metabolism

Question 1

what are adverse drug reactions

Answer 1

any undesired or unintended effect of a drug beyond its anticipated therapeutic effect

does not include therapeutic failures, poisoning, accidental or intentional overdoses

most adverse reactions can be predicted on the basis of the pharmacological actions of the drug and are dose dependent resulting from exceeding minimum toxic levels of a drug, they are therefore readily reversible by reducing the dose or withdrawing treatment with the drug

can occur in up to 15% of all drug administrations, they account for about 6-7% of hospital admissions in the uk

Question 2

how might genetics play a role in adverse reactions, what is another factor that may contribute to an adverse reaction

Answer 2

the cause of an ADR could be pharmacogenomic where polymorphisms in drug metabolising enzymes create poor metabolisers who experience accumulation of parent drug to toxic concentrations in the body

polymorphisms in phase 1 CYP450 enzymes and phase 2 conjugating enzymes can cause ADRs, in these cases an individual patient effectively experiences higher doses of a drug than required at its site of action, and exceeds minimum toxic concentration

in some cases ultrarapid metabolisers may convert too much of a prodrug or less active drug into the potent active form e.g codeine to morphine

also possible due to interactions between drugs and foodstuffs or other xenobiotics, where one drug inhibits or induces expression of drug metabolising enzymes, effecting the bioavailability of the other drug

Question 3

what is bioavailability

Answer 3

is the proportion of an orally administered drug that is absorbed across the GI tract which is then distributed in the systemic circulation and survives metabolism and excretion to be able to exert effects at the site of drug action

it is determined by ADME, bioavailability differs between different routes of administration such as between orally and intravenously

Question 4

what is the therapeutic range

Answer 4

the range between the MEC and MTC

the MEC is the minimum effective concentration, the concentration required to produce desired pharmacological effect

MTC is the maximum tolerated conc, the upper limit of conc beyond which concentration related adverse effects are intolerable (there is toxicity)

Question 5

what is the onset time

Answer 5

the time required for a drug to reach MEC after administration, this varies depending on route of admin

Question 6

what is the duration of action

Answer 6

the difference between the onset time and the time for the drug to decline back to the MEC

Question 7

what is the Cmax, the tmax and the AUC

Answer 7

Cmax; max conc of drug in blood, is a function of the rate and extent of drug absorption

tmax is time required to achieve Cmax, indicates rate of absorption

AUC; area under the curve is a function of the extent of absorption and metabolism and represents the overall systemic exposure of the drug

Question 8

what is the half life of the drug

Answer 8

time taken for 50% of serum drug conc to be eliminated or cleared

Question 9

what are different sorts of enzymes drugs interact with in the body

Answer 9

normal enzymes of intermediary metabolism: drugs encounter enzymes which metabolise natural substrates involved in cell metabolism, the extent to which drug metabolism occurs by these enzymes depends on the enzyme specificity and similarity of the drug to the normal substrate

microsomal drug metabolising enzymes: these are enzymes in the liver which are capable of metabolising many foreign compounds by oxidations, reducations, hydrolyses and conjugations, the enzymes of phase 1 and 2 drug metabolism are contained in the endoplasmic reticulum

Question 10

describe the structure of the liver

Answer 10

composed of 2 major lobes containing many lobules, each lobule is a hexagonal structure made up of hepatocytes arranged in irregular, branching, interconnected channels around a central vein

blood supply to liver comes from hepatic artery which supplies oxygeated blood and hepatic portal vein which carries deoxygenated blood containing nutrients and drugs from GI tract

blood from branches of both vessels flows through spaces between hepatocytes called sinusoids, finally draining into branches of hepatic vein

Question 11

how are sinusoidal endothelial cells adapted for their role

Answer 11

they are highly fenestrated which allows virtually unimpeded flow of plasma from sinusoidal blood into the space of disse, thus hepatocytes are bathes in plasma derived in large part from venous blood returning from small intestine

Question 12

what is the most importat system in the ER of the liver

Answer 12

The most important system in the endoplasmic reticulum is a HYDROXYLATION
system which accounts for the metabolism of 60-70% of all lipid-soluble foreign
compounds. This is better termed a mono-oxygenase as it inserts an oxygen atom
into its substrates to form a hydroxyl group.

Question 13

what is the mechanism of microsomal hydroxylation

Answer 13

The microsomal mono-oxygenase system has been found to have unusual
requirements - isolated microsomes will hydroxylate drugs and other foreign
compounds if the following are present:
1. Nicotinamide Adenine Dinucleotide Phosphate in its reduced form i.e. NADPH
2. Molecular oxygen.

