DRUG METABOLISM

• What is Metabolism?

• Drug metabolism specific organs

• Role/purpose of drug biotransformation

• Principles of drug biotransformation

Biotransformation a series of enzyme-catalyzed processes—that

alters the physiochemical properties of foreign chemicals (drug/xenobiotics) from those that favor absorption across biological membranes (lipophilicity) to those favoring elimination in urine or bile (hydrophilicity )

Drug Metabolism (Biotransformation)

• Humans/Animals exposure: throgh air, water and food -xenobiotics, including Drugs

• Metabolism/ Biotransformation / Detoxification . • The process describes attenuation or loss of pharmacological

activity of a drug/xenobiotic , due to enzymatically controlled chemical activity or toxicity of the resulting metabolite

• Metabolized in the body mainly to inactive substances• Metabolism makes the compound

less lipid soluble and more polar and thus hydrophilic.

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Significance of drug metabolism

• Drugs are mostly lipid soluble, easy to cross membranes, bind to plasma proteins & reabsorb from renal tubules

• Metabolism changes them to water soluble & easily excretable products; also become inactive or less active

• Sometimes, a drug when taken is inactive (Pro-drugs) and is metabolizing to active compound in the body

• Occasionally, drugs may be changed to toxic compounds• important in therapy in Age Group/ Organ Dysfunction• therapeutic index, adverse drug effects/ toxicity• pregnancy status/ neonates s

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Site/Organs of drug metabolism

The major site of drug metabolism is the liver (microsomal enzyme systems of hepatocytes)

Secondary organs of biotransformation • kidney (proximal tubule) • lungs (type II cells) • testes (Sertoli cells)• skin (epithelial cells); plasma. nervous tissue

(brain); intestines

Factors Affecting Drug Metabolism1. Species differences : eg in phenylbutazone, procaine and

barbiturates.2. Genetic differences – variation exist with species3. Age of animal –feeble in fetus,aged, newborn. 4.sex: under the influence of sex hormones.5. Nutrition: starvation and malnutrition6. Patholigical conditions: Liver/Kidney dysfunction

Xenobiotic • PHASE I Expose or Add Biosynthetic functional groups conjugation

Oxidation Reduction Hydrolysis

• Primary products

• PHASE II : biosynthetic conjugation

• Secondary products

• Elimination/excretions

TYPES OF BIOTRANSFORMATION

Two phases in the metabolism of drugs:-Phase 1 reaction. (Non synthetic phase).• a change in drug molecule.• oxidation, reduction or hydrolysis.• result in activation, change or inactivation of drug.Phase II reaction. (Synthetic phase)• Last step in detoxification reactions and almost always

results in loss of biological activity of a compound. • May be preceded by one or more of phase one reaction • involves formation of conjugates with drug or its

metabolites formed in phase 1reaction. • The conjugate is formed with an endogenous substance

such as carbohydrates and amino acids.

( First 15 ) PRINCIPLES OF XENOBIOTIC BIOTRANSFORMATION

PRINCIPLES OF XENOBIOTIC BIOTRANSFORMATION

POINT : 1 • The process of converting lipophilic (fat

soluble) chemicals, which are readily absorbed from the gastrointestinal tract and other sites, into hydrophilic (water soluble) chemicals, which are readily excreted in urine or bile.

Exception : • Acetylation and methylation: can actually

decrease the water solubility of certain xenobiotics

POINT : 2 The biotransformation of xenobiotics is catalyzed by various

enzyme systems that can be divided into four categories

Based on the reaction they catalyze:1. Hydrolysis (e.g., carboxylesterase)2. Reduction (e.g., carbonyl reductase)3. Oxidation (e.g., cytochrome P450) 4. Conjugation (e.g., UDP-glucuronosyltransferase)

THE MAMMALIAN ENZYMES INVOLVED IN THE HYDROLYSIS, REDUCTION, OXIDATION, AND

CONJUGATION OF XENOBIOTICS

Exceptions to Point 2. • Hydrolysis of certain carboxylic and phosphoric acid esters,

reduction of sulfoxides to sulfides (e.g., rabeprazole), isomerization involving enol–keto tautomerization (e.g., thalidomide)

• Conjugation of certain xenobiotics with glutathione can occur nonenzymatically at an appreciable rate.

• Certain reactions are catalyzed by gastric acid, such as the hydrolysis of esters and the conversion of indole-3-carbinol to a dimer that is a potent agonist of the aryl hydrocarbon receptor (AhR) and consequently, an inducer of various enzymes including ytochrome P450 enzymes (CYP1A1, 1A2, 1B1, and 2S1).

