12.

Drug Metabolism: Phase I and Phase II Metabolism

Introduction:
Lipophilic drug properties that promote passage through biological membranes
and facilitate reaching site to drug action inhibit drug excretion.
Note: renal excretion of unchanged drug contributes only slightly to
elimination, since the unchanged, lipophilic drug is easily reabsorbed
through renal tubular membranes.
Biotransformation of drugs to more hydrophilic molecules is required for
elimination from the body
Biotransformation reactions produces more polar, hydrophilic,
biologically inactive molecules -- that are more readily excreted.
 Sometimes metabolites retain biological activity and may be toxic.
Drug biotransformation mechanisms are described as either phase I or
phase II reaction types.
Phase I and Phase II Reactions
Phase I characteristics:
Parent drug is altered by introducing or exposing a functional group (-
OH,-NH2, -SH)
Drugs transformed by phase I reactions usually lose pharmacological
activity
Inactive, prodrugs are converted by phase I reactions to biologically-active
metabolites
Phase I reaction products may:
 be directly excreted in the urine
 react with endogenous compounds to form water soluble
conjugates.
Phase II characteristics:
Parent drug participates in conjugation reactions that:
form covalent linkage between a parent compound functional group and:
 glucuronic acid
 sulfate
 glutathione
 amino acids
 acetate
Conjugates are highly polar, and generally biologically inactive. One exception to this
rule is a morphine metabolite, morphine glucuronide which is a more potent analgesic
compared to the parent compound. Conjugates tend to be rapidly excreted in the urine.
High molecular weight conjugates are more likely excreted in the bile. The
conjugate bond may be cleaved by intestinal flora with the parent compound
released back to the systemic circulation. This process, "enterohepatic
recirculation" results in delayed parent drug elimination and a prolongation of
drug effects.

Principal Organs for Biotransformation:

 The Principal Organ for biotransformation is the liver, although other organs participate
in metabolism. These other systems include lungs, skin, kidney, and the gastrointestinal
tract.
Other metabolizing organs:
Sequence I could be as follows:
(1) Oral administration (isoproterenol (Isuprel), meperidine (Demerol),
pentazocine (Talwain), morphine)
(2) The drug is absorbed intact by the small intestine.
(3) The drug is transported to the liver (portal system) where it might be
extensively metabolized by the liver, an example of a first-pass effect.
Sequence II might be as follows:
(1) Oral administration (e.g. clonazepam (Klonopin), chlorpromazine
(Thorazine)) and
(2) the agent is absorbed intact by the small intestine.
(3) Extensive intestinal metabolism might ensue, contributing to overall first-pass
effects.
Issues in bioavailability: Reduced bioavailability might result from several factors
including (a) the first pass effect in which the bioavailability of orally administered drugs
become so limited that alternative routes of administration must be employed. (b)
Intestinal flora might metabolize the drug. (c) The drug itself is unstable in gastric acid;
an example of this effect would be penicillin. (d) the drug might be metabolized by
digestive enzymes; an example of this effect would be insulin. (d) Finally, the drug might
be metabolized by intestinal wall enzymes; sympathomimetic catecholamines represent
examples of this effect.
First pass effect: bioavailability of orally administered drugs -- so limited --
alternative routes of administration must be used
Intestinal flora may metabolize drugs
unstable in gastric acid-- penicillin
metabolized by digestive enzymes -- insulin
metabolized by intestinal wall enzymes-- sympathomimetic catecholamines

