Midterm 1: Drug Metabolism Introduction Flashcards

1
Q

non-specific enzymes that carry out biotransformation reactions on lipophilic compounds

  • What 3 processes are dominated by the action of these enzymes?
A
  • Biotransformation
  • Clearance
  • Elimination
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2
Q

What rxns do drug metabolism enzymes carry out? Why do they do this?

A
  • oxidation
  • hydrolysis
  • conjugation
  • convert non-polar drugs into polar metabolites
  • polar drug metabolites are more readily cleared from the systemic circulation by the kidneys than the parent drugs
  • also see some metabolites in the bile.
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3
Q

Definition of **drug metabolism **

A
  • process of enzymatic biotransformation of drugs
  • single drug converted into multiple metabolites by multiple enzymes
  • Usually these metabolites are inactive and rapidly eliminated
  • Sometimes the metabolites are also active.
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4
Q

Why does drug metabolism play a critical role in achieving therapeutic drug concentrations?

A
  • concentration of a drug at the site of action determines effect
  • steady state levels of drugs depends on **input **
    • dose size
    • formulation
    • frequency
    • site of administration
  • and output
    • rate of metabolism and renal clearance
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5
Q
  • What do families of drug metabolizing enzymes do?
  • Variability in the concentrations of the enzymes in any given tissue?
  • Where is the primary site of drug metabolism?
A
  • There are families of drug metabolizing enzymes where each family carries out characteristic types of reactions.
  • There is a wide inter-individual variability in the concentrations of the enzymes in any given tissue.
  • The liver is richly endowed with drug metabolizing enzymes and is the primary site of drug metabolism.
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6
Q

metabolism of the beta-blocker propranolol

  • 3 note worthy things
A
  1. 90% of a dose is eliminated as metabolites in the urine and <1% as unchanged drug. (think about were the other 9% went).
  2. Most of the metabolites are more polar (glucuronides (gluc) and sulfates (sulf) are charged).
  3. At least 6 different enzymes participate in metabolite formation and we observe both primary and secondary metabolites in the urine and feces. Thus the parent drug is converted to many metabolites. This is a good thing as it reduces the likelihood that a metabolite will be toxic.
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7
Q

Path of an orally administered drug through the body

A
  • absorbed through the cells lining the small intestine (major) or stomach (minor) into the blood
  • immediately enters the liver via the portal vein.
  • Transits liver and enters the systemic blood circulation
  • Undergoes many passes through the liver until it is finally captured by an enzyme and metabolized.
  • The cells that line the intestinal lumen are called enterocytes.
  • Enterocytes have families of drug transporters and drug metabolising enzymes that can act to promote or reduce the absorption of a given drug into the portal circulation.
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8
Q

Definition of pre-systemic metabolism

A
  • drug metabolized by enzymes in the enterocytes or hepatocytes before it can enter the systemic circulation and travel to the site of action
    • aka: first pass effect
  • reduces the amount of absorbed drug that enters the systemic circulation and reduces **bioavailablity **
    • the fraction of an oral dose that reaches the systemic circulation
  • This set up prevents upwanted dietary compenents from reaching systemic circulation
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9
Q

Diagram of Drug Pathway

A
  • Clearance: anything that take drug to metabolite
  • Every drug has to pass through the liver
  • In high clearance, portal vein concentration is higher than plasma concentration
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10
Q

2 reasons why 1st pass metabolism is something to be avoided

A
  • leads to
    • (1) highly interindividual variability in blood levels of drugs
    • (2) an exaggerated sensitivity to drug-drug interactions.
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11
Q

When a drug molecule drug molecule enters the liver what are it’s possible fates?

A
  • evading metabolism and transiting the liver to enter the systemic circulation via the central vein unchanged
  • being transformed to a metabolite. Usually the metabolite will also exit the liver via the central vein and then enters the systemic circulation for eventual excretion by the kidneys
  • metabolites of drugs and drugs themselves will exit the liver via the bile duct for excretion in the feces.
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12
Q

Definition of hepatic clearance

A
  • drug is converted to a metabolite in the liver
  • designed to provide lipophilic molecules with maximum access to the drug metabolizing enzymes
  • rapid hepatic metabolism is a high extraction ratio
    • ratio of the concentration of drug entering the liver divided by the concentration of drug that leaves the liver.
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13
Q

