Download Pharmacogenetics

CLASS: 10:00 – 11: 00
DATE: December 2, 2010
PROFESSOR: Johnson
I.
II.
III.
IV.
V.
VI.
VII.
PHARMACOGENETICS/PHARMACOGENOMICS
Scribe: Adam Baird
Proof:
Page 1 of 5
PHARMACOGENETICS [S1]
ADVERSE DRUG REACTIONS [S2]
a. This poem is from a 1954 textbook.
b. Many years ago, if a patient died due to a drug, it was called an “idiosyncratic response”. The name has now
changed to “adverse drug reaction” (ADR). It basically means that there was an unanticipated toxicity to a
drug.
PHARACOLOGY [S3]
a. There are two main ways of drug administration (orally, injected).
b. If taken orally, the drug moves through the hepatic metabolism (through the liver, through circulating drug
concentrations, and onto various drug targets, resulting in a response and eventually, excretion). All of these
factors are specific for whatever the drug is.
PHARMACOGENETICS/PHARMACOGENOMICS [S4]
a. Both terms may be used.
b. Most diseases that are trying to be treated are genetically based (cancer, lupus, arthritis, etc.). Drugs can’t
change genetic problems. Instead, drugs treat symptoms.
c. The term “pharmacogenetics” was coined in the 1950s. It referred to inheritable difference in the way people
handle drugs. Idiosyncratic responses, or ADRs, run families (and some ethnic groups), and so a genetic link
was suspected in association with how people responded to a particular drug (but what it was or how it
specifically worked was unknown). About 10 years ago though, we finally understood what was going on
(thanks to the Human Genome Project). The term pharmacogenetics morphed into pharmacogenomics.
d. The older term, pharmacogenetics, referred to a single gene that was abnormal and caused a toxic response.
The newer term, pharmacogenomics, recognizes abnormalities might be found in other places (like
promoters, for example).
1. Example: Response to temozolomide (alkylating agent) in a tumor. There is a gene that repairs DNA
damage. If the promoter is methylated, that gene is turned off, and a response to the drug will result.
If the promoter is not methylated, the gene is turned on, and a response to the drug will not result.
e. Pharmacogenomics considers epigenetic factors (like promoter methylation or alkylation).
f. There are two major areas of pharmacogenetics (and they are almost exclusively in the cancer area):
1. Designing new treatments based on the molecular profile of the tumor.
a. Normal tissue and tumor tissue are genetically compared (like a fingerprint). If there is a
genetic difference between the two, then they develop targets for treatment.
b. Staging people before chemotherapy is given (based on the molecular profile of a tumor). A
biopsy of a tumor can be genetically profiled and can be predicted whether or not it would
respond to a particular drug.
g. Pharmacogenetic syndromes: unanticipated toxicity to a drug (and what the molecular basis of that drug is).
PHARMACOLOGY [S5]
a. Knowing pharmacology terms and definitions is important!
b. All pharmacogenomic syndromes affect one of these parameters (and in turn, affect all of these parameters).
1. Elimination
2. Clearance
3. Half Life
4. Bioavailability
c. One of the parameters may be altered, thus affecting all other parameters, and ultimately affecting efficacy or
toxicity of the drug.
PHARMACOLOGY [S6]
a. All of these pharmacogenomic drugs have something in common: they all have a narrow therapeutic window
(meaning that the difference between an effective dose and a toxic dose is relatively small).
b. Example: acetaminophen requires a large dose for it to become toxic, while other drugs require only a very
little dose for it to become toxic.
c. Another example: cancer chemotherapy drugs, wafarin, etc.