POINT : 3 In general, individual xenobiotic-biotransforming enzymes are

located in a single organelle. • Two sub cellular organelles are quantitatively the most

important: the endoplasmic reticulum and the cytosol. The phase I oxidative enzymes : endoplasmic reticulum. Phase II enzymes: cytosol However, some enzymes• are listed with two or more subcellular locations.• In such cases, the enzyme name generally refers to two or more

enzymes, each with its own distinct subcellular location.

• Eg: Epoxide hydrolase located in microsomes is a different enzyme from the epoxide hydrolase located in cytosol (i.e., they are distinct gene products).

POINT : 4

• In general, xenobiotic Biotransformation: by a limited number of enzymes with broad substrate specificities.

• Eg: CYP2D6 and CYP3A4—metabolize over half the orally effective drugs in current use

• The broad/overlapping substrate specificities of xenobiotic biotransforming enzymes preclude the possibility of naming the individual enzymes after the reactions they catalyze (which is how most other enzymes are named)..

Enzymes naming• based on the primary amino acid sequence of the

individual enzymes. • The same name across all mammalian species or

named in a species-specific manner.• Eg: CYP1A1, CYP1A2, CYP1B1, so named in all

mammalian species • Cytochrome P450 enzymes in CYP2, CYP3, and CYP4

families are named in a species-specific manner. (convention of using italic and normal letters to distinguish between the gene and gene products (mRNA and protein) lower case letters to designate mouse genes and gene products )

HOWEVER• The amino acid sequence may differ among individuals,

which can give rise to differences in rates of drug metabolism.

• A variant form of a xenobiotic-biotransforming enzyme (known as an allelic variant or an allelozyme) has diminished enzymatic activity compared with that of the wild-type enzyme, although this is not always the case .

• The impact of amino acid substitution(s) on the catalytic activity of a xenobiotic biotransforming enzyme may be substrate dependent, such that an allelic variant may interact normally with some substrates (and inhibitors) but interact atypically with others

POINT : 5 • Hydrolysis, reduction, and oxidation expose or

introduce a functional group (such as –OH, –NH2, –SH, or –COOH) • that can be converted to a water-soluble conjugate

reaction enzyme or specific reaction localization• Hydrolysis, reduction, and oxidation: Phase 1

reactions - that resulted in either a decrease or increase in xenobiotic toxicity

• Conjugation reactions : Phase 2 reactions. - that resulted in only a decrease in toxicity

EXCEPTIONS:

• First : xenobiotic undergoes oxidation after it has been conjugated (phase 2 precedes Phase 1 metabolism). Eg: gemfibrozil : onjugated with glucuronic acid before it undergoes oxidation by cytochrome P450 (Ogilvie et al., 2006).

• Second: xenobiotics that are conjugated directly. Eg: acetaminophen is conjugated directly with glucuronic acid and, to a lesser extent, sulfonic acid

Point 5 …contd

• Third : Phase 2 (detoxifying) enzymes : resulting in only detoxication is incorrect.

• Eg: conjugation of carboxylic acid-containing drugs with glucuronic acid to form acyl glucuronides- NSAIDS hepatotoxicity

• Enzymes which do not fit neatly/fit both into the Phase 1 or Phase 2 category. Epoxide hydrolase,Phase 1 enzyme ( it introduces a functional group (-OH) for subsequent conjugation reactions) or Phase 2 enzyme ( catalyzes the addition of water to aliphatic epoxides and arene oxides that are often formed by cytochrome P450.)

POINT : 6

• Not all biotransformation reactions are catalyzed by the mammalian enzymes as listed

Exceptions: • Reactions catalyzed by gut microflora (largely

anaerobic bacteria in the colon) • Kinase and alkaline phosphatase ( anti HIV drug

zidovudine- )not usually considered to be xenobiotic-biotransforming enzymes.- Endobiotic metabolising enzymes

POINT : 7 • xenobiotics : biotransformed by the so-called endobiotic-

metabolizing enzymes (Point 6)• Certain endobiotics : biotransformed by the so-called

xenobiotic-metabolizing enzymes. Eg: • cytochrome P450 enzymes, also contribute to the hepatic

catabolism of steroid hormones,• UDP-glucuronosyl transferases that conjugate xenobiotics

also glucuronidate bilirubin, thyroid hormones, and steroid hormones.

