metabolism 2 Flashcards
describe genetic variation in phase 2 metabolic enzymes
Glucuronidation is the major Phase 2 conjugation pathway in the body catalysed by UDPglucuronyltransferases (UGTs). Only a small number of UGTs catalyze the glucuronidation
of thousands of drug metabolites.
At least 63 UGT1A1 variant alleles have been described, including single base pair changes,
frame shift mutations, insertions, and deletions in the promoter region, 5 exons and 2 introns
of the gene. Most are associated with absent, reduced or inactive UGT1A1 enzyme; one is
associated with increased enzyme, and some are unknown.
The UGT1A128 SNP has been identified in the promoter region of the gene and reduces
promoter activity to 30% of normal.
A heterozygous UGT1A62 phenotype results in reduced activity against phenolic compounds
such as acetaminophen, salicylates and beta-blockers.
describe how genetic variation effects irinotecan metabolism
irinotecan is a chemotherapeutic prodrug used in combination treatments of solid tumours -
mainly colorectal cancer. Irinotecan is converted to its active form SN-38 by carboxylesterases.
SN-38 is a topoisomerase inhibitor and promotes apoptosis.
UGT1A1 glucoronidates and inactivates SN-38 prior to excretion.
Excessive levels of SN-38 can lead to severe leukopenia/neutropenia or diarrhea that is occasionally fatal. Numerous UGT1A1 alleles can lead to deficient UGT1A1 activity. The most common allele that occurs in many populations occurs in the promoter region of the gene.
The UGT1A1 promoter contains a TA repeat region that can have 5-8 TA repeats. Normal or
wild-type activity of UGT1A1 is associated with 6 TA repeats. Deficient UGT1A1 activity occurs
with 7 or 8 TA repeats, also known as *28 and *37, respectively, and these alleles carry an
extended promoter repeat causing reduced UGT1A1 transcription and activity.
This leads to reduced SN-38 glucuronidation, increased SN-38 and irinotecan toxicity.
what are tests offered for UGT1A1 genetic analysis
Mayo Medical Laboratories offers 3 tests for
UGT1A1 genetic analysis:
UGT1A1 TA Repeat Genotype analyzes the
promoter specifically for the TA repeat region.
UGT1A1 gene sequence, irinotecan is a
sequence analysis of the entire UGT1A1 gene,
including the promoter region.
Both of these tests aid in determining an
individual’s potential hypersensitivity response to
irinotecan.
UGT1A1 gene sequence, hyperbilirubinemia
is a sequence analysis of the entire UGT1A1
gene, including the promoter region. This test is
useful in the evaluation of individuals with
unconjugated hyperbilirubinemia to determine if
there is a genetic basis for their high bilirubin
levels.
A fourth UGT1A1 test is also available but not
listed here. This test is the UGT1A1 Known
Mutation test and is mainly used for patients who
would like to be tested for a known familial
UGT1A1 mutation.
how does thiopurine methytransferase genetics effect treatment with azathioprine
Azathioprine is used for the treatment of acute
lymphoblastic leukemia in children. The active
metabolite, 6-mercaptopurine (6-MP) is formed in liver.
6-MP is activated to 6-thioinosine monophosphate (6-
TIMP) by hypoxanthine phosphoribosyltransferase
(HPRT), and subsequently converted to 6-thioguanine
nucleotides (6-TGNs) that prevents nucleotide
formation and thus blocks DNA replication and cell
division.
Alternatively 6-MP is metabolised and S-methylated to
inactive 6-methylmercaptopurine nucleotides (6-MMPN)
by thiopurine methyltransferase (TMPT).
1 in 300 patients inherit TMPT deficiency as an
autosomal recessive trait. Three common SNPs in the
TPMT gene (G238C, G460A and A719G) result in 3
alleles TMPT2, 3A and 3C that produce an enzyme that
is rapidly degraded and therefore produces enzyme
deficiency.
These patients would not metabolise and inactivate 6-
MP and effectively receive a massive overdose of 6-MP
toxic to other blood cells.
Now have to genotype patients and reduce dose of
drug accordingly. This can be 10-15% of conventional
dose in patients with TMPT deficiency.
what is the therapeutic index
dose that produces toxicity/dose that produces desired effect, TD50/ED50
both TD50 and ED50 are calculated from quantal dose response curves from responses within a population
how can combined polymorphisms effect drug metabolism and action
We have an polymorphism in a gene for CYP450 that reduces metabolism and therefore drug clearance. We have a polymorphism in a gene for a receptor that decreases sensitivity to drug. The efficacy:toxicity ratio varies from 65%: 5% (therapeutic index = 13) down to 10%: 80% (therapeutic index = 0.125)
describe polymorphisms in drug targets
Genetic variation in drug targets (e.g., receptors) can have profound effects on drug efficacy.
