Lecture 3 Flashcards
What is an early example of pharmacogenetics in clinical practice?
Butyrylcholinesterase (BCHE)
Succinylcholine muscle relaxants developed in the 1950’s and are still widely used for rapid short term skeletal muscle paralysis - eg intubation. However, some patients experience life-threatening prolonged apnea – due to genetic variation in the BCHE gene.
- Kalow et al (1956) developed phenotypic assays (the dibucaine number) to detect “Atypical” or the A variant, assay still used today.
- Subsequently, genetic basis of the Atypical (A) and Kalow (K) variants defined. More than 60 variants now described, mostly very rare and associated with the Silent (S) or deficient phenotype
- Phenotype and genotype are used to characterise patients, warning cards issued where BCHE activity is low, cascade testing is done in at risk family members
- Note: testing is completed after toxicity has occurred thus not a true pharmacogenetic test.
Silent (S) phenotype in genetics does not have an effect, what does this mean in pharmacogenetics?
Enzyme inactive and thus completely deficient.
What are three aims of pharmacogenetic testing?
Benefit patient – reduced morbidity and mortality
Optimise use of proven first line therapies
Cost saving to the NHS by reducing adverse drug reactions and hence hospital admissions
Why is pharmacogenetics important?
With ageing population, number of those diagnosed with cancer increases and thereby need effect therapies that cause little toxicity.
What are examples of pharmacogenetics variation?
- Coding region single nucleotide polymorphisms (SNPs) and indels which change an amino acid sequence.
- Variants affecting splicing – new exons created, exon skipping
- Repeat variation in regulatory elements.
- Gene duplications and deletions.
- May be multiple rare variants within a single gene
Normal genetic polymorphism is only revealed when?
There is a drug challenge
In what scenario does one not need to be aware of what the cause of a variant is?
When the Markers are in disequilibrium with a causative variant
What is used when exploring pharmacogenetic associations?
What does this require and what becomes mores useful?
ADME
Absorption of drug- often poorly understood
Distribution of drug- First pass metabolism – gut and liver, uptake by cells
Metabolism of drug- Complex -may be incompletely understood
Excretion of drug- Conjugation reactions, solute carriers (SLCs).
Requires a good understanding of how the drug is metabolised, often knowledge is incomplete. Thus WGS approaches to investigating associations between genetic variation and drug metabolism become more useful.
The same enzymes and pathways that clear dietary and environmental toxins also work on drugs.
What is an example
Xenobiotics- chemicals derived from diet and overall environment, and are potentially harmful. Enzymes evolved to remove them.
Xenobiotic metabolism historically classified as Phase I and II, converting hydrophobic lypophilic compounds into derivatives that can easily be excreted in urine or the bile
Phase I enzymes carry out oxidation, reduction, hydrolytic reactions – usually drug inactivation, may activate pro-drugs.
Phase II enzymes – conjugation reactions at site of Phase I leading to increased water solubility and elimination
This is a “convenient” classification of metabolism with many exceptions
Phase 1 metabolism involves which pathways?
Clue: Huge, ostriches, run
Hydrolysis
Oxidation
Reduction
Phase 2 metabolism involve which pathways?
Getting, animals, sugar, guides, summer
Glucuronidation
Acetylation
S-methylation
Glutathion conjugation
Sulfo conjugation
Phase 2 metabolism aim to increase what?
Molecular weight to aid excretion through biliary tract or urine
Phase 1 metabolism mostly involve what enzymes?
Cytochrome P450 (CYP)
How many Cytochrome P450 (CYP) genes exist?
How does these genes differ?
57 functional genes, 58 pseudogenes
CYPs 1-3: 22 different isoforms, high degree of polymorphism
• CYPs 4-51: metabolism of mostly endogenous compounds, little polymorphism.
What are pseudogenes?
Inactive copies of the gene
What is the nomenclature for CYP2E1?
CYP- Abbreviation for mammalian cytochrome P450
2- Gene family
E- Sub family
1- Individual enzyme
How are CYP families 1-3 characterised?
22 different isoforms, high degree of polymorphism
How are CYP families 4-51 characterised?
Metabolism of mostly endogenous compounds, little polymorphism.
What are the CYP phenotypes?