• Benzoic acid, a xenobiotic, is conjugated with glycine , and so are bile acids (endobiotics).

• Certain leukotrienes are glutathione conjugates.

Point 6 and 7• No clear-cut distinction between endobiotic- and

xenobiotic-biotransforming enzymes.• The human genome project : once thought to be

two distinct enzymes, (involved in the metabolism of endobiotics and xenobiotics) are in fact one and the same enzyme.

• For example, the microsomal enzyme known as 11β-hydroxysteroid dehydrogenase is identical to the xenobiotic-metabolizing enzyme known as microsomal carbonyl reductase.

POINT : 8• Several xenobiotic-biotransforming enzymes are inducible,

meaning their expression can be increased (upregulated) usually in response to exposure to high concentrations of xenobiotics.

• Induction is mediated by ligand-activated receptors (socalled xenosensors) that are activated by xenobiotics (ligands) to DNA-binding proteins that upregulate the transcription of various genes encoding xenobiotic-biotransforming enzymes

• Especially cytochrome P450 (CYP) enzymes, which are usually induced to the greatest extent (in terms of fold increase).

The major xenosensors• aryl hydrocarbon receptor (AhR), induces CYP1 enzymes,• constitutive androstane receptor (CAR), which induces CYP2B, 2C,

and 3A• the pregnane X receptor (PXR),induces CYP2B, 2C, and 3A enzymes • peroxisome proliferator activated receptor alpha (PPARα), - CYP4

enzymes. • CAR and PXR are closely related, and they tend to be activated by

the same ligands and bind to the sameDNA-response elements • Certain xenosensors are activated by endogenous ligands (e.g.

bilirubin, bile acids, and fatty acids activate CAR, PXR, and PPARα, respectively)

• Certain nuclear receptors, such as the vitamin D receptor (VDR) can mimic PXR and induce CYP3A4, which inactivates the active metabolite of vitamin D.

• Xenosensors are not just involved in xenobiotic disposition but also play a role in endobiotic homeostasis.

FACTORS• Induction is a reversible, adaptive response to xenobiotic

exposure. • The induced enzymes usually accelerate the elimination of

the xenobiotic that triggered the induction process, in which case the xenobiotic is said to be an auto-inducer

• However, xenobiotics often induce enzymes that are not capable of metabolizing them, in which case the induction is said to be gratuitous.

• Induction is a pleiotropic response: Activation of AhR, CAR, PXR, PPARα, : result in alterations in the expression of numerous genes, some of which are upregulated (or induced) and some of which are downregulated (or suppressed).

POINT : 9

• The ability of enzymes to metabolize hormones and xenobiotics (Point 7) and the ability of certain xenobiotics to induce xenobiotic biotransforming enzymes (Point 8): the mechanism by which certain xenobiotics can alter homeostasis or cause toxicity.

• Persistent exposure to xenobiotics that are enzyme inducers can increase

• the rate of steroid hormone oxidation by cytochrome P450 • increase the rate of thyroid hormone glucuronidation and

sulfonation which, in rodents, can lead to the development of Leydig cell tumors (due to elevated levels of LH and FSH) • thyroid follicular tumors (due to elevated levels of TSH),

respectively (Grasso et al., 1991). • Liver tumors –activation of certain xenosensors

POINT : 10

• Biotransformation can alter the biological properties of a xenobiotic.

• It can make the xenobiotic less toxic (detoxication; Eg: oxidation of acetaldehyde to acetic acid ), but in some cases it can make it more toxic (activation ; eg: oxidation of ethanol to acetaldehyde ).

The biotransformation of drugs can result in (1) a loss of pharmacological activity e.g: acetaminophen to acetaminophen glucuronide morphine to morphine 3-glucuronide (2) no change in pharmacological activity e.g: fluoxetine to its N-demethylated metabolite norfluoxetine) (3) an increase in pharmacological activity codeine to morphine mhorpine to morphine 6-glucuronide

POINT : 11

• In many cases, the toxicity of a xenobiotic is due to the parent compound (the compound that was absorbed), in which case xenobiotic biotransformation serves as a detoxication mechanism.