NADPH + H+ + O2 + Drug-H → NADP + H2O + Drug-OH

The molecular oxygen has been split with one atom going into the substrate, the other
to form water. The mechanism is complex involving carriers which catalyse oxidation
and reduction using electron transfer.
Microsomes contain a high concentration of a protein called a cytochrome. This
cytochrome was discovered by reacting reduced microsomes with carbon monoxide
which gave a new spectrum with a peak at 450nm. This new cytochrome was named
cytochrome P-450

Question 14

describe cytochrome P450

Answer 14

Large and diverse group of enzymes that catalyze the oxidation of organic substances
such as lipids, steroid hormones and drugs.

The most common reaction catalyzed by cytochromes P450 is a monooxygenase reaction:
RH + O2 + 2H+ + 2e– → ROH + H2O

Cytochromes P450 contain a heme iron cofactor to achieve this reaction

The endoplasmic reticulum contains as much as 20% of its protein as cytochrome P-450
as well as other electron carriers. These together form an electron transport chain which
accomplishes the overall reaction i.e. the transfer of electrons from NADPH and the
splitting of oxygen to form hydroxylated substrate and water.

Human CYPs are
associated with the
membrane of the
endoplasmic reticulum
and are most
concentrated in liver
cells. CYPs metabolize
thousands of
endogenous and
exogenous compounds..
The reducing power is
supplied by NADPH
oxidase/P450 reductase.

Question 15

what is phase 2 drug metabolism

Answer 15

The major site of drug metabolism is the smooth endoplasmic reticulum of the liver cell.
• Phase I metabolism reactions can lead either to activation or inactivation of the drug
and can involve reduction or hydrolysis but mostly involve oxidation catalysed by a
cytochrome P450 monooxygenase (often abbreviated CYP), NADPH and oxygen.
•
• If the metabolites of phase I reactions are sufficiently polar, they may be readily
excreted. However, many phase I products are not eliminated rapidly and undergo a
subsequent phase II metabolism conjugation reaction.
• Here the phase I product combines with a functional group (eg. glucuronic acid,
sulphonate, glutathione or amino acid) to form a highly polar conjugate that is rapidly
excreted.
• Sites on drugs where conjugation reactions occur include carboxyl (-COOH), hydroxyl (-
OH), amino (NH2), and sulfhydryl (-SH) groups. Products of conjugation reactions have
increased molecular weight and are usually inactive.

Question 16

what are phase 2 reactions

Answer 16

oxidations, reductions and hydrolyses catalysed by phase 1 enzymes effectively provide a reactive centre in the molecule

a functional group can then be conjugated from a coenzyme to this reactive centre to increase the polarity of the metabolite, if a suitable reactive centre already exists in a drug it may be conjugated without prior metabolism, some normal body constiuents are disposed of in this way e,g some steroids, the conjugating enzymes are found in many tissues e.g liver and kidnet and in different cell compartments e.g ER, mitochondria within those organs

conjugation reactions are synthetic and require energy supplied directly or indirectly by ATP and a donor of the conjugating functional group that is often a coenzyme

more than one conjugation reaction may occur

Question 17

what are different types of conjugation reaction

Answer 17

sulphation, acetylation, methylation, glucuronidation

Question 18

how do genetics interact with phase 2 enzymes

Answer 18

Just as there are multiple forms of CYP 450s (Phase I enzymes) due to a gene
superfamily, there are also multiple forms of the Phase II conjugation enzymes,
such as;
UDP-glucuronyltransferases (UGTs) - 19 human genes encoding the UGT
proteins (9 in UGT1 locus and 10 encoding UGT2 family).
Sulphotransferases (SULTs) - in humans, 11 SULT isoforms have been identified
Glutathione-S-transferases (GSTs) - over 20 human GSTs have been identified
and are divided into two subfamilies; the microsomal and cytosolic forms.
N-Acetyltransferases (NATs) - 2 functional NAT genes in humans, NAT1 and
NAT2.
Methyltransferases (MTs) - humans express 3 different MTs metabolising
different substrates.

Question 19

what is the major phase 2 conjugation pathway

Answer 19

Glucuronidation is the major Phase 2 conjugation pathway in the body which requires the
presence of UDP-glucuronyltransferases (UGTs). Only a small number of UGTs catalyze
the glucuronidation of thousands of drug metabolites often using an O atom introduced
during phase 1 metabolism.

Question 20

what is used in most redox metabolism reations

Answer 20

Glutathione (GSH) is the most abundant
low-molecular-weight thiol in the body, and
GSH/glutathione disulfide is the major redox
couple in mammalian cells. Metabolites are
coupled to reduced glutathione by
glutathione S transferases.

