PHMY 621: Drug metabolism and pharmacogenomics case studies

FdUMP is a ___________ inhibitor of ____________ ____________.

suicide; thymidylate synthase

Several _____________ drugs are converted to FdUMP

fluoropyrimidine

The enzyme (suicide inhibitor) is bound to what active site?

Thiol grp on Cys

5-Fluorouracil (5-FU)

used to treat breast and colorectal cancer

Drugs that contain 5-FU

- capecitabine (Xeloda, oral prodrug of 5-FU)

- Carac, cream for keratosis

5-FU is converted to _________ then ________

FdUrd; FdUMP

5-FU is the __________ ingredient in drugs

active

Thymidine phosphorylase

- a naturally occurring substrate

- converts thymine and deoxyribose-1-phosphate to thymidine with loss of Pi

What is considered the activation step in Pyrimidine salvation?

Thymidine phosphorylase converts 5-FU to 5-gluoro deoxyuridine (FdUrd)

FdUrd is considered a...

nucleoside

Thymidine kinase (TK)

activates FdUrd by adding a phosphate to the 5' position on the ribose

what does the "MP" represent in "FdUMP"?

mono phosphate

Dihydropyrimidine dehydrogenase (DPYD)

- converts uracil to dihydrouracil

- ALSO converts 5-FU to 5-dihydrofluorouracil

5-FU is considered a...

base

5-dihydrofluorouracil is an _________ metabolite

inactive

DNA encodes amino acids in groups of ___ bases

3

Heterozygous mutations naturally occur in 3-5% of patients. What does a mutation mean in terms of genetics?

- change in sequence

- downstream biology determines if mutation is positive or negative

Homozygous __________ mutations naturally occur in 0.2% of patients

inactivating

If there is no DYPD present, there is NO __________

product

If you dose your patient with 5-FU, you will get _______ substrate leading to ______ inactive metabolite produced

More; more

DYPD polymorphism affects dosing of 5-FU, capecitabine and carac

all produce the SAME active site that the suicide inhibitor inhibits

What does 1/1 mean?

two copies of normal function (*1 = normal function)

What does *2 or higher mean?

variant alleles, which can be

- known non-functional or functional

- unknown impact on function

Genetic screening to identify patients with inactivating mutations __________ dosing

BEFORE

homozygote indicates a ________ metabolizer

poor

Thiopurine Therapeutics

- 6-mercaptopurine (antineoplastic) ****

- azathioprine (immunosuppressant)

- 6-thioguanine (antineoplastic)

Thiopurine mechanism of action and breakdown

1. Drug enters the body (azathioprine is the prodrug that is CONVERTED into 6-meraptopurine or (6-MP))

2. (Activation pathway) 6-MP gets metabolized by thioguanine nucleotides (TGNs) which can be incorporated into DNA and RNA, disrupting replication and transcription.

3. Overall rapidly dividing cells are slowed down (like immune cells) ***MAIN MECHANISM OF ACTION

4. (Inactivation pathway) 6-MP must be turned off to prevent too much toxicity

What are the 3 different inactivation pathways of 6-MP

- Xanthine oxidase: breaks 6-MP into 6-thio-uric acid which inactivates it and excretes it

- Thiopurine methyltransferase (TPMT): methylates 6-MP into inactive forms (S-methylated products)

- NUDT15 (nucleotide hydrolase): breaks down active thioguanine nucleotides (TGNs)

Main action of thiopurine (Antiproliferative action)

Azathioprine → 6-MP → TGNs → DNA incorporation → cell death (main action).

Azathioprine is turned into 6-MP, which gets activated into TGNs that mess up DNA and stop cells from dividing. The body inactivates 6-MP and TGNs through XO, TPMT, and NUDT15. If these inactivation pathways don't work properly, the drug can build up and cause dangerous toxicity.

TPMT ___________ 6-mPu and 6-TG by adding a methyl group onto the sulfur

inactivates

Hematopoietic cells _______ xanthine oxidase activity

lack

TPMT is the major __________ route in hematopoietic cells

inactivation

Enzymes depend on ___________ cells for inactivation

Hematopoietic

Heterzygotes start on ____-____ of normal dose, and are more likely to require later dose reductions than those with two normal function alleles

30-80%

Patients who carry the inactivating mutant alleles in TPMT should be doses with ____-______% of the dose used for wild-type patients, or severe hematological toxicity occurs

6-10%

Poor metabolizer recommendations

genotype: two no function alleles

implication: fatal toxicity possible with dose decrease (therapeutic index)

recommendation: consider alternative non-tiopurine immunosuppressant therapy

NUDT15 breaks down _________ to_________

d6-TG-TP; d6-TG-MP

More of drug present indicates it is capable of _______, meaning what for hematopoetic cells?

activation; hematopoeitc cells are killed

NUDT15 polymorphisms are much more common in what two populations?

Asian and Latin

Which of the following enzymes is primarily responsible for metabolizing thiopurine drugs like azathioprine and 6-mercaptopurine?

a) CYP2C19

b) TPMT (Thiopurine methyltransferase)

c) Xanthine oxidase

d) NUDT15

b) TPMT (Thiopurine methyltransferase)

TPMT inactivates thiopurines. Variants in TPMT strongly affect toxicity risk.(Note: XO and NUDT15 also contribute, but TPMT is the classic enzyme tested for in pharmacogenomics.)

Inactivating mutations in TPMT would most likely lead to which outcome in a patient taking standard doses of 6-mercaptopurine?

a) Enhanced drug clearance with little therapeutic effect

b) Accumulation of active metabolites and severe bone marrow toxicity

c) No effect on drug metabolism

d) Increased conversion of drug into inactive uric acid

b) Accumulation of active metabolites and severe bone marrow toxicity

Without TPMT, too many active TGNs build up, causing life-threatening hematologic toxicity.

Approximately 1 in 300 individuals carry homozygous inactivating mutations in TPMT. What is the safest clinical approach when prescribing thiopurines for these patients?

a) Use full standard dose but monitor closely

b) Avoid therapy altogether

c) Start at ~6-10% of the normal dose to prevent hematological toxicity

d) Switch to double the normal dose to overcome slow metabolism

c) Start at ~6-10% of the normal dose to prevent hematological toxicity

Homozygotes have no TPMT activity; they need very low doses to avoid toxicity.

Which statement is TRUE regarding heterozygous carriers of TPMT mutations?a) They should never receive thiopurines.

b) They start at about 30-80% of the normal dose and may need later reductions.

c) They are at no increased risk compared to patients with normal TPMT activity.

d) They metabolize thiopurines faster than normal patients.

b) They start at about 30-80% of the normal dose and may need later reductions.

Heterozygotes have reduced TPMT activity — more than homozygotes, but still risky if dosed normally.

Which of the following best describes the consequence of inactivating mutations in a drug-metabolizing enzyme?

a) Reduced drug exposure and no clinical response

b) Increased or decreased active drug levels, leading to toxicity or treatment failure

c) No impact since most drugs are excreted unchanged

d) Stronger receptor binding independent of metabolism

b) Increased or decreased active drug levels, leading to toxicity or treatment failure

Mutations can cause either too much active drug (toxicity) or too little (treatment resistance), depending on the enzyme's role.