2. Single nucleotide polymorphisms
(SNPs)
• SNPs are single base pair
positions in genomic DNA
at which different
sequence alternatives
(alleles) exist wherein the
least frequent allele has
an abundance of 1% or
greater.
• SNPs are the most
commonly occurring
genetic differences.
3. • SNPs are very
common in the
human
population.
• Between any
two people,
there is an
average of one
SNP every ~1250
bases.
4. • Mutation: difference in the DNA code that
occurs in less than 1% of population
–Often associated with rare diseases
• Polymorphism: difference in the DNA code
that occurs in more than 1% of the population
–A single polymorphism is less likely to be
the main cause of a disease
–Polymorphisms often have no visible clinical
impact
5. Factors Contributing to Interindividual
Variability in Drug Disposition and Action
• Age
• Race/ethnicity
• Weight
• Gender
• Concomitant Diseases
• Concomitant Drugs
• Social factors
• GENETICS
6. Factors that influence medication
effects
• Absorption
GIT
P-glycoprotein
• Drug metabolism
phase-1
phase-2 reactions
7. GIT
- Antacids-> alter stomach pHdecreases
ketoconazole absorption.
- GI transit time have a small role in drug
absorption variability.
Anticholinergics slow down transit
times, crohn’s and ulcerative collitis can
markedly increase transit times .
8. drug Absorption site remarks
Ketoconazole GI tract Acidic environment
improves absorption
cyclosporine PGP Affects bioavailability
Mycophenolete mofitel GI tract Not to be given with
Fe(inhibits absorption)
9. P-GLYCOPROTEIN
• Is a membrane-bound transport protein affects
absorption in GIT.
• Functions as a part of first pass effect by
actively pumping the drugs out from cell, there
by decreasing bioavailability.
• High levels of PGP also found in liver and
kidneys(drug elimination).
• Decreased intestinal P-gp function
–increased amount absorbed
–increased plasma concentration
10. DRUG METABOLISM
process that facilitates drug clearance by
- Increasing solubility
- Prodrugs to their active drugs form
Can be divided into two components
a) phase -I : intra molecular modifications(reductases, oxidases,
hydrolases)
-create a site for attachment of larger polar side chains (in P-II
rxn)
b) phase-II: results in conjugation of drug with an endogenous
substance by glucuronidase, sulphatase, acetylases, methylases.
Despite what the nomenclature suggests, there is no
order in which these reactions take place.
12. CYTOCHROME P-450 ENZYME SYSTEM OVERVIEW
• The cytochrome P-450 mono-oxygenase system
(CYP-450) is largely responsible for catalysing
phase1 reactions(70-80%).
• Complex supergene family: at least 40 enzymes
expressed in human tissues.
• CYP1A2, 3A4, 2C9, 2C19, 2D6, 2E1 exert a major role in
drug metabolism.
• Enzymes are located on smooth endoplasmic
reticulum of most cells, but are found in variable
concentrations.
• Hepatocytes have the greatest concentration of CYP
enzymes.
ARE RESPONSIBLE FOR THE METABOLIC ELIMINATION OF MOST
DRUGS CURRENTLY USED IN MEDICINE
13. CYTOCHROME P-450 ENZYME SYSTEM
NOMENCLATURE
Eg: CYP3A4
• Classified by amino acid similarities
– 1ST No. family number
– 2nd subfamily letter
– Final number for each gene within the subfamily
– asterisk followed by a number (and letter) for each
genetic (allelic) variant
o allele *1 is the normal function gene (wild allele)
o CYP2D6*1a gene encodes wild-type protein CYP2D6*1
14. Drugs metabolized by various CYP isoforms
Isoform % of all drugs
metabolism
CYP3A4 40-45%
CYP2D6 20-30%
CYP2C9 10%
CYP2C19 5%
CYP1A2 5%
15. Polymorphisms –
• Approximately 40% of human CYP-dependent
drug metabolism is carried out by polymorphic
CYP enzymes.
• This polymorphism expressed as variable enzyme
activity, thus effecting metabolism of drugs.
• Depending on enzyme activity, individuals
designated as:
-poor metabolisers(PM)
-Intermediate metabolisers(IM)
-Extensive metabolisers(EM)
-Ultrarapid metabolisers(URM)
20. CYP-3A4 VARIABILITY
• Responsible for 40-50% of all phase-1 reactions
and accounts for up to 7% of GIT CYP activity.
• Is co-expressed with P-glycoprotein in liver and
intestine.
• Despite little genetic variability between
populations , there appears to be as much as 20
fold interindividual variability of enzyme activity.
• CYP-3A4*1B appears to be the most common
variant allele decreased activity.
21.
