Section 'a' of last chapter in Clinical Pharmacokinetics and Phrmacotherapeutic Drug Monitoring. This is useful for 5th year PHARM D students.

1. Genetic Polymorphism in
Drug Metabolism:
Cytochrome P-450
Isoenzymes

2. Drug metabolism
• Drug metabolism is otherwise known as
biotransformation.
• Biotransformation mainly takes place in Liver.
• R.T. Williams constituted this metabolism into two
phases known as Phase-I and Phase-II.
• Phase-I reactions consist of oxidation, reduction and
hydrolysis.
• Phase- II reactions consist of conjugation, acetylation,
methylation etc.

3. Microsomal enzymes
• Liver the principal organ of metabolism consists of
two group of enzymes known as microsomal
enzymes and non-microsomal enzymes.
• Microsomes are derived from rough endoplasmic
reticulum of liver which shed their ribosomes to form
smooth surface.
• Microsomal enzymes are involved in wide variety of
oxidative, reductive, hydrolytic and glucuronidation
reactions. Example: CYP enzymes.

4. Non microsomal enzymes
• These enzymes are present in soluble form in
cytoplasm and they are attached to mitochondria of
liver cells.
• They are involved in few oxidative, reductive and
conjugation reactions.
• They act on relatively water soluble xenobiotics
(drugs) and endogenous compounds. Example:
Alcohol dehydrogenase.

5. Cytochrome P-450 (CYP-450)
Isoenzymes
• As a rule of thumb, all enzymes are proteins and all
proteins are not enzymes.
• Cytochrome is also a haem protien which exploits the
abiltity of iron metal to gain or lose electrons. All P450
enzymes use iron to catalyze the reaction with the
substrate.
• CYP450s are the families of oxidases. Over 17 CYP
families have been identified in humans. The families are
numbered as CYP1, CYP2, CYP3 etc.
• The sub-families are identified as CYP1A, CYP2A etc.

6. Continuation of CYP
• Finally the individual isoforms or isoenzymes which
orginate from a single gene, are given a number such
as CYP1A1, CYP1A2, CYP3A4, CYP2D6 etc.
PHARMACOGENETICS:
The study of genetic variations in drug
response is called pharmacogenetics when
studying an individual gene, or
pharmacogenomics when studying all genes.

7. GENETIC POLYMORPHISM
Genetic polymorphisms are naturally occurring
variants in gene structure that occur in more than 1
percent of the population. Polymorphisms may
influence a drug’s action by changing its
pharmacokinetics or its pharmacodynamics. It is also
known as intersubject variability. It paves the path for
individualized rational dosage regimen. Differences
observed in the metabolism of a drug among different
races are called as ethnic variations.

8. GENETICS
Genotype: A genotype is an individual's collection of
genes. The term also can refer to the two alleles
inherited for a particular gene. The genotype is
expressed when the information encoded in the
genes' DNA is used to make protein and RNA
molecules.
• Phenotype: The expression of the genotype
contributes to the individual's observable traits
(genetically expressed characteristics), called the
phenotype.

9. GENOTYPING
Genotyping is the process of determining differences in
the genetic make-up (genotype) of an individual by
examining the individual's DNA sequence using biological
assays and comparing it to another individual's sequence or
a reference sequence. It reveals the alleles an individual
which were inherited from their parents.
Allele, also called allelomorph, any one of two or more genes
that may occur alternatively at a given site (locus) on a
chromosome. Alleles may occur in pairs, or there may be
multiple alleles affecting the expression (phenotype) of a
particular trait.

10. FACTORS INFLUENCING CYP ENZYMES
LEVEL
Diet
• The enzyme content and activity is altered by a
number of dietary components.
• Low protein diet decreases and high protein diet
increases the drug metabolising ability. This is
because the enzyme synthesis is promoted by protein
diet which also raises the level of amino acids for
conjugation with drugs.

11. Factors continued..
• Grapefruit inhibits metabolism of many drugs and
improve their oral availability.
• Starvation results in decreased amount of
glucuronides formed than under normal conditions.
• Malnutrition in women results in enhanced
metabolism of sex hormones.
• Alcohol ingestion results in a short-term decrease
followed by an increase in the enzyme activity.

