering the
blood is termed first-pass metabolism or pre-
systemic metabolism; ; it may in some cases remove
up to 100% of the administered dose so that none of
the parent chemical reaches the general circulation.
The intestinal lumen contains a range of hydrolytic
enzymes involved in the digestion of nutrients. The
gut wall can perform similar hydrolytic reactions and
contains enzymes that can oxidize many drugs
١٩

٢٠

Absorption and Bioavailability
Irrespective of the reason that is responsible for the incomplete
absorption of the chemical as the parent compound, it is
essential that there is a parameter which defines the extent of
transfer of the intact chemical from the site of administration
into the general circulation. This parameter is the
bioavailability, , which is simply the fraction of the dose
administered that reaches the general circulation as the parent
compound. (The term bioavailability is perhaps the most
misused of all kinetic parameters and is sometimes used
incorrectly in a general sense as the amount of drug available
specifically to the site of toxicity.)
٢١

Calculation of Bioavailability
The fraction absorbed as the intact compound or bioavailability (F)) is
determined by comparison with intravenous (i.v(
i.v.) .) dosing (where F = 1 by
definition). The bioavailability can be determined from the area under the
plasma concentration–time time curve (AUC) of the parent compound , or the
percentage dose excreted in urine as the parent compound, i.e. for an oral
dose:
٢٢

٢٣

2. Distribution
Distribution is the reversible transfer of the
chemical between the general circulation and
the tissues. Irreversible processes such as
excretion, metabolism, or covalent binding are
part of elimination and do not contribute to
distribution parameters. The important
distribution parameters relate to the rate and
extent of distribution.
٢٤

Rate of Distribution
The rate at which a chemical may enter or leave a tissue may be
limited by two factors:
(i) the ability of the compound to cross cell membranes and
(ii) the blood flow to the tissues in which the chemical
accumulates.
The rate of distribution of highly water-soluble compounds may
be slow due to their slow transfer from plasma into body
tissues such as liver and muscle; water-soluble compounds do
not accumulate in adipose tissue. In contrast, very lipid-soluble
chemicals may rapidly cross cell membranes but the rate of
distribution may be slow because they accumulate in adipose
tissue, and their overall distribution rate may be limited by
blood flow to adipose tissue
٢٥

The rate of distribution is indicated by the distribution
rate constant, which is determined from the decrease
in plasma concentrations in early time points after an
intravenous dose. The rate constants refer to a mean
rate of removal from the circulation and may not
correlate with uptake into a specific tissue. Once an
equilibrium has been reached between the general
circulation and a tissue, any process which lowers the
blood (plasma) concentration will cause a parallel
decrease in the tissue concentration.
٢٦

Factors affecting distribution
1. Blood flow
Drugs are readily distributed to highly perfused tissue
like brain, liver, and kidneys
2. Permeability limitations
Many drugs do not readily enter the brain due to the
blood brain barrier
3. Protein binding
Acidic drugs are bound to the most abundant plasma
protein (albumin); while basic drugs bind to -1-
acid glycoprotein.
٢٧

4. Effect of pH
The pH of the blood or tissue affect the
ionization of the drug and thus its distribution
5. Age
In old people, Protein binding and body water
will decrease, thus increasing the concentration
of the drug per unit time
6. Existence of storage sites:
These include: Adipose tissue, plasma proteins,
liver and kidneys, and bone
٢٨

Extent of Distribution
The extent of tissue distribution of a chemical depends
on the relative affinity of the blood or plasma
compared with the tissues. Highly water-soluble
compounds that are unable to cross cell membranes
readily are largely restricted to extracellular fluid
(about 13 L per 70 kg body weight). Water-soluble
compounds capable of crossing cell membranes (e.g.(
caffeine, ethanol) are largely present in total body
water (about 41 L per 70 kg body weight).
٢٩