The β2-adrenoreceptor (coded by the ADRB2 gene) is the target receptor of β-agonists, such as
albuterol, for bronchodilation in the treatment of asthma. The ADRB2 gene illustrates the link
between SNPs in drug targets and clinical responses. Three SNPs in the ADRB2 gene are
associated with altered expression, down-regulation, or coupling of the receptor in response to
β2-agonists.
Two SNPs of ADRB2 result in mutations of Arg-to-Gly at codon 16 (R16G) and a Gln-to-Glu at codon
27 (Q27E). Both SNPs are common with allele frequencies of 0.4 to 0.6.
The β-agonist albuterol evokes a larger and more rapid bronchodilation response in Arg16/Arg16
homozygotes than in carriers of the Gly16 allele (Arg16/Gly16 and Gly16/Gly16). The maximal %
increase in albuterol-evoked forced expiratory volume in 1sec is 18% for individuals carrying the
Arg16/Arg16, but only 6% for those with the Gly16 allele after an oral dose of 8 mg of albuterol.
However, the influence of genotype was different in patients receiving long-term, regular therapy with
inhaled β -agonists. Patients homozygous for Arg at ADRB2 codon 16 have nearly complete
desensitization after continuous infusion of isoproterenol, with venodilatation decreasing from
44% at base line to 8% after 90 min of infusion. In contrast, patients homozygous for Gly at codon
16 had no change in venodilatation.
In addition, morning peak expiratory flow deteriorated dramatically after cessation of therapy in
patients with the Arg/Arg genotype, but not in those with Gly/Gly. These findings are consistent
with the desensitization of the receptor in patients with a codon 16 Arg/Arg genotype.
Thus a codon 16 Arg/Arg genotype may identify patients at risk for nonbeneficial effects of regular
therapy with inhaled β -agonists.
The bronchodilator response to inhaled
βagonist therapy in asthma pa
what are ABC transporters
ATP-binding cassette transporters (ABC-transporter) are transmembrane proteins that use
energy from ATP hydrolysis in nucleotide-binding domain (NBD) to translocate substrates
across extra- and intracellular membranes through channel formed by alpha helices of
transmembrane domain (TMD).
ABC transporters have a broad substrate specificity and handle a large number of diverse,
hydrophobic compounds including metabolic products, lipids and sterols, and drugs..
describe human ABCB1/MDR1 P glycoprotein (Pgp)
Pgp is an ABC efflux transporter associated with multi-drug
resistance
• It can transport cationic, electrically neutral, and a broad spectrum
of amphiphilic substrates including bilirubin, anticancer drugs,
cardiac glycosides, glucocorticoids, immunosuppressive agents,
(HIV) type 1 protease inhibitors, and other medications.
• Pgp was first recognized for its ability to actively export anticancer
agents from cancer cells and promote multidrug resistance to
cancer chemotherapy.
• Pgp is widely distributed in normal cell types, and serves a protective role by transporting
toxic substances or metabolites out of cells into urine, bile, and the intestinal lumen.
• Increased intestinal expression of Pgp can limit the absorption of Pgp substrates and reduce
their bioavailability preventing attainment of therapeutic plasma concentrations. Conversely,
decreased Pgp expression may result in supratherapeutic plasma concentrations of relevant
drugs and drug toxicity.
describe polymorphisms in human Pgp transporter gene
At least 50 SNPs have been identified in the promoter
and exon regions of the ABCB1 gene.
The diagram of human PgP shows a circle for each
amino acid and each colour is a different exon. Two of
the most common SNPs in the human ABCB1 gene
are associated with altered drug disposition or altered
drug effects .
The SNP that does not alter the amino acid encoded
in exon 26 (the 3435C→T, Ile unchanged) has been
associated with variable expression of PgP. Patients
homozygous for T allele (TT), have 50% lower
duodenal expression of P-glycoprotein compared with
the CC genotype. Hence the oral bioavailability of
digoxin is higher (cardiac stimulant) in patients
homozygous for TT (Cmax denotes maximal
concentration). However, TT homozygotes have lower
plasma concentrations after oral doses of
fexofenadine (antihistamine) and nelfinavir (HIV
protease inhibitor).
This SNP (TT) is also linked to better CD4 cell
recovery in HIV-infected patients who are treated with
nelfinavir and other antiretroviral agents .