Cutaway diagram of liver

A
  • Blood flow 1.2 L/min
  • Blood volume 6-7 L
  • RBC transits every 5 min or so
  • Liver good at exposing blood molecules to enzymes and also putting molecules in the blood
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14
Q

Representation of Hepatocytes

A
  • ​Capillary bed in liver is efficient in exposing molecules in blood to membrane and allowing them to enter the haptocytes
  • Every cell has border on the bile. Metabolites secreted into the bile. Molecules in the bile go back to the GI tract. Pancreatic juices are also in the bile. Also bile salts produced by the liver to help us emulsify fats.
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15
Q
  • concentration of drug in hepatocyte available for metabolism
  • Where does drug enter and hepatocytes?
A
  • drugs in the blood are bound to plasma proteins
    • albumin
  • fraction that is not bound is called the free drug concentration
  • [drugs] in the hepatocytes available to drug metabolizing enzymes ~ [free drug]blood
  • Drug enters and leaves the hepatocyte by passing through the sinusoidal membrane.
  • Passage through membranes is normally much faster than drug metabolism rates
    • (Some drugs are also taken up into the heptaocytes via transporters.)
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16
Q

Drugs and their metabolites leave the hepatocyte via:

A
  • Passing through the sinusoidal membrane where they eventually enter the hepatic vein and systemic circulation (major route)
  • Passing through the cannicular membrane into the bile. Passage into the bile usually uses transporters so it is much more selective than entry into the hepatocyte from the blood (minor).
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17
Q

Two Factors in Bioavailability

A
  1. Absortion
    • ​Poorly absorbed go into feces
  2. ​First Pass Metabolism
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18
Q

Explain This Figure

A
  • Area under curve smaller for oral dose of propanol (high 1st pass metabolism)
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19
Q

Big Picture of Liver in Circulatory System

A
  • Note: bile flow from the liver into the GI tract (green)
  • Placement of the liver so that it gets the first look at orally absorbed drugs before they enter the systemic circulation.
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20
Q
  • drug metabolizing enzymes in the liver hepatocytes are located in one of two places
A
  • ER
    • contains the membrane-bound P450 enzymes
    • some forms of glucuronyl transferases.
    • Microsomes are the workhorse of drug metabolism research.
  • Cytosol
    • glutathione S-transferases (GST)
    • epoxide hydrolases (EH)
    • various dehydrogenases
    • esterases
    • 50% of the cell volume is cytosol.
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21
Q

Making microsomes

A
  • homogenize liver which
    • lyses the cells and shears the ER into small donut shaped vesicles
  • centifuged at 10,000 x g and the pellet is discarded
    • supernatant contains the enzymes.
  • centrifuged again a 100,000 x g
    • cytosolic enzymes are in the supernatant
    • microsomal enzymes are in the pellet
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22
Q
  • cytosolic enzymes (supernatant) and the microsomal enzymes (pellet) for metabolic studies.
A
  • P450 enzymes and glucuronyl transferases are in the microsomal fraction.
  • can study metabolism in each type of fraction
    • 10,000 x g supernatent contains all of th enzymes of interest and all cofactors
  • can identify the number and concentrations of metabolites upon incubation with drugs.
  • can measure the rate of loss of drug and estimate how rapidly the drug will be metabolised
  • in vivo by scaling rates of metabolism observed in vitro. The in vitro systems we use provide lots of useful information about interindividual variablity and drug-drug interactions.
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23
Q
  • two basic types of drug metabolism reactions which can occur in cytosol or microsomes
A
  • Phase I reactions
    • create a polar functional groups
      • more readily excreted in urine and bile
      • Cytochrome P450 enzymes
      • Oxidation, reduction or hydrolysis
  • ​**Phase II reactions **
    • generate highly polar derivatives known as conjugates of drugs and metabolites of drugs
    • deactivates highly reactive products of P450 reactions
      • epoxide hydrolase (EH)
      • glutathione-S-transferases (GSTs)
    • Highly reactive products can bind to protein/DNA, causing hepatic necrosis and cancer
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24
Q

Phase II Transformations

A
  • Drugs and/or their metabolites are combined in covalent reactions with endogenous hydrophilic compounds. The products are substances with sufficient hydrophilic character for excretion.
    • aka: stick on something polar
  • Conjugation reactions can occur with a variety of substances, usually intermediates in the organism’s metabolism (e.g. glucuronic acid, sulfate and glutathione are common added groups).
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25
Q