12. Factors continued..
• The protein-carbohydrate ratio in the diet is also
important; a high ratio increases the microsomal
mixed function oxidase activity.
• Fat free diet depresses cytochrome P-450 levels
since phospholipids, which are important
components of microsomes, become deficient.
• Dietary deficiency of vitamins (e.g. vitamin A,
B2, B3, C and E) and minerals such as Fe, Ca, Mg,
Cu and Zn retard the metabolic activity of
enzymes.

13. Factors continued..
Pregnancy:
In women, the metabolism of promazine and pethidine
is reduced during pregnancy or when receiving oral
contraceptives. Higher rate of hepatic metabolism of
anticonvulsants during pregnancy is thought to be due
to induction of drug metabolizing enzymes by the
circulating progesterone.

14. Disease states
• Congestive cardiac failure and myocardial
infarction which result in a decrease in the
blood flow to the liver, impair metabolism of
drugs having high hepatic extraction ratio e.g.
propranolol and lidocaine.
• In diabetes, glucuronidation is reduced due to
decreased availability of UDPGA (Uridine
DiPhospho Glucuronic Acid).

15. Influence of different isoenzymes on
drug response:
Cytochrome P450 2C9 (CYP2C9)
The CYP2C9 enzyme is involved in the metabolism of many
common drugs such as glipizide , tolbutamide , losartan,
phenytoin and warfarin . The phenotypes CYP2C9*2 and
CYP2C9*3 are the two most common variations and are
associated with reduced enzymatic activity. CYP2C9 is the
principal enzyme responsible for the metabolism of warfarin.
Persons who are CYP2C9 poor metabolizers have reduced
warfarin clearance. Clinical studies have shown that these
persons require lower dosages of warfarin and are at an
increased risk of excessive anticoagulation.

16. Cytochrome P450 2C19 (CYP2C19)
• The CYP2C19 enzyme metabolizes many drugs,
including proton pump inhibitors, citalopram,
diazepam and imipramine. More than 16 variations of
CYP2C19, associated with deficient, reduced,
normal, or increased activity, have been identified.
Genotyping for CYP2C19*2 and CYP2C19*3
identifies most CYP2C19 poor metabolizers.
• The CYP2C19*17 variant is associated with
ultrarapid metabolizers and seems relatively common
in Swedes (18 percent), Ethiopians (18 percent), and
Chinese (4 percent).

17. CYP2C19 continued
The proton pump inhibitor omeprazole is primarily
metabolized by CYP2C19 to its inactive metabolite,
5-hydroxyomeprazole. Persons who are CYP2C19
poor metabolizers can have fivefold higher blood
concentrations of omeprazole and experience superior
acid suppression and higher cure rates than the rest of
the population.
Conversely, blood concentrations of omeprazole are
predicted to be 40 percent lower in ultrarapid
metabolizers than in the rest of the population, thus
putting persons with the CYP2C19 ultrarapid
metabolizers phenotype at risk of therapeutic failure.

18. Cytochrome P450 2D6 (CYP2D6)
• The enzyme CYP2D6 is involved in the metabolism
of an estimated 25 percent of all drugs. More than 75
allelic variants have been identified, with enzyme
activities ranging from deficient to ultrarapid.
• The most common variants associated with
poor metabolizer phenotype are CYP2D6*3,
CYP2D6*4, CYP2D6*5, and CYP2D6*6 in whites and
CYP2D6*17 in blacks. Codeine is metabolized by
CYP2D6 to its active metabolite, morphine.

19. CYP2D6 continued
• Clinical studies have shown that CYP2D6 poor
metabolizers have poor analgesic response as a
result of the reduced conversion of codeine to
morphine.
• Conversely, CYP2D6 ultrarapid metabolizers
quickly convert codeine to morphine and have
enhanced analgesic response.

20. CYP2D6 continued
The activity of drug-metabolizing enzymes
may be induced or inhibited by many other
intrinsic and extrinsic factors, including
comorbid conditions, use of other medications,
smoking, alcohol intake, and dietary factors.

21. Ethnic variations in N-acetylation of
Isoniazid
• An example of polymorphism is the
acetylation of isoniazid (INH) in humans. A
bimodal population distribution was observed
comprising of slow acetylator or inactivator
phenotypes (metabolise INH slowly) and rapid
acetylator or inactivator phenotypes
(metabolise INH rapidly).

