Week 4 - HPLC and Drug Metabolism Flashcards
What is biotransformation?
conversion of free drug into metabolites or excreted product
What does ADME stand for?
Absorption
Distribution
Metabolism – anabolism, catabolism
Excretion
What part of ADME is drug elimination
Metabolism – anabolism, catabolism
Excretion
WHY IS THE STUDY OF DRUG METABOLISM &
ELIMINATION IMPORTANT?
- The route of metabolism of a drug can determine its ultimate
pharmacological or toxicological activity - May impact on dose and frequency
Where does a lot of the biotransformation processes happen?
liver
What is anabolism?
adding components
What is catabolism?
breaking down
Characteristics of intravenous drug administration?
- active straight away
- likely to ass liver/kidney
What is the first pass effect?
first thing that happens to drug which is metabolism of drug occurring before entering the systemic circulation
What is the drug metabolised by in the first pass effect?
- Enzymes in GIT (also not all will be absorbed)
- Liver
What are the consequences of the first pass effect?
means that there is not 100% bioavailability of the drug and less than 100% of drug enters the circulatory system
- therefore we need more of the drug
What type of variation in humans affect drug absorption?
metabolic rate and enzyme rate
drugs can also be slowed down when intestinal blood flows changes - during eating etc
having two different drugs at the same time can also cause incomplete drug absorption
How does body remove polar/hydrophilic drugs from blood?
urine and faeces
What needs to be done to non-polar and lipophilic drugs?
transform in liver
What are the characteristics of most drugs?
they are non-polar and lipophilic
Where is the primary location where drug metabolism occurs?
Primarily in the liver but can occur in all organs
What is metabolism?
process of converting chemicals to more
polar metabolites
What happens to the polarity of drugs/metabolites after phase 1 and 2?
polarity increases
What are the two phases of drug metabolism?
phase 1 and 2
What is the purpose of phase one?
activation of the drug - to make it more active
How does the drug become more active in phase 1?
undergoes
oxidation
hydroxylation
dealkylation
deamination
hydrolysis
What is formed after phase 1?
a derivative
What is a derivative?
a compound that is derived from a similar compound by a chemical reaction.
What is the purpose of phase 2?
to form a conjugate from a derivative through conjugation
What is a conjugate?
A compound formed by chemically joining two or more different substances
What is conjugation?
The process of covalently linking drugs to various natural or synthetic molecule carriers for specific applications
What does oxidation do to drug molecules?
increases water solubility
introduces functional group to make it easier to metabolise
What is an electrophile?
electron acceptor
What is a nucleophile?
provide electron pair to get new covalent bond
What process in phase one produces electrophiles?
oxidation of lipophilic molecules
What process in phase one produces nucleophiles?
hydrolysis and reduction
What is the purpose of phase 1 and 2?
convert lipophilic molecules into hydrophilic
What do electrophiles produced from phase 1 undergo?
glutathione conjugation to produce hydrophilic molecule
What do nucleophiles produced from phase 2 undergo?
sulfation
acetylation
glucuronidation
What type of reactions occur in phase 1?
reactions are catabolic
- Oxidation –most important, catalysed by cytochrome P450 enzymes
- Reduction
- Hydrolysis
Where is P450 from?
liver
- lots of genetic variation in cytochrome - which influences drug metabolism
What is a feature of the products produced from phase one?
metabolites can be more toxic or carcinogenic than parent drug
- this is when they need extra metabolic processes
What occurs in phase one?
introduction of reactive group
- known as a process of functionalisation
- group acts as a point of attack for conjugating system
What type of reactions occur in phase two?
synthetic/anabolic
What does phase two reactions involve?
conjugation - attachment of substituent group
- Glucuronic acid
- Sulphate
- Amino acids
- Glutathione
- Acetylation
What is the aim of phase 2?
to activate end product
What are the exceptions of phase 2?
exceptions -active sulphate metabolite of minoxidil (K+channel activator)
Where does phase 2 mainly take place?
