Pharmacogenetics

Question 1

Describe pharmacogenomics.

Answer 1

The genome wide approach to prediction of drug responsiveness.

Question 2

What is the systemic drug concentration?

Answer 2

The systemic drug concentration is the concentration of drug that gives a therapeutic effect. Too high and it will be toxic, too low and it will be ineffective.

Question 3

What might determine the systemic drug concentration?

Answer 3

The absorption of drug and its bioavailability,
The subsequent distribution of the drug in various body compartments,
The metabolism and excretion of the drug.

Question 4

Genetic studies have contributed most to what determinant of systemic drug concentration?

Answer 4

From the point of view of pharmacogenetics, the area where genetic studies have contributed most is to those genes that encode enzymes which are responsible for the metabolism of the drugs and those genes that encode target cell receptors.

Question 5

Why is avoiding adverse drug reactions important?

Answer 5

6.5% of hospital admission in the UK are due to adverse drug reactions.

It is estimated that the annual cost (in 2004) is approximately £500 million to the NHS.

The estimated death rate is more than 10,000 per year.

Question 6

What is one example of where pharmacogenetics could be used for the avoidance of adverse drug reactions.

Answer 6

Detection of thiopurine methyl transferase deficiency which accounts for some cases of thiopurine drug toxicity.

Question 7

What can cause some cases of thiopurine drug toxicity?

Answer 7

Thiopurine methyl transferase deficiency accounts for some cases of thiopurine drug toxicity.

Question 8

Give three examples of thiopurine drugs.

Answer 8

1) . Azathioprine
2) . 6-mercaotopurinol
3) . Thioguanine

Question 9

What are thiopurines used for therapeutically?

Answer 9

Thiopurine drugs are used as myelosuppressants in patients that have had organ transplants and patients with inflammatory bowel disease, rheumatoid arthritis and eczema that are unresponsive to other treatments.

Question 10

Azothiprine is a prodrug. Describe the metabolism of Azothioprine into its active form.

Answer 10

Azothioprine is metabolised to 6-mercaotopurine. Thiopurine methyl transferase (TPMT) then methylates 6-mercaptopurine creating methyl-mercaptopurine.

If 6-mercaptopurine is not metabolised by TPMT into methylmercaptopurine then it is converted into Thioguanine nucleotides which can then go on to cause inhibition of DNA and RNA synthesis.

Question 11

What happens when patients with TPMT deficiency are given thiopurine drugs?

Answer 11

In patients with TPMT deficiency the thiopurines cannot be metabolised efficiently and this is associated with severe bone marrow toxicity due to the accumulation of unmetablised drug.

Question 12

What happens when patients with very high levels of TPMT are given Thiopurines?

Answer 12

There is a rapid metabolism of the thiopurines which is associated with a risk of ALL relapse and may be associated with hepatotoxicity.

Question 13

What can cause TPMT deficiency?

Answer 13

There are a number of single nucleotide mutations that can cause amino acid substitutions within the enzyme TPMT.

This results in an increased degradation rate of the enzyme which accounts for the failure to efficiently metabolise the thiopurines.

About 1 in 300 individuals are homozygous for these single nucleotide mutations (or compound heterozygotes) and these individuals have absent TPMT activity.

Heterozygotes bearing one mutant allele are much more common (approximately 1 in 10 individuals) and they have intermediate TPMT activity.

Question 14

What are the 4 most common TPMT variant alleles?

Answer 14

TPMT1,
TPMT2,
TPMT3A,
TPMT3C.

Have various polymorphisms at 238, 460, 719.

1 and 3A are most common in Caucasians and Africans.

1 and 3C are most common in Indians.

See the table in notes for more info.

Question 15

How can the TPMT genotype guide Thiopurine treatment?

Answer 15

Homozygote or compound heterozygotes individuals have absent TPMT activity and have a high risk of marrow suppression. They should not be treated with thiopurine drugs.

Individuals that have heterozygosity for a mutant allele have intermediate TPMT activity. These individuals have increased risk of marrow suppression. These individuals may be treated with low dose thiopurines and achieve a therapeutic response.

Question 16

How can TPMT activity be measured?

Answer 16

TPMT activity can be measured in whole blood within the red cells which is its main location. Nobody knows what the physiological function of TPMT is. Measuring the red cell activity has certain caveats. Firstly the activity decreases with red blood cell age. Therefore, anything that alters the red cell lifespan will likely affect the measured TPMT activity. Measured TPMT activity will also be affected by blood transfusions.

There is evidence that TPMT activity is increased in patients with chronic renal failure who are are a group of patients who are likely to be treated with immunosuppressants.