22. N-acetylation variation continues
• Approximately equal percent of slow and rapid
acetylators are found among whites and blacks
whereas the slow acetylators dominate Japanese and
Eskimo populations.
• Dose adjustments are therefore necessary in the latter
groups since high levels of INH may cause peripheral
neuritis. Other drugs known to exhibit
pharmacogenetic differences in metabolism are
debrisoquine, succinyl choline, phenytoin, dapsone
and sulphadimidine.

23. Induction of Drug Metabolising
Enzymes
• The phenomenon of increased drug metabolising
ability of the enzymes (especially ofmicrosomal
monooxygenase system) by several drugs and
chemicals is called as enzyme induction and the
agents which bring about such an effect are known
as enzyme inducers.
• Mechanisms involved in enzyme induction are –
Increased synthesis of cytochrome P-450.
Decreased degradation of cytochrome P-450.

24. ENZYME INDUCTION
• Some drugs such as carbamazepine, meprobamate,
cyclophosphamide, rifampicin, etc. stimulate their own
metabolism, the phenomenon being called as auto-induction
or self induction.
• The most thoroughly studied enzyme inducer is phenobarbital
which can increase enzyme activity up to 4 times. An example
which shows that enzyme induction can have serious
consequences in clinical practice is the inducing effect of
phenobarbital on dicoumarol levels.
• Extreme caution must be exercised when phenobarbital and
dicoumarol are co-administered to avoid either failure of the
anticoagulant therapy or haemorrhagic crises.

25. ENZYME INHIBITION
A decrease in the drug metabolising ability of an
enzyme is called as enzyme inhibition. The process
of inhibition may be direct or indirect.
Direct Inhibition: may result from interaction at the
enzyme site.
Direct enzyme inhibition can occur by one of the 3
mechanisms –

26. Competitive Inhibition
It results when structurally similar compounds
compete for the same site on an enzyme. Such
an inhibition due to substrate competition is
reversible and can be overcome by high
concentration of one of the substrates, e.g.
methacholine inhibits metabolism of
acetylcholine by competing with it for
cholinesterase.

27. Non-competitive Inhibition:
• It results when a structurally unrelated agent
interacts with the enzyme and prevents the
metabolism of drugs. Since the interaction is
not structure-specific, metals like lead,
mercury and arsenic and organophosphorus
insecticides inhibit the enzymes non-
competitively. Isoniazid inhibits the
metabolism of phenytoin by the same
mechanism.

28. Product Inhibition
• It results when the metabolic product competes
with the substrate for the same enzyme. The
phenomenon is also called as autoinhibition.
• Certain specific inhibitors such as xanthine
oxidase inhibitors (e.g. allopurinol) and MAO
inhibitors (e.g. phenelzine) also inhibit the
enzyme activity directly. Direct enzyme inhibition
is usually rapid; a single dose of inhibitor may be
sufficient to demonstrate enzyme inhibition.

29. INDIRECT INHIBITION
Indirect Inhibition: is brought about by one of the
two mechanisms –
a. Repression: is defined as the decrease in enzyme
content. It may be due to a fall in the rate of enzyme
synthesis as affected by ethionine, puromycin and
actinomycin D or because of rise in the rate of
enzyme degradation such as by carbon tetrachloride,
carbon disulphide, disulphiram, etc.
b. Altered Physiology: due to nutritional deficiency or
hormonal imbalance.

30. Enzyme inhibition continues
• Enzyme inhibition is more important
clinically than enzyme induction, especially
for drugs with narrow therapeutic index, e.g.
anticoagulants, antiepileptics, hypoglycaemics,
since it results in prolonged pharmacological
action with increased possibility of
precipitation of toxic effects.

31. CONCLUSION
• Use of genotyping is more accurate than race or
ethnic categories to identify variations in drug
response.
• Unlike other influences on drug response, genetic
factors remain constant throughout life.
• The use of pharmacogenetic information to support
drug selection and dosing is emerging.
• Commercial testing is available for drug metabolizing
enzymes and some pharmacodynamic targets such as
VKORC1, stromelysin-1, and apolipoprotein E .

32. Conclusion continues
• Prospective genetic testing would be beneficial
for drugs for which a clear genotype-response
relationship as been demonstrated, such as
warfarin (CYP2C9) and proton pump
inhibitors (CYP2C19).
• The U.S. Food and Drug Administration has
suggested relabeling warfarin to include
genetic information to guide initial dosing.