7- Drug Metabolism/Detoxification Flashcards Preview

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Flashcards in 7- Drug Metabolism/Detoxification Deck (41)
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therapeutic dose

ideal concentration of drug in blood that gives desired clinical effect

too high- toxic
too low- no desired clinical effect

*this depends on RATE of specific drug metabolism


Overview of Detoxification

meant to rid our body of drugs and xenobiotics

Phase I
-enzymes: cytochrome P450s

Phase II
-enzymes: transferases
-adds molecule to drug to inactivate it and make it more soluble


Phase I metabolism


-these enzymes are adding a polar group (hydroxyl or amino or sulfhydryl)

-enzymes exhibit low specificity (overlapping substrate specificities) and low catalytic rates



-exposed to ~1 million in a lifetime

-hydrophobic/nonpolar/lipophilic/small molecules that easily cross membranes of skin and gut.

-Once absorbed, usually poorly excreted due to protein binding, renal tubular reabsorption, and accumulation in lipid-soluble tissues.

-Accumulation in body may pose serious health problems

"foreign to life"
--chemicals that are not utilized by cells for generation of energy for catalysis or for structural features of the cell
--animals have evolved systems designed for the limitation of xenobiotics--bacteria utilize xenobiotics for energy


Phase II metabolism

-enzymes are performing conjugation reactions

-modifies what cyt P450 put on it

-increase hydrophilicity of molecule so they can be excreted via bile or urine

-enzymes exhibit low specificity (overlapping substrate specificities) and low catalytic rates


Phase I Enzymes

Cytochrome P450 (CYP)

-Contain the prosthetic group heme (Heme is ESSENTIAL for activity of P450s)
---heme is bound to a cystine residue in the active site and the 6th and final attachment to the heme group is either H2O, CO, NO

-integral membrane proteins

-Absorbs light maximally at 450nm when complexed to CO

-Electron transferring protein

RH + O2 + NADPH + H+ -----> ROH + H2O + NADP+

-Super gene family (CYP)

-P450 acts as an oxygenase rather than simply an e- carrier

-a variety of reactions are carried out by the family of P450s, focus on hydroxylation reactions

-The mass of substrates recognized by P450’s ranges from that of ethylene (MW = 28 Da) to that of cyclosporin A (MW = 1201 Da)



-CYP’s are a family of heme-containing proteins that catalyze the monooxygenation of a large variety of structurally diverse, lipophilic compounds of endogenous or exogenous origin.

-There are 57 human P450 isoforms, of which 50 are found in the endoplasmic reticulum (ER) membrane and carry out the detoxification of drugs (i.e., xenobiotics).

-Endogenous substrates include sterols and steroid hormones, fatty acids, eicosanoids, vitamin D, and arachadonic acid.


Which CYP P450 isoforms are clinically important

-involved in metabolism of many drugs

-CYP3A4 (metabolism of ~40% of prescription drugs)


Structure and Tissue Distribution of CYPs

Majority of P450’s are found in the ER – exception are mitochondrial P450 enzymes involved in steroidogenesis

-P450’s found in all tissues, enriched in LIVER, lung, intestinal mucosa

-integral membrane proteins

-smooth ER has a ton of these and forms vesicles or microsomes when isolated, enriched in hepatocytes (active site is facing cytosol)


Substrate Binding Pocket of CYP

-long, hydrophobic channel

-highly dynamic

-van der Waals surface

-active site-- HEME at the bottom of the pocket



has O2 and one goes water while the other goes to the substrate


Hydroxylation Reaction (shows how important heme iron molecule is to doing the enzymatic reaction)

REQUIRES: Fe and e-

1. substrate binding to heme group

2. e- comes in and reduces iron to ferrous form

3. molecular O2 binds to enzyme at heme iron
-THIS STEP FORMS A SUPEROXIDE (1e- reduction of O2)

4. another 1e-
electron reduction of oxygen so a peroxide anion is formed

5. splitting of O-O bond where one atom of O2 goes to water

6. activated oxygen molecule is when you have 1O bound to the heme. Production of transient carbon radical

7. formation of hydroxylated substrate

8. hydroxylated substrate is now released from the enzyme so the enzyme can bind another substrate


where do cytochrome P450 acquire e- to carry out the hydroxylation reaction?

Electron Transport Chain.

-get e- from NADPH and mainly from pentose phosphate shunt and malic enzyme

-reductase is an integral membrane protein (on smooth ER) that mediates electron transfer b/w NADPH and cytochrome P450s


1. NADPH donates electron to cyt P450 reductase (flavo protein) to then activate cyt P450

2. cyt P450 (heme protein) then e- used to activate O2 for the hydroxylation reaction


Where does the NADPH required get created? 2 different ways

-In hepatocytes and adipocytes, cytosolic NADPH is largely generated by the pentose phosphate pathway and by malic enzyme.

Main way
1. Pentose Phosphate Pathway
-Glucose 6-phosphate to Ribulose 5-phosphate
-creates 2NADPH

2. Malic enzyme
-malate to pyruvate
-enzyme: malic enzyme
-creates 1NADPH


NADPH-Cytochrome P450 Oxidoreductase

-The 50 different cytochrome P450’s in the endoplasmic reticulum all use a single NADPH cytochrome P450 oxidoreductase (CYPOR).
---has a crystal structure
---accepts electrons from NADPH and it then goes to FAD and FMN

-It currently is not known how a single CYPOR can interact with so many different cytochrome P450’s.