Lipid-soluble compounds frequently show extensive
uptake into tissues and may be present in the lipids of
cell membranes, adipose tissue.
The partitioning between circulating lipoproteins and
tissue constituents is complex and may result in
extremely low plasma concentrations. A factor which
may further complicate the plasma/tissue partitioning
is that some chemicals bind reversibly to circulating
proteins such as albumin (for acid molecules) and
acid glycoprotein (for basic molecules).
٣٠

The extent and pattern of tissue distribution can
be investigated by direct measurement of
tissue concentrations in animals. Tissue
concentrations cannot be measured in human
studies and, therefore, the extent of distribution
in humans has to be determined based solely
on the concentrations remaining in plasma or
blood after distribution is complete.
٣١

The parameter used to reflect the extent of distribution is the
apparent volume of distribution (V),(
which relates the total
amount of the chemical in the body (Ab(
Ab) ) to the circulating
concentration (C)(
) at any time after distribution is complete:
The volumes of distribution of tubocurarine and caffeine are
about 13 and 41 L per 70 kg because of their restricted
distribution
٣٢

Caffeine
tubocurarine
٣٣

when a chemical shows a more extensive reversible
uptake into one or more tissues the plasma
concentration will be lowered and the value of V will
increase. For highly lipid-soluble chemicals, such as
organochlorine pesticides, which accumulate in
adipose tissue, the plasma concentration may be so
low that the value of V may be many litres for each
kilogram of body weight. This is not a real volume of
plasma and therefore V is called the apparent volume
of distribution.
٣٤

It is an important parameter because extensive
reversible distribution into tissues, which will
give a high value of V, , is associated with a low
elimination rate and a long half-life life . It must be
emphasized that the apparent volume of
distribution simply reflects the extent to which
the chemical has moved out of the site of
measurement (the general circulation) into
tissues, and it does not reflect uptake into any
specific tissue(s).
٣٥

Elimination
The parameter most commonly used to describe
the rate of elimination of a chemical is the
half-life life . Most toxicokinetic processes are
first-order reactions, i.e. the rate at which the
process occurs is proportional to the amount of
chemical present. High rates (expressed as
mass/time) occur at high concentrations and
the rate decreases as the concentration
decreases; in consequence the decrease is an
exponential curve.
٣٦

The usual way to analyze exponential changes is to use
logarithmically transformed data which converts an
exponential into a straight line. The slope of the line
is the rate constant (k)(
) for the process and the half-life
life
for the process is calculated as 0.693/k. . Rate
constants and half-lives lives can be determined for
absorption, distribution, and elimination processes.
The clearance of a chemical is determined by the
ability of the organs of elimination (e.g.(
the liver,
kidney, or lungs) to extract the chemical from the
plasma or blood and permanently remove it by
metabolism or excretion.
٣٧

The mechanisms of elimination depend on the
chemical characteristics of the compound:
• volatile chemicals are exhaled,
• water-soluble chemicals are eliminated in the
urine and/or bile and
• lipid-soluble chemicals are eliminated by
metabolism to more water-soluble molecules,
which are then eliminated in the urine and/or
bile.
٣٨

٣٩

If a chemical undergoes metabolic activation then
toxicokinetic studies should measure both the parent
chemical and the active metabolite. If the metabolite
is so reactive that it does not leave the tissue in which
it is produced (e.g.(
alkylating metabolites of chemical
carcinogens), then toxicokinetic studies should define
the delivery of the parent chemical to the tissues, and
the process of local activation should be regarded as
part of tissue sensitivity (toxicodynamics(
toxicodynamics) ) because it
is not part of toxicokinetics, i.e. the movement of the
chemical and/or metabolites around the body.
٤٠

• Clearance = a ratio relating the rate of
elimination of a chemical from an appropriate
reference fluid (usually plasma) to its
concentration in the same reference fluid.
Clearance has the units of flow rate in
milliliters per minutes (mL(
mL/min).
• A clearance of 100 mL/minute of a chemical
means that 100 mL of blood/plasma is
completely cleared of the compound in each
minute.
٤١