Question 21

how is aspiring metabolised

Answer 21

metabolised by liver to:

salycylic acid which is then

glycine conjugated (75%) to salicyluric acid

salicyl (phenyl) glucuronide 10%

salicyl (acyl) glucuronide 5%

trace levels of 2,3 dihydroxy salicylic acid

<1% gentisic acid which is converted to trace levels of 2,3,5 trihydroxy salicylic acid

since the liver and perhaps enzymes of the gut wall metabolises drugs such as aspirin and salbutamol so efficiently a much larger dose is needed when given orally, variations can occur due to genetic differences of metabolising enzymes

Question 22

describe the genetics of CYP450 enzymes

Answer 22

The Human Genome Project has identified 57 human genes coding for the various
cytochrome P450 enzymes.
• The CYPs are grouped into 18 families where each has at least 40% homology in
amino acid sequence – each family has a number eg. CYP3.
• These families are divided into sub-families each with at least 60% amino acid
sequence homology – each sub-family is given a letter eg CYP3A.
• Individual members of sub-families are identified by a number eg CYP3A4.
• CYP450s metabolize thousands of endogenous and exogenous chemicals. Some
CYPs metabolize only one (or a very few) substrates, while others may metabolize
multiple substrates.
• Cytochrome P450 enzymes are present in most tissues of the body, and play
important roles in steroid hormone synthesis and breakdown, cholesterol synthesis,
and vitamin D metabolism.
• CYP450 are most concentrated in liver where they metabolize potentially toxic
compounds, including drugs and products of endogenous metabolism e.g.bilirubin.
• These isoforms have different substrate specificities (which overlap) and behave
differently with inhibitors and inducers.
• Most CYP450s have a low substrate specificity. The large number of CYP450s
together with their low specificity is responsible for our ability to metabolise an
extremely wide variety of foreign compounds (including drugs).

Although humans have 57 unique cyP450 genes, only a few in families 1-
3 are responsible for the metabolism of the vast majority of drugs
CYP1A2, CYP2C19, CYP2C9, CYP2D6, CYP2E1 & CYP3A4 are
responsible for metabolising 70-80% of currently used drugs.

Question 23

describe the different sequences of CYP450 enzymes, how does this relate to structure

Answer 23

Sequence identity between CYP450s is low (<20%). Only 3 amino acids are totally conserved, the Glu and Arg
of the domain that stabilizes the core and heme-binding, and the heme-binding Cys.

however crystal structures show overall structure is highly conserved, they contain conserved domains essential for structure and function, and variable regions that mediate substrate binding

All CYPs have a well-conserved heme-binding core formed out of aD, aE, aI, and aL and aJ and aK. The bbulge region contains the thiolate heme ligand and is referred to as a Cys- pocket. Between aK and the Cyspocket, a structurally conserved region is called the ‘meander’ loop that plays a role in heme binding and
stabilization of the tertiary structure The reductase interaction face of CYPs mainly comprises the aJ/aJ’ and
the insertion following the meander loop

the different biochemical properties of CYPs are mediated by the diverse regions, which vary in sequence and structure

Question 24

what are types of polymorphism

Answer 24

A polymorphism is a mutation that occurs in >1% of the population

Single Nucleotide Polymorphisms (SNPS) account for approx 90% of
all polymorphisms. They are single base changes in the genome

Microsatellite repeats: this is a locus where different numbers of
copies of a short repeat sequence are upstream of a particular gene.
The number of repeats can be different for different alleles (even
within the same person). The number of repeats often influences the
level of gene expression.

Copy Number Variants (CNVs) are gains or losses in genetic material
among individuals of the same species. Large chromosome deletions
or additions can explain why different individuals can have different
numbers of copies of a particular gene.

Question 25

where are SNPs likely to occur in CYP450 enzymes

Answer 25

in polymorphic ones (2D6, 2A6, 2B6 and 3A4), these four account for >60% of all drugs metabolised

CYP 2D6 is a major polymorphic CYP and is the greatest contributor of polymorphic alleles in caucasians

Question 26

how might polymorphic CYPs influence adverse reactions

Answer 26

Families 1-3 are polymorphic ie. have single nucleotide polymorphisms
that affect activity.
This leads to differences in drug metabolism and can be the cause of
many adverse drug reactions.
This polymorphism results in distinct phenotypes with respect to drug
metabolism:
1. Poor Metabolisers – have abolished CYP activity due to lack of
functional gene. They are homozygous for autosomal recessive
allele.
2. Intermediate Metabolisers – have reduced CYP activity because
they are heterozygous for the defective allele.
3. Efficient Metabolisers – have normal CYP activity due to 2
functional gene copies as they are homozygous for autosomal
dominant allele.
4. Ultrarapid Metabolisers – increased CYP activity due to more than
2 functional gene copies through gene amplification.
The therapeutic effect of the same dose may vary from patient to patient. In
an ultrarapid metaboliser the drug may be metabolised so rapidly it has no
effect at all, in a poor metaboliser the usual dose may lead to high
concentrations and have toxic side-effects.