Mixed function oxidase System (cytochrome 450 System)--Phase I Reactions

Microsomes have been used to study mixed function oxidases

Drug metabolizing enzymes are located in lipophilic, hepatic endoplasmic
reticulum membranes. Smooth endoplasmic reticulum contains those enzymes
responsible for drug metabolism.
The reaction:
one molecule oxygen is consumed per substrate molecule
one oxygen atom -- appears in the product; the other in the form of water
Oxidation-Reduction Process:
Two important microsomal enzymes:
A. flavoprotein--NADPH cytochrome P450 reductase
B. Cytochrome P450: -- terminal oxidase
1. multiple forms
2. named cytochrome P450 because:
  the reduced (ferrous) form, binds carbon monoxide:
-- the resulting complex exhibits of absorption
maximum at 450 nm.
3. NOTE in the Figure Below the CONVERSION OF RH to
ROH representing DRUG OXIDATION

Cytochrome p450 cycle (diagram by Matthew Segall, 1997)

1. "The binding of a substrate to a P450 causes a lowering of the redox potential by

approximately 100mV, which makes the transfer of an electron favourable from its redox
partner, NADH or NADPH.
2. The first reduction -The next stage in the cycle is the reduction of the Fe3+ ion by an
electron transfered from NAD(P)H via an electron transfer chain.
3. Oxygen binding An O2 molecule binds rapidly to the ion Fe2+ forming Fe2+-O2
4. Second reduction A second reduction is required by the stoichiometry of the reaction.
This has been determined to be the rate-determining step of the reaction
5. O2 cleavage: The O2 reacts with two protons from the surrounding solvent, breaking the
O-O bond, forming water and leaving an Fe-O3+ complex.
6. Product formation The Fe-ligated O atom is transferred to the substrate forming an
hydroxylated form of the substrate.
7. Product release The product is released from the active site of the enzyme which returns
to its initial state."--Matthew Segall, 1997

"The active site of substrate-free cytochrome p450: Note the water molecule (which can
be seen as a single oxygen atom) that forms the sixth axial ligand of the haem iron.
Oxygen atoms are shown in red, nitrogen in light blue, sulphur in yellow and iron in
dark blue. Carbon atoms are shown in grey as bonds only and hydrogens have been
omitted from this figure for clarity."
"The active site of camphor-bound cytochrome p450cam , an example of a substrate-bound
system. Note the absence of the water molecule which formed the sixth axial ligand of
the haem iron in the substrate-free enzyme."
" A representation of with bound camphor. The enlarged active site region shows the
camphor substrate, haem moiety and cysteine residue which forms the distal haem ligand.
In the representation of the full enzyme the protein backbone is shown in green, the haem
moiety in blue and the substrate is coloured according to atomic species. Oxygen atoms
are shown in red, carbon in grey, nitrogen in light blue, sulphur in yellow and iron
in dark blue."-diagrams and text by Matthew Segall, 1997
Cytochrome P450 Enzyme Induction:
Following repeated administration, some drugs increase the amount of P450
enzyme usually by:
increase enzyme synthesis rate (induction)
 reduced enzyme degradation rate
Cytochrome P450 enzyme inhibition:
Certain drugs, by binding to the cytochrome component, act to competitively
inhibit metabolism. Examples:
Cimetidine (Tagamet) (anti-ulcer --H2 receptor blocker) and Ketoconazole
(Nizoral) (antifungal) bind to the heme iron a cytochrome P450, reducing
the metabolism of:
 testosterone
 other coadministered drugs
 Mechanism of Action: competitive inhibition
Catalytic inactivation of cytochrome P450.
Macrolide antibiotics (troleandomycin, erythromycin estolate (Ilosone)),
metabolized by a cytochrome P450:
 metabolites complex with cytochrome heme-iron: producing a
complex that is catalytically inactive.
Chloramphenicol (Chloromycetin): metabolized by cytochrome P450 to
an alkylating metabolite that inactivates cytochrome P450
Other inactivators: Mechanism of Action: -- targeting the heme moiety:
 steroids:
 ethinyl estradiol (Estinyl)
 norethindrone (Aygestin)
 spironolactone (Aldactone)
 others:
 propylthiouracil
 ethchlorvynol (Placidyl)