The best measure of the ability of the organs of
elimination to remove the compound from the
body is the clearance (CL(
CL):
Because the rate of elimination is proportional to the concentration,
clearance is a constant for first-order processes and is independent of
dose. It can be regarded as the volume of plasma (or blood) cleared of
compound within a unit of time (e.g. mL/ min).
٤٢

Renal clearance depends on the extent of protein
binding, tubular secretion and passive reabsorption in
the renal tubule; it can be measured directly from the
concentrations present in plasma and urine:
The total clearance or plasma clearance (which is the
sum of all elimination processes, i.e. renal metabolic,
etc.) ) is possibly the most important toxicokinetic
parameter.
٤٣

It is measured from the total amount of compound available for
removal (i.e.(
an intravenous dose) and the total area under the
plasma concentration–time time curve (AUC) extrapolated to
infinity.
Plasma clearance reflects the overall ability of the body to remove
permanently the chemical from the plasma. Plasma clearance is the
parameter that is altered by factors such as enzyme induction, liver
disease, kidney disease, inter-individual or inter-species differences in
hepatic enzymes or in some cases organ blood flow.
٤٤

Once the chemical is in the general circulation, the same volume
of plasma will be cleared of chemical per minute (i.e.(
the
clearance value) applies irrespective of the route of delivery of o
chemical into the circulation. However, the bioavailability (F)(
will determine the proportion of the dose reaching the general
circulation. Therefore, bioavailability has to be taken into
account if clearance is calculated from data from a non-
intravenous route (e.g.(
oral):
٤٥

Measurement of dose/AUC for an oral dose determines CL/F, , which contains
two potentially independent variables – the amount of chemical delivered
to the blood from the site of administration and the clearance of chemical
present in the blood. The overall rate of elimination, as indicated by the
terminal half-life life (t(
), is dependent on two physiologically related and
independent variables:
where CL is the ability to extract and remove irreversibly the compound from f
the general circulation, and V the extent to which the compound has left the
general circulation in a reversible equilibrium with tissues.
٤٦

Chemicals that are extremely lipid-soluble and
are sequestered in adipose tissue are
eliminated slowly. Lipid soluble
organochlorine compounds, which are not
substrates for P450 oxidation, due to the
blocking of possible sites of oxidation by
chloro-substituents
substituents, , are eliminated extremely
slowly: for example, the half-life life of 2,3,7,8-
tetrachlorodibenzodioxin (TCDD) is about 8
years in humans.
٤٧

Summary of Toxicokinetic
Parameters
• Apparent volume of distribution (V(
d )
• A way to express the apparent space in the body that a chemical
occupies
• Expressed as a proportionality constant (in units of volume [e.g.,
.,
liters of blood] or volume/body weight)
• Relates the total amount of chemical in the body to the
concentration in plasma
• For a 70-kg human,
• Plasma volume ≈ 3 L (~0.045 L/kg body weight)
• Total blood volume ≈ 5 L (~0.07 L/kg body weight)
• Total extracellular fluid volume ≈ 12 L (~0.2 L/kg body weight)
• Total body water ≈ 42 L (~0.6L/kg body weight)
٤٨

• Example – If you know that 3 mg of chemical has been
injected into the body, and the concentration of the
chemical in the plasma is 1 mg/L, then you can calculate
that the apparent V d to be 3 L. This suggests that the
chemical is confined to the plasma space, and is not
significantly absorbed into the tissues or into the
particulate components (i.e., blood cells) in blood.
3 mg ÷ 1.0 mg/L = 3.0 L
٤٩

• Example – If you know that 3 mg of chemical has been
injected and the concentration of the chemical in the
plasma is 0.55 mg/L, then the apparent V d = 5.5 L. This
suggests that the chemical may be distributed evenly
between the plasma and the blood cells. More likely,
the reality is that the chemical is somewhat absorbed
into the tissues.
3 mg ÷ 0.55 mg/L = 5.5 L
٥٠