Clue: Puppies, immediately, eat, ultrasounds
Poor metabolisers – two defective alleles
Intermediate metabolisers – heterozygous for
a defective allele
Extensive metabolizers – two functional alleles
Ultra-rapid metabolisers >2 copies or promoter variants
Multiple CYPS can metabolise the same drug
True or false
Give an example
True
Caffeine
There is a strong association between CYP2C8 and what drug?
Is this clinically useful?
CYP2C8 and ibuprofen plasma levels. If CYP2C8 33 homozygous maintain drug levels for considerable length of time
Not clinically useful, self medicate- individuals will not be tested prior to use of this
What serious side effect does Ibuprofen cause?
What was found?
Is this introduced into clinical practise?
Gastroduodenal bleeding
CYP2C9*3 genotyping may identify subgroups of persons who potentially are at increased risk of gastroduodenal bleeding when treated with NSAIDs.
Further studies are needed before genotyping is introduced into clinical practice..
In clinical practice what is focused upon?
Focus on drugs with serious toxicity or a clear therapeutic benefit in optimising therapy
What enzyme subfamily are the most important drug-metabolizing enzymes in liver and gut, and amount to ~30 and 70% of total CYP content?
CYP3A5*3
CYP3A5*3 is an intronic noncoding SNP which creates what?
A splice site resulting in a mRNA insertion and frame shift. Some normally spliced transcripts still formed
What is CYP3A subfamily able to metabolise
Tacrolimus (an immunosuppressant drug) used in transplantation
CYP3A53/3 genotypes CYP3A5 expression is…
reduced to 4% of total CYP3A in the liver and 3% of total CYP3A in the jejunum
Heterozygous CYP3A51/3 subjects CYP3A5 expression is…
decreased to 50% of total CYP3A in the liver, and 61% of CYP3A in the jejunum
Tacrolimus is metabolised primarily where?
In the liver and intestine mainly by CYP3A4 and CYP3A5 - hydroxylation and demethylation reactions - estimated to be at least 15 metabolites
CYP3A5*3 has marked ethnic diversity, with a frequency of 32% in African–Americans, but 90– 93% in Caucasians.
True or false
True
What do transplant recipients with the CYP3A5*1 allele require?
A higher daily tacrolimus dose compared with CYP3A53/3 genotypes to maintain both the target trough level and area under the curve
Why has the CYP3A5*3 marker not yet entered routine clinical care despite overwhelming research support?
In transplantation initial drug dosing computed using therapeutic dose monitoring- area of curve measured and this is used for dosage, no impact of genotyping CYP3A5*3 prior to start of therapy
What is Tamoxifen used to treat
Breast cancer
The WHO estimated 522,000 deaths worldwide due to breast cancer in 2012
True or false
True
What percentage of tumours will be hormone receptor positive and will respond to tamoxifen therapy?
60-70%
What is the active metabolite of tamoxifen?
What is it produced by?
Endoxifen
Action of CYP2D6
CYP2D6 poor metaboliser genotypes result in what?
Poor responders to therapy
In a systematic review into Pharmacogenetics of Tamoxifen metabolism, 25 studies, 6 showed what?
What three factors were concluded from this study?
Extensive metaboliser (EM) CYP2D6 genotypes associated with better outcomes than poor metabolisers
- data are limited and conflicting
- not possible to recommend pharmacogenetic testing
- further research needed
Glucuronidation and sulfation increase what?
hydrophilicity.
What do glutathione-S-transferases (GSTs) transfer?
Glutathione to reactive electrophiles, protects cellular macromolecules from damage.
What does glucuronidation catalyse?
The transfer of glucuronic acid from UDP-glucuronic acid to a substrate forming glucuronides
What markedly increases molecular weight favouring biliary excretion?
Glucuronidation
How many UGT genes are there known to be?
19 genes, nine encoded by the UGT1 locus and 10 are encoded by the UGT2 family of genes.
What is Gilbert’s syndrome?
What is this due to?
What is seen in normal individuals compared to those with Gilberts syndrome?
Up to 10% of the population have circulating bilirubin levels which are 60% to 70% higher than those seen in normal subjects.
Variation in the UGT1A1 gene promoter, leading to reduced expression levels of UGT1A1.
