Drug Metabolism 1 + 2 + 3 Flashcards
What are the two principal biological functions of drug metabolism and what does drug metabolism lead to?
TWO PRINCIPAL BIOLOGICAL FUNCTIONS OF DRUG METABOLISM ARE
- DRUG DETOXIFICATION
- AID TO DRUG ELIMINATION/REMOVAL
DRUG METABOLISM LEADS TO:
- DRUG INACTIVATION
- H 2O-SOLUBLE DRUG METABOLITES
What does the type, extent and rate of drug metabolism depend on?
- CHEMICAL STRUCTURE OF THE DRUG
- Patient health status and genetic ‘make-up’
> Deactivation or reducing drug bioactivity is achieved by modification/masking of drug chemical groups
> Increasing drug aqueous solubility (increase drug hydrophilicity/ polarity) is undertaken by the introduction of polar groups
What is phase 1 drug metabolism? Where does it occur?
INVOLVES THE ADDITION OF SMALL POLAR FUNCTIONAL GROUP(S) TO THE DRUG MOLECULE --> ALSO MAY INVOLVE MODIFYING OR EXPOSING A DRUG FUNCTIONAL GROUP(S) TO GENERATE A MORE POLAR FUNCTIONAL GROUP(S) IN THE DRUG
- Occurs in the liver
- Metabolism of drugs is catalyzed by hepatic cytochrome P450 (CYP450) enzymes
- CYP450 enzymes comprise of a large grouping of ‘microsomal’ membrane-bound monooxygenases
> Polar functional groups include: carboxylic acid, hydroxy group, amino, thiol (increase drug hydrophilicity)
Phase 1 reaction types include:
- OXIDATION (most are oxidative by nature)
- HYDROXYLATION
- HYDROLYSIS
- REDUCTION
Discuss the properties of cytochrome P450 enzymes. What are the three basic components?
- Commonly referred to as CYP ENZYMES
- Represent a type of MONOOXYGENASE enzyme
- Also function as electron (e-) transport systems
- Perform most Phase 1 ‘oxidative’ metabolism
- Consist of a ‘superfamily’ of HAEM-based enzymes
- HAEM is an IRON [III] (Fe3+, Ferric cation) PORPHYRIN based catalytic centre in the CYP active site
CYP450 enzymes contain three basic components:
- CYTOCHROME PROTEIN (CYP) - Haem-based OXYGEN BINDER
- FLAVOPROTEIN - NADPH-CYP450 REDUCTASE (CYPR) that acts as an ELECTRON CARRIER from NADPH (bioreductant) to CYP
- PHOSPHOLIPID - PHOSPHATIDYL CHOLINE that aids electron (e-) transfer from the flavoprotein to CYP
Discuss the haem center of the cytochrome P450 enzyme
- Fe3+ CENTRE OF THE HAEM UNIT IS REDUCED TO Fe2+ (Ferrous cation) BY NADPH/REDUCTASE (Fe3+ gains e-)
- Fe2+ CENTRE BINDS TO AN OXYGEN (O2) MOLECULE
- BOUND O2 IS USED FOR THE ‘MONOOXYGENATION’ REACTION OF THE DRUG ‘SUBSTRATE’ MOLECULE
- CYP oxidative process is a highly complex catalytic cycle but can be simply expressed as follows:
Discuss CYP enzymes
CYP1A2
1 = family and A = sub family and 2 = gene
- CYP ENZYMES GENERALLY BIND ‘NON-SPECIFICALLY’ TO LIPOPHILIC COMPOUNDS (i.e. MOST DRUGS) VIA HYDROPHOBIC BINDING INTERACTIONS
- Almost 30% of drugs metabolized by CYP3A4
- CYP3A4 IS THE MOST ABUNDANT hepatic CYP enzyme (~ ⅓ ) and extrahepatic CYP enzyme (intestines, ~ ⅔ )
- In the GIT it accounts for poor drug oral bioavailability
- CYP enzymes exhibit POLYMORPHISM
- Polymorphism results in the differing capability of certain PHENOTYPES (sub-groups) within the general population to perform the metabolism of some drugs
- Knowledge of polymorphic enzymes and phenotypes is key to PHARMACOGENETICS i.e. study of how gene variations determines drug efficacy and side-effects
What is the result of polymorphic enzymes?