Glucuronidation by glucuronyl transferases (GTs):

A
  • Phase II Reaction
  • These enzymes are microsomal
  • Conjugation of a polar group ( alcohol, phenol, carboxylic acid, amine) of a drug or a metabolite with glucuronic acid
    • metabolic clearance of NSAIDs dominated by glucuronidation
  • ​substrate is uridine diphosphate glucuronic acid (UDPGA)
    • UDPGA is co-substrate for the enzyme-catalyzed rxn
    • uridine diphosphate UDGP is a good leaving group.
      • recycled after rxn
  • ​Glucuronide conjugates are anions
    • readily excreted into the bile and/or the urine
      • heme metabolite bilirubin diglucuronide which is made from heme and excreted in the bile.
      • inability to make these glucuronides due to a deficiency in the enzyme or hepatic failure leads to accumulation of unconjugated bilirubin (jaundice, yellow babies, kernicturis).
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26
Q

Sulfate conjugation by sulfotransferases (SULTs)

A
  • Phase II Reaction
  • Phenolic drugs and metabolites are converted to highly polar sulfate conjugates by sulfotransferases
    • co-factor in these reaction is an endogenous compound 3’- phosphoadenosine-5’-phosphosulfate (PAPS)
    • product PAP is also “recycled” to PAPS.
  • Sulfate conjugates (anions at body pH) are excreted mainly in the urine.
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27
Q

Glutathione conjugation by glutathione-S- transferases (GSTs) (Mercapturic acid formation)

A
  • Phase II Reaction
  • GSTs catalyze the attack of the sulfhydryl group of the co-substrate glutathione at electrophillic sites on drugs (sometimes), their metabolites (often) as well as environmental toxins and their metabolites.
    • cytosolic enzymes
  • Glutathione (GSH) is a novel highly polar (nucleophilic) tripeptide (γ-glutamylcysteinylglycine) substrate that is present at high levels in cells (5 mM). Glutathione can react with substrates directly or via GST catalysis.
  • The main purpose of GSH and the family of GSTs appears to be “deactivate” reactive groups on molecules (such as epoxides, quinones and halides) and in so doing to create a water soluble metabolite. Conjugation with glutathione is a major defense pathway for reactive compounds created by oxidative metabolism.
  • Glutathione conjugates are rarely excreted intact into the urine due to their high molecular weight. Instead they converted to N-acetyl-L-cysteine (mercapturic acid) conjugates. Thus the glycine and glutamate of glutathione are recycled but the cysteine is excreted as the mercapturate.
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28
Q

Aromatic Hydroxylation

A
  • Phase I Reaction
  • Substrate on Left, Metabolite on Right
29
Q

Aliphatic Hydroxylation

A
  • Phase I Reaction
  • Substrate on Left, Metabolite on Right
30
Q

Epoxidation

A
  • Phase I Reaction
  • Substrate on Left, Metabolite on Right
31
Q

N-Hydroxylation

A
  • Phase I Reaction
  • Substrate on Left, Metabolite on Right
32
Q

O-dealkylation

A
  • Phase I Reaction
  • Substrate on Left, Metabolite on Right
33
Q

N-dealkylation

A
  • Phase I Reaction
  • Substrate on Left, Metabolite on Right
34
Q

S-dealkylation

A
  • Phase I Reaction
  • Substrate on Left, Metabolite on Right
35
Q

deamination

A
  • Phase I Reaction
  • Substrate on Left, Metabolite on Right
36
Q

S-Oxidation

A

Phase I Reaction
Substrate on Left, Metabolite on Right

37
Q

dechlorination

A

Phase I Reaction
Substrate on Left, Metabolite on Right

38
Q

Oxidative Desulfuration

A

Phase I Reaction
Substrate on Left, Metabolite on Right

39
Q

II amine oxidation

A

Phase I Reaction
Substrate on Left, Metabolite on Right

40
Q

III dehydrogenation

A

Phase I Reaction
Substrate on Left, Metabolite on Right

41
Q

Azoreduction

A

Phase I Reaction
Substrate on Left, Metabolite on Right

42
Q

Nitroreduction

A

Phase I Reaction
Substrate on Left, Metabolite on Right

43
Q

Carbonyl Reduction

A

Phase I Reaction
Substrate on Left, Metabolite on Right

44
Q

Ester Hydrolyses

A

Phase I Reaction
Substrate on Left, Metabolite on Right

45
Q

Amide Hydrolyses

A

Phase I Reaction
Substrate on Left, Metabolite on Right

46
Q

Putting one story together (Phase I and Phase II metabolism and toxicity): Acetaminophen Overdose

