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IMCP I > Drug Biotransformation I and II > Flashcards

Drug Biotransformation I and II Flashcards

1
Q

What is biotransformation?

A

A metabolic conversion of endogenous or exogenous substances

Its purpose is to deactivate or improve elimination of exogenous substances and xenobiotics

Can also activate a pro-drug as well as produce actie metabolites

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2
Q

What factors influence metabolism?

A

Genetic

Physiologic

Pharmacodynamic

Environmental

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3
Q

What is the difference between Phase I and Phase II metabolism?

A

Phase I metabolism deals with the biotransformation of core reactions by adding, exposing, or modifying functional groups.

Phase I Rxns: oxidation, reductions, hydroxlation, hydrolysis.

Phase I reactions may provide new sites for Phase II metabolism.

Phase II metabolism is made up of conjugation (addition) reactions.

There are only a limited number of donor molecules in Phase II: glucouronidation, sulfation, methylation, acetylation, etc.

Functional Groups in Phase II are masked by addition of auxiliary groups.

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4
Q

What type of enzymes are in Phase I metabolism?

A

Oxygenases

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5
Q

What type of enzymes are in Phase II metabolism?

A

Transferases

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6
Q

List the Pase I enzymes and their reactions.

A

Cytochrome P450s (P450 or CYP) - C and O oxidation, dealkylation, others

Flavin-containing monooxygenases (FMO) - N, S, and P oxidation

Epoxide hydrolases (mEH, sEH) - Hydrolysis of epoxides

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7
Q

List the Phase II enzymes and their reactions.

A

Sulfotransferases (SULT) - addition of sulfate

UDP-glucouronosyltransferases (UGT) - addtion of glucouronic acid

Glutathione-S-transferases (GST) - addition of glutathione

N-acetyltransferases (NAT) - addition of acetyl group

Methyltransferases (MT) - addtion of methyl group

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8
Q

What are the additional metabolism enzymes and their reactions?

A

Alcohol dehydrogenases - reduction of alcohols

Aldehyde dehydrogenases - reduction of aldehydes

NADPH-quinone oxidoreductases (NOO) - reduction of quinones

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9
Q

What are the characteristics of the Phase I superfamily Cytochrome P450 (CYP450)?

A

Bound to smooth ER

Major metabolic enzyme class

Binds substrate, then molecular oxygen

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10
Q

What are the characteristics of the Phase I superfamily Flavin Monooxygenases (FMO)?

A

Bound to smooth ER

Minor metabolic enzyme class

Binds/activates molecular oxygen, then binds substrate

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11
Q

What are the characteristics of the Phase I superfamily Hydrolases?

A

Bound to smooth ER and free in cytosol

Minor metabolic enzyme class

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12
Q

True or false: Phase I metabolism must come before Phase II reactions.

A

False

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13
Q

Where are the CYPs found?

A

Found in liver and extrahepatic tissue

• Bound to smooth endoplasmic reticulum
– Microsomal fraction: homogenized tissue contains smooth ER

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14
Q

CYPs have an absolute requirement for what?

A

P450 has absolute requirement for NADPH & molecular O2

• Molecular oxygen is consumed

– Oxidation – 1 oxygen atom incorporated into substrate

– Reduction – 1 oxygen atom is reduced to H2O

  • Endogenous: steroids, fatty acids, prostaglandins, bile acids
  • Exogenous: drugs, carcinogens, toxins
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15
Q

What are CYP450 Isoforms?

A
  • Different amino acid sequence within the same enzyme class
  • Results in distinct structure and functions

Nomenclature based on sequence homology not function

  • CYP|FAMILY (number) | SUBFAMILY (letter) | GENE(number)
  • Example: CYP1A1
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16
Q

Explain CYP450 Isoform specificity.

A

Substrates predicted by membrane binding CYP

  • Lipophilic substrates bound tightly to ER
  • Hydrophilic substrates bound weakly to ER

Specificity based on active site structure

  • Wide range of specificity between isoforms
  • May be highly constrained, restricting to specific 3D shape
  • Flexibility or large lipophilic pockets accept diverse structures

– CYP3A4 is capable of metabolizing nearly 50% of drugs

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17
Q

What are the most common CYP Isoforms?