FOUR ‘nonstandard’ phenotypes (patient types) can exist:
- POOR METABOLISERS (PM): possess less enzyme or an enzyme form which has reduced catalytic activity
- NULL METABOLISERS (NM): possess no enzyme or an enzyme form with no catalytic activity
- EXTENSIVE METABOLISERS (EM): possess more enzyme or an enzyme form with increased catalytic activity
- ULTRA-RAPID METABOLISERS (UM): possess much more enzyme or a form with tremendous activity
> For certain drugs PM/NM phenotypes may experience SLOW/DECREASED/ZERO metabolism i.e. decreased or no drug clearance –> ADVERSE EFFECT (overdose) or TOXICITY
> For certain drugs EM/UM phenotypes may experience RAPID/INCREASED metabolism i.e. increase drug clearance –> THERAPEUTIC FAILURE (decreased or lack of drug efficacy)
What can CYP enzymes can be INDUCED by various substances i.e. enzyme activity or its biosynthesis can be stimulated or increased?
CYP enzymes can be INDUCED by various substances i.e. enzyme activity or its biosynthesis can be stimulated or increased
- CYP INDUCERS include various drugs (e.g. Barbiturates), cigarette smoke and herbals (e.g. St John’s Wort)
- CYP INHIBITORS include various drugs (e.g. Antifungals, Statins), caffeine and grapefruit juice
ABOVE EFFECTS MAY ACCOUNT FOR THE MAJOR TYPES OF DRUG INTERACTIONS - NAMELY DRUG-DRUG, FOOD-DRUG + HERBAL-DRUG INTERACTION
> EXISTENCE OF VARIOUS DRUG INTERACTIONS AND POLYMORPHIC ENZYMES SHOULD BE ACCOUNTED FOR IN THE USE/PRESCRIBING OF ALL DRUGS
What are the types of phase 1 metabolism? What drugs and chemical group types does it occur in?
Phase 1 metabolism:
- OXIDATION
- HYDROXYLATION
- DEALKYLATION
- DEAMINATION
- DEHALOGENATION
- HYDROLYSIS
- REDUCTION
Mostly occurs to lipophilic drugs with the below compound/chemical group types (see attached image)
Discuss the oxidation of aliphatic groups
- Aliphatic/alkyl groups are uncommon metabolic sites
- Typically undergo slow hydroxylation and often only metabolised in the absence of other groups
- After hydroxylation, the alkyl group is often further oxidised to a carbonyl or carboxylic acid group
- Methyl (Me) unit is the most prone to metabolism but if >1 is present only one is hydroxylated
- Alkyl group is metabolised to hydroxyalkyl and may be further oxidised to -CO2H by a non-CYP enzyme
- Large alkyl groups usually undergo hydroxylation at the terminal C-atom ( Oxidation) or at the penultimate C-atom (-1 Oxidation)
- Alicyclic groups are usually hydroxylated at the least hindered methylene (-CH2-) group
- Non-aromatic heterocycles undergo oxidation at the C-atom adjacent to the heteroatom i.e. the α C-ATOM
- Benzylic group is prone to oxidation at the methylene C-atom (-CH2-) - if NOT adjacent to a heteroatom:
- In addition to above C-hydroxylations, aliphatics may be converted to alkenes by dehydrogenation –> Involve complex reactions catalysed by CYP3A4 via carbon radical and/or carbocation intermediates
Discuss oxidation of alkene/alkyne groups
- Alkenyl groups are normally oxidised to intermediary EPOXIDE metabolites of variable stability