PHARMACOGENETICS/GENOMICS [S7]
a. Pharmacogenetic syndrome examples:
1. Ethanol
a. Alcohol
b. Some people can drink a lot of ethanol while others can drink very little.
c. Ethnic differences in clearance (alcohol dehydrogenase)
d. Example: Asians and American Indians can’t tolerate lots of alcohol
CLASS: 10:00 – 11: 00
Scribe: Adam Baird
DATE: December 2, 2010
Proof:
PROFESSOR: Johnson
PHARMACOGENETICS/PHARMACOGENOMICS
Page 2 of 5
e. Another example: women tend to have higher alcohol dehydrogenase in later ages than men
(meaning that women can tolerate more alcohol)
2. Primaquine
a. Anti-malaria drug
b. A fraction of the people that took it had a safe reaction while it cause hemolysis for others
c. Deficiency in G6PD results in primaquine sensitivity
3. Succinylcholine
a. Called the “perfect poison” (because it is virtually undetectable)
b. Muscle relaxer (of all muscles), used in surgery
c. Clearance of the drug was found to be much different between populations
d. Associated with defects in cholinesterase function
4. Isoniazid
b. These are all Phase I enzymes (oxidation-reduction) where the body is trying to modify the drug. Recall:
Phase II is where the body is trying to attach something to it so that it can be eliminated.
VIII.
CLASSIC PHARCOGENETIC SYNDROMES [S8]
a. The only way pharmacogenetic syndromes were identified: clinical observation of an infected individual,
followed by population observations. (This is why it took nearly 30 years for some of these syndromes to be
fully determined.)
b. Links between drug response and genetics were slowed to be recognized, but were gradually recognized
beginning in the 1950s.
c. It wasn’t until the 1970s that an entire compound was tracked from an idiosyncratic response.
IX.
DEBRISOQUINE [S9]
a. This is an anti-hypersensitive drug (and a relatively good one, at that, especially in the 1970s).
b. The only problem: about 5 – 10% of the people who took it would faint. Nobody knew why until it was further
researched. Pharmacogenetic studies showed that some people had increased half-life to this drug (meaning
that the drug was not being properly needed). This meant that a standard dose, for example, was actually too
much. Research tracked the genetics all the way down to a mutation in CYP2D6 (one of the cytochrome
P450 drugs that actually metabolizes nearly every drug).
X.
CYTOCHROME P450s [S10]
a. This simply shows the abundance of P450s in the liver. Notice that CYP2D6 makes up only a portion of the
total P450s in the liver, but it’s contribution to drug metabolism is nearly 25%.
b. This means, “more doesn’t necessarily mean more important”. Example: You can have a small amount of a
particular enzyme in the liver and just because it is over/under expressed doesn’t mean that it is more/less
important.
XI.
CODEINE [S11]
a. Codeine is activated by CYP2D6 (which is the enzyme that, when there is a deficiency, will cause a increase
in debrisoquine). Codeine itself is not actually active; it has to be converted into morphine (which crosses the
blood-brain barrier and attaches to the opiate receptors).
b. If you are taking debrisoquine and you have low CYP2D6, you have increased levels of debrisoquine in your
blood plasma and you have an exaggerated response. If you’re taking codeine and you have low CYP2D6,
less of the drug is activated and you have a reduced response.
c. One syndrome can cause two different effects: it can cause an exaggerated response in one drug, and less
of a response in another drug.
d.
XII.
WARFARIN (COUMADIN) [S12]
a. Warfarin (rat poison) was introduced in the mid-1950s. One of the first recipients was D. Eisenhower.
b. It has a narrow therapeutic window. It’s a great blood thinner, but to go from an effect dose to a toxic dose
takes very little. Even still, it is commonly used today to treat thrombosis.
c.
d.
XIII.
MECHANISM OF ACTION [S13]
a. Here is the metabolism of warfarin.
b. It comes into anti-verse RNS (it’s mixed). The “S” enantiomer is 5x more potent than the “R’ enantiomer.
70% of the response from warfarin comes from the “S” enantiomer.
c. Warfarin binds to an enzyme called “Vitamin K Epoxide Reductase”. The gene for this was identified just
about 8 years ago.
d. Vitamin K Epoxide Reductase simply reduces Vitamin K. Reduced Vitamin K is a cofactor and it’s necessary
for the carboxylation of glutamic acid residues (on proteins that cause coagulation in the blood).
