CLASS: 10:00 11: 00 Scribe: Adam Baird s1

CLASS: 10:00 – 11: 00 Scribe: Adam Baird

DATE: December 2, 2010 Proof:

PROFESSOR: Johnson PHARMACOGENETICS/PHARMACOGENOMICS Page 5 of 5

I.  PHARMACOGENETICS [S1]

II.  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.

III.  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.

IV.  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).

V.  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.

VI.  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.

VII.  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

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.

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).

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 (5-FU): thymidine phosphorylase (TP).

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.