- Unstable epoxide may undergo enzymatic or chemical hydrolysis to give dihydrodiol metabolites
- Alkyne groups are readily oxidised and form various products (dependent on which C-atom is attacked)
- Initially form unstable ketenes that can hydrolyse (to CO2H) or damage nearby proteins by alkylation
Discuss the oxidation of aromatic groups
- Aromatics undergo RING HYDROXYLATION via highly reactive ARENE OXIDE (EPOXIDE) intermediates
- ARENE OXIDES REARRANGE TO FORM PHENOLS
- May also react with water, glutathione (GSH) or cell biomolecules (e.g. DNA) causing damage/toxicity
- Hydroxylation is influenced by ring substituents:
> SUBSTITUENT TYPE ( e-donating/withdrawing)
> SUBSTITUENT SIZE (steric factor)
> NUMBER OF SUBSTITUENTS (steric factor)
- NORMALLY HYDROXYLATES AT THE LEAST HINDERED AND MOST ‘ELECTRON DENSE’ RING C-ATOM
- para-HYDROXYLATION IS THE MOST COMMON
- If more than one aromatic group is present normally only one is hydroxylated
- HETEROAROMATIC ring systems may also undergo RING OXIDATION/HYDROXYLATION via CYP enzymes
Discuss oxidation at α-C to heteroatoms
INVOLVES OXIDATION (HYDROXYLATION) AT AN α-C ATOM ADJACENT TO OXYGEN, NITROGEN OR SULFUR
Occurs mostly in the following groups
- ETHERS
- THIOETHERS
- AMINES
Overall process can lead to drug molecule:
- N-DEALKYLATION
- OXIDATIVE DEAMINATION
- O-DEALKYLATION
- S-DEALKYLATION
α-C ATOM OXIDATION INVOLVES THE FORMATION OF AN UNSTABLE HYDROXY INTERMEDIATE THAT DECOMPOSES TO YIELD POLAR METABOLITES
See attached image for dealkylation general mechanism
Discuss heteroatom oxidation
- DEALKYLATION PROCESSES OFTEN COMPETE WITH HETEROATOM (X = N AND S ONLY) OXIDATION IN AMINES, AMIDES, AND THIOETHERS
- HETEROATOM OXIDATION IS BIOCATALYSED BY CYPs AND FLAVIN MONOXYGENASES (FMO)
- FMOs are relatively similar to CYP enzymes i.e. they require oxygen (O2) and NADPH substrates
- N-OXIDATION via FMO occurs mostly in tertiary AMINES (ACYCLIC, CYCLIC and AROMATIC) to give N-OXIDES
- CYP/FMO N-Oxidation only occurs in primary/secondary amines if α-H is absent to generate a variety of hydroxylamine, nitroso, imine, nitrone and/or oxime metabolites
- Thioethers can be FMO S-oxidised to sulfoxides and usually then further oxidised to give sulfones
Discuss paracetamol N-Hydroxylation
- REPRESENTS AN IMPORTANT EXAMPLE OF AN ‘NOXIDATIVE METABOLISM OF AN AMIDE
- Catalysed mostly by CYP2E1 and NOT FMO
- GENERATES AN UNSTABLE N-HYDROXYLAMIDE THAT DEHYDRATES TO A VERY REACTIVE N-ACETYL-paraBENZOQUINONE IMINE = NAPQI (reactive quinonimine)
- NAPQI IS AN ELECTROPHILIC METABOLIC SPECIES
- NAPQI can react with H2O giving a diphenolic catechol and then eliminated via PHASE 2 CONJUGATION
- NAPQI USUALLY REACTS WITH GLUTATHIONE (GSH) TO FORM H2O-SOLUBLE METABOLITES - THIS REPRESENTS A MAJOR NAPQI DETOXIFICATION PATHWAY
- BUT LIMITED IN VIVO RESERVES OF ENDOGENOUS GSH EXIST THAT CAN BE PROBLEMATIC IN OVERDOSE CASES
- IN THE ABSENCE OF THE GSH THE NAPQI MAY REACT WITH NEARBY ‘NUCLEOPHILIC’ CELLULAR MACROMOLECULES (e.g. PROTEINS) i.e. REACT WITH VITAL CELL APPARATUS
- ‘DAMAGING’ BIOMOLECULAR NAPQI REACTIONS MAY INDUCE CYTOTOXICITY LEADING TO HEPATIC CELL DEATH