• Illustrated by the clinical observation that the incidence of adverse drug events is often higher in individuals with a poor metabolizer phenotype

Exceptions: • Enzymes can convert certain xenobiotics to reactive (electrophilic)

metabolites, resulting in • chemical toxicity (covalent binding of electrophilic metabolites to

critical cellular nucleophiles like proteins) • chemical mutagenicity/carcinogenicity ( involves covalent binding to

one or more purine or pyrimidine bases in DNA) • Cytochrome P450 : convertproximate carcinogens to ultimate

carcinogens by converting the former to electrophilic metabolites that bind to and mutate DNA, thereby leading to mutations and tumor initiation

• Egs. of proximate carcinogens that require metabolic activation to form electrophilic, DNA-binding metabolites (ultimate carcino gens)

• Polycyclic aromatic hydrocarbons (combustion pollutants and components of tobacco smoke), aromatic amines (industrial chemicals), aflatoxin,tobacco-specific nitrosamines

POINT : 12 • The toxicity and potential carcinogenicity of electrophilic

metabolites produced by cytochrome P450 and other xenobiotic-biotransforming enzymes is reduced and often altogether eliminated

by their conjugation with glutathione• In the majority of cases, conjugation with glutathione

represents a detoxication reaction; one that protects critical cellular nucleophiles, such as protein and DNA, from covalent modification

Exceptions: • Conjugation with glutathione actually produces a DNA-

reactive (mutagenic) metabolite ( eg: Dibromoethane). • Conjugation with glutathione can occur both enzymatically (by

glutathione Stransferase ) and nonenzymatically.

POINT : 13

• The biotransformation : can result in the production of reactive oxygen species (ROS), which can cause cell toxicity (including DNAdamage) through oxidative stress and lipid peroxidation.

• Glutathione, glutathione transferases, and glutathione peroxidases : all limit the toxic effects of ROS just as they limit the toxicity of reactive metabolites formed directly from xenobiotics

Two proteins : • KEAP-1 (Kelch-like ECHrelated protein)• Nrf2 (nuclear factor E2 p45-related factor-2) • Function as xenosensors - induce enzymes in response to

oxidative stress,(often associated with xenobiotic biotransformation) and subsequent formation of electrophilic metabolites that generate reactive oxygen species (ROS), reduce

• glutathione transferase , microsomal epoxide hydrolase, aldo-keto reductase , NAD(P)H-quinone oxidoreductase and glutamate-cysteine ligase (GCL), which catalyzes the rate-limiting step in glutathione synthesis

• All contributing to reduction in glutathione levels (Lee and Johnson, 2004).

POINT : 14 • The enzymes involved in xenobiotic activation and detoxication

play important roles in determining the susceptibility of mammals to the chemical toxicity, and is often the basis for organ or species differences in toxicity.

eg: Aflatoxin cyp450 aflatoxin epoxide (hepatotoxic and hepatocarcinogenic) Reaction rate Detoxification susceptibility Mice FAST FAST LESSRats SLOWER SLOWER MORE

Coumarin: More Hepatotoxic to rats (reactive epoxide that rearranges to a

reactive aldehyde ) than to humans (nontoxic metabolite 7-hydroxycoumarin )

• The additional factors can also play a role in determining organ and species differences in xenobiotic toxicity.

Eg: increased susceptibility of mice to acetaminophen hepatotoxicity

• Deletion of glutathione transferase-Pi, Jun Kinase-2 DNAse 1, interferon-γ (IFN-γ ), lipopolysaccharide-binding protein (LPSBP), Toll-like receptor-4 (TRL4), neutrophil and NK/NKT cells, osteopontin,( in knock out mice study) – enhance susceptibility

• Conversely: other gene deletions : Nrf2, interleukin-6 (IL-6), interleukin-10 (IL-10), cyclooxygenase-2 (Cox-2), chemokine receptor-2 (CCR2), chemokine receptor-5 (CCR5), Kupffer cells- protect mice from the hepatotoxic effects of acetaminophen

POINT : 15

First Pass Effect/ Elimination ( presystemic metabolism/elimination)

• Exposure to xenobiotics (especially drugs) is largely through oral ingestion, and the small intestine and liver are highly developed to limit systemic exposure to orally ingested xenobiotics

• The enterocytes at the tips of the small intestinal villi express the efflux transporter P-glycoprotein (also known as MDR1 or ABCB1), which serves to limit xenobiotic absorption.

Point 15….contd

Intestines• The enterocytes: high levels of certain cytochrome P450 and

UDP-glucuronosyltransferase enzymes Liver• Uptake transporters- remove xenobiotics from the blood.• Efflux transporters - discharge xenobiotics or their

metabolites (especially conjugates) into the bile canaliculus for biliary excretion, or that actively discharge xenobiotic metabolites (especially conjugates) across the sinusoidal membrane back into the blood for urinary excretion.