A

3A4/3A5 and 2D6

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18
Q

Types of Interactions with CYP450 Isoforms:

A

Isoforms can be induced/inhibited/shared

• Common drug-drug interaction

• Induction by drug activating gene transcription – Increased expression of enzyme
– Carbamazepine induces CYP 1A2, 3A4, 2C8/9, 2D6

• Inhibition by drug binding to active site of CYP enzyme

– Faster onset and recovery (if reversible)
– Erythromycin inhibits CYP 1A2 and 3A4

• Shared when two different substrates compete for binding to pharmacophore (CYP enzyme)

– Donepezil & tramadol are substrates for CYP 2D6

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19
Q

What are common CYP inducers?

A

Phenobarbital - CYP 3A4/CYP 2C (CYP 2B6, 2D6, and 1A2)

Phenytoin/carbamazepine - CYP 3A4/CYP 1A2/CYP 2C

Rifampin - CYP 3A4/CYP 2C/CYP 2B6/2D6

St. John’s wort - CYP 3A /CYP 2C9/CYP 1A2

Cigarette smoke - CYP 1A1/2

Alcohol - 2E1 and CYP 2D6

Dietary substances  indole‐3‐carbinol  CYP 1A2

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20
Q

What is Reversible inhibition?

A

Interactions at heme‐iron complex

Lipophilic site on the apoprotein

Ex: norfloxacin, cimetidine, diltiazem, and voriconazole

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21
Q

What is the Metabolite‐P450 complexation?

A

Alkylamine oxidation to nitrosoalkane metabolite

High‐affinity to reduced P450 intermediates of CYP 2B, 2C, and 3A

Essentially an irreversible complexation requiring new synthesis

Must possess dimethylamino sugar and compound is lipophilic

Ex: erythromycin and clarithromycin

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22
Q

What is Mechanism‐based inhibition (“suicide inhibition”)?

A

• Parent drug is not inhibitor of CYP but functional groups on the drug are oxidized and irreversibly binds to the enzyme

Requires new enzyme synthesis

Ex: 17α‐ethinyl estradiol, norethindrone, chloramphenicol, cyclophosamide, and spironolactone

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23
Q

What are General CYP450 Rxns adding oxygen?