- LARGE DRUG OVERDOSE CAN LEAD TO DEPLETION OF GSH AND UNCHECKED HEPATOCYTE NECROSIS
- THIS MAY IN TURN RESULT IN SEVERE LIVER DAMAGE (NECROSIS/CIRRHOSIS) AND POSSIBLY DEATH
Discuss oxidative deamination by mao (monoamine oxidase)
- INVOLVES THE OXIDATIVE REMOVAL OF AN ENTIRE AMINO GROUP BY MONOAMINE OXIDASE (MAO)
- MAO is a flavin-based mitochondrial enzyme
- In the presence of oxygen MAO enzyme catalyses the oxidative deamination of amines to give aldehydes
- MAO usually only deaminates secondary and tertiary amines which possess an adjacent CH2 methylene group(s
- MAO is mostly involved in metabolism of endogenous amino substrates (e.g. dopamine, serotonin, epinephrine)
- MOST DRUGS ARE DEAMINATED BY CYP ENZYMES
Discuss metabolism of carbonyl groups
- ALDEHYDES AND KETONES MAY UNDERGO IN VIVO OXIDATIVE AND REDUCTIVE METABOLISM
- ALDEHYDE DEHYDROGENASES (ALDHs) CATALYSE THE OXIDATION OF ENDOGENOUS ALDEHYDES MOSTLY AND THE METABOLIC PRODUCTS OF ALCOHOL OXIDATION
- ALDEHYDES UNDERGO OXIDATION TO GENERATE THE CORRESPONDING CARBOXYLIC ACIDS
- REDUCTIVE METABOLISM OF CARBONYL GROUPS TO THEIR CORRESPONDING ALCOHOLS MAY ALSO OCCUR - MOSTLY BY CARBONYL REDUCTASES
What occurs during dehalogenation?
- MANY HALOGENATED DRUGS TYPICALLY UNDERGO OXIDATIVE DEHYDROHALOGENATION
- Various reactive intermediates and metabolites can be generated e.g. radicals, aldehydes, acyl halides
- Process is catalysed by CYP enzymes (e.g. CYP2E1)
- Oxidative dehydrohalogenation requires the presence of at LEAST ONE HALOGEN AND ONE a-HYDROGEN
- BIOACTIVITY OF REACTIVE ACYL HALIDE METABOLITES CAN LEAD TO HEPATO- AND NEPHROTOXICITY
- Acyl halides may also act as HAPTENS which can trigger an immune response and hypersensitivity
- his is common with FLUORINATED ANESTHETICS - patients may become sensitised to future exposure for these drugs and develop a hepatitis-like condition
- Fluoride (F-) formed may also induce NEPHROTOXICITY
Discuss azo and nitro bioreduction
- AZO compounds are bioreduced to 1 AMINES by hepatic NADPH-dependent AZOREDUCTASE
- NITRO compounds are bioreduced to 1 AMINES (via nitrosamines) by NITROREDUCTASE
- Bioreduction is also known to occur in bacterial flora within the intestines
Discuss hydrolysis
- MAJORITY OF ESTERS, PHOSPHATES, CARBAMATES AND AMIDES ARE HYDROLYSED IN VIVO BY A VARIETY OF ‘UBIQUITOUS’ ESTERASE TYPE ENZYMES
- Found in the liver, GIT, kidney, plasma and skin
- HYDROLYSIS USUALLY RESULTS IN THE FORMATION OF INACTIVE POLAR (HYDROPHILIC) METABOLITES
- NUMEROUS ESTER PRODRUGS RELY ON IN VIVO BIOACTIVATION BY ESTERASE HYDROLYSIS
- Sterically hindered esters are more stable (e.g. Atropine)
- Phosphate ester prodrugs are rapidly bioactivated by phosphatase hydrolysis
- Normally AMIDES ARE MORE STABLE to hydrolysis than esters and are largely UNMETABOLISED
- But peptide amide groups within peptide and protein drugs are rapidly degraded orally by enzymatic (peptidases) and chemical (gastric acid) hydrolysis
What is phase 2 metabolism? What polar groups does it require the presence of?