CLASS: 10:00 – 11: 00
Scribe: Adam Baird
DATE: December 2, 2010
Proof:
PROFESSOR: Johnson
PHARMACOGENETICS/PHARMACOGENOMICS
Page 3 of 5
e. There is inhibition of Vitamin K Epoxide Reductase with warfarin; there is no reduced Vitamin K and there is
no activation of the proteins (no coagulation).
f. Warfarin is cleared by two mechanisms. The “S” enantiomer is cleared by CYP2C9. The “R” enantiomer is
cleared by several mechanisms.
g. The first genetic variations here were seen in CYP2C9. And there’s not just one mutation – there is about 50!
(See next slide.)
h. CYP2C9*1 is the wild type. CYP2C9*2, CYP2C9*3, CYP2C9*4, CYP2C9*5, etc. are mutations.
XIV.
VARIATION IN RESPONSE TO WARFARIN [S14]
a. There are also mutations in CYP2C9, 34A, 1A2, and 1A1 (which metabolize the “R” enantiomer).
b. When these mutations were identified, how people respond to warfarin could be determined (which was
important because that is what caused people to clear the drug). If there is a mutation and there isn’t effective
clearance of the drug, less warfarin can be given. Unfortunately, the more this was studied, the more
heterogenous it was determined to be (and it turned out that there were 50 mutations in the gene, all of which
act a little bit differently).
XV.
CYP2CP MUTATIONS [S15]
a. The most common: the CYP2C9*2 (which actually affects Vmax and turn-over of the enzyme). The next most
common: the CYP2C9*3 (which actually affects Km and binding of the substrate).
XVI.
CLINICAL IMPLICATIONS [S16]
a. Different mutations affect the enzyme in different ways.
b. If you do have any of the mutations, and CY2C9, you have decreased clearance, increased half-life, and an
exaggerated response.
c. Overdosing on warfarin can occur very easily.
XVII. VKORC1 [S17]
a. When the gene for the Vitamin K Epoxide Reductase (VKORC1) was finally identified, genetic variance was
observed. Mutations 1173 and 3730 are the most common.
b. If you have genetic variation in the target of warfarin, it also affects how warfarin works (because it changes
how warfarin is broken down and removed from the body or because it affects what warfarin binds to or
inhibits).
c. Because of this, a genetic profile of VKORC1 and CYPs can be made to predict how people will respond to
warfarin, but this only covers about half of the mutations. There are other mutations that affect warfarin
response.
XVIII. OVERALL CONTRIBUTION OF GENETIC VARIATIONS [S18]
XIX.
ETHNICITY [S19]
a. This is a classic pharmacogenomic syndrome because the Asian population nearly completely lacks
mutations in the CYP2C9 gene (the gene that clears the warfarin) but they have a very high proportion of
mutations in the mutations in the target of the drug (VKORC1). The mutations in the Asian population are so
prevalent that Asians are typically given a lower starting dose (2.5 – 3 mg, as opposed to the normal 5 mg,
and are then titrated up to an effective dose).
XX.
WARFARIN – FUTURE DIRECTIONS [S20]
a. There are many studies attempting to determine the genetic dose required, but there is just too much genetic
variance from one individual to the next.
XXI.
CHEMICAL STRUCTURE OF URACIL, 5-FU, AND CAPECITABINE [S21]
a. In the 1950s, the treatment of cancer was unknown.
b. The first anti-cancer drug: mustard gas (alkylating agent). It was determined that if someone survived
mustard gas exposure, their WBC dropped dramatically, so they gave it to patients with lymphoma.
c. Soon afterwards, 5-Fluorouracil (5-FU) was introduced.
d. It was determined that cancer cells use a large amount of uracil. Why? Because uracil is a building block for
pyrimidines (DNA synthesis) and cancer cells have a high turnover rate (they have to replicate their genome
frequently).
e. Capecitabine was introduced about 7 years ago (for breast cancer). It has now been approved for colorectal
cancer. It is a derivative of 5-FU (which is still commonly used today).
f. The difference between 5-FU and capecitabine is that 5-FU cannot be taken orally due to bioavailability (it is
a small molecule and how much gets into the plasma can be affected by diet, the state of GI tract, etc.). 5-FU,
then, must be administered with an IV. Capecitabine can be given orally (so it’s more cost effective and
easier on the patient). Some studies also show that capecitabine has higher efficacy too.
