Download Drug Metabolism

Drug Metabolism
Dr. Mohammad Khanfar
Drug Metabolism
• When drugs enter the body, they are subject to attack from a range
of metabolic enzymes. The role of these enzymes is to degrade or
modify the foreign structure, such that it can be more easily
excreted. As a result, most drugs undergo some form of metabolic
reaction resulting in structures known as metabolites. Very often
these metabolites loss the activity of the original drug, but in some
cases they may retain a certain level of activity.
• Why we study metabolism?
• Detoxifications : Drugs, plant toxins, food additives, environmental
chemicals, insecticides, and other chemicals foreign to the body
undergo enzymatic transformations that usually result in the loss of
pharmacological activity
• Bioactivation: enzyme-catalyzed reactions may lead to the
formation of a metabolite having therapeutic or toxic effects (as in
prodrugs)
Drug Metabolism
• Liver is the major site of drug metabolism, although other
xenobiotic-metabolizing enzymes are found in nervous tissue,
kidney, lung, plasma, and the gastrointestinal tract (digestive
secretions, bacterial flora, and the intestinal wall) .
• The ability of the liver and extrahepatict tissues to metabolize
substances to either pharmacologically inactive or bioactive
metabolites before reaching systemic blood levels is termed “
first -pass metabolism” or the “presystemic first -pass effect.”
Pathways of Metabolism
• Phase 1 Reactions: a new functional group is introduced into
the substrate molecule, an existing functional group is
modified, or a functional group or acceptor site for Phase 2
transfer reactions is exposed, thus making the xenobiotic
more polar and, therefore, more readily excreted.
• Reactions include : oxidation, hydroxylation, reduction, and
hydrolysis
• Phase 2 Reactions (Conjugations): a functional group, such as
alcohol, phenol, or amine, is masked by the addition of a new
group, such as acetyl, sulfate, glucuronic acid, or certain
amino acids, which further increases the polarity of the drug
or xenobiotic.
Factors affecting metabolism
1- Genetic factors:
Pharmacogenetics: study the relation between genetic variation
and different response of drugs
• Polymorphisms (mutations) responsible for interindividual
differences in drug metabolism and disposition
• For example, mutations in the CYP2D6 gene resulting poor,
intermediate, or ultrarapid metabolizers of more than 30
cardiovascular and central nervous system drugs
• Incorporating pharmacogenomics to drug treatment , into
drug therapy will alter the way in which drug regimens are
chosen for patients based on their individual genetic makeup
Factors affecting metabolism
2- Physiologic factors. Age is a factor because the very young
and the old have impaired metabolism. Hormones ( including
those induced by stress), sex differences, pregnancy, changes in
the intestinal microflora, diseases (especially those involving the
liver ), and nutritional status also can influence drug and
xenobiotic metabolism
3- Pharmacodynamic factors. Dose, frequency, and route of
administration, plus tissue distribution and protein binding of
the drug, affect its metabolism
4- Environmental factors. Competition of ingested
environmental substances with other drugs and xenobiotics for
the metabolizing enzymes and poisoning of enzymes by toxic
chemicals, such as carbon alter metabolism by induction or
inhibition
Phase I Metabolism
Human Hepatic Cytochrome P450 Enzyme System
• Oxidation probably is the most common reaction in
xenobiotic metabolism. This reaction is catalyzed by a group
of membrane-bound monooxygenases found in the smooth
endoplasmic reticulum of the liver and other extrahepatic
tissues, termed the “ cytochrome P450 monooxygenase
enzyme system” (CYP450)
• CYP450 are haemoproteins (containing haem and iron) and
they catalyse a reaction that splits molecular oxygen, such
that one of the oxygen atom is introduced into the drug and
the other ends up in the water:
• Drug-H + O2 + NADPH + H+
Drug-OH + NADP+ + H2O
Phase I Metabolism
Human Hepatic Cytochrome P450 Enzyme System
• There are at least 33 different CYP450 enzymes, grouped into
4 main families (homology > 40%) CYP1 – CYP4. Within each
family there are various subfamilies designated by letter
(homology > 55%), and each enzyme within that subfamily is
designated by a number.
