7- Drug Metabolism/Detoxification

Question 1

therapeutic dose

Answer 1

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

Question 2

Overview of Detoxification

Answer 2

meant to rid our body of drugs and xenobiotics

Phase I

• enzymes: cytochrome P450s
• modification

Phase II

• enzymes: transferases
• adds molecule to drug to inactivate it and make it more soluble

Question 3

Phase I metabolism

Answer 3

• P450s
• these enzymes are adding a polar group (hydroxyl or amino or sulfhydryl)
• enzymes exhibit low specificity (overlapping substrate specificities) and low catalytic rates

Question 4

xenobiotics

Answer 4

• 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

Question 5

Phase II metabolism

Answer 5

• 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

Question 6

Phase I Enzymes

Answer 6

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

-Monooxygenase
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)

Question 7

CYP

Answer 7

• 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.

Question 8

Which CYP P450 isoforms are clinically important

Answer 8

-involved in metabolism of many drugs

• CYP3A4 (metabolism of ~40% of prescription drugs)
• CYP2D6
• CYP2C9
• CYP2C19
• CYP1A2

Question 9

Structure and Tissue Distribution of CYPs

Answer 9

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)

Question 10

Substrate Binding Pocket of CYP

Answer 10

• long, hydrophobic channel
• highly dynamic
• van der Waals surface
• active site– HEME at the bottom of the pocket

Question 11

monooxigenases

Answer 11

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

Question 12

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

Answer 12

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

Question 13

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

Answer 13

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

STEPS

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

Question 14

Where does the NADPH required get created? 2 different ways

Answer 14

-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

Question 15

NADPH-Cytochrome P450 Oxidoreductase

Answer 15

• 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.

Question 16

3 major families of Phase II enzymes

Answer 16

1. UGT
2. GST
3. SULT

Question 17

UDP Glucuronyl Transferase

Answer 17

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.

Question 18

How is UDP Glucuronyl Transferase clinically relevant?

Answer 18

• 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.

Question 19

bilirubin

Answer 19

breakdown product of heme (happens when RBC get degraded)

Question 20

Glutathione S-Transferase

Answer 20

SO ESSENTIALLY…
-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.

Question 21

Sulfotransferase

Answer 21

• 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.

Question 22

Activation (Carcinogenesis)

Answer 22

• 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.

Question 23

Regulation of P450 gene expression

Answer 23

most common regulation is through gene transcription

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

Question 24

Xenobiotic response elements (XREs)

Answer 24

function as transcriptional enhancers for P450 gene expression regulation

Question 25

Drug interactions (general info)

Answer 25

• 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

Question 26

Drug interactions (induction and inhibition)

Answer 26

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

Question 27

CYP 3A4

Answer 27

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

Question 28

Inhibition of P450

Answer 28

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.

Question 29

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

Answer 29

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

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

Question 30

Dioxin (TCDD)

Answer 30

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

Question 31

Mechanism of Induction for Dioxin and CYP1A1

Answer 31

Dioxin = aromatic hydrocarbon

• Analogous to action of steroid hormones
• can easily get into cells
• when in cells it binds to Ah receptor which is a transcription factor
• this complex then enters into the nucleus and gets with another protein called XRE (xenobiotic response element- act as transcriptional enhancers: found in CYP1A1, UDPGT, & GST genes - promoter region)
• increases expression of P450
• Dioxin induces Phase I & II enzyme levels via XRE
• very toxic: competes with endogenous ligand for receptor and is VERY slowly metabolized 7-15yrs

Question 32

Dietary influences of expression of P450 and phase II metabolism

Answer 32

Food & dietary components may affect the fate of a drug by affecting phase I & phase II metabolism:

1) Grapefruit juice (rich in furanocoumarins) effective inhibitors of CYP3A4
~40% of prescription drugs are metabolized by CYP34A
-CYP3A4 metabolizes many cardiovascular drugs (Ca++ channel blockers, HMGCoA reductase inhibitors), ingestion of grapefruit juice would result in increased levels of cardiovascular drugs in the blood

2) Cruciferous vegetables—broccoli, brussels sprouts, cabbage, cauliflower (rich in indoles & isothiocyanates)
effective inducer of CYP1A1 in the intestine
increased levels of CYP1A1 in intestine may aid in the metabolism and elimination of dietary polyaromatic hydrocarbons, thus reducing the exposure of internal organs to these carcinogens

3) Iron deficiency
Decreased activity of P450’s; results in increased levels of drugs in the circulation