• Example – If you know that 3 mg of chemical has been
injected and the concentration of the chemical in the
plasma is 0.25 mg/L, then the apparent V d = 12 L. This
suggests that the chemical may be distributed evenly
within the total extracellular water. More likely, the
reality is that the chemical is somewhat absorbed into
the tissues, and perhaps somewhat metabolized.
3 mg ÷ 0.25 mg/L = 12 L
• It is possible to have a V d greater than the total body
water
• A high V d is an indication that one or both of two things
has occurred,
• The chemical has been absorbed from the blood and
concentrated in the tissues.
• The chemical has been metabolized.
٥١

• Clearance (Cl(
Cl)
• A ratio relating the rate of elimination of a chemical
from an appropriate reference fluid (usually plasma) to
its concentration in the same reference fluid.
• Has the units of flow rate in volume cleared per unit
time (e.g., mL/min)
• A clearance of 100 mL/minute of a chemical means that
100 mL of blood/plasma is completely cleared of the
compound in each minute .
٥٢

• Zero- versus first-order kinetics
• For zero order kinetics, the rate of elimination of a compound is a
constant, and is independent of the concentration of the chemical l in
the blood. For example, the average human body is able to
eliminate ~10-15
15 mL of ethanol per hour, regardless of the amount
of ethanol consumed. This may be higher or lower depending on
the factors previously discussed (e.g., induction of alcohol
dehydrogenase by repeated alcohol exposure).
• For first-order kinetics, the rate of elimination of a compound is
dependent on the concentration of the chemical in the blood. The
higher the concentration, the more rapidly the chemical is
eliminated, unless the elimination mechanisms have been saturated.
At that point, the kinetics become zero-order. order. This is known as
saturation kinetics.
٥٣

• Half-life life (t 1/2
1/2 )
• The time required for the concentration of a chemical in
the plasma to decrease by 50%.
• This is a constant for all but zero-order order kinetics. For
example, for a compound eliminated by first-order
kinetics, if the concentration at time 0 is 4 mg/L, and the
t 1/2 is 6 hours, then at the end of 6 hours, the plasma
concentration will be (0.5 x 4 mg/mL
mL =) 2 mg/L, and at
the end of the next 6 hours, the concentration will be
(0.5 x 2 mg/mL
mL =) 1 mg/L, and so on.
٥٤

• Area under the Curve (AUC)
• A measure of the total amount of chemical present in
the body over a defined period. This is defined as the
area under the concentration vs time curve for the
defined period.
• Example: The shaded area in the figure below
represents the AUC(0-6hr). You can express the AUC
for any given time period. It is often expressed as the
AUC(0-∞).
•
Concentration
in
plasma
0
Time (hrs)
6
٥٥

• Classical toxicokinetics
• One compartment model
• The elimination of a chemical is said to follow a one-
compartment model when the elimination phase of the
log concentration vs time curve is a straight line.
Conceptually, this is as though the chemical were
evenly distributed throughout a single body
compartment (e.g., total body water), and is eliminated
at a constant rate over time. That is, a constant
percentage of the chemical present is eliminated over
any given time period.
٥٦

• The rate at which a chemical is eliminated at any time is directly
proportional to the amount of that chemical in the body at that time.
• A one-compartment model indicates that no one tissue has a high affinity
for the chemical. Changes in the plasma reflect changes in the tissue.
Plasma
Concentratio
n
Log of
Plasma
Concentratio
n
Time
Time
٥٧

• Two compartment model
• The elimination of a chemical is said to follow a two-
compartment model when the elimination phase of the
log concentration vs time curve is not a straight line.
Conceptually, this is as though the chemical were
unevenly distributed throughout two body
compartments (e.g., highly concentrated in tissues and a
lower concentration in the plasma). The net elimination
is a sum of the rates of elimination from the various
compartments.
• The appearance of a two-compartment curve suggests
that the chemical is distributed out of the blood into
tissues.
٥٨