• For example, CYP3A4 is enzyme 4 in the subfamily A of the
main family 3.
• Most drugs in current use are metabolized by five primary CYP
enzymes (CYP3A, CYP2D6, CYP2C, CYP1A2, and CYP2E1).
• CYP3A4 is particularly important and responsible for
metabolism of most drugs, increasing the probability of drug –
drug interaction
• CYP1A2 is predominantly involved in the bioactivation of
environmental substances
Phase I Metabolism
Human Hepatic Cytochrome P450 Enzyme System
• CYP1A1. expressed primarily in extrahepatict issues, small
intestine, placenta, skin, and lung as well as in the liver in
response to the presence of CYP1A1 inducers such as
polycyclic aromatic hydrocarbons (PAH) in cigarrete smoke
• Interindividual variation in the inducible expression of CYP1A1
explain differences in individual susceptibility to cigarette
smoke–induced lung cancer
Phase I Metabolism
Human Hepatic Cytochrome P450 Enzyme System
Phase I Metabolism
Human Hepatic Cytochrome P450 Enzyme System
• CYP1A2. It is expressed in the liver, intestine, and stomach
• CYP1A2 is primarily responsible for the activation of the carcinogen
aflatoxin B1
• The expression of the CYP1A2 gene in the stomach becomes an
important issue for gastric carcinogenesis induced by smoking
• CYP2A6. Involved in the metabolism of nicotine. Smokers with a
defective CYP2A6 gene smoke fewer cigarettes, implicating a
genetic factor in nicotine dependence.
• CYP2D6. Responsible for at least 30 different drug oxidations,
representing approximately 21% of the clinically important drugs.
• It also appears to preferentially catalyze the hydroxylation of a
single enantiomer (stereoselectivity) in the presence of
enantiomeric mixtures.
Phase I Metabolism
Human Hepatic Cytochrome P450 Enzyme System
• CYP2E1. plays a major role in the metabolism of numerous
halogenated hydrocarbons (including volatile general
anesthetics) and a range of low-molecular -weight organic
compounds, including dimethyformamide, acetonitrile,
acetone, ethanol, and benzene
• Induced in alcoholics
• CYP3A4. most abundantly expressed CYP450s in the human
liver and intestine
• Responsible for the metabolism of more than one- third of
the clinically important drugs.
Induction and Inhibition of CP450
Enzyme induction
• Enzyme induction. Describe the process by which the rate of
synthesis of an enzyme is increased relative to the rate of
synthesis in the uninduced organism.
• Many drugs, environmental chemicals, and other xenobiotics
enhance the metabolism of themselves or of other
compounds, thereby altering their pharmacological and
toxicological effects.
• Enzyme induction is a dose-dependent phenomenon.
• Enzyme induction can alter the PK and PD of a drug, with
clinical implications for the therapeutic actions of a drug and
increased potential for drug interactions
Induction and Inhibition of CP450
Enzyme induction
• Specific Inducers
• Phenobarbital and rifampin are the most common enzyme
inducres
• Cigarette smoke, cabbage and cauliflower
• Alcohol, induce CYP2E1
Induction and Inhibition of CP450
Enzyme Inhibition
• CYP450 inhibitors can be divided into three categories
according to their mechanism of action:
• Reversible inhibition
• Metabolite intermediate complexation of CYP450
• Mechanism-based inactivation of CYP450
Induction and Inhibition of CP450
Enzyme Inhibition
• Reversible Inhibition.
• Reversible interactions at the heme–iron active center of
CYP450, the lipophilic sites on the apoprotein, or both.
• The interaction occurs before the oxidation steps of the
catalytic cycle, and their effects dissipate quickly when the
inhibitor is discontinued.
Induction and Inhibition of CP450
Enzyme Inhibition
• Metabolite intermediate complexation of CYP450
• The complexed-CYP450 is unavailable for further oxidation,
and synthesis of the new enzyme is required to restore
CYP450 activity.
