24

It is important to remember that many nongenetic factors such as age, weight, diet,

smoking status, and medication interactions contribute to a great variability of

warfarin dosing among patient populations. Drug–drug interactions in which the

CYP2C9 enzyme may be induced or inhibited will affect the rate of warfarin

metabolism. Additionally, a diet high in vitamin K will make warfarin less effective,

because this facilitates increased synthesis of vitamin K–dependent clotting factors.

Algorithms for determining appropriate starting doses for patients over the age of 18

years based on both nongenetic and genetic factors are available at

www.warfarindosing.org. Close monitoring of INR is also typically recommended

to ensure proper anticoagulation and reduced risk of bleeding.

CASE 4-2, QUESTION 2: What other drugs are significantly affected by the CYP2C9 pathway?

CYP2C9 is estimated to play a role in the metabolism of up to 20% of commonly

used medications.

25 From these medications, several associations have been found

linking variants in the CYP2C9 pathway due to both increased rates of adverse drug

events and variable medication response. Examples of such drugs include phenytoin,

certain nonsteroidal anti-inflammatory drugs (such as celecoxib and diclofenac),

sulfonylureas, losartan, and certain statins (such as fluvastatin and simvastatin).

Patients with *2 or *3 alleles may be at increased risk of toxicities at standard doses

of drugs that are processed through the CYP2C9 pathway decreased due to

metabolism and increased parent drug concentrations and may require reduced

dosing or increased monitoring.

TOXICITY IMPLICATIONS

Many known variants exist within the genes that code for enzyme proteins, affecting

the rate at which the enzyme is able to metabolize drugs through its pathway. These

variants may result in either reduced enzyme function and increased concentration of

parent drug, or increased enzyme activity with decreased concentrations of a parent

drug. The clinical effects of the variants depend on whether the parent drug is

pharmacologically active or a prodrug.

CASE 4-3

QUESTION 1: T.B. is a 28 kg, 5-year-old male admitted for status epilepticus treated with IV lorazepam and

IV fosphenytoin on presentation, aborting the seizures. Past medical history is significant for congenital

hydrocephalus, a ventriculoperitoneal shunt and refractory epilepsy secondary to an in utero right middle

cerebral artery stroke. On day 5 of his hospitalization, he is still extremely lethargic and his free phenytoin level

is high, peaking at 3 mcg/mL on day 2 and 1.4 mcg/mL on day 5 (see Fig. 4-2). Looking at his records, he

received an appropriate initial loading dose of IV fosphenytoin18 mg phenytoin sodium equivalents (PE)/kg (500

mg PE) followed by a single maintenance dose of IV fosphenytoin140 mg PE (5 mg PE/kg) 5 hours later on

day 1 of the hospitalization. He has no clinically relevant drug–drug interactions and his albumin is normal. What

is a possible reason for the toxicity T.B. is experiencing?

Phenytoin/fosphenytoin serum concentrations can be difficult to control and are

complicated by multiple factors including Michaelis–Menton kinetics, also known as

capacity limited metabolism. Drugs that follow Michaelis–Menton kinetics go from

first to zero order, meaning that metabolism increases with increasing concentration

until enzyme saturation takes place.

26 Once saturation is reached, drug plasma

concentrations can increase to toxic levels in a fast and unpredictable manner.

Additionally, many factors affect an individual’s safe and effective phenytoin dose,

including albumin levels, other medications in the patient’s regimen, and

pharmacogenetics.

Phenytoin has many chronic effects associated with long-term use, including

hepatotoxicity, osteoporosis, megaloblastic anemia, gingival hyperplasia, hirsutism,

and peripheral neuropathy.

27

In the acute setting of toxic plasma concentrations or an

overdose, phenytoin toxicity can manifest with a variety of signs and symptoms,

including CNS effects (dizziness, confusion, drowsiness, and ataxia) as well as GI

upset and nausea. Phenytoin is also associated with severe cutaneous reactions such

as Stevens–Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) (see

section with Case 4-6).

Fosphenytoin is a prodrug, converted by plasma esterases to the active drug

phenytoin. Phenytoin is further metabolized by CYP2C9 to phenytoin arene-oxide,

which is then broken down to multiple metabolites that are eventually excreted.

27 The

contribution of these various metabolites to toxicity and efficacy of phenytoin is not

well understood.