- PHASE 2 METABOLISM USUALLY INVOLVES ADDING A WATER-SOLUBLE UNIT TO THE DRUG MOLECULE OR MORE COMMONLY TO A PHASE 1 DRUG METABOLITE
- INVOLVES A VARIETY OF CONJUGATION PROCESSES
- USUALLY CONVERTS A POLAR DRUG OR PHASE 1 DRUG METABOLITE TO A VERY H 2O-SOLUBLE METABOLITE
- TYPICALLY INVOLVES THE ADDITION OF H 2O-SOLUBLE GROUP TO A DRUG OR PHASE 1 DRUG METABOLITE TO GIVE AN ‘INACTIVE CONJUGATED PRODUCT’
- PHASE 2 (P2) DRUG METABOLISM ULTIMATELY LEADS TO DRUG DETOXIFICATION
- P2 REQUIRES THE PRESENCE OF POLAR GROUPS i.e. carboxylic acid, amino, hydroxyl group, thiol
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What metabolic processes are there for phase 2 metabolism?
- Necessitates a variety of metabolic ‘CONJUGATING REAGENTS’ and enzymes - mostly TRANSFERASES
- Include the following metabolic processes
> GLUCURONIC ACID CONJUGATION
> SULFATE CONJUGATION
> GLUTATHIONE/MERCAPTOPURIC ACID CONJUGATION
> AMINO ACID CONJUGATION
> ACETYLATION
> METHYLATION
What is glucuronic acid conjugations?
- ALSO KNOWN AS GLUCURONIDATION
- MOST IMPORTANT PHASE 2 METABOLIC ROUTE
- Occurs to wide variety of DRUGS and PHASE 1 DRUG METABOLITES which possess the below groups:
> CARBOXYLIC ACID
> HYDROXY GROUP (ALCOHOL OR PHENOL)
> AMINO
> THIOLS
- GENERATES A HIGHLY H 2O-SOLUBLE GLUCURONIDE METABOLITE i.e. HIGHLY EXCRETABLE (in urine)
- Relies on a readily available in vivo supply of hepatic GLUCURONIC ACID (GA)
- INVOLVES THE ENZYMATIC CONJUGATION OF A DRUG OR A PHASE 1 DRUG METABOLITE WITH AN ACTIVE FORM OF GA
- ACTIVE FORM OF GA IS URIDINE DIPHOSPHATE GLUCURONIC ACID (Glucuronate) = UDPGA
- UDPGA = GA TRANSFER/CONJUGATION REAGENT
- Derived from GLUCOSE and URIDINE TRIPHOSPHATE
- GLUCURONIDATION CATALYSED BY TWO FAMILIES OF UDP-GLUCURONOSYL TRANSFERASES (UGTs)
- UGTs are found in the LIVER and intestines mostly
- Generally glucoronidation involves (see attached image)
Discuss sulfate conjugation
- ALSO KNOWN AS SULFATION
- AN IMPORTANT PHASE 2 METABOLIC PROCESS FOR PHENOLS MOSTLY AND ALCOHOLS
- PROCESS GENERATES A VERY H2O-SOLUBLE SULFATE METABOLITE i.e. HIGHLY EXCRETABLE (in urine)
- INVOLVES THE ENZYMATIC CONJUGATION OF A DRUG OR PHASE 1 DRUG METABOLITE WITH AN ACTIVATED SULFATE BIOREAGENT