22. CYP-2D6 VARIABILITY
• Shows significant pharmacological variation
and is integral in metabolism of numerous
medications especially psychiatric and cardiac
medications.
• almost 80 allelic variants.
• 20-30% drugs are metabolised through this
pathway.
23. CYP-2C9 polymorphisms
• 10% drug metabolism.
• Although there have been over 100 SNP
identified, only 2 variants have been shown to
significantly reduce substrate affinity through
inhibiting CYP activity.
• Homozygote CYP-2C9*3/*3, comprising 0.5%
of populations , is considered to have marked
clinical significance with very low CYP-2C6
activity.
24. • CYP-2C9*3- role phenytoin induced cutaneous
adverse drug reactions.
• Warfarin is the most clinically significant
substrate.
• Fluconazole inhibition of CYP-2C9 can result in
markedly elevated levels of warfarin.
25. CYP2C19
• 5% of all drug
metabolism.
• PPI and numerous
anticonvulsants are
primarily substrates
metabolized.
CYP1A2 polymorphism
• Metabolize several
antipsychotic medications
and theophylline.
• Environment and genetic
factors are shown to
influence the activity of
CYP1A2.
• Inducers- Tobacco, OCP,
26. Dihydropyrimidine dehydrogenase(DPD)
• In metabolism of 5-FU.
• More than 80% of given drug is metabolised by
DPD.
• Deficiency of DPD have been reported to have
severe neurotoxicity from 5-FU treatment.
• Severe gastrointestinal and hematological
toxicity reported with DPD deficient patient
who applied it topically on scalp.
Therefore contraindicated in DPD deficient
patients.
27. Polymorphisms
• Many genetic variants in DPD gene have been
described, most common DPD*2A allele,
which leads to an enzymatically deficient DPD.
• This allele is associated with 5-FU induced
toxicity specially leucopenia and mucositis.
28. PHASE-2 REACTIONS
P-GLYCOPROTEIN(permeability glycoprotein)
• Is an ATP-activated pump, increased attention
because of its role in multidrug resistance, in
particular to chemotherapeutic agents.
• PGP involves pumping molecules from
intracellular to extracellular spaces,
countering the effects of passive diffusion,
most notably in GIT, resultant decrease in net
drug absorption.
29.
30. Polymorphisms
• Identified in the multidrug resistance1(MDR1)
gene that encodes PGP.
• This decreased expression is associated with
increased drug concentrations(digoxin).
Importance-
Significant overlap between PGP and CYP3A4,
limiting assessment of PGP polymorphisms.
31. THIOPURINE METHYLTRANSFERASE(TPMT)
• Is a catalyst for the metabolism and inactivation of
azathiopurine, 6-mercaptopurine(6-MP) and
thioguanine.
• Function- converting 6-MP to active methyl
mercaptopurine(6MMP) nucleotides and
converting thioguanine to inactive metabolites.
• decreased activity results in increased 6-
thioguanine levels, leading to increased
toxicity(myelosuppression)
32. 6-MMP
• is correlated with
azathiopurine-
induced
hepatoxicity.
• Decreased levels of
formation in TPMT
deficient
individuals.
• Therefore, deficient
individuals are at
lower risk of
developing
hepatotoxicity.
33. TPMT polymorphism
• normal allele homozygous TPMT*1high
activity(89-90% popl’tn)
• Mutant alleles TPMT*3C, *3A *2 decreased
activity
• Heterozygous expression along with TPMT*1 –
results in intermediate TPMT activity(10-11%).
• Low to no TPMT activity0.3%{decreased
hepatotoxicity & increased myelosuppression}
34. Testing methods for TPMT polymorphisms
• TPMT activity in erythrocytes
• DNA micro-array studies
Clinical application
i. TPMT*1(normal)– 2 to 2.5ml/kg/day
(standard dose)
ii. Heterozygous with one mutant allele - dose
reduced by 15-50%.
iii. Two mutant alleles – recommended that not
to be treated; if must, @ dose reduced by
90%.
35. N-ACETYL TRANSFERASE (NAT2)
• Acetylates numerous xenobiotics making the
parent compound water soluble, thus
facilitating drug elimination.
• 25 allelic variants of NAT2 gene have been
identified.
• Enzyme activity often reported as rapid,
intermediate, or slow (analogous to
EM,IM,PM).
36. Rapid acetylators
• homozygous for NAT2*4, NAT2*12 and NAT2*13.
• Require higher doses of medications to minimize
the likelihood of treatment failure.
Intermediate acetylators NAT2*5, *6, *7, *14
Slow acetylators
• More likely to develop toxic adverse effects,
including
drug induced lupus - procainamide and hydralazine
neuropathy –isonaizd
TEN – sulfonamides
• Have increased risk certain solid tumors and for
some IgE-mediated food allergies seen in children
37. GLUCOSE-6-PHOSPHATE DEHYDROGENASE
• Catalyses the first reaction in the pentose
phosphate pathway(PPP), leading to reduction
of NADP to NADPH.