• Alkylamine drugs have the ability to undergo CYP450mediated oxidation to nitrosoalkane metabolites, which have
a high affinity for forming a stable complex with the reduced
(ferrous) heme intermediate for the CYP450
Induction and Inhibition of CP450
Enzyme Inhibition
• Mechanism-Based Inhibition
• Certain drugs that are noninhibitory of CYP450 contain
functional groups that, when oxidized by CYP450, generate
metabolites that bind irreversibly to the enzyme. This process
is termed “mechanism-based inhibition” ( “suicide inhibition'')
• Alkenes and alkynes were the first functionalities found to
inactivate CYP450 by generation of a radical intermediate that
alkylates the heme structure
Oxidation Catalyzed by CYP450 Isoforms
Aliphatic and Alicyclic hydrocarbons
• Methyl oxidation
• Methyl group is oxidized to the hydroxymethyl derivative
followed by its nonmicrosomal oxidation to the carboxylic acid
• On the other hand, some methyl groups are oxidized only to
the hydroxymethyl derivative, without further oxidation to
the acid.
• For aromatic methyl groups, the para methyl is the most
vulnerable
Oxidation Catalyzed by CYP450 Isoforms
Aliphatic and Alicyclic hydrocarbons
• Alkyl side chain oxidation
Oxidation Catalyzed by CYP450 Isoforms
Aliphatic and Alicyclic hydrocarbons
• Alkyl side chain oxidation
Oxidation Catalyzed by CYP450 Isoforms
Aliphatic and Alicyclic hydrocarbons
• Benzylic methylene oxidation
Oxidation Catalyzed by CYP450 Isoforms
Aliphatic and Alicyclic hydrocarbons
• Alicycle Oxidation
• The methylene groups of an alicycle are readily hydroxylated,
generally at the least hindered position, or at an activated
position—for example, α to a carbonyl, α to a double bond, or
α to a phenyl ring. The products of hydroxylation often show
stereoisomerism.
• Nonaromatic heterocycles generally undergo oxidation at the
carbon adjacent to the heteroatom
Alicycle Oxidation
Oxidation Catalyzed by CYP450 Isoforms
Aliphatic and Alicyclic hydrocarbons
• Dehydration
• In addition to hydroxylation reactions, CYP450s can catalyze
the dehydrogenation of an alkane to an alkene (olefin)
Valproic acid
• Hydroxylation generally is favored over dehydration
Oxidation Catalyzed by CYP450 Isoforms
Alkene and alkyne hydroxylation
• The oxidation of alkenes yields primarily epoxides and a
series of products.
• The epoxides can differ in reactivity:
1- Those that are highly reactive either undergo pHcatalyzed hydrolysis to excretable vicinal dihydrodiols or
react covalently (alkylate) with macromolecules, such as
proteins or nucleic acids, leading to t issue necrosis or
carcinogenicity.
2- The epoxide hydrolase can catalyze the rapid hydrolysis of
epoxides to nontoxic vicinal dihydrodiols.
3- Many drugs maintain stable epoxide
Oxidation Catalyzed by CYP450 Isoforms
Alkene and alkyne hydroxylation
Oxidation Catalyzed by CYP450 Isoforms
Alkene and alkyne hydroxylation
Oxidation Catalyzed by CYP450 Isoforms
Alkene and alkyne hydroxylation
• With monosubstituted, unconjugated alkenes, the
epoxidation is accompanied by the mechanism-based
(“suicide'') N-alkylation of the heme–porphyrin ring.
Oxidation Catalyzed by CYP450 Isoforms
Aromatic hydroxylation
• Aromatic hydroxylation is a major route of metabolism for many
drugs containing phenyl groups
• Aromatic hydroxylation proceeds initially through an epoxide
intermediate called an "arene oxide” which rearranges rapidly and
spontaneously to the arenol (phenol) product in most instances
Arene
arene oxide Arenol
• Mostly hydroxylation occurs at the para position
• Most phenolic metabolites formed from aromatic oxidation
undergo further conversion to polar and water-soluble glucuronide
or sulfate conjugates
Possible reaction pathways for arene oxides
Spontaneous
rearrangement
epoxide
hydrolase
Epoxide
hydrolase
trans dihydrodiol
GSH
Glutathione adduct
Macromolecules
Macromolecule adduct
Covalently bound
M: DNA, RNA, protein
Oxidation Catalyzed by CYP450 Isoforms
Aromatic hydroxylation
• The position of hydroxylation can be influenced by the type of
substituents on the ring
• Electronic effect: Electron-donating substituents enhance
hydroxylation, whereas electron-withdrawing substituents
reduce or prevent hydroxylation.