Normal: UGT1A11 = A(TA)6TAA
Those with Gilberts Syndrome: UGT1A128 = A(TA)7TAA with 8-23% homozygous
Those with UGT1A1*28 are predisposed to what?
Adverse drug reactions resulting from a reduced capacity to metabolize drugs by UGT1A1.
What is used for the treatment of colorectal cancer?
TA7/7 or TA6/7) carriers experience what?
Irinotecan
Severe grade 3/4 neutropenia and/or diarrhea
What do United States Food and Drug Administration recommend in the package insert for irinotecan that UGT1A1*28?
However…
That homozygous patients should receive a lower starting dose of irinotecan.
Evidence is contradictory – testing has not entered widespread clinical practice
What is warfarin?
Anticoagulant used for the prevention of thrombosis
What is a major side effect of warfarin?
Major side effect – life threatening bleeding
What explains 30% of the warfarin dose variation?
Vitamin K epoxide reductase subunit 1 (VKORC1) polymorphisms
What explains 10% of the warfarin dose variation?
CYP2C9 poor metabolises
What did Kimmel et al. N Engl J Med. 2013 Dec 12;369(24):2283-93 find?
What did Pirmohamed et al N Engl J Med. 2013 find?
What is the reasoning behind the outcome of Kimmel et al?
Genotype-guided dosing of warfarin did not improve anticoagulation control during the first 4 weeks of therapy
Pharmacogenetic-based dosing was associated with a higher percentage of time in the therapeutic INR range than was standard dosing during the initiation of warfarin therapy.
Negative outcome may be explained by the practise of regular testing and monitoring of clotting in the clinic when patients are initiated on warfarin, this is used to set warfarin dose - if carried out often does not lead to a difference between patients whose doses are set by genetics or therapeutic drug monitoring
What is Allopurinol used to treat?
Gout
Allopurinol is considered to be a what?
A safe drug, tonnes prescribed every year
What is the active metabolite of Allopurinol?
Oxypurinol- A potent xanthine oxidase inhibitor preventing theformation of uric acid
Is Allopurinol completely safe?
Why?
No
Some experience allergic reaction known as toxic epidermal necrolysis - HLA-B*5801 allele is associated with toxic epidermal necrolysis in Han Chinese populations (odds ratio 580, 95% confidence interval 34.4 to 9780.9; P<0.001).
Not all carrier experience TEN, but testing is proposed as clinically useful and cost effective (Ko et al BMJ. 2015 Sep 23;351). Avoid allopurinol, prescribe febuxostat
Consequence: testing not done, febuxostat prescribed for all patients
HLA-B*5801 allele is rare in Caucasians
Phase I drug metabolism. Role of CYPs in drug metabolism: drug activation - tamoxifen, drug degradation - ibuprofen, warfarin
Phase II drug metabolism. Glucuronidation and irinotecan
Dose independent hypersensitivity reactions: HLA related – allopurinol
Are all examples of what?
Pharmacogenetic influences on drug metabolism, but none are currently used in routine clinical practice
What makes a good pharmacogenetic test?
- Replication, replication, replication
- The marker must have a large effect / high penetrance, but need not explain all toxicity!
- Predictive not prognostic: alternate therapies or therapeutic strategies must be available
- Clear patient benefit by reducing toxicity or improving efficacy
- Testing should not delay therapy
- Testing must be cost effective
What is Abacavir used to treat?
HIV
What side effect is Abacavir associated with?
What is the rate of this?
How long does it take for these side symptoms to present?
What is the issue if Abacavir is re-challenged?
Hypersensitivity Reaction (HSR) an idiosyncratic multi- organ system syndrome Common symptoms are fever, rash, GI, constitutional and respiratory.
HSR rate 8% - this is high
Median time to onset 9 days; >90% of cases occur within first 6 weeks with an HSR worsens with continued therapy and may be life-threatening but resolves with prompt diagnosis and discontinuation of abacavir
Contraindicated due to potential for severe or fatal HSR
What did Mallal et al. N Engl J Med. 2008 Feb find?
What was the outcome of this
HLA-B*5701 screening reduced the risk of hypersensitivity reaction to
abacavir.