T.B. is found to have a loss of function variant status in CYP2C9 *1/*2. With loss

of CYP2C9 enzyme function comes decreased breakdown of the active drug

phenytoin at standard doses. Given the narrow therapeutic index of phenytoin and the

drug’s propensity to cause side effects, T.B. ultimately experienced drug toxicity and

accompanying symptoms. The evidence relating genotypes such as T.B.’s to the

development of adverse drug events is strong, and the Clinical Pharmacogenetics

Implementation Consortium (CPIC, pronounced “See-Pick”) has published dosing

guidelines based on CYP2C9 genotype summarized in Table 4-4.

28 Note that only the

maintenance, not the loading dose, has adjustment recommendations ensuring that

acute seizure activity can adequately be terminated.

Figure 4-2 Free phenytoin levels (Case 4-3).

p. 55

p. 56

CASE 4-4

QUESTION 1: J.P. is a 50-kg, 17-year-old female, status post kidney transplant. She is currently receiving

azathioprine 100 mg PO daily to prevent rejection. She is brought to the emergency department 5 days after

starting the medication complaining of lethargy, fever, and malaise. A white blood cell count comes back as a

critically low value at 900 cells/μL (normal range,3.8–9.8 × 10

3

/μL), with an absolute neutrophil count (ANC)

of 760 cells/μL (neutropenia defined as an ANC ≤ 2,000 cells/μL). What factors might explain the severe

neutropenia and what test should be ordered for J.P.?

Azathioprine is an immunosuppressive agent in the thiopurine class, acting as a

prodrug of 6-mercaptopurine. These drugs are purine analogs and antagonize purine

synthesis, inhibiting synthesis of DNA, RNA, and proteins.

29 Thiopurines are used in

a variety of conditions including renal transplant, rheumatoid arthritis, certain

cancers, and inflammatory bowel disease.

Azathioprine is metabolized to an active drug 6-mercaptopurine via glutathione Stransferase (GST) reduction. 6-mercaptopurine is then converted into the active 6-

methylmercaptopurine ribonucleotide (6-MMPR) and several inactive metabolites

such as 6-methylmercaptopurine (6-MMP) through multiple pathways.

30 The two

primary enzymes contributing to 6-mercaptopurine breakdown are TPMT and

hypoxanthine-guanine phosphoribosyltransferase (HPRT). Although TPMT

metabolism results in the generation of the inactive metabolite 6-MMP, HPRT

contributes to a pathway that leads to the generation of active 6-MMPR and 6-

thioguanine nucleotide (6-TGN) metabolites.

31 Furthermore, active 6-TGN

metabolites are then inactivated by TPMT. The accumulation of active 6-TGN

metabolites via HPRT is associated with myelosuppression with thiopurine therapy.

In summary, TMPT acts as the detoxifying enzyme for drugs in the thiopurine class

and its activity is directly linked to risk of drug toxicities.

The severe neutropenia J.P. experienced 5 days after beginning azathioprine is

very likely the result of starting a full dose (2 mg/kg/day) in the setting of a

homozygous variant TPMT genotype, such as *3B/*3C. Because of the near-absent

enzyme level produced by homozygous variants, the TGN metabolites build up,

resulting in severe and sometimes life-threatening neutropenia.

Table 4-4

CYP2C9 Genotype-Based Dosing Recommendations for

Phenytoin/Fosphenytoin

CYP2C9

Metabolizer Status Sample Genotype(s) Recommendation

Extensive

metabolizer

*1/*1 Initiate therapy with recommended maintenance dose

Intermediate

metabolizer

*1/*2, *1/*3 Consider 25% reduction of recommended starting

maintenance dose and adjust according to therapeutic drug

monitoring and response

Poor metabolizer *2/*2, *3/*3, *2/*3 Consider 50% reduction of recommended starting

maintenance dose and adjust according to therapeutic drug

monitoring and response

It is important to note that the genotype *3B/*3C is nearly impossible to distinguish

from the less clinically impactful heterozygous genotype of *1/*3A on most

commercially available TPMT assays. In cases of suspected homozygous variant

status, parental studies may be necessary for absolute determination. Alternative

therapy or a 90% reduction in the azathioprine dose is advised for J.P.’s TPMT

genotype.

32

In the case of a heterozygous genotype such as *1/*3C, the dose would be

decreased by 30%–70% (Table 4-5).

32

DRUG TARGET IMPLICATIONS

CASE 4-5

QUESTION 1: L.K. is a 45-year-old woman referred for interpretation of pharmacogenomic results provided

by a certified clinical lab as part of a research study. The report states that the called genotype for SLCO1B1 is

CC. L.K. asks if she is at risk for severe muscle pain, or myopathy, which she heard about on a recent

television malpractice commercial. What information do you need to answer her question?