XXII. FLUOROPYRIMIDINE METABOLISM [S22]
a. The capecitabine is taken orally and is activated in the gut by carboxyesterase (CE) and cytodine-deaminase
(CD) into 5’deoxyfluoruridine (5’FUrd). The final (and rate-limiting step) that activates it into 5-fluorouricil (5FU): thymidine phosphorylase (TP).
CLASS: 10:00 – 11: 00
Scribe: Adam Baird
DATE: December 2, 2010
Proof:
PROFESSOR: Johnson
PHARMACOGENETICS/PHARMACOGENOMICS
Page 4 of 5
b. If 5-FU is given, it is rapidly degraded by an enzyme called dihydropyrimidine dehydrogenase (DPD). The
half-life is relatively short. 85% of the drug is cleared. What is not cleared by DPD is then broken down and
eliminated. Anything that is not actively catabolized goes into the anabolic pathway. (Note: For 30 years, they
anabolic steroid was studied because that is where the anti-tumor effect came from.) After several metabolic
steps, it gets converted into fluorodioxyuridine monophosphate (FdUMP), which binds to thymidylate
synthase (TS). TS is responsible for the conversion of uracil monophosphate to dexoythymadine
monophosphate (dTMP), which is a precursor for DNA synthesis. If this enzyme is shut down,
deoxythymidine monophosphate can’t be made, so dTMP pools are dried up and DNA synthesis is shut down
(and cells can’t live if there isn’t active DNA synethsis).
XXIII. PHARMACOGENTIC SYNDROME [S23]
a. Clinical presentation: A patient is diagnosed with breast cancer. She is given cyclophosphamide,
methotrexate, and 5-FU (the CMF Regimen). She is given a standard dose (one cycle) and her WBC drops
dramatically (almost too much). So during the second cycle of treatment, a 2/3 dose of the first dose is given
(and was given 5 days past normal to allow for WBC counts to increase). The WBC levels dropped even
further and the patient becomes lethargic and confused. For the third cycle of treatment, 2/3 of the second
dose was given and the patient is taken to the ICU five days later (comatose). The patient almost died. She
was given an IV, antibiotics, and allowed her to rest (and waited to see what would happen).
XXIV. PYRIMIDINE LEVELS [S24]
a. Clinical presentation (continued): After many tests, only one significant observation was made: the patient
had elevated levels of thymine and uracil in her plasma. Recall: 5-FU is a derivate of uracil. It was thought
that there was an abnormality to how the patient metabolizes pyrimidines.
XXV. CLINICAL PHARMACOKINETIC PROTOCOL [S25]
a. Clinical presentation (continued): In the 1980’s, this patient would be given a “loading dose” (a small amount
of radioactive 5-FU) and monitored the patient’s clearance of the drug by collecting 15 samples of plasma
over 24 hours.
XXVI. 5-FU PHARMACOKINETICS IN DPD DEFICIENCY [S26]
a. Clinical presentation (continued): She eliminated half of the drug in about 6 hours. The only problem: the halflife of this drug is about 11 minutes. So, this patient was basically getting a massive overdose of this drug.
She couldn’t clear it quickly enough.