Alpha distribution
Log of Plasma
Concentration
Beta distribution
Time
٥٩

Chronic Administration
The kinetic concepts and parameters of a single dose (as
discussed above) apply to chronic administration, but
the exposure has to allow for the fact that not all of
the previous dose(s) ) may have been eliminated when
the subsequent dose is given. Therefore, there may be
an increase in plasma concentration (and body load)
until an equilibrium is reached in which the rate of
elimination balances out the rate of input, in other
words, the daily dose is eliminated each day.
٦٠

٦١

٦٢

The equations above assume that CL is not
altered by repeated exposure; the assumption is
not correct if the chemical induces or inhibits
its own elimination because clearance would
be increased or decreased, respectively, after
the period of chronic intake.
٦٣

Because the plasma and therefore tissue concentrations
increase during chronic intake until an equilibrium is
reached , the amount in the body (Ab(
Ab) ) will also
increase to reach a steady state. The time taken to
reach steady state is 4–54
5 times the elimination half-
life and, therefore, the true duration of steady-state
state
exposure in a toxicity study is the study duration
minus 4–54
5 half-lives lives of the chemical (needed to reach
the steady state). This is particularly important for
chemicals that have a very long half-life; life; for example
in rodents the steady-state state body load of TCDD, which
has a half-life life in rats of about 1 month, will not be
reached until after about 4–54
5 months of continuous
treatment.
٦٤

Saturation Kinetics
All the parameters described above relate to first-order
processes and, therefore, are independent of dose at
low doses. However, at high doses and/or during
chronic studies it is possible to overload or saturate
compound–protein protein interactions. Under such
circumstances any increase in the concentration of the
compound cannot give a proportional (first-order)
increase in the rate of the process. When a process is
saturated the rate is at the maximum possible and is
essentially independent of concentration.
٦٥

In simple mathematical terms this means that the reaction
changes from first to zero order. This is best described by
Michaelis–Menten
Menten kinetics, i.e.
٦٦

3. Metabolism
• 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
٦٧

• Example: Cholinesterase is an enzyme that normally metabolizes
acetylcholine, a neurotransmitter found in the synapse.
Cholinesterase can also metabolize the local anesthetic agent
procaine and the muscle-paralyzing agent succinylcholine. . If a
person has high levels of cholinesterase activity, the effectiveness
of these drugs can be compromised. If a person is exposed to
anything that inhibits cholinesterase activity, the pharmacologic
effects of the drugs can be enhanced, resulting in toxicity.
• 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 i
MAO activity (like many blood pressure medications), it can be
dangerous for that person to take other drugs that can be
metabolized by MAO.
٦٨

• The products of biotransformation can be either less toxic
than, more toxic than, 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
• 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 – many of the drug-metabolizing enzymes are located in
the smooth endoplasmic reticulum (SER). These are called
“microsomal” enzymes.
٦٩

• Types of Biotransformation Reactions
• Basically, two types of reactions, nonsynthetic (Phase I) and
synthetic (Phase II)
• Phase I reactions
• Involve modification of the basic structure of the substrate
• Do not involve covalent binding of the substrate to an endogenous
compound
• Examples include hydrolysis, oxidation, and reduction reactions
• Phase I enzymes are often membrane-bound bound (e.g.,
microsomal). This is because they generally act on more lipid-
soluble (nonpolar(
nonpolar) ) substrates, and their purpose is to make the
compounds MORE POLAR and therefore, MORE EASILY
EXCRETABLE by the kidney and biliary tract. Think of the
active transport mechanisms in the kidney.
٧٠

• 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 448-450 450 nm.
٧١

• Anything that affects the activity of any one of the steps can affect a
the way the body reacts to a given drug or other xenobiotic.
• Examples of the various types of oxidation reactions are in the
textbook,
• Deamination – replacement of an amine group (NH 2
) with an oxygen
(O) atom
• N-, , O-, O , or S-DealkylationS
– replacement of an alkyl group (e.g., CH 3
)
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)
٧٢