A

Aromatic hydroxylation

Aliphatic hydroxylation

N-Oxidation

Sulfoxidation

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24
Q

Aromatic hydroxylation

A
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25
Aliphatic hydroxylation
26
N-Oxidation
27
Sulfoxidation
28
General CYP450 Rxns Cleaving Bonds | (Deamination)
29
General CYP450 Rxns Cleaving Bonds | (O-Dealkylation)
30
General CYP450 Rxns Cleaving Bonds | (N-Dealkylation)
31
Describe Flavin Monooxygenase Metabolism.
Flavin monooxygenase (FMO) * Five isoforms with tissue specificity (FMO1-5) * Expressed in liver (FMO3) and bound to ER * Converts N-/S- lipophilic drugsmore polar molecules • Minor contribution to metabolism * Not prone to induction or inhibition Metabolites are typically benign * Does not produce a lot of drug-drug interactions Mechanism * Catalytic cycle is different from CYP type of reactions • Exists as activated hydroperoxyflavin (FAD-OOH) species * Peroxide reactive species, not radical based * Oxidation reactions, but not dealkylation reactions
32
Describe the FMO mechanism.
Oxygenates sulfur and nitrogen compounds Non-radical nucleophilic displacement * Binding of reduced FMO to molecular oxygen (5) Nucleophilic attack by heteroatom at peroxide (1) * Generates hydroxyflavin and oxidized drug
33
General FMO N-oxidation reactions
34
General FMO N-oxidation reactions
35
General FMO S-oxidation reactions
36
What are Hydrolases?
Carboxyl hydrolases Hydrolyzes esters & amides Found in cytosol and bound to ER Epoxide hydrolases Two forms: – soluble (sEH) and – microsomal (mEH) Hydrolyze epoxides (ex: carbamazepine epoxide)
37
Additional Phase I metabolism
Peroxidases * Hemoproteins utilizing the [FeO]+3 reactive intermediate * Heteroatom oxidation; aromatization of 1,4-dihydropyridines Other monooxygenases • Copper-based – Dopamine b-monooxygenase (mammalian) – Carbon hydroxylation, epoxidation, S-oxidation, and N-dealkylation Iron-based (non-heme) • Various from bacteria and plants
38
Additional Phase I metabolism
Reductases • Disulfides, sulfoxides, N-oxides, nitro, olefins, and carbonyls Dehydrogenases Protons * Alcohol dehydrogenase * Aldehyde dehydrogenase Oxidases • Monoamine and diamine oxidase – 1°, 2°, and 3° amines: – must have a proton on adjacent atom! – Regulation of neurotransmitters – Oxidation to imines, then hydrolysis to an aldehyde – MAO vs. CYP deamination: MAO is selective methylene (CH2)
39
Mechanism: Non-CYP450 hydroxylases
Enzyme • Molybdenum hydroxylases – Xanthine oxidase (oxidoreductase) GOUT • Electron transfer system – Molybdenum (one); – Fe-S complex (two); – FAD (one) • Oxygen incorporated from water – Other hydroxylases (CYP450) use molecular oxygen • Catalytic species Mo(V)-OH
40
Mechanism: Non-CYP450 hydroxylases
Enzyme Molybdenum hydroxylases Different from aldehyde dehydrogenase • Aldehydeoxidase – Metabolizes: aldehydes and azaheterocycles Aza-heterocycles are oxidized 2-hydroxy, 2-amino purine nucleosides are more efficiently metabolized – Does not metabolize: Thia- and oxa-heterocyles – Carbon alpha to nitrogen (generatesalactam) • Known drug substrates: – Zaleplon – Brimonidine – Thioguanine • Known drug inhibitors: – SERMs (Raloxifene) and estradiol
41
Mechanism: Non-CYP450 oxidases
Peroxidases • Cyclooxygenase (COX) – Closely related to CYP450; hemoprotein – Two-step process oxidizes arachidonic acid via H-atom abstraction; forming endoperoxide intermediate – Substrate: arachidonic acid/Product: prostaglandins NAD-specific dehydrogenase enzymes • Alcohol Dehydrogenases (ADH) – Oxidizes: 10 alcohols  aldehydes or 20 alcohols  ketones • Aldehyde Dehydrogenase (ALHD) – Oxidizes: aldehydescarboxylic acids Monoamine Oxidase (MAO) MAO • Oxidative deamination – 10 aminealdehyde + ammonia – 20 aminesaldehyde + primary amine
42
Phase II conjugation auxiliary groups:
43
Phase II conjugation auxiliary groups:
44
Phase II conjugation auxiliary groups:
45
What are the Glucuronic acid basics?
Glucuronidation * Predominant phase II reaction * Increases water solubility * Large supply of glucuronic acid is needed
46
What are the Glucuronic acid: isoforms?
UGT1 and UGT2 Substrates • 1A1: bilirubin, estrogenic steroids • 1A3/1A4: tertiary amines • 2B4: bile acids (6α-hydroxyl) • 2B7: largest number of substrates, morphine, codeine * UGT1: 3-methylcholanthrene, cigarette smoke * UGT2: barbiturates
47
Glucuronic acid: distribution
Location • Endoplasmic reticulum of liver – Facilitates detoxification of phase I (CYP450) metabolites • Epithelial cells of intestines – Substrates face phase II metabolism prior to phase I • Extrahepatic tissues – Kidney, brain, and lung Rearrangement • Acyl conjugates may undergo transesterification
48
Glucuronic acid: Bioactivation
Activity modulation * Most metabolites are inactive or less reactive than the parent substance * Some metabolites have increased activity – Morphine(3-O-gucuronide): Majormetaboliteofmorphine 20-fold greater [plasma] \> morphine Antagonistatopiatereceptors – Morphine(6-O-glucuronide): μ-receptoragonist 2-fold greater [plasma] \> morphine 650-foldgreateractivity\>morphine
49
Glucuronic acid: Toxicity