return to Table of Contents

Phase II Metabolism

Some Phase II Reactions

Type of Endogenous Transferase Types of
Examples
Conjugation Reactant (Location) Substrates
morphine,
UDP phenols, alcohols, acetaminophen,
UDP glucuronic glucuronosyl carboxylic acids, diazepam,
Glucuronidation
acid transferase hydroxylamines, digitoxin,
(microsomal) sulfonamides digoxin,
meprobamate
Acetylation Acetyl-CoA N-Acetyl Amines sulfonamides,
transferase isoniazid,
(cytosol) clonazepam,
dapsone,
 mescaline
GSH-S-
epoxides, nitro
Glutathione transferase ethycrinic acid,
glutathione groups,
conjugation (cytosolic, bromobenzene
hydroxylamines
microsomes)
estrone, 3-
hydroxy
Sulfate Phosphoadenosyl Sulfotransferase phenols, alcohols,
coumarin,
conjugation phosphosulfate (cytosol) aromatic amines
acetaminophen,
methyldopa
dopamine,
catecholamines, epinephrine,
S-Adenosyl- transmethylases
Methylation phenols, amines, histamine,
methionine (cytosol)
histamine thiouracil,
pyridine
Adapted from Table 4-3, Correia, M.A., Drug Biotransformation. in Basic and Clinical
Pharmacology, (Katzung, B. G., ed) Appleton-Lange, 1998, p 57.

Overview: Phase II reactions involve non-microsomal enzymes

Reaction types:
1. conjugation
2. hydrolysis
3. oxidation
4. reduction
Location (non-microsomal enzymes): primarily hepatic (liver); also plasma &
gastrointestinal tract
Non-microsomal enzymes catalyze all conjugation reactions except
glucuronidation
Nonspecific esterases in liver, plasma, gastrointestinal tract hydrolyzed drugs
containing ester linkages, including succinylcholine (Anectine), Atricurium
(Tracrium), Mivacurium (Mivacron), esmolol (Brevibloc) as well as ester-type local
anesthetics.

Conjugation reactions are usually "detoxification reaction". Conjugates tend to be more

polar compared to the parent compound, more easily excreted, and usually
pharmacologically inactive.
Conjugation reactions require "high-energy" intermediates in specific transfer enzymes
which include both microsomal and cytosolic transferases.
Conjugation with glucuronic acid: Glucuronic acid is available from glucose and
its conjugation with lipid-soluble drugs results in a lipophilic glucuronic acid
derivative which is typically pharmacologically inactive and more water-soluble
compared to the parent compound. Therefore, the glucuronic acid derivative
molecule is more readily excreted in both urine or bile.
Transferases are enzymes which catalyzes the coupling of an endogenous
substance with the drug.
 For example, transferase which catalyzes the "transfer" of uridine-5'-
diphosphate (UDP) derivative of glucuronic acid and a drug.
A transferase may catalyze an inactivated drug with an endogenous
substrate. For example a S-CoA derivative of benzoic acid with an
endogenous substrate.
Toxicity:
Certain conjugation reactions form toxic reactive species (hepatotoxicity). For
example, acyl glucuronidation of nonsteroidal anti-inflammatory drugs may result
in toxicity. Another example would be N-acetylation of isoniazid.
Drugs metabolized to toxic products:
Acetaminophen hepatotoxicity -- normally safe in therapeutic doses
Therapeutic doses:
 glucuronidation + sulfation to conjugates (95% of excreted
metabolites); 5% due to alternative cytochrome P450 depending
glutathione (GSH) conjugation pathway
At high doses:
 Glucuronidation and sulfation pathways become saturated
 Cytochrome P450 dependent pathway becomes now more
important.With depletion of hepatic glutathione, hepatotoxic,
reactive, electrophilic metabolites are formed. In this circumstance
antidotes would include N-acetylcysteine and cysteamine. N-
acetylcysteine protects patients from fulminant hepatotoxicity and
death following acetaminophen overdose.