HLA-B*5701 genotyping is standard of care for treatment naive HIV patients
If positive for HLA-B*5701, alternative therapy to abacavir must be prescribed
Definition and management of this drug safety risk has enabled more widespread and effective use of abacavir containing anti-HIV treatment regimens
Is inflammatory bowl disease (IBD) the same as IBS
NO IBS is not an autoimmune disease
What are two forms of IBD?
Ulcerative Colitis
Crohn’s Dosease
What is used to treat IBD?
Azathioprine
What is Azathioprine?
Thiopurine drug analogue
Standard 1st line immunosuppression in IBD
What is Azathioprine used for?
Remission induction and maintenance
What is the response to Azathioprine defined as?
Variable: 40%-65%
Therefore significant individual variation in therapeutic benefit
What is the step-wise treatment of Crohn’s disease
- 5-ASA, Antibiotics. If this does not control disease step 2 is implemented.
- Corticosteroids
- Azathioprine- steroid sparing agent and to maintain remission
- Methotrexate
- Biologics - anti-tumor necrosis factor-alpha agents/ TNF blockers
- Surgery
What is becoming a first line treatment for Crohn’s disease?
What is used in conjunction with this and why?
Biologics
Azathioprine. As biologics are antibodies against TNF, the body begins to produce antibodies against these antibodies. To decrease this reaction and prolong life Azathioprine are used also.
Describe the biotransformation/metabolisation of azathioprine to thioguanine nucleotides
Azathioprine- pro drug of mercaptopurine
imidazole group is attached to Azathioprine to increase solubility when
Azathioprine is absorbed. imidazole split off non enzymatically to methylmercaptopurine (6 MP)
6MP has three metabolic phases. The first is that degraded to thyer uric acid through Xanthine Oxidase. 2. It can be methylated by enzyme TMPT 3. or activate through normal purine salvage pathways catalysed by HPRT to form activate metabolites thioguanine nucleotide (6-TGN)
What does TPMT deficiency =
Increased drug activation and over-dosing
Main enzyme for regulating of Azathioprine which is converted to thioguanine nucleotide is what?
How does this do it?
Thiopurine methyltransferase (tpmt)
Transfers methyl group to mercaptopurine to for methyl mercaptopurine
What happens if TMPT is deficient?
more methylmercaptopurine (6 MP) is shuttered toward metabolic pathway to form active thioguanine nucleotides and this leads to increase immunosuppression, increase of cytotoxicity and increased risk of adverse effects
What determines the activity of TMPT?
Genetic polymorphism
What did a London IBD Forum prospective study investigating TPMT activity vs. Azathioprine withdrawal in IBD in 189 patients find?
What does this indicate?
80% of TMPT variants carriers experienced toxicity
Wild type (normal TMPT) 36% withdrew due to toxicity
This does not indicate that it is a poor therapeutic test, but rather that TPMT accounts for a subset of patients whom experience toxicities, but does not all toxic events
Everyone is tested prior to Azathioprine dosing and how does this inform dosing?
As TPMT accounts for a subset of patients whom experience toxicities, but does not all toxic events, what is monitored?
TPMT zero:
Use alternative therapy
Carrier range:
Initiate at 1/3rd standard dose, dose increment to a target dose of 50% standard dose
Normal Range
Initiate at standard dose
FBC (Full blood count) and LFT (Liver function tests )
Azathioprine target dose is adjusted on the basis of TPMT result - patients are not excluded from therapy
True or false
True
What is the main purpose of TPMT testing?
What is the added value of TPMT testing?
To detect all completely deficient patients.
Detect carriers and dose adjust accordingly
It is not currently cost effective to test for all possible TPMT variants therefore testing is pursued for what?
The most common variants TPMT3C/3A/*2)
Why is TPMT genotyping considered better than phenotyping?
Discordance in the TPMT carrier range- There is no clear distinction between who is a carrier and who is wild type in terms of phenotype
When is phenotyping better in relation to TPMT?
Rare and private TPMT variants which are not included in genotyping better- phenotyping is better at identifying all possible mutations and all combinations of mutations particularly in those patients who are completely deficient due to compound heterozygous genotypes.
Is TPMT genotyping or phenotyping better for blood transfusions and why?