Toxicity • Drug-acyl glucuronide metabolites may pose toxicity to tissues due to being reactive conjugates ~ biological pH Protein modification Acyl glucuronide can transesterifyproteins forming hapten Drug-protein conjugate can induce an immune response – Result in hypersensitivity, anaphylactic response, or immunotoxicity – Reaction with NSAID class of drugs Cellular/Tissue injury * Problematic when primary route of metabolism is acyl-glucuronide, with patients that have impaired renal function * elimination of acyl-glucuronides prolongs plasma circulation of these reactive (electrophilic) conjugates
50
What are the basics of Sulfate Metabolism?
Minor phase II reaction * Limited sulfate stores * Can produce reactive electrophiles Two types of sulfotransferases • Cytosolic – Phenolic steroids, neurotransmitters, xenobiotics • Membranebound(golgiapparatus) – Glycosaminoglycans, glycoproteins, tyrosinyl groups of peptides – Not typical for xenobiotic metabolism
51
Where are Sulfonation Enzymes located?
Location • Liver, small intestine, brain, kidney, platelets Examples • Intestine distribution is significant for oral administration – First pass: isoproterenol, albuterol, steroids, acetaminophen • Competition for sulfotransferases can enhance bioavailability
52
Basics for Amino acid Reactions:
Enzyme(s) * Acyl CoA synthetase * Transacetylase Donor molecule(s) • CoA; amino acids Acceptor groups • Electrophiles – Carbon (activated CoA thioesters of carboxylic acids)
53
Amino acid: basics
Important for carboxylic acid xenobiotics • Glycine metabolites are less toxic / more readily excreted Carboxylic acids • Metabolism depends on substitution – Branched: resistant to β-oxidation; predominantly undergo glucuronic acid / glycine conjugation – Unbranched: typically do not form conjugates – α-substituted: favor glucuronic acid over glycine conjugation – Benzoic and heteroaromatic: favor glycine conjugation Concentration High [drug] favors glucuronate conjugation Low [drug] favors glycine conjugation
54
CoEnzyme A: basics
Enzyme • Acyl CoA Synthetase Donor molecule • CoA Acceptor groups • Electrophiles – Carbon • activated carboxylic acids
55
CoEnzyme A: basics
Initiation step for amino acid conjugation * Precedes coupling to amino acids Bioactivation * Chiral inversion of 2-arylpropionic acid NSAIDs – Stereoselective thioesterification of inactive R-enantiomer – Unidirectional conversion to active S-enantiomer Toxicity * Reactive, potential non-specific protein modification * Unpredictable allergic reactions
56
Basics of Acetylation Metabolism
Enzyme • Transacetylase Donor molecule • AcetylCoA Acceptor groups • Nucleophiles – Nitrogen (primary
57
Acetylation: basics
Very significant reaction for amines * 1° aliphatic, aromatic, amino acids, hydrazines, sulfonamides * Secondary amines are not acetylated Predominantly takes place in liver * Also in extrahepatic tissues * N-acetyl metabolites are typically non-toxic
58
Acetylation: Toxicity
Slow acetylation phenotype • Prone to drug-induced toxicity because of reduced elimination – Hydralazine and procainamide: lupus erythematous – Isoniazid: peripheral nerve damage – Sulfasalazine: hematologic disorders Fast acetylation phenotype • Rapid build up of toxic metabolites from accelerated metabolism
59
Glutathione: basics
Common conjugation pathway for electropilic species Enzyme • Glutathione S-transferase (GST) Donor molecule • Glutathione – (γ-glutamyl-cysteinyl-glycine) Acceptor groups • Electrophiles – Carbon (unsaturated esters, halogens, epoxides)
60
Glutathione: basics
High intracellular concentration of GSH • Ranges from 2 to 5mm Detoxification of electrophiles * Thiol reacts as a nucleophile * First step in mercapturic acid conjugation GSH: GSTM1 and GSTT1 substrates • GSTM1: – nitrosourea drugs, mustard anticancer drugs • GSTT1: – small organics (solvent, halocarbons, electrophiles)
61
Glutathione: Toxicity
1,2-dihaloalkanes • Thioether metabolite reacts to form epi-sulfonium ion Acetaminophen • CYP450 oxidation -\> N-acetyl p-benzoquinonimine – Depletion of GSH stores when taken at high doses – Prevents metabolism (detoxifying) of other toxic species
62
Mercapturic acids: basics
Enzyme * GST, glutamyl transferase * Cysteinyl glycinase, acetylase Donor molecule • Glutathione Acceptor groups • Electrophiles – Carbon (unsaturated carbonyls, haloalkanes)
63
Mercapturic acids: synthesis
Further metabolism of GSH adduct Cleavage of glutamine, and glycine Final acetylation of terminal amine – Increases solubility
64
O- / N- / S- Methylation: basics
Enzyme * O-Methyltransferases * N-Methyltransferases * S-Methyltransferases Donor molecule • S-adenosylmethionine (SAMe; SAM) Acceptor groups • Nucleophiles – Oxygen (alcohols and phenols) – Nitrogen (amines) – Sulfur (thiols)
65
O-/N-Methylation: isoforms
O-Methyl transferases * Catechol O-MT: meta and para phenol of catecholamines * Hydroxyindole O-MT: N-acetylseratonin, serotonin N-Methyl transferases Phenylethanolamine N-MT: norepinephrine norephedrine Histamine N-MT: inactivates histamine Nicotinamide N-MT: 1° and 2° amines; – Drugs and neurotransmitters

IMCP I (32 decks)

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