Table 4-5

Summary of CPIC Guidelines for TPMT Genotype-Based Thiopurine Dosing

Phenotype

(Genotype)

Sample

Genotype(s)

Implication for Thiopurine

Pathway Thiopurine Dosing Recommendations

Normal/High

Activity

(Homozygous

wild-type)

*1/*1 Lower concentrations of TGN

metabolites

Start with normalstarting dose and adjust

doses of thiopurine based on diseasespecific guidelines. Allow 2 weeks to reach

steady state after each dose adjustment.

Intermediate

activity

(Heterozygous)

*1/*2, *1/*3A,

*1/*3B,

*1/*3C, *1/*4

Moderate to high concentrations

of TGN metabolites

Consider starting at 30-70% of target dose

for azathioprine and 6-mercaptopurine and

30-50% of target dose for thioguanine.

Titrate based on tolerance. Allow 2-4

weeks to reach steady state after each

dose adjustment.

Low/deficient

activity

(Homozygous

variant/mutant)

*3A/*3A,

*2/*3A,

*3C/*3A,

*3C/*4,

*3C/*2,

*3A/*4

Extremely high concentrations

of TGN metabolites

Consider alternative agents. If using

thiopurine, start with drastically reduced

doses (reduce daily dose by 10-fold and

dose thrice weekly instead of daily) and

adjust doses based on degree of

myelosuppression and disease-specific

guidelines. Allow 4-6 weeks to reach

steady state after each dose adjustment.

Source: MV Relling, EE Gardner, WJ Sandborn, et al. Clinical Pharmacogenetics Implementation Consortium

Guidelines for Thiopurine Methyltransferase Genotype and Thiopurine Dosing. Clin Pharmacol Ther. 2011

Mar;89(3):387–91.

p. 56

p. 57

Table 4-6

SLCO1B1 Genotype-Based Dosing Recommendations for HMG-CoA

Reductase Inhibitors

SLCO1B1

rs4149056

Genotype

Myopathy Risk

with Simvastatin

Use Simvastatin Dosing Recommendation

TT Normal Prescribe standard starting dose and modify based on tolerance

and disease response

TC Intermediate Prescribe a lower dose or consider alternative statin (such as

pravastatin or rosuvastatin). Routine monitoring of CK may be

considered

CC High Prescribe a lower dose or consider alternative statin (such as

pravastatin or rosuvastatin). Routine monitoring of CK may be

considered

“Statins” or HMG-CoA reductase inhibitor drugs are associated with muscle

toxicity, ranging from mild aches to the development of severe debilitating myopathy

and rhabdomyolysis in a segment of the population.

33 Regardless of severity, this

adverse drug event is a common cause for drug discontinuation.

34 Recent published

literature links this risk of developing these muscle-related side effects to certain

variants in the SLCO1B1 gene.

Keeping in mind that a raw result from DNA analysis is essentially a string of A’s,

C’s, T’s, and G’s within each individual gene, the CC genotype for SLCO1B1

information provided by L.K. is insufficient. In order to review available evidence, it

is essential to know the actual variant location that the CC call was based upon.

Although there are multiple polymorphisms that have been identified in the SLCO1B1

gene, only few are linked to clinical effect.

35 The majority of laboratory

interpretation will call out the relevant rs numbers needed to make a

pharmacogenomic determination of drug selection and dose alteration. An rs number,

or rsID, is used to point to a specific nucleotide location within a gene. Known SNPs

within a gene are defined by rsID for the purpose of clinical guidelines and research

reporting. This ensures standardization and proper assessment of variants. For

SLCO1B1, the rsID most commonly associated with development of myopathy with

HMG-CoA reductase inhibitors is rs4149056.

35 This rsID should be provided with

any result in order to make the proper assessments and recommendations.

CASE 4-5, QUESTION 2: The clinical lab confirms the SLCO1B1 genotype CC call was based on

rs4149056. What is your answer to L.K.’s question regarding her risk of developing severe myopathy?

The C allele at SLCO1B1 rs4149056 has been associated with decreased statin

intracellular transport and clearance.

34 SLCO1B1 is a transporter protein with a

primary function of drug uptake into the liver. Variants affecting the hepatic uptake of

drugs via SLCO1B1 ultimately increase overall area under the curve and drug

exposure, resulting in higher risks of adverse drug events such as myopathy. Patients

with homozygous variant status such as L.K. are at significantly increased risk of

developing muscle toxicity with statin use. Although all statins may have adverse

event profiles linked to this variant, the evidence is strongest for simvastatin.

