Drug Biotransformation I and II Flashcards
What is biotransformation?
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
What factors influence metabolism?
Genetic
Physiologic
Pharmacodynamic
Environmental
What is the difference between Phase I and Phase II metabolism?
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.
What type of enzymes are in Phase I metabolism?
Oxygenases
What type of enzymes are in Phase II metabolism?
Transferases
List the Pase I enzymes and their reactions.
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
List the Phase II enzymes and their reactions.
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
What are the additional metabolism enzymes and their reactions?
Alcohol dehydrogenases - reduction of alcohols
Aldehyde dehydrogenases - reduction of aldehydes
NADPH-quinone oxidoreductases (NOO) - reduction of quinones
What are the characteristics of the Phase I superfamily Cytochrome P450 (CYP450)?
Bound to smooth ER
Major metabolic enzyme class
Binds substrate, then molecular oxygen
What are the characteristics of the Phase I superfamily Flavin Monooxygenases (FMO)?
Bound to smooth ER
Minor metabolic enzyme class
Binds/activates molecular oxygen, then binds substrate
What are the characteristics of the Phase I superfamily Hydrolases?
Bound to smooth ER and free in cytosol
Minor metabolic enzyme class
True or false: Phase I metabolism must come before Phase II reactions.
False
Where are the CYPs found?
Found in liver and extrahepatic tissue
• Bound to smooth endoplasmic reticulum
– Microsomal fraction: homogenized tissue contains smooth ER
CYPs have an absolute requirement for what?
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
What are CYP450 Isoforms?
- 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
Explain CYP450 Isoform specificity.
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
What are the most common CYP Isoforms?
3A4/3A5 and 2D6
Types of Interactions with CYP450 Isoforms:
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
What are common CYP inducers?
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
What is Reversible inhibition?
Interactions at heme‐iron complex
Lipophilic site on the apoprotein
Ex: norfloxacin, cimetidine, diltiazem, and voriconazole
What is the Metabolite‐P450 complexation?
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
What is Mechanism‐based inhibition (“suicide inhibition”)?
• 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
What are General CYP450 Rxns adding oxygen?
Aromatic hydroxylation
Aliphatic hydroxylation
N-Oxidation
Sulfoxidation
Aromatic hydroxylation
Aliphatic hydroxylation
N-Oxidation
Sulfoxidation
General CYP450 Rxns Cleaving Bonds
(Deamination)
General CYP450 Rxns Cleaving Bonds
(O-Dealkylation)
General CYP450 Rxns Cleaving Bonds
(N-Dealkylation)
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 drugsmore 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
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
General FMO N-oxidation reactions
General FMO N-oxidation reactions
General FMO S-oxidation reactions
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)
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
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)
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
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
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: aldehydescarboxylic acids
Monoamine Oxidase (MAO)
MAO
• Oxidative deamination
– 10 aminealdehyde + ammonia
– 20 aminesaldehyde + primary amine
Phase II conjugation auxiliary groups:
Phase II conjugation auxiliary groups:
Phase II conjugation auxiliary groups:
What are the Glucuronic acid basics?
Glucuronidation
- Predominant phase II reaction
- Increases water solubility
- Large supply of glucuronic acid is needed
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
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
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
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 transesterifyproteins 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
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
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
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)
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
CoEnzyme A: basics
Enzyme
• Acyl CoA Synthetase
Donor molecule
• CoA
Acceptor groups
• Electrophiles
– Carbon
• activated carboxylic acids
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
Basics of Acetylation Metabolism
Enzyme
• Transacetylase
Donor molecule
• AcetylCoA
Acceptor groups
• Nucleophiles
– Nitrogen (primary
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
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
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)
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)
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
Mercapturic acids: basics
Enzyme
- GST, glutamyl transferase
- Cysteinyl glycinase, acetylase
Donor molecule
• Glutathione
Acceptor groups
• Electrophiles
– Carbon (unsaturated carbonyls, haloalkanes)
Mercapturic acids: synthesis
Further metabolism of GSH adduct
Cleavage of glutamine, and glycine
Final acetylation of terminal amine
– Increases solubility
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)
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