in the liver
- If drug molecule or Phase 1 product has a suitable ‘handle’, it is susceptible
to conjugation - Handle -hydroxyl, thiol or amino group
What type of drugs are more easily transported?
lipophilic are more easily transported than hydrophilic
Why are polar molecules a problem?
they need to be transported actively rather than passively
What organ is important in phase 1?
liver
Where are drug metabolising enzymes located?
embedded in the smooth ER
What type of enzymes are embedded?
microsomal enzymes
What happens to the drug to interact with these enzymes?
crosses the plasma membrane
What happens to polar drugs?
Polar molecules move less readily unless transport system – polar drugs partly
excreted unchanged in urine
What does CYP450 use as cofactor?
heme as a cofactor in their catalytic activity
What phase is CYP more involved in?
phase 1 of metabolism
Characteristics of CYP450
Superfamily
Differ in sensitivity to inhibition/induction and specificity of reaction they
catalyse
Distinct but overlapping substrate specificities
MIXED-FUNCTION OXIDASE REACTION
(CYTOCHROME P450; CYP) EQUATION
NADPH + O2 + RH NADP+ + H2O + ROH
Where is P450 found in?
endoplasmic rectilium
liver, kidney, lungs, intestine
What does a mixed-function oxidase reaction require?
substrate, enzyme, molecular oxygen
What is a mixed-function oxidase reaction catalysed by?
- Cytochrome P450
- NADPH-cytochrome P450 reductase
What is an example of species differences of P450?
rats and humans developed colon cancer but not monkeys when eating bbq food
- dietary amines and genotoxic products
What are characteristics of P450 expression?
polymorphism
environment – enzyme inducers and inhibitors e.g. grapefruit
juice inhibits enzymes, brussels sprouts induce P45. Clinically
important
CYP2D6 : GENOTYPE/PHENOTYPE VARIABILITY
poor metabolizer
– little or no CYP2D6 function
intermediate metabolizers
– metabolize drugs at a rate somewhere
between the poor and extensive metabolizers
extensive metabolizer
– normal CYP2D6 function
ultrarapid metabolizer
– multiple copies of the CYP2D6 gene are expressed, so greater-than-normal CYP2D6 function occurs
Is P450 the only enzyme involved in drug metabolism?
no there are others
- Suxamethonium hydrolysed by plasma cholinesterase
- Ethanol metabolised by alcohol dehydrogenase + CYP2E1
- Xanthine oxidase inactivates 6-mercaptopurine
- MAO – inactivates biologically active amines – e.g. NA, tyramine, 5-HT
Hydrolysis –
Ester and amide (less readily) bonds susceptible to hydrolytic cleavage
Warfarin – reduction of ketone to OH by CYP2A6
PHASE II REACTIONS
Insertion of a chemical group
Glucuronyl
Sulphate
Methyl
Acetyl
What happens if there is an enzyme that is missing?
its find because other enzymes have different functional groups that can make up for it
What drugs create the R -OH glucuronide conjugation?
paracetamol, aspirin, morphine
What drugs create the R- COOH glucuronide conjugation?
clofibrate, nicotinic acid
What drugs create the R- NH2 glucuronide conjugation?
dapsone, sulphafurazole, meprobamate
What drugs create the R- SH glucuronide conjugation?
2-mercapto-benzothiazole
What are the endogenous substrates of glucoronide conjugation?
bilirubin, steroid hormones, thyroxine and catechols
Characteristic of glucose
energy rich donor
- replacement of CH for H molecule of carbon 6
What are the two molecules glucose is turned into?
glycolysis; glycogenesis
glucuronic acid
What is the enzyme required when converting glucose into other molecules?
UDP glucuronosyl transferases
GLUTATHIONE (GSH) CONJUGATION:
GLUTATHIONE S-TRANSFERASES - DOES IT REQUIRE ACTIVATION?
no
- they are electrophilic substrates
Why is steroselectivity important?