The measurement of TPMT activity does not predict all cases of marrow suppression.

Question 17

Before prescribing thiopurine drugs should patients be genotyped, phenotyped or both?

Answer 17

Genotyping for SNPs is technically easy. It is unaffected by medication and illness but a problem is that it will not detect rare variants as current methods only target specific mutations. On the whole only common mutations will be affected. Certain variants are more prominent in some racial groups and there may be variants that we haven’t detected in some racial groups. Therefore it may be difficult to provide a completely comprehensive screen for all genetic variants.

Enzyme activity/phenotyping is technically tricky. It is subject to interference. However, this method will detect reduced TPMT activity due to any variant including rare ones.

The approach that has been adopted in centres that have taken up TPMT testing is to carry out enzyme activity measures as a first test and then follow up patients with low enzyme activities with genotyping tests.

TPMT genotyping/phenotyping can assist in deciding starting does of thiopurines.

Monitoring blood count and liver function remains mandatory whichever method is used.

Question 18

Why might we want to use pharmacogenetics to assist in choosing an appropriate drug?

Answer 18

The use of an inappropriate drug delays effective treatment and exposes patients to unnecessary risk of adverse drug reactions. It also incurs unnecessary expense.

Question 19

Give an example of one situation in which pharmacogenetics can be used to assist in the choice of appropriate drugs.

Answer 19

Herceptin and breast cancer.

Question 20

How can pharmacogenetics be utilised in selecting breast cancer patients for which Herceptin will be an appropriate treatment?

Answer 20

The selection of the drug Herceptin for the treatment of breast cancer can be guided by genetic testing of the tumour tissue.

Her2 is a tyrosine kinase growth factor receptor that responds to EGF and other growth factors. Binding of the relevant growth factor to the Her2 receptor triggers gene activation and cell division and thus growth of the tumour.

Clinically, the importance of this is that there is Her2 gene amplification in 25-30% of breast tumours. Her2 is positively associated with more aggressive tumours. Her2 positive tumours are responsive to Herceptin.

In detecting patients for treatment with Herceptin, Her2 can be detected initially by IHC of tumour tissue and in selected cases the increased copy numbers of the Her2 gene can be established either by FISH or Her2 PCR.

There are ethical issues that arise from the utilisation of genetic tests to select an appropriate treatment. For example, is withholding a drug deemed to be ineffective on genetic testing acceptable? Will pharmacogenetics cause unacceptable disparity in prescribing to different ethnic groups? Will pharmaceutical developments be adversely affected by market segmentation as those with rarer genotypes are neglected in drug development because the market is smaller?

Question 21

Why might you want to use pharmacogenetics for rapid optimisation of drug dose?

Answer 21

There is wider inter-individual variation in plasma levels and during drug responsiveness which are the result of complex interactions between genetic and environmental factors.

Currently we use a trial and error approach to drug dosing.

Question 22

Describe an example of a situation in which pharmacogenetics can guide optimisation of drug dose.

Answer 22

Anticoagulantion with Warfarin.

Question 23

Describe who warfarin acts and how optimisation of warfarin dose can be achieved with pharmacogenetic assistance.

Answer 23

Warfarin is prescribed to patients who are at risk of thromboembolism due to pulmonary embolus, DVT or atrial fibrillation.

Warfarin acts by inhibiting the enzyme Vitamin K reductase, which recycles oxidised vitamin K to its reduced form following use in carboxylation of prothrombin and coagulation factors VII, IX and X. Carboxylation is essential for the activation of these coagulation factors. Therefore if warfarin inhibits vitamin k reductase, vitamin k does not return to its reduced form and the coagulation factors cannot be carboxylated and so remain non-functional.

Warfarin is metabolised by cytochrome p450 enzyme CYP2C9. Optimising the warfarin dosage is extremely important because under treatment will fail to achieve reduction in risk of thromboembolism. Patients are often given a loading dose to achieve therapeutic levels rapidly. Overdose of warfarin increases the risk of haemorrhage.

Patients on warfarin are monitored using the International Normalised Ratio to assess the activity of the clotting factors. Typically the target INR is between 2 and 3.

The first gene that was of interest in the pharmacogenetics of warfarin was the CYP2C9 gene which encodes the cytochrome p450 enzyme. Cytochrome p450 enzyme metabolises not only warfarin but many other drugs glipizide and Ibuprofen.

Single base changes at aa144, 359 and 360 can result in amino acid substitutions which render the CYP2C9 enzyme less active.

The main allele of CYP2C9 is *1 on Caucasian and Asian populations, *2 is found in both populations also. *3 is only found in Caucasians. *4 and *5 is found in Japanese and African American populations only.