3 major families of Phase II enzymes

1. UGT

2. GST



UDP Glucuronyl Transferase

Phase II enzyme

-Conjugation with (“glucuronidation”) is the most common conjugation reaction and occurs with compounds containing hydroxyl, amino, or sulfhydryl groups.

-The source of glucuronic acid is uridine diphosphate-α-D-glucuronic acid (UDPGA).

-The reaction is catalyzed by UDP-glucuronyl transferase (UGT), a family of enzymes (19 UGTs in humans) associated with the endoplasmic reticulum of many cell types, especially hepatocytes.


How is UDP Glucuronyl Transferase clinically relevant?

-UGT1A1 = important role in drug metabolism since
----the glucuronidation of bilirubin by UGT1A1 is the rate-limiting step in efficient bilirubin clearance, and this rate can be affected both by genetic variation and competing substrates (e.g., drugs).

Gilbert’s syndrome
-benign condition present in up to 10% of the population
-diagnosed b/c circulating bilirubin levels are ~60-70% higher than observed in normal individuals
-reduced expression levels of UGT1A1
-predisposed to adverse drug reactions resulting from a reduced capacity to metabolize drugs by UGT1A1

For example, if a drug undergoes selective metabolism by UGT1A1, competition for drug metabolism with bilirubin glucuronidation will exist, resulting in pronounced hyperbilirubinemia as well as reduced clearance of the metabolized drug.



breakdown product of heme (happens when RBC get degraded)


Glutathione S-Transferase

-glutathione gets rid of potentially harmful epoxide intermediates

-Glutathione S-transferase (GST, over 20 human GST’s) accounts for ~10% of the soluble proteins in the hepatocyte which has a concentration of glutathione approaching 10 mM.

-Catalyzes the nucleophilic attack of the thiolate anion of glutathione (GSH, γGlu -Cys-Gly) on the electrophilic atom of any lipophilic compound to which the enzyme will bind.

-Its biological importance is related to the formation of highly reactive oxidation products, such as epoxides, by cytochrome P450's.
----These epoxide intermediates have the potential to cause cell injury due to their ability to form covalent adducts with proteins, DNA, or RNA.



-Sulfotransferases (SULT, 13 human SULT genes) catalyze the transfer of sulfate from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to a nucleophilic acceptor, either a hydroxyl or amino group.

-PAPS is an “activated” form of sulfate and is the universal donor for all sulfation reactions in the cell.

-Sources of inorganic sulfate include the diet, with tap water contributing ~10% of the total dietary source

-catabolism of sulfur-containing amino acids (methionine and cysteine); recycling of released sulfate following lysosomal degradation of macromolecules by lysosomal sulfatases.


Activation (Carcinogenesis)

-P450’s may oxygenate a molecule in a conformationally hindered position – inaccessible to Phase II enzymes

-Reactive intermediates can interact nonenzymatically with intracellular nucleophiles, such as protein, DNA or RNA to form covalent adducts, resulting in necrosis, mutations, malignancy.

Benzo[a]pyrene, a weak carcinogen, is metabolized to the potent carcinogen (epoxide derivative). Benzo[a]pyrene is a common environmental contaminant produced from the burning of coal and is present in cigarette smoke.


Regulation of P450 gene expression

most common regulation is through gene transcription

-subject to tissue-specific patterns of expression resulting in differences in isoform composition & activities in various tissues


Xenobiotic response elements (XREs)

function as transcriptional enhancers for P450 gene expression regulation


Drug interactions (general info)

-Defined as a measurable modification of the action of one drug by prior or concomitant administration of another drug – a drug becomes more or less active in the presence of a 2nd drug

-An average patient during a hospital stay receives 9 different drugs

-~2-3% of patients experience adverse drug interactions

-Many commonly used drugs alter cytochrome P450 activity and therefore can potentiate or inhibit the metabolism of other drugs


Drug interactions (induction and inhibition)

Exposure of a drug or dietary chemical can result in either stimulating the drug metabolizing capacity of the liver (shorter half-life) or inhibiting the drug metabolizing capacity (cyt p450) of the liver (longer half-life) of certain xenobiotics in the body



enzyme that metabolizes estrogen in oral contraceptives
-results in decrease in effective levels of oral contraceptive (unplanned pregnancies)

-this occurs as a result of 2 drugs (antibiotic) and herbal remedy for depression interacting with each other which INDUCES CYP 3A4


Inhibition of P450

Inhibition results in an increase in the half-life of certain xenobiotics in the body.

1) Mechanism-Based Inhibition
Certain drugs contain functional groups that are oxidized by
P450’s to metabolites that bind irreversibly to the enzyme resulting in the inactivation of the enzyme (“suicide inhibitor”)

2) Disease States
liver disease (cirrhosis) – most commonly associated with
Impaired drug elimination (decreased capacity of the liver
to metabolize xenobiotics due to decreased expression of P450’s)
process of aging

3) Multiple Drug Therapy
Drugs compete for the same P450.


what happens to half-life of drug if cyt P450 is inhibited or induced?

inhibited- slower breakdown of drug, so longer half-life

induced- faster breakdown of drug, so shorter half-life


Dioxin (TCDD)

Induces P450

-product of pulp and paper mills (in manufacturing process)

-element in agent orange (herbicide sprayed during vietnam war (also found in Times Beach, Missouri) to clear vegetation

-tons of health problems