Clinically important drugs e.g. warfarin, cyclophosphamide – mixtures of
stereoisomers
Different pharmacological effects
Differences in metabolism & pathways
Toxicity – linked to one stereoisomer – impact on new drugs
Glutathione (GSH) conjugation - What happens to the conjugates?
conjugates can either be excreted or processed further
What base is P450?
haem based
What can enzyme induction increase?
drug toxicity or carcinogenicity
What do phase one drugs tend to be?
reactive - toxic - carcinogenic
How can we exploit the fact that phase 1 drugs tend to be reactive?
sometimes we need very reactive phase on metabolites
Can some drugs act as inhibitors?
yes
What type of inhibition can P450 do?
competitive inhibition
non-competitive inhibition
mechanism-based
P450 - Competitive Inhibition Example
eg. quinidine - not substrate
P450 Non-Competitive Inhibition
- reversible
- eg. ketoconazole
- complex with Fe 3+ from haem iron of CYP3A4
P450 Mechanism-Based
- requires oxidation by P450 enzyme
eg. oral contraceptive gestodene - oxidation product (epoxide intermediate of gestodene) binds covalently enzyme which destroy itself - suicide inhibiton
Microsomal enzyme induction
Rifampicin, ethanol, carbamazepine - incr. activity of enzymes with repeated
admin
Carcinogenic chemicals have this effect e.g. benzypyrene
Can increase drug tox/carcinogenic can be exploited therapeutically
ACTIVE DRUG METABOLITES
Pro-drugs
?deliberate design – drug delivery
e.g. aspirin inhibs platelet function and has anti-inflammatory activity but
hydrolysed to salicylic acid (anti-inflammatory not anti-platelet)
Metabolites may have similar effects to parent compound e.g.
benzodiazepines
toxicity eg. methanol and ethylene glycol
How can other drugs interact with other drugs?
Slow onset of induction
Slow recovery,
Selective induction
it may affect how fast drug is metabolised and increase tolerance
What can enzyme inhibition in the context of drugs?
Slow metabolism, increases action of drugs normally inactivated by enzyme
What is a consequence of inhibiton?
therapeutic effects
- eg. disulfiram (aldehyde dehydrogenase inhib)
-> produces adverse reaction to ethanol, also inhibits metabolism of other drugs
What does inhibition to the conversion of a prodrug to active metabolite result in?
loss of activity
eg. proton inhibitors (such as omeprazole) and anti platelet drug clopidogrel which is widely co-prescribed
What do liver cells do?
transports substances from plasma to bile using transport system
eg. organic cation transporters (OCTs), organic anion transporters (OATs) and P-gylcoproteins (P-gps)
Enterohepatic circulation steps
hydrophilic drug conjugates (esp glucuronides) concentrated in bile and delivered to the intestine
glucuronide can be hydrolysed, regenerating active drug;
free drug
reabsorbed
cycle repeated - reservoir of recirculating drug
eg morphine
Renal clearance
volume of plasma containing the amount of substance that is removed from the body by kidneys in unit time
What are the three processes that account for renal drug excretion?
- glomerular filtration
- active tubular secretion
- passive reabsorption
- diffusion from the concentrated tubular fluid back across tubular epithelium
What are the mechanisms by which one drug can affect the rate of renal excretion of another are:
altering protein binding (hence filtration)
inhibiting tubular secretion
altering urine flow and/or urine pH
INDIVIDUAL VARIATION
Ethnicity – ethanol, propranolol
Age - hepatic microsomal enzyme activity declines
Pregnancy - Cardiac output increases - increased renal blood flow and
GFR, increased renal elimination of drugs
Disease
Drug interaction
Pharmokinetics interaction
GENETIC VARIATION
Plasma cholinesterase deficiency – suxamethonium
Drug acetylation deficiency
Drug targeting
What does the entroepatic circulation transport?
different conjugates