A
  • glutathione levels in the liver can become depleted
  • reactive metabolites can react with cellular macromolecules and cause hepatic toxicity
  • acetominophen (Tylenol) overdose
  • Note the two reactions (glucuronidation and sulfation) (phase II pathways to conjugate metabolites of parent drug).
  • Note also the Phase I oxidation reaction that is catalyzed by a P450 enzyme (CYP2E1). The immediate metabolite of this reaction is highly reactive (electrophillic) metabolite NAPQI. The NAPQI formed after a normal dose is readily deactivated by conjugation with GSH. This sequence is a Phase I followed by a Phase II reaction. In overdose situations the amount of NAPQI that is formed is sufficient to deplete glutathione levels leading to alkylation of proteins by NAPQI and cell death.
  • Alchol ingestion induces more CYP2E1, makes more of the GSH. Double the amount of this pathway, increase likelihood of overwhelming GSH pathway.
  • Give n-acetyl cysteine, a glutathione like substance for an overdose.
47
Q

What are P450 enzymes?

A
  • present in virtually all mammalian cells. membrane bound enzymes that have a heme prosthetic group. The iron atom of heme is the catalytic center of the enzyme and does most of the heavy chemistry.
  • The active site of the enzyme is located in a lipophilic region in the interior of the enzyme. The active site of these enzymes readily binds a broad range of of lipophilic compounds.
  • Over 1000 different P450 enzymes are known at this time. P450’s play important biological roles in all species including mammals, fish, bacteria, fungi and plants. In plants for instance flower color is controlled by various activities of P450’s. In fungi the composition of the cell wall depends on a P450 enzyme that we inhibit with the antifungal drugs (e.g. fluconazole).
48
Q

Picture of P450 Enzymes

A
49
Q

There are families of human P450 enzymes (CYPs) with different functions and classes of substrates (note overlaps, eg CYP1, 2 and 3 enzymes will oxidize some fatty acids)

A
50
Q

Major functions of the human P450 enzymes

A
  • Chemoprotective ‘housekeeping” function ( CYP1-3): Lipophilic compounds that have been absorbed from the diet that would otherwise accumulate to unacceptably high concentrations in membranes are oxidized to more polar metabolites for excretion. Thus it makes sense that P450’s are at high concentrations in the liver, that the liver gets a first look at compounds that have been taken up from the intestine and that blood is processed rapidly and continuously by the liver. These are the drug metabolizing P450 enzymes and the ones we will focus on.
  • Endogenous compound metabolism function (all other CYPs): Specific P450 enzymes located in the liver and elsewhere play an critical role in the biosynthesis and degradation of endogenous compounds such as the steroid hormones, arachidonic acid, vitamins (vitamin D and cholesterol. These enzymes are tailored to a specific purpose such as the conversion of one substrate to one product. The production of steroids from cholesterol uses many different P450 enzymes which are located either on the inner mitochondrial membrane or the endoplasmic reticulum. Steroid production occurs in many tissues (adrenal glands).
  • In some cases a single enzymes will carry out multiple rounds of catalysis without releasing the intermediate products. In the example below cholesterol undergoes 3 successive oxidation reactions to produce pregnenolone which is the precursor to all of the steroid hormones (androgens, estrogens, glucocorticoids and mineralcorticoids). Also note that the hydroxylation reactions are stereospecific.
51
Q

Cholesterol Example

A
  • Cholesterol bind P450 in mitochondria, it hydroxylates, then another hydroxylates, then cleavage (3 successive oxidations)
52
Q

Where in the cell are the membrane-bound P450’s located ?