4) Vitamin deficiencies can affect P450 activity
- Deficiency of riboflavin (vitamin B2, water soluble):
- –riboflavin is converted to FMN and then to FAD
- –in severe deficiencies, the activity of P450’s is decreased
- ——–decrease is due to decreased levels of FMN and FAD which are the prosthetic groups of NADPH cytochrome P450 oxidoreductase (CYPOR)

Question 33

Polymorphism (genetic factors that influence expression of P450)

Answer 33

simultaneous occurrence in a population of different alleles producing different phenotypes (i.e., genetic differences – expression of the different isoforms of P450 varies from individual to individual)

• one size does not fit all
• many drugs do not work similarly in all patients
• optimum dose requirement may differ among individuals

Expression of P450’s influenced by:

• Disease
• Xenobiotics (drugs, environmental compounds) -Genetic factors
• -each person can have a different

Question 34

Polymorphism: 4 types of metabolizers

Answer 34

-multiple alleles for ~40% of human P450 genes

1. Poor metabolizers
   - 2 defective alleles resulting in complete lack of certain P450 enzyme activity
2. Intermediate metabolizers
   - either heterozygous for defective allele or have 2 defective alleles resulting in decreased activity
3. Extensive metabolizers
   - carry two functional alleles
4. Ultrarapid metabolizers
   - carry more than 2 active gene copies resulting in P450 enzyme with increased activity

Question 35

CYP2D6 Deficiency

Answer 35

• Important to screen for this P450 polymorphism since adverse reactions (death) have been experienced by individuals genetically deficient in CYP2D6 who use certain drugs.
• poor metabolizer phenotype (autosomal recessive trait)
• all some sort of deletion mutation
• Phenotype is silent in the absence of drug challenge.

Question 36

if prozac inhibits activity of CYP2D6 then…

Answer 36

drugs that are metabolized by CYP2D6 require a lower dose in the presence of Prozac.

Question 37

CYP2C19 Polymorphism

Answer 37

• Ethnic differences: 2-5% Caucasians, 4% African- Americans, and 13-23% Asians are “poor metabolizers”
• Clopidogrel (Plavix) is converted to its active form by CYP2C19.
• This highly prescribed anti-clotting drug is used for the prevention of strokes and heart attacks.

Question 38

NADPH-Cytochrome P450 Oxidoreductase (CYPOR) Polymorphism

Answer 38

-completely knocking out NADPH- cytochrome P450 reductase gene in animal model is embryonic lethal

Antley-Bixler syndrome

• mutation that alters activity of CYPOR
• steroidogenic abnormalities

Question 39

Acetaminophen (APAP)

Answer 39

-Acetaminophen has been approved for OTC (OTC = over the counter) use since 1960 (analgesic)

-Although the drug is remarkably safe, toxicity can occur with high doses, resulting in liver
injury and even death.

• Alcoholics are particularly susceptible to hepatotoxicity
• Therapeutic dose of acetaminophen is 10-15 mg/kg/ dose in children and 325-1000 mg/dose every 4-6 hours in adults, with a maximum of 4g/day for adults.
• VERY common in multiple different types of drugs (so now they need to label more clearly and limit amount in mixed drugs (325mg) to prevent overdose)

Question 40

Acetaminophen (APAP) and liver disease

Answer 40

1998 to 2003: leading cause of acute liver failure in the United States, with 48% of acetaminophen-related cases (131 of 275) associated with accidental overdose.

2007: nationally 1600 cases of acute liver failure (ALF) each year (all causes). Acetaminophen-related ALF was the most common etiology.
- estimated 56,000 emergency room visits, 26,000 hospitalizations, and 458 deaths related to acetaminophen-associated overdoses per year during the 1990-1998 period

Question 41

Acetaminophen hepatotoxicity

Answer 41

Occurs as a result of metabolism through a cytochrome P450-dependent pathway.

• 5% of ingested acetaminophen is metabolized by CYP2E1. The product of oxidation is N-acetyl-p- benzoquinoneimine (NAPQI), a highly reactive/toxic in many organs, including the liver.
• NAPQI is usually rendered nontoxic by conjugation with the intracellular tripeptide glutathione.
• Hepatotoxicity occurs at high doses of acetaminophen because the glutathione stores are exhausted by the generation of large amounts of NAPQI.

-Treatment with the antidote N-acetylcysteine (NAC): NAC serves as the precursor in the synthesis of glutathione.

Enhanced acetaminophen hepatoxicity occurs:

1) In the fasting state (UDPGA becomes limiting)
2) with chronic alcohol use (ethanol induces CYP2E1 expression, thereby increasing the production of NAPQI at rates that cannot be detoxified due to the limited stores of glutathione).