• Largest number and, with few exceptions, the highest concentrations of xenobiotic-biotransforming enzymes.

• The number of hepatocytes in the liver > enterocytes in the small intestine, assumption: the small intestine would make only a small contribution to first-pass metabolism, IS NOT CORRECT

Point 15…contd Examples: • Furanocoumarins in grapefruit juice inhibit intestinal but not

hepatic CYP3A4 and yet grapefruit juice increases systemic exposure to felodipine and other drugs that undergo first-pass metabolism by CYP3A4

• The impact of ketoconazole on the disposition of the CYP3A4 substrate midazolam depends on whether midazolam is given orally or intravenously.

Midazolam• IV: (clearance is dependent only on hepatic metabolism)

ketoconazole (the CYP3A4 inhibitor) causes a three- to fivefold increase in AUC

• Oral: (clearance is dependent on hepatic and intestinal metabolism) ketoconazole causes a three- to fivefold increase in AUC ketoconazole causes a 10-to 15-fold increase in AUC, the difference reflecting the significant role of intestinal metabolism to the presystemic clearance

Liver and Small Intestine • Possess high levels of the enzymes.• Possess protective mechanisms (enzymes and transporters)

to minimize the risk of toxicity and carcinogenicity.

Liver : • High levels of glutathione (5–10 mM)• A large proportion of diploid (binucleated) cells• high regenerative capacity• Severely damaged hepatocytes can undergo apoptosis (cell-programmed

death) to eliminate precancerous cells (i.e., cells with extensive DNA damage).

SI: the enterocytes at the villus tips –turnover,; the mature cells that are exposed to high levels of xenobiotics and/or reactive metabolites are quickly lost (exfoliated) and replaced in a matter of days

• Several biotransforming enzymes and transporters are inducible: can enhance the rate of xenobiotic biotransformation and elimination.

PIONT 16- Exposure of Xenobiotics

• Mechanism protect the Brain and reproductive organs

Brain

• Blood Brain barrier- tight junction

• Biochemical barrier- efflux of Transporter p- glycoprotein (MDR1 or ABCB1)

Reproductive organs

• Testis (Germ cells) Blood- testis Barrier Glutathione ( Glutathine transferases & Glutathione

peroxidases) High cell turnover

OVUM No cell turn Over High level Of glutathione (> 8mM) Protects from the DNA reactive electrophiles & Oxidative

stress- fertilization

TUMOR CELLS

• Chromosomal rearrangements over-expression of a transporter and/or glutathione transferase can confer resistance to certain cancer chemotherapeutic agents (reactive metabolites - damage DNA and kill tumor cells).

Point 17- Enzyme Induction/ Activation• Cytochrome P450 - Metabolic activation of proximate carcinogens -

ultimate carcinogens, • Defense mechanisms• Activation by Cytochrome P450 - Carcinogenic effects• Induction of Cytochrome P450 - increase in the toxicity of certain

xenobiotics. • Treatment of rodents with a Cytochrome P450 inducer prior to

treatment with a known proximate carcinogen (such as aflatoxin, various nitrosamines, or polycyclic aromatic hydrocarbons) is generally associated with a decrease, not an increase, in tumor incidence (Parkinson and Hurwitz, 1991).

• The route of exposure - the impact of enzyme induction;• Oral route : increase the tumorigenicity of carcinogens applied

directly to their site of action (Nebert et al., 2004).• Rodent - Prolonged treatment enzyme inducers - tumor promoters .• Enzyme induction - protection against chemical carcinogenesis.

POINT 18 – LOCATION OF ENZYMES

• Concentration

• The amount of enzyme on a per-mg-protein basis.

• Small intestine and liver

• Nasal epithelium - Volatile xenobiotics.

• Lung - Inhalation,

• Eye - Eye drop

• Skin - Topical application.

• Kidney - metabolizes glutathione conjugates formed in the liver.

POINT 19 - SPECIES DIFFERENCES

• Qualitative and quantitative aspects of xenobiotic biotransformation and Toxicity.

• Coumarin metabolism and toxicity (activation by epoxidation in rats, detoxication by aromatic hydroxylation in humans).

• Glutathione transferase - rats and mice- aflatoxin-induced liver toxicity.

• Foetal expression - - Human fetal liver expresses CYP3A7- aflatoxin - reactive metabolites- Human teratogenicity whereas no such enzyme is expressed in the fetus of rodents (Li et al., 1997).