A
  • The endoplasmic reticulum (microsomal P450’s); the drug metabolizing P450s are here.
  • The inner mitochondrial membrane (mitochondrial P450’s); specialized P450.
  • Generally the organs of greatest importance in the metabolism of drugs are located in (in order of importance) Liver>>Intestine> Lung, Kidney.
  • P450 enzymes dominate drug metabolism
53
Q

P450 catalyzed monooxygenation

A
  • molecular oxygen is bound by the enzyme heme iron and eventually, one oxygen atom is incorporated into the substrate (R-H) and the other oxygen atom is converted to water.
    • Mixed function oxidase because oxygen goes to 2 places
  • NADPH + H+ + O2 + R-H →NADP++ H2O + R-O-H
54
Q

P450 catalytic cycle (word overview)

A

O2 is split at the end of the oxygen activation steps by addition of two protons.

One of the oxygen atoms from dioxygen is incorporated into the substrate and the other oxygen atom is reduced to water.

The reactive oxygen atom bound to the heme iron is the perferryl oxygen. Thus the P450 cycle produces a high reactive oxygen atom that is bound to iron but is readily transferred to substrates.

55
Q

What provides 2 electrons that drive the oxygen activation process of P450 catalytic cycle?

A
  • NADPH (a cosubstrate)
  • The electrons of NADPH are first transferred to cytochrome P450 reductase and then on to the dioxygen oxygen bound to the P450 enzyme heme iron. NADPH and oxygen concentrations in the healthy cell are always high and never rate limiting.
56
Q

conversion of substrate (R-H) to oxidized product (R-O-H) in P450 catalytic cycle

A
  • two electron oxidation reaction of the substrate
  • last step in the catalytic cycle
57
Q

P450 catalytic cycle (steps)

Not memorize whole cycle. Memorize the stoichiometry and where the oxygens go

A
  • sulfur anion of cysteine promotes dioxygen cleavage
    1. Structure 8 is the resting state of the enzyme where either water or the product of the last cycle is bound to the ferric heme iron.
    2. Substrate (R-H) binds to the enzyme and displaces the water molecule to give 3.
    3. Structure 3 is reduced to the ferrous state (4) which binds oxygen to give the ferrous dioxygen species 5.
    4. Species 5 accepts a second electron from the reductase and a proton to produce the ferrous hydroperoxy species 6. (This is the RDS)
    5. A second protonation of 6 causes heterolytic (both electrons in the O-O bond leave with the water) O-O bond cleaveage to produce a molecule of water and the active oxidizing species called P450 Compound 1 (7).
    • ​Compound I is a hypervalent iron-oxygen, porphyrin radical cation
      1. Compound I reacts with functional groups on bound substrates to produce metabolites.
58
Q

Metabolic specificity has two components:

A
  • Which molecules are accepted as substrates by a particular P450 (substrate selectivity)
  • What site or sites on a particular molecule are oxidized by a particular P450 (product profile).
59
Q

Any given P450 can oxidize hundreds to thousands of different substrates because…

A
  • Any given P450 can oxidize hundreds to thousands of different substrates because the active sites are very large and flexible.
  • Note that the active site pocket is must larger than the substrate.
  • The substrates can move around in the active site and present different oxidizable locations to Compound I. This means that the crystal structures do not necessarily predict the sites of metabolism.
  • The e_ntrance and exit channels for substrates and products are not well understood_ and there are major differences in the structures of the substrate-free and substrate bound forms.
60
Q

Oxidation of sp3 carbon hydrogen bonds

A
  • Hydroxylation reactions that occur at sp3 hybridized carbons involves insertion of the Compound I oxygen atom into a carbon-hydrogen bond to make an alcohol.
  • Formation of simple alcohols of alkyl groups present on substrates. Often the alcohol products are substrates for the glucuronyl transferases.
  • The net hydroxylation reaction is insertion of atomic (six electron) oxygen into a carbon hydrogen bond. Alcohols from alkanes is the simplest example here.
  • ω−1 hydroxylation is usually preferred over ω hydroxylation. Note that hydroxylation at prochiral carbons can generate stereoisomeric products (R,S 2-hexanol). Hydroxylation reactions are usually steroselective and sometime stereospecific.
  • Hydroxylation reactions are very common since almost all drugs have sp3 carbons. These carbons may be aliphatic or benzylic (midazolam). Also synthesis of the steroid hormones involves many specific hydroxylation reactions on the steroid backbone. The pattern of hydroxyl groups is important for targeting the steroid hormones to their receptors.
61
Q