Blood transfusion will mask carrier and complete TPMT deficiency, genotyping better
Ideally all patients should be TPMT genotyped and phenotyped, but why is this not possible?
• Phenotyping costs pennies, genotyping costs pounds
• Children and young adults with acute lymphoblastic leukaemia – poor concordance between genotype and
phenotype, national guidelines say genotype Lennard L, Br J Clin Pharmacol. 2013 Aug 21. doi: 10.1111/bcp.12226. [Epub ahead of print]
For most patients why is therapeutic drug monitoring to identify levels of active metabolites or methylated metabolites to further optimise dosing Azathioprine used?
Discordance in the TPMT carrier range- There is no clear distinction between who is a carrier and who is wild type in terms of phenotype. Therefore some may be miscalled as normal or carrier. Thus method determines if patients are TPMT carrier.
Metabolite monitoring and pharmacogenetics are important to optimise therapy
True or false
True
What does therapeutic drug monitoring do?
Measures levels of thioguanine nucleotide (6-TGN) and levels of methylmercaptopurine (6 MP)
If thioguanine nucleotide (6-TGN) levels are high and methylmercaptopurine (GMP) very low what does this mean?
Patient is a TPMT carrier treated with normal dose that can be adjusted based on that
If thioguanine nucleotide (6-TGN) are therapeutic and methylmercaptopurine (6 MP) is moderate. The patient is….
Adequately dosed
Subset of patients with high methylmercaptopurine (6 MP).
What is this associated with
Hepatic toxicity and can be associated with lack of response
At the pharmacogenetics basis of very high methylmercaptopurine (6 MP) metabolities is currently not known
True or false
True
What has entered into clinical practise of azathioprine and mecatopurine and how often is this used?
Therapeutic drug monitoring
Most patients TGNS measured at least one or twice a year, this is also particularly measured after any dosage change
TPMT testing is accepted in clinical practice and guides initial dosing strategies
True
What variants are more important than TPMT in Asian ethnicities and should be included in genotyping panels?
NUDT15 variants
TGN levels summarise genetic and epigenetic effects – further optimise azathioprine dosing strategies
True or false
True
There are other multiple pharmacogenetic TPMT markers with large effect sizes influencing drug metabolism
True or false
False
There are other multiple pharmacogenetic TPMT markers with small effect sizes influencing drug metabolism
Fluoropyrimidine drugs developed in the 1950s
True or false
True
Fluoropyrimidine drugs are used as a first line treatment for what?
Solid tumours including colorectal and
breast cancer
Approximately how many patients receive Fluoropyrimidine?
Approximately 2 million patients pa receive these antimetabolites worldwide
What percentage of patients will develop severe (Common Terminology Criteria for Adverse Events (CTCAE) grade ≥3) toxicity from Fluoropyrimidine?
What does this lead to?
What does this impact?
10-20%
Leads to treatment interruption, delays of subsequent cycles and termination of treatment altogether.
• Impacts negatively on efficacy and prognosis as well as posing a significant cost burden to health care providers. 5FU grade 3-4 toxicity to Guy’s and St Thomas’ Hospitals - Significant more bed stay
What is recognised as a cause of 5FU toxicity (5-Fluorouracil)?
Dihydropyrimidine dehydrogenase deficiency (DPD) - – first case of severe toxicity due to DPD deficiency reported in 1985 (Tuchman et al N Engl J Med 313(4), 245-249 (1985).
What did the FDA warn in 2003 regarding 5FU?
What would benefit patients?
That 5FU and the pro-drug capecitabine are contra-indicated in patients with DPD deficiency
Predicting and avoiding severe toxicity
Fluoropyrimidine metabolism and DPD involves what
Capecitabine is a prodrug activated by enzymatic steps to form 5FU (the active metabolite)
5-FU is degraded through Dihydropyrimidine dehydrogenase deficiency (DPD) and 80-90% of the drug follows this route. Therefore a small change in DPD can cause a large change in 5-FU blood levels.
5-FU that isn’t degraded is converted into 5F-deoxyuridine and this is a potent inhibitor of thymidylate synthase and for thymidylate synthase to be inhibited folate binding is required.