The SNP at nucleotide 2677 (G→T, Ala becomes Ser)
has been associated with lower plasma fexofenadine
concentrations in patients homozygous for the T
nucleotide at position 2677 (lower panel)
describe the role of ABC transporter in multidrug resistance and ADRs
Role of ABC transporters in multidrug resistance and ADRs
ABC transporters play a crucial role in the development of multidrug resistance (MDR).
In MDR, patients develop resistance not only to the drug they are taking but also to several
different types of drugs. This is caused by several factors, one of which is increased excretion of
the drug from the cell by ABC transporters such as the ABCB1 protein (P-glycoprotein).
ABC transporter single nucleotide polymorphisms (SNPs) can have an impact on their transport
activity and thus on pharmacokinetics (PK) of the drugs transported. They can alter drug
concentrations at therapeutic sites of action and have important clinical implications for
therapeutic index and ADRs.
Human ABCG2 also known as breast cancer resistance protein (BCRP) contributes to
multidrug resistance (MDR) in cancer chemotherapy. Overexpression of ABCG2 correlates with
the MDR phenotypes of numerous cancer cell lines and tumour types.
Two polymorphisms of the ABCG2 gene
• ABCG2 34G>A , resulting in a Val12Met substitution in exon 2
• ABCG2 421C>A , resulting in a Glu141Lys substitution in exon 5
have shown to be related to the adverse effect of many drugs transported by ABCG2.
The ABCG2 G34A allele, resulting in a Val12Met substitution, causes the apical plasma
membrane dislocalization of ABCG2 and produces a protein with significantly reduced ability to
transport several drugs
The ABCG2 C421A polymorphism has been associated with similar levels of mRNA but low
levels of ABCG2 expression and altered sensitivity to anticancer drugs in vitro, compared with
the wild type. Several groups have reported that the C421A genotype is associated with
altered pharmacokinetic parameters of ABCG2/BCRP substrates.
HEK293 human embryonic kidney cells were transfected with wild-type ABCG2 and mutant
ABCG2 421C>A genes.
HEK293 cells transfected with 421C>A demonstrate reduced expression of ABCG2 and
reduced transport of gefitinib (EGF receptor tyrosine kinase inhibitor – anticancer drug) .
The presence of the variant has been associated with greater gefitinib plasma accumulation
at steady state in patients receiving gefitinib therapy.
how might polymorphisms in drug metabolism and drug targets in combination effect 5-FU treatment
5-Fluorouracil (5-FU) is a common chemotherapeutic agent.
In normal patients about 5% is converted to Fluorodeoxyuridine
monophosphate (FdUMP) which is a cytotoxic antitumour agent.
80-95% is catabolised to inactive metabolites by
Dihydropyrimidine Dehydrogenase (DPD) and these
metabolites are excreted in urine and bile.
A SNP that is a G to A change at a splice site for exon 14 results
in the formation of non-functional DPD (lacks exon 14).
Patients are poor metabolisers of 5-FU and 5-FU toxicity results.
5-Fluorouracil (5-FU) is converted to Fluorodeoxyuridine monophosphate (FdUMP) which
inhibits Thymidylate Synthase (TS).
Thymidylate synthase is the critical enzyme in thymidylate synthesis which is the
precursor of thymidine triphosphate needed for DNA synthesis and DNA replication for cell
division. Hence the antitumour activity of FdUMP.
Clinical resistance to 5-FU is linked to overexpression of TS in tumours.
TS expression is linked to the number of polymorphic tandem repeats in the TS enhancer
region (TSER) of the promoter for the TS gene.
Individuals homozygous for TSER 3 (3 tandem repeats) have increased TS expression
compared to TSER 2 (2 tandem repeats). Thus TSER 3 have poor response to 5-FU.
how might polymorphisms in drug metabolism and targets effect warfarin treatment
Warfarin inhibits the vitamin K-dependent
synthesis of biologically active forms of the clotting
factors II, VII, IX and X, as well as the regulatory
factors protein C, protein S, and protein Z. These
factors require carboxylation of Glu amino acids in
their structure to allow binding to phospholipid
membranes inside blood vessels, on the vascular
endothelium.
The enzyme gamma-glutamyl carboxylase carries
out the carboxylation only if it is able to convert a
reduced form of vitamin K (vitamin K
hydroquinone) to vitamin K epoxide at the same
time. The vitamin K epoxide is recycled back to
vitamin K and vitamin K hydroquinone by vitamin K
epoxide reductase (VKOR).