• Furafylline - Inhibitors• Omeprazole – Inducers • Human CYP1A2, • Do not inhibit or induce CYP1A2 in rodents. • Humanized mice- substituting the murine genes that

encode xenobiotic-metabolizing enzymes with their human counterparts (Gonzalez, 2003; Gonzalez and Yu, 2006).

Xenobiotic Biotransformation - low or absent.• Cats - Glucuronidation • Dogs – Acetylation Metabolism by Gut microflora • Quinic acid Benzoic acid • Humans and old-world monkeys but not in new-world

monkeys, mice, rats, and most other laboratory animals (Adamson et al., 1970).

POINT 20 – GENDER DIFFERENCES

• Women appear to have lower CYP1A2 and higher CYP3A4 activity than men (reviewed in Parkinson et al., 2004).

• Women - a higher incidence of idiosyncratic drug toxicity (Walgren et al.,2005).

• Drug withdrawals and black box warnings

POINT 21- IDIOSYNCRATIC REACTION

• Rare adverse events (<0.1%) - an exaggerated pharmacological response,

• do not occur immediately after exposure - after weeks or months of repeated administration (Walgren et al., 2005).• Type B reactions, • Allergic and nonallergic IDRs. • Allergic - develop relatively quickly (in days or weeks) and, after the

drug is discontinued, patients respond robustly when rechallenged with the same or a closely related drug.

• Nonallergic - develop relatively slowly (6 months or more of drug treatment) and patients may or may not respond to rechallenge with the drug.

• Hepatotoxicity - a reactive metabolite

• Rosiglitazone and Pioglitazone –

• Troglitazone - idiosyncratic hepatotoxicity.

• Uetrecht (2001) - drugs administered at a daily dose of 10 mg or less do not cause idiosyncratic toxicity

Closely related drugs- One does and one does not cause hepatotoxicity include

• Tolcapone/Entacapone,

• Ticlopidine/Clopidogrel,

• Amineptine/Tianeptine,

• Tamoxifen/Toremifene,

• Ibufenac/Ibuprofen,

• Ebrotidine/Famotidine,

• Niperotidine/Ranitidine

• Labetrol /dilevalolIn

POINT 22 – INDIVIDUAL DIFFERENCES

• The same dose of drug administered to humans - large interindividual differences in exposure to the parent compound

• Plasma concentration–time curve or AUC

• Genetically determined differences : Xenobiotic-biotransforming enzymes Transporters (Genetic polymorphisms) Environmental differences Drug– Drug interactions.

POINT 23 – PHARMACOGENETICS/ GENETIC POLYMORPHISM

• Prevalence, and impact of heritable differences in xenobiotic-biotransforming ezymes

• Dosage of drugs with a narrow therapeutic index (such as anticancer drugs) is adjusted in accordance with an individual’s genotype (i.e., drug-metabolizing potential).

• Genetic variation in a xenobiotic-metabolizing enzyme produces Four discernable phenotypes:

Poor metabolizers (PMs), Intermediate metabolizers (IMs), Extensive metabolizers (EMs) Ultrarapid metabolizers (UMs),

PMs - no functional alleles (−/−) for, IMs - one nonfunctional allele and one partially

functional allele or two partially active alleles (−/* or */*) EMs - one or two functional alleles (+/−, +/*, or +/+) UMs - Gene duplication ([+/+]n).

• An activity score

• Functional activity value from 1 (for the wild type or *1 allele) to zero (for any completely nonfunctional allele) (Zineh et al., 2004).

• PM- CYP2D6 genotype is common among Caucasians but not Asians (CYP2C19).

• Clearance of a drug is largely determined by a polymorphically expressed enzyme, - CYP2D6, exposure follows the rank order:

• Poor metabolizers (PMs) > Intermediate metabolizers (IMs) > extensive metabolizers (EMs) > Ultrarapid metabolizers (UMs).

PM phenotype - requires dosage adjustment to prevent drug toxicity or an exaggerated pharmacological response.

• EX: PMs - CYP2D6 are at increased risk for Perhexiline hepatotoxicity and at increased risk for an exaggerated pharmacological response to Debrisoquine and Sparteine,

Drugs are converted to active metabolites by CYP2D6, then PMs derive inadequate therapeutic effect.