• Reduction
• Azo reduction – reduction of an azo bond (N=N) to two
amines (NH 2 )
• Nitro reduction – reduction of a nitro group (NO 2 ) to an
amine
• Hydrolysis
• Addition of water (H 2 O) to an ester bond (C-O-O-C) C) to
form an alcohol (C-OH) and a carboxylic acid (COOH)
R-C-O-O-C-R R + H-O-H H = R-C-OH + R-COOHR
٧٣

• Phase II reactions
• 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.
٧٤

• Sulfation
• 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
• Glucuronidation
• Replacement of a hydrogen atom with a glucuronic acid
• Uses the enzyme UDP-glucuronosyl
transferase (UDP-GT)
• Uses the cofactor called UDPGA (uridine(
diphosphate glucuronic
acid)
• One of the major Phase II enzymatic pathways
٧٥

• Acetylation
• 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
• Remember the discussion of slow versus fast acetylators.
• Methylation
• 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
٧٦

• Glutathione conjugation
• 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
• Amino acid conjugation
• Adds an amino acid to the parent compound.
• Mercapturic acid formation
• Formed by cleavage of the glycine and glutamic acid substituents
from a glutathione conjugate, followed by N-acetylationN
of the
resulting product
٧٧

• 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.
٧٨

• Good example – metabolism of acetaminophen
• 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 able in
only limited concentrations, so these are rate-limiting.
• As long as the amount of acetaminophen in the liver is relatively y 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.
• 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
les
destroys the activity of those molecules, and can lead to compromised
mised
cell function and, ultimately, cell death.
٧٩

• Another good example – metabolism of carbon
tetrachloride
• 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.
٨٠

• The activity of drug metabolizing enzymes is
dependent on numerous factors
• 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
٨١

• 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)
• 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.
• Induction may result in increases in the amount of cellular
protein, hypertrophy (increases in size) of the cells, and
increases in weight of the affected organs (especially liver).
• One effect of induction of microsomal enzymes is an
increase in the amount of smooth endoplasmic reticulum in
a cell. This can be seen microscopically.
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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
• Classic example of an inhibitor is SKF-525A, which
inhibits microsomal enzymes
• Inhibition may be either competitive or non-competitive.
• Competitive inhibition
• 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 l be able
to find and bind to an available enzyme molecule.
• Reversible, since the binding of the inhibitor to the active site e 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 life for diazepam.
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• Non-competitive inhibition
• 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 t 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.
• A special subset of non-competitive inhibition is “suicide inhibition”, , in
which a compound binds to the active site of an enzyme and is
metabolized, but the product then binds irreversibly to the active site. An
example of this is the binding of organophosphate insecticides to the
enzyme acetylcholinesterase (AchE). This results in a prolonged inhibition
of AchE activity.
• Inhibition may result in increases or decreases in toxicity, depending ending on
whether the metabolite is more or less toxic than the parent compound.
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Factors affecting metabolism
1. 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
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4. Excretion
• Excretion
• The removal of materials from the body
• Routes of excretion
• Bile – through the liver
• Urine – through the kidneys
• Feces – through the intestines
• Expired air – through the lungs
• Sweat – through the skin
• Saliva – through the mouth
• Milk
• Hair
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Factors affecting excretion
1. Age
Many of the functions of the kidney are not well developed in
neonates, toxic drugs will be slowly excreted. In the elderly,
lower renal plasma flow will also decrease excretion
2. Disease states
Renal and gastrointestinal diseases can considerably slow the
excretion
3. Recirculation
Some drugs that are excreted as their more water soluble
metabolites may be back transformed to the parent
compound by some bacteria
4. Ion trapping
Weakly acidic drugs trapped in the tubules are excreted at higher
urinary pH as the ion form, thus preventing reabsorption.
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