• NADPH plays important role in reducing
glutathione, which plays pivotal role preventing
cell damage by oxidative stress.
• Since erythrocytes lack mitochondria, the PPP
is the only source of NADPH, thus making
erythrocytes sensitive to oxidative stress,
resulting in hemolysis.
38.
39. In a study of 3166 Indian’s, g6Pd
deficiency is about 10.5%
40. Glutathione S-transferase(GST)
• Involved in detoxification of carcinogenic
derivatives of coal tar.
• 50% Europeans have low to absent activity
owing to the presence of the GSTM1-null
genotype.
• Hence , when these individuals are treated
with topical coal tar have a greater mutagen
exposure.
41. Folate pathway polymorphisms
• Methotrexate
- structural analouge of folic acid which competitively
inhibits dihydrofolate reductase (DHFR).
- Also directly inhibits thymidylate synthase(TS)
- Influences the activity of methylene
tetrahydrofolate reductase(MTHFR), which converts
homocysteine to methonine.
- The adverse drug reactions have been associated
with polymorphisms of TS and MTHFR.
42. • Thymidylate synthase (TS) 5’-untranslated region (TS
5’-UTR 3R)
- This allele has been significantly linked with ADR in
psoriasis patients taking MTX when folic acid is not
administered.
- associated with a poor therapeutic response to MTX.
- Lower risk for 5-FU induced ADR.
• TS 5’-UTR 6 bp deletion allele has also been
associated with increased MTX-induced toxicity ,
including up to an 8-fold increase of developing
elevated ALT transaminase levels in absence of folic
acid supplementation.
43. • C677T polymorphism of MTHFR, observed in
8% of normal population, leads to a
thermolabile variant subsequently reducing its
activity to about 30% of wild type.
• This has been associated with an increased
risk of discontinuing MTX because of elevation
in liver enzymes.
45. • HLA-B*1502 & Carbamazepine (chinese)
- Drug induced SJS and TEN
- Not associated with hypersensitivity syndrome
• HLA-B*5801- Allopurinol induced severe
cutaneous ADR.
• Abacavir hypersensitivity
- HLA-B*5701(severe and fatal)
- HLA-DQ3, HLA-DR7
46. • Nevirapine (nnrti)
- HLA-B*3505 is predictor for all types of
nevirapine induced cutaneous drug
reactions(thai patients).
- HLA-DRB1*0101 hepatitis, fever, and rash, but
not with isolated rash.
- ADR occur more frequently in patients with
higher pre-treatment CD4 levels
- Therefore, it is now recommended that it
should be avoided with CD4 count
women- >250/microL
men- >400/microL
47. Tests
a) CYP2D6 polymorphisms- DNA microarray
analysis
b) HLA polymorphisms- specific HLA typing
c) TPMT-
- phenotyping:- measures enzyme activity in
peripheral RBC lysates.
- Genotyping:- DNA microarray, PCR,
prometheus TPMT genetics
- genetic allele testing.
48. d) G6PD
• Fluorescent spot test- enzyme activity in
erythrocytes
• Methemoglobin or nile blue sulfate reduction
G6PD studies
• G6PD genotyping
e) Thymidylate synthase
• Genotyping of 5’UTR repeats
f) GSTM1- PCR analysis
51. Drugs and liver injury
• Drugs are estimated to be responsible for 10%
of cases of hepatitis in adults.
• 40-50% of hepatits in adults over 50yrs of
age.
• 25% of fulminant hepatitis.
• Liver failure, when drug induced on an
idiosyncratic basis, is fatal 75-80% of the time.
52. Hepatic drug metabolism
- Major site of metabolism for most drugs
- Overall goal of hepatic drug metabolism is to
convert pharmacologically active relatively
lipophilic drugs into inactive relatively
hydophilic metabolites(biotransformation) and
conversion of reactive metabolic intermediates
to more stable molecular compounds
(detoxification).
- Reasonable balance between the two process
keeps liver away from toxicity.
53. Biotransformation
• goal- lipophilic to hydrophilic
• Increased hydrophilicity --> excreted by either
renal or billiary routes.
Detoxification
• Goal- To avoid local or distant damage from
reactive intermediates(electrophilic
compounds) during biotranformation.
These systems are adequate to allow safe drug
administration without significant risk to liver.
54.
55. Time period
• Majority occur between 15-90 days after the initiation
of therapy.
• Potentially serious hematologic drug reactions have a
much more gradual onset.
within 15 days after cessation
- Marked improvement(mostly)
- may lead to hepatocellular toxicity, hypersensitivity
syndrome and cholestasis type reactions.