• Steric effect: steric factors also must be considered, because
oxidation usually occurs at the least hindered position. For
monosubstituted benzene compounds, para hydroxylation
usually predominates, with some ortho product being
formed. When there is more than one phenyl ring, usually
only one is hydroxylated
Oxidation Catalyzed by CYP450 Isoforms
N-dealkylation, oxidative deamination, and N-oxidation
• N-dealkylation
• The dealkylation of secondary and tertiary amines to yield
primary and secondary amines, respectively
• Typical N-substituents removed by oxidative dealkylation are
methyl, ethyl, n-propyl, isopropyl, n-butyl, allyl, and benzyl
• Dalkylation initially occurs with the smaller alkyl group.
Substituents that are more resistant to dealkylation include
the tert -butyl and the cyclopropylmethyl
• Tertiary amines are dealkylated to secondary amines faster
than secondary amines are dealkylated to primary amines.
This difference in rate has been correlated with lipid solubility
• N-dealkylation of substituted amides and aromatic amines
occurs in a similar manner .
Tertiary amine
Carbinolamine
Carbonyl
Aldehyde or
Secondary amine
keton
Oxidation Catalyzed by CYP450 Isoforms
N-dealkylation, oxidative deamination, and N-oxidation
• Oxidative deamination
• The mechanism of oxidative deamination follows a pathway similar
to that of N-dealkylation.
• Oxidative deamination can occur with α-substituted amines
(amphetamine).
Oxidation Catalyzed by CYP450 Isoforms
N-dealkylation, oxidative deamination, and N-oxidation
• Disubstitution of the α-carbon inhibits deamination
Direct
oxidative
deamination
Propranolol
Aldehyde metabolite
Carbinolamine
Oxidative deamination
through primary amine
Carbinolamine
Primary amine metabolite
Oxidation Catalyzed by CYP450 Isoforms
N-dealkylation, oxidative deamination, and N-oxidation
• N-oxidation
• In general , N-oxygenation of amines form stable N-oxides
with tertiary amines and amides, and hydroxylamines with
primary and secondary amines, when no α-protons are
available
• In general N-hydroxylamines are chemically unstable and
susceptible to spontaneous or enzymatic oxidation to the
nitroso and nitro derivatives.
Hydroxylamine
Nitroso
Nitro
Oxidation Catalyzed by CYP450 Isoforms
N-dealkylation, oxidative deamination, and N-oxidation
• O-Dealkylation
• Mechanistically, the biotransformation involves an initial acarbon hydroxylation to form either a hemiacetal or a
hemiketal. which undergoes spontaneous carbon—oxygen
bond cleavage to yield the dealkylated oxygen species (phenol
or alcohol) and a carbon moiety (aldehyde or ketone). Small
alkyl groups (e.g., methyl or ethyl) attached to oxygen are Odealkylaced rapidly.
Ether
Hemiacetal or
hemiketal
Carbonyl
Alcohol
or phenol (aldehyde or keton)
Oxidation Catalyzed by CYP450 Isoforms
• Oxidation involving carbon-sulfur systems
• Carbon—sulfur functional groups are susceptible to metabolic
S-dealkylation, desulfuration, and S-oxidation reactions.
• The first two processes involve oxidative carbon— sulfur bond
cleavage. S-dealkylation is analogous to O- and N-dealkylation
mechanistically (i.e.. it involves carbon hydroxylation) and has
been observed for various sulfur xenobiotics.
