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What is Metabolism?
Drug metabolism specific organs Role/purpose of drug biotransformation Principles of drug biotransformation
Biotransformation a series of enzyme-catalyzed processesthat 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 )
Factors Affecting Drug Metabolism 1. Species differences : eg in phenylbutazone, procaine and barbiturates. 2. Genetic differences variation exist with species 3. Age of animal feeble in fetus,aged, newborn. 4.sex: under the influence of sex hormones. 5. Nutrition: starvation and malnutrition 6. Patholigical conditions: Liver/Kidney dysfunction
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.
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 enolketo 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 CYP3A4metabolize 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 glucuronidesNSAIDS 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 endobioticmetabolizing 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 11hydroxysteroid 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 P450 (CYP) enzymes, which are greatest extent (in terms of fold
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 (PPAR), - 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 PPAR, 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, PPAR, : 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 ratelimiting 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 LESS Rats SLOWER SLOWER MORE
Coumarin: More Hepatotoxic to rats (reactive epoxide that rearranges to a reactive aldehyde ) than to humans (nontoxic metabolite 7hydroxycoumarin )
The additional factors can also play a role in determining organ and species differences in xenobiotic toxicity. Eg: increased susceptibility hepatotoxicity of mice to acetaminophen
Deletion of glutathione transferase-Pi, Jun Kinase-2 DNAse 1, interferon- (IFN- ), 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 cellsprotect 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 15contd
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
Several biotransforming enzymes and transporters are inducible: can enhance the rate of xenobiotic biotransformation and elimination.
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).
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).
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
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 CriglerNajjar syndrome and Gilberts 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.
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 drugdrug 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
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 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_-phosphoadenosine5_-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
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
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
of
molecule
has
undergone
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.
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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