Thymidylate synthase is needed for thymidine nucleotide synthesis which enters DNA
Other routes of drug action for fluro-deoxy UTP can enter DNA, fluro UTP can enter RNA
5FU has a narrow therapeutic window with what percentage of 5FU catabolised through DPD?
80-90%
Complete DPD deficiency is classified as what?
A rare metabolic disorder characterised by increased thymine and uracil in urine – usually fatal toxicity if treated with 5FU.
Most patients in clinics experience what kind of DPD deficiency?
Partial (heterozygous) DPD deficiency - 4-6% of the population - is associated with severe Grade 3-4 toxicity to 5FU
Why is partial or heterozygous DPD deficiency is much more difficult to diagnose?
Thymine and uracil not elevated in urine, carriers are asymptomatic.
Why is there no enzyme DPD assay?
DPD cannot be assayed in red cells
- White cell radiochemical assay not suitable for high throughput screening or referred samples
- Assay is not linear at low protein concentrations and is affected by the white cell differential count
- Genotype-phenotype correlation in the carrier range is very poor.
Complete DPD deficiency involves many, mostly rare mutations scattered across 23 exons
True or false
True
What is the first common DPYD variant to be associated with toxicity and has a heterozygous genotype frequency of 1.8%.?
DPYD IVS14+1G>A splice junction variant
How many DPD variants are generally accepted as a panel to predict toxicity and have a combined genotype frequency of ~6%?
Approximately what percentage of cases grade 3-4 toxicity were explained by these DPYD variants?
How well is evidence for such variants considered?
4 DPYD variants
25%
Extremely well
What did Behnke et al 2002 find in concern to the cost of DPYD IVS14+1G>A variant toxicity in the US?
.
- Estimated 250,000–300,000 patients treated annually
- 2350–2820 patients estimated to carry the IVS14+1G>A variant, 1210–1452 will suffer severe toxicity, and some will die from their treatment.
- Estimated annual cost $36.3 million to $145 million.
- Costs for screening all patients ($100/test) between $25-30 million, Savings $11.3–115 million
What did Deenen MJ et al, J Clin Oncol. 2016 Jan find in relation to the cost of DPYD IVS14+1G>A variant toxicity in Europe?
- 2,038 patients were prospectively screened for IVS14 (DPYD*2A), of whom 22 (1.1%) were heterozygous
- The risk of grade 3 toxicity was reduced from 73% in historical controls to 28% by genotype-guided dosing
- Death due to toxicity was reduced from 10% to 0%.
- Average total treatment cost per patient was lower for screening (€2,772) than for non-screening (€2,817), outweighing screening costs
Pharmacogenetic directed dosing (CPIC) advice for the DPD variants that are generally accepted as a panel to predict toxicity is what?
IVS14+1G>A, c.1905+1G>A - 50%
c. 2846A>T (p.D99V)- 50-75%
c. 1679T>G (p.A560S)- 50%
c. 1236G>A/HapB3 c.1129-5923C>G deep intronic variant- 50-75%
Heterozygous and compound heterozygous genotypes of DPD variants exist.
In relation to compound heterozygous genotypes, the effect of variant alleles is considered as what?
Additive
A study investigating the impact of rare DPYD variants found what?
What are two conclusions based from this finding?
All three patients died.
Phenotypic screening tests may be useful to ID these patients.
Co-drugs and other genes likely to contribute to unexplained toxicity.
What are the 2020 healthcare related changes relating to DPYD variants?
- EMA recommends testing for four DPYD variants prior to therapy
- NHSE commissions testing for four DPYD variants in August
- In August UK Chemotherapy Board issues dosing guidelines for DPYD variants
What are other approaches to personalising 5FU therapy?
Pharmacokinetic dosing – clearance of 5FU from blood Works for infusional 5FU
• Plasma levels of 5FU checked towards end of the infusion, up to three dose adjustment cycles
• Doses increased as well as decreased, clinical response and tolerability both improved
Pre-treatment endogenous metabolites in serum
• Uracil/dihydrouracil ratio – used in France , evidence contradictory
• Raised serum uracil – correlated wih increased risk of toxicity in alarge Dutch study
Loading tests:
• Thymine loading test – correlates with DPD deficiency and non-DPD related toxicity
