FACTORS AFFECTING METABOLISM

• 1. Genetic factors: Individual differences in drug effectiveness (drug sensitivity or drug resistance), drug–drug interactions, and drug toxicity can depend on racial and ethnic characteristics with the population frequencies of the many polymorphic genes and the expression of the metabolizing enzymes.

• 2. Physiologic factors: Age, hormonal level, gender, pregnancy, disease state, nutritutional status, etc...

• 3. Pharmacodynamic factors: Dose, frequency, and route of administration, plus tissue distribution and protein binding.

• 4. Environmental factors: Competition of ingested environmental substances with other drugs and xenobiotics for metabolizing enzymes, and poisoning of enzymes by toxic chemicals such as carbon monoxide or pesticide synergists alter metabolism. Induction of enzyme expression (in which the number of enzyme molecules is increased, while the activity is constant) by other drugs and xenobiotics is another consideration.

Human Hepatic Cytochrome P450 Enzyme System

• Oxidation is probably the most common reaction in

xenobiotic metabolism. This reaction is catalyzed by a

group of membrane-bound monooxygenases found in

the smooth ER of the liver and other extrahepatic

tissues, termed the “cytochrome P450 onooxygenase

enzyme system”.

• The most important function of P450 is to “activate”

molecular oxygen (dioxygen), permitting the

incorporation of one atom of oxygen into an organic

substrate molecule concomitant with the reduction of

the other atom of oxygen to water.

Ferric heme thiolate catalytic center of P450. The porphyrin side chains are deleted for clarity.

Central to the functioning of this unique superfamily of heme proteins is an iron protoporphyrin. The iron protoporphyrin is coordinated to the sulfur of cysteine and has the ability to form a complex with carbon monoxide, resulting in a complex that has its primary absorption maximum at 450 nm .

Total human P450 isoforms expressed in the liver that metabolize drugs.

Cyclic mechanism for P450. The substrate is RH, and the valence state of the heme iron in P450 is indicated.

Oxidations (Phase I) Catalyzed by Cytochrome P450 Isoforms: ALIPHATIC AND ALICYCLIC HYDROXYLATIONS

Privileged Hydroxylation Positions: Effect of alpha-Activating Factors

Oxidations (Phase I) Catalyzed by Cytochrome P450 Isoforms: ALKENE AND ALKYNE HYDROXYLATION

Oxidations (Phase I) Catalyzed by Cytochrome P450 Isoforms: AROMATIC HYDROXYLATION

Oxidations (Phase I) Catalyzed by Cytochrome P450 Isoforms: N-dealkylation

Oxidations (Phase I) Catalyzed by Cytochrome P450 Isoforms: Oxidative Deamination

Oxidations (Phase I) Catalyzed by Cytochrome P450 Isoforms: N-oxidation

Oxidations (Phase I) Catalyzed by Cytochrome P450 Isoforms: O- AND S-DEALKYLATION

Oxidations (Phase I) Catalyzed by Cytochrome P450 Isoforms: DEHALOGENATION

Reductions (Phase I) Catalyzed by Cytochrome P450 Isoforms: AZO AND NITRO REDUCTION

Oxidations Catalyzed by Flavin Monooxygenase (FMO)

• Flavin-containing monooxygenase (FMO) oxidizes drugs, xenobiotics, and environmental chemicals containing a “soft nucleophile,” usually nitrogen or sulfur. Unlike P450, FMO does not catalyze epoxidation reactions or hydroxylation at unactivated carbon atoms of xenobiotics.

• FMO does not require a reductase to transfer electrons from NADPH. The catalytic cycles of FMO and P450 are very different.

• Another distinction is the lack of induction of FMOs by xenobiotics.

• In general, P450 is the major contributor to oxidative xenobiotic metabolism. However, FMO activity may be of significance in a number of cases.

N-Oxidations Catalyzed by FMO

Example: Amitriptyline N-Oxidation catalyzed by FMO

FMO

S-Oxidations Catalyzed by FMO

Example: Cimetidine S-Oxidation (to sulfoxide metabolite) catalyzed by FMO

FMO

Example: Omeprazole S-Oxidation (to sulfone metabolite) catalyzed by FMO

FMO

Oxidation of Alcohols Alcohol dehydrogenases and Aldehyde Dehydrogenase

Monoamine Oxidases (MAOs) • MAO is a mitochondrial membrane flavin-containing enzyme that

catalyzes the oxidative deamination of monoamines where oxygen

is used to remove an amine group from a monoamine substrate,

resulting in the formation of the corresponding aldehyde and

ammonia according to the following equation:

Substrates for this enzyme include monoamines and 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. Because of the vital role that MAOs have in the inactivation of neurotransmitters, MAO dysfunction (too much or too little MAO activity) is thought to be responsible for a number of neurologic disorders.