O- dealkylation

A
  • reactions cleave alkyl or aryl-alkyl ethers to make alcohols or phenols
  • sp3 carbon-hydrogen bond of the substrate is attacked by compound I to make an alcohol. The carbon is attached to an oxygen in an ether linkage rather than another carbon.
  • This hydroxylation reaction initially produces a hemiacetal or hemiketal product. Hemiacetals and hemiketals rapidly and spontaneously cleave to give the corresponding alcohol or phenol product and a small aldehyde or ketone fragment. Often the alcohol and phenol products are substrate for the glucuronyl transferases.
  • These are common reactions since many drugs have ether linkages. They are named for the small group that is cleaved off from the “parent” molecule. Examples are:
  • O-de-methylation
  • O-de-ethylation
  • O-de-isopropylation
62
Q

N-dealkylation reactions

A
  • cleave alkyl groups from alkylamines to make smaller alkylamines or arylamines.
  • very common since many drugs incorportate alkylamine functional groups that are essential for proper binding to receptors.
  • initiated by oxidation of an sp3 carbon hydrogen bond to form a carbinolamine followed by spontaneous cleavage of the C-N bond to give the amine and the aldehyde or ketone fragment.
63
Q
  • Oxidation of isolated carbon-carbon double bonds to make epoxides
A
  • pi bond of the substrate is attacked by Compound I to give an epoxide. Epoxides are usually unstable, electrophilic molecules and are sometimes toxic.
  • For isolated, non aromatic double bonds the epoxide metabolite will eventually react with water to give the corresponding diol as the final metabolite. Since epoxides can be reactive towards nucleophiles other than water such as cellular macromolecules, some epoxides can be toxic.
  • Perhaps for this reason enzymes called epoxide hydrolases are present in cytosol that readily catalyze water addition to epoxides to give diols.
64
Q

Oxidation of double bonds in aromatic systems to make arene oxides and phenols

A
  • Again the most common product of attack on the pi bond of an aromatic ring is an epoxide. However these types of epoxides have a special name, arene oxides.
  • Arene oxides, as opposed to simple aliphatic epoxides, rapidly rearrange to phenols by a mechanism called the NIH shift. The driving force for rearrangement of arene oxides is the return to a stable, low energy aromatic product. Phenolic metabolites are often further converted to their glucuronide or sulphate metabolites prior to excretion.
  • The arene oxides themselves are highly electrophilic and reactive. They can bind covalently to cellular macromolecules such as DNA and protein. Reaction with DNA is of concern because the modified DNA can be misread and lead to mutations and cancer.
  • aromatic hydroxylation reactions are not due to a simple carbon-hydrogen bond insertion reactions like aliphatic hydroxylation even though the final product looks like that is what has happened.
  • The realization the aromatic hydroxylation involves an arene oxide reactive intermediate was key to our understanding of the bioactivation and toxicity of polycyclic aromatic hydrocarbons.
65
Q

NIH Shift

A

involves a hydrogen shift from one carbon to another during rearrangement of the arene oxide to the phenol.

66
Q

metabolites of caffeine

A
  • This enzyme is responsible for approximately 90% of the metabolic clearance of caffeine. The half-life of caffeine in the body is approximately 7 hours. If CYP1A2 were not present in the liver the half-life of caffeine would increase to greater than 50 hours. A problem for Starbucks.
  • These metabolites are produced by N-dealkylation reactions (carbinolamine intermediates that spontaneously fall apart to formaldehyde and the demethylated xanthine. Note that the enzyme reacts around the entire periphery of the substrate to produce it’s metabolic profile.
67
Q
  • substrates can move in the active site of a given enzyme after Compound I is formed by the cycle so the oxidant often has a “choice”.
A
  • This movement in the active site is responsible for the formation of multiple metabolites from a single substrate by a single P450 enzyme. Typically one major metabolite and some minor metabolites are formed for the drug metabolizing P450 enzymes
  • The fact that a single P450 enzyme can make multiple metabolites of a given substrate provides some insight into catalysis and helps us understand substrate and product specificity
68
Q

A second example is the formation of two different metabolites of the procarcinogen aflatoxin B1

A

by a P450 CYP3A4 and CYP1A2.

Here the epoxide metabolite can be deactivated by reaction with glutathione (Phase II) or travel to the nucleus and alkylate DNA leading to mutagenesis and carcinogenesis. Aflatoxin is a very potent mutagen due to the activity of P450 enzymes. Aflatoxin is produced by fungi that grow on improperly stored peanuts and corn. Big problem in developing world and agriculture.