Question 27

what drug is used for phenotyping a specigic CYP enzyme

Answer 27

Debrisoquine is an antihypertensive
drug frequently used for phenotyping
CYP2D6 that hydroxylates the drug in
phase 1 metabolism

Question 28

describe the effects of genetic polymorphism in CYP2D6

Answer 28

Over 100 different alleles in the CYP2D6 gene have been identified.
CYP2D6 metabolises some 25% of clinically used drugs and the dose required to achieve the
same level of drug in different patients can vary 10-30-fold if it is metabolised by CYP2D6.
The anti-hypertensive drug debrisoquine is hydroxylated by CYP2D6 to produce the principal
metabolite, 4-hydroxydebrisoquine. Measuring the concentrations of parent drug and
metabolite in the urine of patients gives a metabolic index (ratio) that defines phenotype.
This ratio is variable but ranges from 0.2 – 12.6 for intermediate or efficient metabolisers (ie.
normal). But a lower ratio corresponds to ultrarapid metabolism and is observed in
patients who do not respond to this drug. Higher ratios correspond to poor metabolisers
who exhibit overdosing and adverse side effects. See next slide.
In Western Europe, 5.5% of the population carry more than 2 functional gene copies of
CYP2D6 and are ultrarapid metabolisers.
In Western Europe, 7.0% of the population carry polymorphisms that produce no functional
CYP2D6 and are poor metabolisers.
It would therefore seem important to give a patient a “personalised dosage” because a
“standard dose” would result in lower concentrations in a UM patient and fail to reach the
Minimum Effective concentration, or would result in a higher concentration in a PM
patient and exceed the Minimum Toxic Concentration

The most frequent polymorphism on
CYP2D6*4 is a G to A in position
1934 between intron 3 and exon 4.
This induces synthesis of a truncated
inactive protein containing only 181
amino acids instead of 457 in the
wild type.
CYP2D6*2 is a polymorphism
caused by duplication of CYP2D6
gene (Copy Number Variants) and
creates Ultrarapid metabolisers.
CYP2D6*5 = deletion of CYP2D6
gene
CYP2D6*17 represent multiple SNPs
that reduces affinity for substrates.
The large number of identified allelic
variants (>70) easily explains the
wide variability of metabolic index
(see previous slide)

Question 29

how do different metabolisers effect the metabolism of codeine

Answer 29

It is a pro-drug
commonly found in cough medicines that is partly (5-10%) metabolized to
morphine in the liver by CYP2D6

An intermediate metabolizer will convert a
reduced amount of codeine to morphine,
decreasing the efficiency of the drug’s
effects.

Poor metabolizers will not be able to
metabolize codeine at all, and will
experience no pain relief.

Ultrarapid metabolizers on the other hand,
may metabolize codeine too efficiently,
resulting in morphine overdose.

Question 30

describe genetic variation of human CYPs between different ethnic populations

Answer 30

The CYP2D6*2 allele producing ultrarapid metabolisers is observed in only 5% of Western
Europeans but 16% of Ethiopians.
Also (not shown) CYP2C19 causes a higher incidence of poor metabolisers (PMs) in asians
(73%) compared with caucasians (3-5%).
75% of caucasians but only 50% of africans have a genetic inability to express functional
CYP3A5.

Question 31

what does CYP2D6 have a high affinity for, how is enzyme expression effected by other factors

Answer 31

CYP2D6 has a very high affinity for
alkaloids
Enzyme expression not regulated by any
known environmental agent and not
inducible by known hormones

CYP2D6 is important for metabolism of selective serotonin reuptake inhibitors
(SSRIs), tricyclic antidepressants and some benzodiazepines and antipsychotics, dosage of commonly used antipsychotics is dependent of CYP2D6 genotype

Question 32

how does CYP2D6 effect tamoxifen

Answer 32

Tamoxifen treats breast cancer as it binds to the oestrogen receptor and prevents its
action as a transcription factor. The permissive action of oestrogen on abnormal breast
growth is thus blocked.
Tamoxifen is metabolized in the liver to various metabolites including 4-hydroxy-Ndesmethyltamoxifen (endoxifen), that is 100 times more potent than the parent
compound.
Tamoxifen can be thought of as a prodrug that is activated by CYP2D6 to produce
endoxifen, the therapeutic molecular effector.
Poorly functioning CYP2D6 isoforms result in lower endoxifen levels and decreased or
absent antioestrogenic activity and thus compromise anti-breast cancer therapy.