Occurs over years- Steatosis progressing to fibrosis is
much slower
56. Outcome
• Most are completely reversible if detected
early(days/ few weeks)
• If detected relatively late, some are not fully
reversible.
• Delayed diagnosis
- Death
- liver failure requiring transplantation
- severe fibrosis or cirrhosis.
LOSS OF SOME DEGREE OF LIVER FUNCTION IS
INDEFINITE
57. Cellular and structural targets involved
with DILD
Cell or structure Category of reaction Representative drug
etiologies
Hepatocytes Hepatocellular
necrosis
Ketoconazole,
minocycline
Bile ducts, bile
canaliculi
Cholestasis Erythromycin
Endothelial cells,
sinusoids
Veno-occlusive Very high dose
cyclophosphimide
Ito cells Steatosis-> fibrosis methotrexate
59. Drug specific risk factors
• Daily dose- retinoid hepatotoxicity.
• Cumulative dose- MTX induced liver disease.
Red flag signs in prescribing recently released
medications
- Presence of significant number of patients in
clinical trails with mild elevation in LFT.
- Report of even 1-2 deaths from drug
hepatoxicity in clinical trails or very soon after
drug’s release.
60. Major classification systems in DILD
1) Based on laboratory test abnormalities(LFT)
- hepatocellular:- elevated transaminases
- Obstructive:- elevated ALP, GGT and bilirubin
values
- Mixed:- combination of both (either type over
time commonly becomes ‘mixed’)
2) Based liver histology
- Steatosis(fatty liver)
- Fibrosis to cirrhosis
- Granulomatous
- Veno-occlusive
61. 3) Based on pathogenesis
- Toxic
- Idiosyncratic
a) metabolic- local toxic reactive
intermediates.
b) immunologic- neoantigens
62. Mechanisms of drug hepatoxicity
• Mainly by
- Idiosyncratic(majority, unpredictable))
- Toxic (dose dependent, predictable)
- Idiosyncratic DILD
• Well accepted hypothesis:- Reactive
electrophilic intermediates with defective
cellular detoxification systems
63.
64. Metabolic targets involved with DILD
molecule consequence comments
Various proteins Neoantigen formation Reactive metabolites
induce a change in
structure or
conformation
CYP proteins Neoantigen formation CYP especially
vulnerable , due to
proximity to reactive
metabolites
DNA Apoptosis or necrosis results in cell death
Lipids Lose membrane
integrity
Result of lipid
peroxidation by
reactive metabolites
65. Hepatocellular Best diagnostic
test
Classic culprit Dermatology drugs
Hepatocellular AST & ALT Halothane Ketoconazole
Dapsone
Minocycline
Azathioprine
Acitretin
MTX
Hypersensitivity AST, ALT &
eosinophil count
Phenytion Dapsone
Minocycline
Azathioprine
Sulfonamides
Cholestasis ALP
GGT
Bilirubin(direct)
Chlorpromazine Rifampin
Erythromycin
TMP/SMX
Steatosisfibrosis Liver biopsy
Transaminases
MTX Acetretin(rarely)
66. Dapsone and azathioprine
All physicians will do careful hematologic
monitering, whereas far fewer will routinely
check transaminase levels, another is the very
‘delayed’ discovery of the potential for
minocycline to induce hepatotoxicity (an
isolated findings) or DHS.
67. Diagnostic algorithm
1) Challenge- circumstances of the original drug course ,
drug timing/reputation, patient history or prior reactions
to the drug in question
2) Dechallenge- expected improvement after the drug is
discontinued
3) Rechallenge- only when it is essential to know the
responsible drug with certainty and the reaction pattern is
a relatively low-risk adverse effect from drugs.
4) Exclusion- other non-drug etiologies for the same adverse
effect.
‘Dechallenge’ and ‘exclusion’ steps are appropriate for
virtually all patients with a 2-3 fold elevation of
transaminase values.
68. Transaminases
1-2 fold elevation-
- Reduce the drug dose and follow up the
transaminase values carefully.
Above 3- fold elevation(critical)-
- Prompt indefinite discontinuation of the drug or
drugs likely to be responsible, unless an
alternative etiology for this elevation is
uncovered.
69. • Hepatocyte integrity-
- Specificity- ALT/SGPT>AST/SGOT
- Greater sensitivity if both are ordered.
Periodic Liver biopsy- psoriasis patients on long
term MTX therapy.
In rhematology, patients with RA and on MTX ;
repeated elevations of transaminases i.e
elevetions in 5 of 9 tests performed every 6
weeks, over a year or albumin levels <3 gm%
are the published standard of care to indicate a
need for a liver biopsy.