Published CPIC guidelines for simvastatin with SLCO1B1 rs4149056 variant status

are summarized in Table 4-6.

NONMETABOLISM IMPLICATIONS

CASE 4-6

QUESTION 1: J.C. is an 18-year-old female with a past medical history of epilepsy. Following a failed course

of levetiracetam, her physician has decided to start her on carbamazepine therapy for seizure control.

Approximately 2 months after starting the drug, J.C. is seizure free but feels as though she is coming down with

what feels like a cold with accompanying headache and fever. She also begins to notice that her skin is

becoming red and itchy in several locations. The rash quickly progresses to the point where she notes swelling

and the formation of blisters on her nostrils, eyes and lips. J.C. realizes that she is experiencing something more

than a common cold and rash, and calls the pharmacy in a panic to ask if it is possible that she is reacting to her

medication, even though she has been on it for some time. Given the known side effects of carbamazepine, is it

possible that J.C.is reacting to the medication?

Adverse drug reactions are often thought of as happening acutely following initial

doses of medications; however, delayed hypersensitivities are also possible with

certain drugs. In the case of carbamazepine, these reactions are linked to DNA

changes within the human leukocyte antigen (HLA) genes. There is a strong

association between positive HLA-B*15:02 gene variant carrier status and increased

risk of severe cutaneous adverse reactions.

36 A less established link has also been

seen with carriers of the HLA-A*31:01 variant, more common in Japanese and

Caucasians.

37

The HLA genes code for proteins that form an immunologic complex on all

nucleated cells. This complex is responsible for presenting both self- and foreign

peptides to immune cells, such as T-cells.

38 When a foreign peptide is presented and

then recognized by a T-cell, an immunologic response results. Examples of peptides

that may be recognized as foreign include viruses, bacteria, tumor antigens, and

drugs. Although this may be the basis for the drug-induced reactions seen with the

described HLA variants, there is still significant uncertainty that this is truly the only

mechanism in play. Evidence has suggested that drug concentrations are also higher

in patients who experience cutaneous reactions, meaning that CYP loss-of-function

alleles as well as HLA variants may work synergistically to increase a patient’s risk

of SJS and TEN.

39 This is supported by the fact that not all patients who carry an

HLA allele will experience a cutaneous reaction.

38

HLA Class I genes are highly polymorphic.

40 Variants within these genes are

thought to result in structural changes in the HLA complex that increase the

recognition and subsequent response of the immune system to certain drugs, resulting

in hypersensitivity. Reactions related to these variants tend to be dermatologic in

nature with or without systemic involvement, ranging from a mild maculopapular rash

or progressing to more severe cases of SJS and TEN.

CASE 4-6, QUESTION 2: What is the appropriate advice for the pharmacist to provide to J.C. given her

presentation?

J.C. should be advised to seek care immediately at the closest and most accessible

hospital emergency department. SJS and TEN have significant associated

complications, and typically require inpatient treatment with fluids, corticosteroids,

analgesics, nutritional supplementation, and antibiotics. Mortality rates with TEN are

estimated to be higher than 30%.

38

In all cases, the suspected offending agent should

be discontinued with no attempt to rechallenge.

CASE 4-6, QUESTION 3: In questioning J.C., the pharmacist learns that she has no prior history of adverse

drug or food reactions. She is of Chinese descent, and has never had genetic testing. What would have been the

more appropriate course of action for a patient with J.C.’s history prior to starting carbamazepine therapy?

p. 57

p. 58

Table 4-7

HLA Variants, Affected Drugs, and Population Incidence

HLAVariant

Drugs with Associated

Adverse Cutaneous

Reactions Highest Variant Frequencies by Ethnicity

HLA-B*57:01 Abacavir Caucasian: 5%–8%

HLA-B*58:01 Allopurinol Han Chinese: 6%–8%

Korean: 12%

HLA-B*15:02 Phenytoin, Carbamazepine Han Chinese: ˜10%44

Populations from Hong Kong, Thailand, Malaysia, Vietnam,

Philippines, India, and Indonesia: >5%

HLA-A*31:01 Carbamazepine Northern European: 2%–5%

Japanese: 9%

Chinese: 3.7%45

Carriers of certain HLA variants are at increased risk of cutaneous adverse drug

reactions; however, recommendations and guidelines for preemptive testing are

either nonspecific or lacking. Other HLA variants have been linked to liver injury,

leading to the theory that the burden of HLA expression may be an important factor in

the organ systems affected.