• Oxidative conversion of carbon—sulfur double bonds (C = S)
(thiono) to the corresponding carbon—oxygen double bond
(C=O) is called desulfuration
Organosulfur xenobiotics commonly undergo S-oxidation to yield
sulfoxide and sulfone derivatives
Sulfoxide
Sulfone
Oxidation Catalyzed by CYP450 Isoforms
Dehalogenation
• Dehalogenation
• Oxidative dehydrohalogenation is a common metabolic
pathway for many halogenated hydrocarbons. The CYP450catalyzed oxidation generates the carbinol intermediate
(analogous to alkane hydroxylation) that can eliminate the
hydrohalic acid to form carbonyl derivatives (aldehydes,
ketones, acyl halides, and carbonyl halides)
Oxidation Catalyzed by CYP450 Isoforms
Azo and Nitro Reduction
• A number of azo compounds, such as Prontosil and
sulfasalazine, are converted to aromatic primary amines by
azoreductase
Oxidation Catalyzed by CYP450 Isoforms
Azo and Nitro Reduction
• Nitro compounds are reduced to aromatic primary amines by
a nitroreductase, presumably through nitroso and
hydroxylamine intermediates
Phase I metabolism
Flavin-containing monooxygenases (FMO)
•
•
•
•
Nonheme, microsomal monooxygenase
Present in the endoplasmic reticulum of liver cells
Like CYP450, FMO requires NADPH and oxygen
Responsible for oxidation at nucleophilic N, S, and P atoms
rather than C atoms
• S-oxidation occurs almost exclusively by FMO
• The FMO does not catalyze epoxidation reactions or
hydroxylation at unactivated carbon atoms of xenobiotics
• Normally, FMO is not inducible by phenobarbital , nor is it
affected by CYP450 inhibitors.
Phase I metabolism
Flavin-containing monooxygenases (FMO)
Phase I metabolism
Flavin-containing monooxygenases (FMO)
Oxidation of Aldehyde
• A NAD+-specific aldehyde dehydrogenase catalyzes the
oxidation of endogenous aldehydes, such as those produced
by the oxidation of primary alcohols or the deamination of
amines, and of exogenous aldehydes to the corresponding
carboxylic acids. By inhibiting this enzyme, disulfuram
(Antabuse®) and metronidazole produce an unpleasant set of
reactions ( flushing, abdominal cramping, and headache)
when small amounts of alcohol are ingested. Antabuse is used
therapeutically in controlling alcohol abuse.
Oxidation of Alcohols
Alcohol dehydrogenase
• Alcohol dehydrogenase is an NAD+-specific
• If not conjugated, most primary alcohols are readily oxidized
to their corresponding aldehydes.
• Some secondary alcohols are oxidized to the ketones,
whereas other secondary and tertiary alcohols are excreted
either unchanged or as their conjugate metabolite
• Oxidation by alcohol dehydrogenase is the principal pathway
for ethanol metabolism (2/3), but the microsomal isoform
CYP2E1 (1/3) also plays a significant role in ethanol
metabolism and tolerance.
Oxidation of Alcohols
Alcohol dehydrogenase
• In the presence of NADH or NADPH, alcohol dehydrogenase
functions as a reductase where it catalyzes the reduction of
an aldehyde or ketone to an alcohol
• 4-Methylpyrazole (Fomepizole®) is an alcohol dehydrogenase
inhibitor that is used as an antidote for the treatment of
methanol or ethylene glycol poisoning.
Oxidative Deamination of Amines
• Monoamine oxidase (MAO) catalyze oxidative deamination of
amines to the aldehydes in the presence of oxygen.
• According to the following equation:
• Two types of MAO are isolated: MAO-A, and MAO-B.
• MAO-A is found mainly in peripheral adrenergic nerve
terminals and selectively and reversibly inhibited by
Minaprine
• MAO-B is found principally in platelets and selectively and
irreversibly inhibited by pargyline and selegiline
Oxidative Deamination of Amines
MAO
• Substrates for MOA include
• Several monoamines, mostly primary amine
• Secondary and tertiary amines in which the amine
substrates are methyl groups.