Dopaminergic system and MAO (topic of P. Chem 2)

Hydrolytic enzymes: Hydrolysis

• In general, esters and amides are hydrolyzed by enzymes in the blood, liver

microsomes, intestine, kidneys, and other tissues. Esters and certain amides

are rapidly hydrolyzed by a group of enzymes termed “carboxylesterases.” The

more lipophilic the amide, the more favorable it is as a substrate for this

enzyme. In most cases, the hydrolysis of an ester or amide bond in a toxic

substance results in bioinactivation to hydrophilic metabolites that are readily

excreted. Some of these metabolites can yield conjugated metabolites (i.e.,

glucuronides). Amides are very common in food as proteins and peptides we

eat. Not surprisingly, there are a large number of proteolytic enzymes in the

gastrointestinal tract called amino endopeptidases and amino exopeptidases

that hydrolyze ingested proteins into amino acids.

• Carboxylesterases include cholinesterase (pseudocholinesterase), aryl-

carboxyesterases, liver microsomal carboxylesterases, and other unclassified

liver carboxylesterases.

Examples of hydrolysis reactions

Review on PHASE 1

DRUG CONJUGATION PATHWAYS (PHASE 2)

• Xenobiotics are as a rule lipophilic, well absorbed from the blood,

but excreted slowly in the urine. Only after conjugation (Phase 2)

reactions have added an ionic hydrophilic moiety, such as glucuronic

acid, sulfate ester, or glycine, to the xenobiotic is water solubility

increased and lipid solubility decreased enough to make urinary

elimination possible. The major proportion of the administered drug

dose is excreted as conjugates into the urine and bile. Conjugation

reactions can be preceded by Phase 1 reactions. For xenobiotics

with a functional group available for conjugation, conjugation can be

its fate.

• The major conjugation reactions (glucuronidation and sulfonation)

were traditionally thought to terminate pharmacologic activity by

transforming the parent drug or Phase 1 metabolites into readily

excreted ionic polar products

Glucuronidation pathway catalyzed by UDP-glucuronosyl transferases (UGTs)

Sulfonation pathways: Sulfotransferases

SULT

Conjugation with Amino Acids

Glutathione Conjugation

Methylation

• Methylation is a common biochemical reaction but appears to be of greater significance in the metabolism of endogenous compounds than for drugs and other xenobiotics. Methylation differs from other conjugation processes in that the O-methyl metabolites formed can, in some cases, have as great or greater pharmacologic activity and lipophilicity than the parent molecule.

• O-Methylation is catalyzed by the magnesium-dependent enzyme catechol-O-methyltransferase (COMT) transferring a methyl group primarily to the m- or 3-hydroxy group of the catechol moiety (3,4-dihydroxylphenyl moiety) of norepinephrine, epinephrine, or Dopamine.

• N-Methylation of various amines is among several conjugate pathways for metabolizing amines. Specific N-methyltransferases catalyze the transfer of active methyl groups from S-adenosylmethionine to the acceptor substance.

Example: Methylation pathways.

Miscellaneous examples

Homework

• For the compounds given below draw the chemical structures of possible phase 1 and phase 2 metabolites.

• Tamoxifen, chlorzoxazone, enalaprile

Reactive Metabolites Resulting from Bioactivation Electrophiles

• The concept that small organic molecules can undergo bioactivation to electrophiles and free radicals and elicit toxicity by chemical modification of cellular macromolecules has its basis in chemical carcinogenicity.

• These reactive metabolites are short-lived, with half-lives of usually less than 1 minute.

• Electrophiles are reactive because they possess electrondeficient centers (polarization-activated double bonds or positive-charge acylators) and can form covalent bonds with electron-rich biologic nucleophiles. They are either soft electrophiles that react directly with soft nucleophiles (:Nuc), such as the thiol groups in either glutathione or cysteine residues within proteins, or hard electrophiles that react with hard nucleophiles, such as basic groups in DNA and lysine ω-amino residues in proteins

Some examples of electrophilic intermediates resulting from bioactivation.

Some examples of electrophilic intermediates resulting from bioactivation.

Some examples of drug bioactivation to their hepatotoxic intermediates.

Some examples of drug bioactivation to their hepatotoxic intermediates.

The effect of structure modification on the drug-induced hepatotoxicity of troglitazone.

The bioactivation of procarcinogens and a proposed mechanism of chemical carcinogenesis.