• EX: Codeine - Morphine (Analegic)

Tamoxifen - Endoxifen (30- to 100X) Genetic polymorphisms that affect drug disposition (Robert et

al., 2005; Relling and Giacomini, 2006)

• CYP2C9 (Warfarin),

• CYP2C19 (Omeprazole),

• CYP3A4 (Clopidogrel, irinotecan)

• UGT1A1 (Irinotecan),

• COMT (Levodopa),

• DPD (5- fluorouracil),

• Efflux transporter P-glycoprotein (Digoxin)

Environmental factors.• Ex. Alcohol dehydrogenase and Aldehdye dehydrogenase

impact the incidence of alcoholism (Li, 2000).

• Genetic polymorphisms - CYP2A6 impact the incidence of cigarette-smoking-induced lung cancer.

• PMs - Individuals lacking CYP2A6 are of nicotine and are poor activators of tobacco-specific mutagens.

Disease.

• For example, the severe and mild hyperbilirubinemia

associated with Crigler–Najjar syndrome and Gilbert’s syndrome respectively,

• Complete and partial loss of the UDP- glucuronosyl

transferase responsible for conjugating bilirubin, namely, UGT1A1.

• Genetic polymorphisms have been described in laboratory animals.

• Ex: Laboratory-bred rabbits have about 50:50 chance of being a poor or rapid acetylator (isoniazid)

• N-acetyltransferase 2 (NAT2) is polymorphically expressed in rabbits.

POINT 24 – ENVIRONMENTAL FACTORS

• Drug– drug interactions. • Ex : PM - CYP3A4 inhibitors (ketoconazole and

Erythromycin), • Ums - Inducers (Rifampin and the Herbal agent St. John’s

wort). • victim and perpetrator perspective.• Victim drug : A drug whose clearance is largely determined by

a single route of elimination, such as metabolism by a single CYP enzyme. (also known as a sensitive or object drug).

• Victim drugs have a high therapeutic index, hence, they are therapeutically effective in PMs to UMs and cause roughly the same incidence of adverse effects regardless of genotype.

• Victim drugs with a narrow therapeutic index, dosage must be increased in UMs to achieve a therapeutic effect and/or it must be decreased in PMs to prevent adverse drug events.

VICTIM DRUGS

• Terfenadine (Seldane),• Cisapride (Propulsid),• Astemizole (Hismanal),• Cerivastatin (Baycol)• Metabolized by CYP3A4. Ketoconazole, and Erythromycin, • Decrease the clearance of terfenadine, cisapride, and astemizole

and increase their plasma concentrations .• Ventricular arrhythmias (QT prolongation) - fatal heart attacks.

Cerivastatin is extensively metabolized by CYP2C8. Its metabolism is inhibited by Gemfibrozil (actually by gemfibrozil glucuronide),

• Fatal, cerivastatin-induced rhabdomyolysis (Ogilvie et al., 2006).

PERPETRATORS

• Inhibit or induce the enzyme that is otherwise responsible for clearing a victim drug.

• perpetrators are drugs that cause drug–drug interactions.

• Precipitants.

• Partial or complete loss of enzyme cause a decrease in the clearance of and an increase in exposure to victim drugs.

• Ex : Mibefradil (Posicor) – Calcium channel -inhibition of

CYP3A4 – irreversible

Herbal remedies + Drugs

• St. John’s wort (Hyperacium perperatum) contains hyperforin, an inducer of CYP3A4 and P-glycoprotein (MDR1 or ABCB1).

• St. John’s wort is not recommended for patients on antirejection drugs (such as Cyclosporine and Tacrolimus), anti-HIV drugs (such as Indinavir and Nevirapine), anticoagulants (such as warfarin and phenprocoumon), or oral contraceptive steroids (Pal and Mitra, 2006).

• Large quantities of Cruciferous vegetables (e.g., Boccoli and Brussels sprouts) can induce hepatic CYP1A2 (Conney, 1982),

• Grapefruit juice can inhibit intestinal CYP3A4 (Paine et al., 2006).

Enzyme mapping

• Is the process of identifying which enzyme are largely responsible for metabolizing a drug candidate (Williams et al., 2003b; Ogilvie et al., 2007).

• Allows an assessment of the victim potential of a drug candidate or other xenobiotic.

POINT 25 DRUG – DRUG INTERACTION

• Increase in the incidence of adverse events or, in the case of induction a loss of therapeutic efficacy.

• Ex: Anti-HIV drugs, like Ritonavir, inhibit CYP3A4 and thereby improve the pharmacokinetic profile of other anti-HIV drugs, like Saquinavir and Lopinavir.