Possession of 1 or 2 copies of a variant CYP2C9 allele predicts low warfarin dosage. Other factors that predict low warfarin dosage include advanced age, low body weight, white race, low serum albumin, tobacco use, cardiac failure and liver disease, drug interactions.

Question 24

What factors predict low warfarin dose?

Answer 24

Possession of 1 or 2 copies of a variant CYP2C9 allele predicts low warfarin dosage. Other factors that predict low warfarin dosage include advanced age, low body weight, white race, low serum albumin, tobacco use, cardiac failure and liver disease, drug interactions.

Question 25

What is warfarin prescribed for?

Answer 25

Warfarin is prescribed to patients who are at risk of thromboembolism due to pulmonary embolus, DVT or atrial fibrillation.

Question 26

How does warfarin function?

Answer 26

Warfarin acts by inhibiting the enzyme Vitamin K reductase, which recycles oxidised vitamin K to its reduced form following use in carboxylation of prothrombin and coagulation factors VII, IX and X. Carboxylation is essential for the activation of these coagulation factors. Therefore if warfarin inhibits vitamin k reductase, vitamin k does not return to its reduced form and the coagulation factors cannot be carboxylated and so remain non-functional.

Question 27

What enzyme metabolises warfarin and why is optimising warfarin dose important?

Answer 27

Warfarin is metabolised by cytochrome p450 enzyme CYP2C9. Optimising the warfarin dosage is extremely important because under treatment will fail to achieve reduction in risk of thromboembolism. Patients are often given a loading dose to achieve therapeutic levels rapidly. Overdose of warfarin increases the risk of haemorrhage.

Question 28

What gene is of most interest in the pharmacogenetics of warfarin? What changes render the the protein product of this gene less active?

Answer 28

The first gene that was of interest in the pharmacogenetics of warfarin was the CYP2C9 gene which encodes the cytochrome p450 enzyme. Cytochrome p450 enzyme metabolises not only warfarin but many other drugs glipizide and Ibuprofen.

Single base changes at aa144, 359 and 360 can result in amino acid substitutions which render the CYP2C9 enzyme less active.

Question 29

What populations are the main alleles of the CYP2C9 gene found in?

Answer 29

The main allele of CYP2C9 is *1 on Caucasian and Asian populations, *2 is found in both populations also. *3 is only found in Caucasians. *4 and *5 is found in Japanese and African American populations only.

Question 30

Describe the study that Gage et al published in Thrombosis and Homeostasis on 2004.

Answer 30

It was a retrospective study in 2004 to identify factors associated with stable maintenance dose of warfarin.

They designed a regression equation for dose incorporating age, body surface area, CYP2C9 genotype, concurrent medication, race and sex.

It was claimed on is study that the use of this regression equation at the start of warfarin therapy had the potential to reduce the percentage of patients who were overdosed at initiation of therapy from 16% to 6.5%.

It also became apparent that there is another gene that is important in determining the inter individual variation on the stabilising dose of warfarin. The VKORC1 gene.

Question 31

What other gene did Gage et al identify as important in determining the inter individual variation on the stabilising dose of warfarin?

Answer 31

The VKORC1 gene. The VKORC1 gene encodes a component of vitamin k epoxide reductase, the main target of warfarin.

There are two main haplotypes of the VKORC1 gene which account for about 30% if the inter individual variation in warfarin dose.

Question 32

Describe the warfarin study carried out by Jorgensen et al.

Answer 32

A more recent study than that of Gage et al in 2004 is the prospective study carried out by Jorgensen et al.

This prospective study looked at 311 patients and assessed the time to and size of stable dose of warfarin.

They found significant associations with CYP2C9 and VKORC1 and 6 of 27 other genes tested had significant but minor effects.

The most important predictor of cost of care is an adverse drug event and therefore predicting optimal dose is financially worthwhile also. However, they warned that further cost-benefit evaluations and robust dosing algorithms were required before genetic testing can be introduced into routine prescribing.

Question 33

What is required before pharmacogenetic testing can be extensively used for drug dosage predictions?

Answer 33

1) . Large prospective studies are required to confirm clinical usefulness.
2) . Rapid genotyping turnaround times may be required for some clinical applications.
3) . Other information needs to be considered in dosing algorithms. Users will likely need some sort of education in the use of pharmacogenetic information. Users will also need easy access to dosing algorithms.

Question 34

What drugs may pharmacogenetics prove most valuable in dosage guidance?

Answer 34

Those that have a narrow therapeutic index.

Question 35

Define pharmacogenetics.

Answer 35

The genetic basis of variation in response to drugs.