38 Much is left to the provider or institution-specific

protocols to determine when and whom to test prior to therapy. An exception to this

is the antiretroviral medication, abacavir, for which CPIC and FDA

recommendations are in place stating that all patients should have HLA-B*57:01

testing before initiating this regimen.

41

Of note, HLA variants are reported as positive (at least one variant present) or

negative (no variant present). Variants of the HLA genes are seen more commonly

within certain ethnicities. In some cases, because of the high prevalence of allele

carrier status in these populations, patients with particular ethnic backgrounds may

be recommended for testing. Certain Asian populations in particular are known to

have higher rates of certain variants, warranting caution when using particular

therapies (see Table 4-7). In the case of carbamazepine and J.C., her physician may

have justifiably ordered pharmacogenomic testing to determine her HLA-B*15:02

carrier status on the fact that she identifies as Chinese. Relying on self-reported

ethnicity can be problematic, however, as many people are not fully knowledgeable

of their complete lineage.

Although recommendations for testing vary, for every drug with established

clinical guidelines, if a patient is a known carrier of an HLA variant, it is

recommended that they avoid the use of drugs for which there is an HLA variant–

associated risk of adverse reaction.

38,42–44

THE PECULIARITIES OF CYP2D6

CASE 4-7

QUESTION 1: P.F. is a 37-year-old female with a history of depression and anxiety who presents to the local

pharmacy with a printed report from a for-profit pharmacogenomic testing company. The report states that P.F.

is an extensive metabolizer for CYP2D6 and she is concerned that her paroxetine is still not providing any relief

of her depressive symptoms despite having taken the drug correctly for the past 3 months. What information do

you want to know about the testing modality the company used prior to answering her question regarding the

interpreted genotype?

There are several pharmacogenomic testing platforms available. The vast majority

of panels that are available commercially are SNP based. The advantages to SNPbased platforms include a lower cost, faster turnaround time, vendor-provided

interpretation software, and reliable calling for known variants. The main

disadvantage to the SNP-based platforms is that the user is limited only to the genes

and specific variants on the panel. If there are any unique or rare variants, the SNPbased panel will not detect them. Therefore, the patient’s reported genotype will not

match their phenotype (how they actually respond to the drug). Additionally, in the

case of CYP2D6, it is a very polymorphic gene that frequently has more than two

copies. This is referred to as copy number variation (CNV) and is extremely

important in the interpretation of a patient’s CYP2D6 genotype.

46,47 All of the copies

should be typed, and this is not always possible on commercially available assays

because it requires extra primers and interpretation algorithms. Another issue is that

CYP2D7 is a pseudogene (a gene with no function) that can be accidentally included

in the CYP2D6 assay if it has not been designed correctly.

48

Pharmacogenetic data can be the by-product of other genetic assays as well. The

exome is considered the workhorse or coding region of the DNA. Whole exome

sequencing or sequencing of targeted regions of interest can provide significant

pharmacogenomic data as long as the level of coverage of the genes of interest is

adequate. This may include variants that may have only been reported once or twice

before in the literature or variants of unknown significance (VUS). These findings

become a challenge to manage and to provide meaningful interpretation to the

patients. Additionally, in both exome and whole genome sequencing, incidental or

secondary findings of disease state markers will become paramount to deal with

making the case for pharmacogenomics (see section with Case 4-8). The hybrid test,

a sequence-based targeted panel of pharmacogenes, has become a hot area of

development, attempting to incorporate the best features of both sequencing and

limited gene interrogation.

In the case of P.F., the report shows a genotype of CYP2D6 *1/*2 but does not

indicate the copy number of the CYP2D6 gene. This is a red flag for interpretation

and should prompt the pharmacist to call the company to obtain a detailed

explanation of the assay used and how the interpretation of extensive metabolizer

was determined.

Table 4-8

CYP2D6 Genotypes, Phenotypes, and Activity Scores

Number of Copies Genotype Activity Score Phenotype

≤2 *1/*2 1.0–2.0 Extensive metabolizer

>2 *1/*2 2.0 Ultrarapid metabolizer

p. 58

p. 59

CASE 4-7, QUESTION 2: Per the company, the copy number was equal to 3. Does this change the

interpretation of P.F.’s phenotype?

With a copy number of 3, a genotype of CYP2D6 *1/*2 is associated with an

ultrarapid metabolizer phenotype.

49 Unique to CYP2D6, an activity score is

calculated based on the number of copies of the gene present as all contribute toward

drug metabolism. The activity score was described by Gaedigk

46

to better categorize

the metabolism status of persons with multiple copies of the gene. An example of

how this can change a phenotype is represented in Table 4-8.