• Tumors often over-express various transporters and glutathione transferases, which limit the effectiveness of several anticancer drugs.

• Drugs that inhibit transporters and glutathione transferase are being developed to enhance the efficacy of anticancer drugs.

POINT 26

• Although the amount of functional enzyme determines whether an individual is a poor or rapid metabolizer.

• Liver disease

• UDP-glucuronic acid (UDPGA) or 3_-phosphoadenosine-5_-phosphosulfate (PAPS) to saturate the enzymes responsible for glucuronidating or sulfonating xenobiotics.

• Fasting lowers hepatic levels of these cofactors, and fasting decreases the extent to which acetaminophen is conjugated.

• Ex : Acetaminophen - fasting – Hepatotoxicity

POINT – 27 STERIOCHEMICAL ASPECTS

• Interaction between a Xenobiotic and its biotransforming enzyme• Xenobiotic biotransforming enzymes can play a key role in

converting one stereoisomer to another, - mutarotation or inversion of configuration.

Xenobiotic - two forms called stereoisomers or enantiomers. • one enantiomer (stereoisomer) is biotransformed faster than its antipode. Ex: the antiepileptic drug Mesantoin®,• which is a racemic mixture of R- and S-mephenytoin, • S-enantiomer is rapidly hydroxylated (by CYP2C19) and eliminated

faster than the R-enantiomer. Xenobiotics- inhibit the biotransforming enzymes Quinidine is a potent inhibitor of CYP2D6, where as Quinine, its

antipode, has relatively little inhibitory effect on this enzyme.

• Inversion of configuration is the process whereby one enantiomer is converted to its antipode via an achiral intermediate.

• Nonenzymatically,

Thalidomide, { R- Sedative , S- Teratogen – Phocomelia congenital defects & angiogenesis(Vasculogenesis)}

Lisofylline (Ketone )

Simvastatin

Paraoxonases

POINT – 28 MASS SPECTROMETRY

Characterize the structure of metabolites Xenobiotic reactions are associated with discrete changes in

mass.• loss of 2 atomic mass units (amu) signifies dehydrogenation,• loss of 14 amu usually signifies demethylation (–CH2). Increases the mass units• Reduction (+2 amu = 2H),• Methylation (+14 amu = CH2)• Oxidation (+16 amu = O), • Hydration (+18 amu = H2O), • Acetylation (+42 amu = C2H2O),

• Glucosylation (+162 = C6H10O5),

• Sulfonation (+80 amu=SO3),

• Glucuronidation (+176 amu=C6H8O6),

• Conjugation with glutathione (+305 amu = C10H15N3O6S),

• Glycine (+74 amu = C2H4NO2),

• Taurine (+42 amu = C2H6NO3S). Routine changes in mass can arise from unexpected reactions. Ex: Ziprasidone

• On which region of a molecule has undergone biotransformation,

• Closely related possibilities- Phenyl group (Hydroxylation)

• Nuclear magnetic resonance (NMR) is required to ascertain whether the hydroxylation occurred at the ortho, meta, or para position.

POINT 29- Distribution of Xenobiotic enzymes

• Widely distributed throughout the body.

• The advent of high-throughput assays based on quantitative real-time reverse-transcription polymerase chain reaction (QPCR) -measurements of the levels of mRNA - encoding numerous xenobiotic-biotransforming enzymes, transporters, and xenosensors in a wide range of human tissues (Nishimura et al., 2003, 2004; Furukawa et al., 2004).

• Ex: • Based on mRNA levels in human liver, the levels of the top-

ten CYP enzymes follow the rank order: CYP2E1 _ 2A6 > 2C8 > 2C18 ≈ 1A2 ≈ 4A11 ≈ 8B1 ≈ 2C9 ≈ 3A4 > CYP51A1 (Nishimura et al., 2003).

• This contrasts substantially from the levels of CYP protein, which follow the rank order: CYP3A4 > 2C9 ≈ 3A5 ≈ 2E1 > 1A2 > 2A6 > 2C8 > 2C19 > 2B6 > 2D6 (Rowland- Yeo et al., 2004; Howgate et al., 2006).

• In the case of CYP2E1, hepatic mRNA levels are more than 17 times higher than the levels of CYP3A4 mRNA, but CYP2E1 protein levels are less than half those of CYP3A4.

• most of the mRNA encoding CYP2E1 is sequestered in

the cytoplasm and is not available for translation (Gonzalez, 2007).

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