Warfarin inhibits the VKORC1 subunit thus
diminishing available vitamin K and vitamin K
hydroquinone. This inhibits the activity of the
glutamyl carboxylase and the coagulation factors
are no longer carboxylated and are thus
biologically inactive. As the body’s stores of
previously-produced active factors degrade over
several days and are replaced by inactive factors,
the anticoagulation effect becomes apparent. The
end result of warfarin treatment is to diminish
blood clotting in patients with atrial fibrillation,
heart valve prosthesis, recurrent stroke, deep vein
thrombosis and pulmonary embolism
SNPs associated with the genes for CYP2C9 that metabolises warfarin or VKORC1 that is the warfarin
target result in diminished warfarin metabolism and increased warfarin sensitivity. This explains the 10-
fold inter-individual variation in warfarin dosing.
SNPs in CYP2C9
CYP2C92 (430C>T) results in an Arg144-Cys (R144C) change in amino acid sequence, and reduces
catalytic activity compared to wild type. This reduces warfarin metabolism by approx 30% and extends
the half-life of the drug. The average daily Warfarin requirement is approximately 17% lower in patients
with one copy of CYP2C92. Allele frequency Caucasians 8-19%, African 3-4%, Asians ~ 1%.
CYP2C93 (1075A>C) results in an Ile359-Leu (I359L) change in amino acid sequence, and reduces
catalytic activity compared to wild type. This reduces warfarin metabolism by approx 80%; extends halflife of drug. Average daily Warfarin requirement is approximately 37% lower in patients with one copy of
CYP2C93. Allele frequency Caucasians 3-17%, African 2%,.Asians 1-7%.
Because warfarin given to patients with *2 or *3 variants will be metabolized less efficiently, the drug will
remain in circulation longer, so lower warfarin doses will be needed to achieve anticoagulation.
SNP in the promoter regions of VKORC1 (1636G>A) reduces expression of the warfarin target
receptor, VKORC1, via a transcriptional mechanism. About 20% lower warfarin doses are needed to
inhibit the lower levels of VKORC1 and to produce an anticoagulant effect in carriers of the A alleles.
Heterozygous (G/A) allele frequency is approx 40% in Caucasians and 14% in Africans.
Combinations of mutations from CYP2C9 and VKORC1 accounts for up to 79% of variability in warfarin
dosing. FDA admits warfarin is among the top 10 drugs with the largest number of serious ADRs and
updated the drug label for Warfarin in 2007 and again in 2010 with recommendations for initiation dose
modifications in carriers of the specific CYP2C9 and VKOR variants. Genetic screening would vastly
improve Warfarin therapy
what are factors affecting drug metabolism through CYP450s
The most important factor determining the duration of action of a drug is the rate
and extent of its metabolism. This is largely due to the amount and activity of the
enzymes responsible for the metabolism of the drug - especially CYP450s of
Phase 1 drug metabolism. The drug metabolic potential of an individual reflects
the spectrum of cytochrome P450 activity present.
The amount and activity of CYP450 enzymes are determined by many factors:
Age There are lower levels of P450s in young compared to adults. UDGT is
low in infants so glucuronidation capacity is decreased. In old age, some CYP450
levels go down but decreases occur at different ages in different people.
Sex Some species, notably the rat, show a marked difference in drug
metabolism in males and females. Usually, the rate of drug metabolism is higher in
males. There is not such a marked difference in humans.
Hormones Cytochrome P450s are involved in steroid metabolism and they
are subject to hormonal control.
Health Diseases, especially those affecting the liver and the kidney, will
have profound effects on the drug metabolising capacity.
Genetics – different individuals have different levels of CYP450/Phase 2 activity.
The activity of CYP450s can be changed by exposure to different foreign
compounds, including drugs, foodstuffs, carcinogens and environmental pollutants.
describe some adverse drug reactions occuring due to drug interactions with CYP450s
Phenytoin, induces CYP1A2, CYP2C9, CYP2C19 and CYP3A4. Substrates for the latter
may be drugs with critical dosage, like amiodarone or carbamazepine, whose blood plasma
concentration may decrease because of enzyme induction
Sertraline (Zoloft) is considered a mild inhibitor of CYP2D6 at a dose of 50 mg, but if the
dose is increased to 200 mg, it becomes an immediate potent inhibitor.
The calcium channel blocker mibefradil (Posicor) was withdrawn from the U.S. market in
1998 because it was a potent CYP450 inhibitor that resulted in toxic levels of other
cardiovascular drugs.