XXVII. 5-FU PHARMACOKETICS IN DPD DEFICIENCY [S27]
a. Clinical presentation (continued): The normal controls had a 13-minute half-life. This patient had a 159-minute
half-life. 90% of the drug was eliminated in the urine unchanged. Recall: 85% of the drug is eliminated as
beta-alanine. This patient wasn’t getting rid of the drug as beta-alanine. This patient couldn’t clear the drug;
she couldn’t break it down. As it turns out, this patient had a deficiency in DPD (affecting the metabolism of
the drug). Note: The enzyme assays for this drug are particularly difficult. Blood is isolated, then the
peripheral nuclear cells out of the blood, break them open, spin down the debris, collect the remaining protein
in a reaction tube with radio-labeled 5-FU, mix it with NADPH (as a proton donor and cofactor), let it react for
varying amounts of time, and inject it on an HPLC (so the radio-labeled 5-FU can be separated from the
reduced 5-FU). This, obviously, takes very long (about a full day).
XXVIII. DPD DEFICIENCY IN 103 PATIENTS WITH TOXICITY TO 5-FU [S28]
a. Later studies asked: If a patient is toxic to 5-FU, is the patient also DPD deficient? Out of 100 people, only
about half of them would be DPD deficient. It’s one of the causes for toxicity of 5-FU, but it’s not the only
cause. The other cause is unknown.
b. This observation is similar to warfarin.
XXIX. DPD DEFICIENCY [S29]
a. DPD deficient individual are phenotypically normal. The drug just can’t be cleared.
b. The original breast cancer patient from the study: #5. She had two children, and when they tested them for
DPD deficiencies, they both were found to have very low DPD enzyme activity.
XXX. PEDIGREE OF C.M. – COMPLETE DPD DEFICIENCY [S30]
a. The original study was done in the 1980s. With new understanding, the same study was done in 2002. The
breast cancer patient had died, but genotyping was done on her children. The patient was a complex,
compound heterozygote (she had one mutation on one allele and a different mutation on another allele) and
each child inherited one mutation and each were partially DPD deficient, making it an autosomal, codominant inherited trait. Don’t worry. This won’t be required material for the exam.
XXXI. PEDIGREE OF C.M. – COMPLETE DPD DEFICIENCY [S31]
a. The point: It takes a long time to determine the trait and the molecular basis of pharmacogentic syndromes
(taking about 40 – 50 years).
XXXII. MOLECULAR BASIS OF DPD DEFICIENCY [S32]
CLASS: 10:00 – 11: 00
Scribe: Adam Baird
DATE: December 2, 2010
Proof:
PROFESSOR: Johnson
PHARMACOGENETICS/PHARMACOGENOMICS
Page 5 of 5
a. With DPD deficiency and toxicity to 5-FU, it took about 14 years. The time frame, then, is decreasing (due to
research and better understandings). As it turns out, it is usually more than one gene that is responsible.
XXXIII. [2-13 C]-URACIL METABOLISM [S33]
a. It was suggested that about 5% of the population is DPD deficient (a relatively large amount when you think
about it). It was thought that DPD deficiencies could be predicted or tested (before the drug was given). At
first, enzyme assays were tried, but this was completely impractical. So, genetic tests were attempted. They
found that there are about 50 mutations in the DPD gene. Genetic test weren’t very practical either. It was
finally determined that if they orally gave people C-13 labeled uracil (which is not a radioactive isotope). If you
have DPD in your system, and it goes through the gut rapidly, there’s a ring-opening step (where the Co2is
broken off). This can be detected.
XXXIV. [2-13 C]-URACIL BREATH TEST PROFILES [S34]
a. A breath test could be given to measure the amount of DPD deficiency by the exhaled CO 2. So before the
drug is given, a breath test is analyzed.
b. This method is similarly used when testing for heliobacter pylori in the gut.
XXXV. CONCLUSIONS [S35]
a. Genetic testing is available, but rarely definitive (there are many, many factors)
1. Complicated metabolism
2. Multiple SNPs in several genes in metabolic pathway
b. The future: eventually, everyone will have their own DNA code. You can do this online (for a high price). The
next 20 to 30 years will likely be spent to determine how genes work together.
[End 48:00 mins]