• The amine must be attached to an unsubstituted
methylene group, and compounds having substitution at
the α-carbon atom are poor substrates for MAO
Drug Conjugation Pathway
Phase 2
• Conjugation reactions have added an ionic hydrophilic moiety,
such as glucuronic acid, sulfate, or glycine, to the xenobiotic
to increase water solubility to make urinary elimination
possible
• Moreover , these terminal metabolites would have no
significant pharmacological activity (i .e., poor cellular
diffusion and affinity for the active drug's receptor), except
morphine 6-glucuronide has more analgesic activity than
morphine in humans and that minoxidil sulfate is the active
metabolite for the antihypertensive minoxidil
Glucuronic Acid Conjugation
• Glucuronide formation probably is the major and most
common route for xenobiotic Phase 2 metabolism
• Mechanism of Glucuronide Conjugation
• The reaction involves the direct condensation of the
xenobiotic (or its Phase 1 product ) with the activated form of
glucuronic acid, UDP–glucuronic acid (UDPGA). The reaction
between UDPGA and the acceptor compound is catalyzed by
UDP–glucuronosyl transferases (UGT)
Glucuronic Acid Conjugation
• Not all glucuronides are excreted by the kidneys. Some are
excreted into the intestinal tract with bile (enterohepatic
cycling) , where β-glucuronidase in the intestinal flora
hydrolyzes the C1-O-glucuronide back to the aglycone
(xenobiotic or their metabolites) for reabsorption into the
portal circulation.
Sulfate Conjugation
• Conjugation of xenobiotics with sulfate occurs primarily with
phenols and, occasionally, with alcohols, aromatic amines,
and N-hydroxy compounds.
• The sulfate conjugation process involves activation of
inorganic sulfate to the coenzyme 3-phosphoadenosine-5phosphosulfate (PAPS).
Sulfate Conjugation
• In adults, the major urinary metabolite of the analgesic
acetaminophen is the O-glucuronide conjugate, with the
concomitant O-sulfate conjugate being formed in small amounts.
Interestingly, infants and young children (ages 3–9 years) exhibit a
different urinary excretion pattern: the O-sulfate conjugate is the
main urinary product. The explanation for this reversal stems from
the fact that neonates and young children have a decreased
glucuronidating capacity because of undeveloped
glucuronyltransferases
Conjugation with Glycine, Glutamine,
and Other Amino Acids
• The amino acids glycine and glutamine are used by
mammalian systems to conjugate carboxylic acids, particularly
aromatic acids and arylalkyl acids.
• The quantity of amino acid conjugates formed from
xenobiotics is minute because of the limited availability of
amino acids in the body and competition with glucuronidation
for carboxylic acid substrates.
GSH or Mercapturic Acid Conjugates
• Glutathione (GSH) protects vital cellular constituents against
chemically reactive species by virtue of its nucleophilic SH
group.
• The SH group reacts with electron-deficient compounds to
form S-substituted GSH adducts
Acetylation
• Acetylation constitutes an important metabolic route for
drugs containing primary amino groups
• This encompasses primary aromatic amines (ArNH2),
sulfonamides (H2NC6H4SO2NHR), hydrazines (—NHNH2),
hydrazides (—CONHNH2), and primary aliphatic amines.
• Because water solubility is not enhanced greatly by Nacetylation, it appears that the primary function of acetylation
is to terminate pharmacological activity and detoxification.
• The acetyl group used in N-acetylation of xenobiotics is
supplied by acetyl-CoA and transferred by Nacetyltransferases
• Acetylation polymorphism
Methylation
• Methylation generally does not lead to polar or water-soluble
metabolites, except when it creates a quaternary ammonium
derivative.
• Methyltransferases of particular importance in the
metabolism of foreign compounds include catechol-Omethyltransferase (COMT), phenol-O-methyltransferase, and
nonspecific N-methyltransferases and S-methyltransferases.
• COMT carries out O-methylation of such important
neurotransmitters as norepinephrine and dopamine and thus
terminates their activity.
• Foreign compounds that undergo methylation include
catechols, phenols, amines, and N-heterocyclic and thiol
compounds.