- ACTIVATED SULFATE IS 3-PHOSPHOADENOSINE-5’- PHOSPHOSULFATE = PAPS
- PAPS = SULFATE TRANSFER REAGENT
- PAPS generated from INORGANIC SULFATE (SO42-) and ADENOSINE TRIPHOPSHATE (ATP)
- Relies on the limited in vivo supply of SULFATE
- SULFATION competes with GLUCURONIDATION
- SULFATION IS CATALYSED BY THREE FAMILIES OF SULFOTRANSFERASES (SULTs)
- SULTs are found in the LIVER, intestines and kidneys
Discuss glutathione/mercaptopuric acid conjugation
- SIGNIFICANT DETOXIFICATION ROUTE IN VIVO FOR ELECTROPHILIC COMPOUNDS AND METABOLITES
- INVOLVES GLUTATHIONE (GSH) - A ‘NUCLEOPHILIC’ TRIPEPTIDE (γ-Glutamylcysteinylglycine)
- GSH REACTS (CONJUGATES) WITH ELECTROPHILES AND REDUCES THEIR REACTIVITY/TOXICITY
- GSH CONJUGATION IS ENZYMATICALLY CATALYSED BY GLUTATHIONE-S-TRANSFERASE
- GSH DRUG CONJUGATES MAY UNDERGO ENZYMATIC HYDROLYTIC DEGRADATION AND ACETYLATION TO YIELD H2O-SOLUBLE MERCAPTOPURIC ACIDS
Discuss amino acid conjugation
- IMPORTANT PHASE 2 METABOLIC PROCESS FOR LIPOPHILIC CARBOXYLIC ACIDS AND INVOLVES CONJUGATION WITH SIMPLE AMINO ACIDS
- COMMONLY INVOLVES USE OF GLYCINE BUT ALSO GLUTAMINE AND TAURINE (a Cysteine derivative)
- INVOLVES A TWO STAGE DETOXIFICATION PROCESS WHICH YIELDS A H2O-SOLUBLE AMIDE CONJUGAT
- REQUIRES INITIAL ENZYME CATALYSED ACTIVATION OF CO2H VIA ITS COENZYME A (CoA) THIOESTER
Discuss acetylation
- COMMON PHASE 2 PROCESS FOR primary AMINES, AMINO ACIDS, SULFONAMIDES AND HYDRAZINES
- INVOLVES THE ENZYMATIC ACETYLATION OF A DRUG OR PHASE 1 DRUG METABOLITE USING ACETYL COENZYME A (ACETYL CoA)
- DETOXIFICATION PROCESS WHICH YIELDS POORLY H2O-SOLUBLE ACETYLATED METABOLITES
- ENZYMATICALLY CATALYSED BY TWO FAMILIES OF HEPATIC N-ACETYL TRANSFERASES (NATs)
- NATs are polymorphic with TWO phenotypes known as FAST and SLOW ACETYLATORS
Discuss methylation
- ‘MINOR’ PHASE 2 METABOLIC PROCESS - MOSTLY FOR ENDOGENOUS PHENOLS, AMINES AND THIOLS
- DETOXIFICATION PROCESS WHICH YIELDS POORLY H2O-SOLUBLE METHYLATED METABOLITES
- INVOLVES AN ENZYMATIC METHYLATION WITH AN ACTIVATED METHYLATING BIOREAGENT = SAM
- SAM = S-ADENOSYLMETHIONINE = METHYL (Me) TRANSFER REAGENT from METHIONINE and ATP
- METHYLATION catalysed by METHYL TRANSFERASES
