Pharmacogenomics of DMEs PGEN II

CYP3A, TPMT, ALDH2, UGT, NAT2, HLA

MEDCH 527

Molecular Pharmacogenetics of Phase I Enzymes

Phenotype - 1977 (Debrisoquine/Sparteine) Genotype (Null) -1988

Phenotype - 1982 (S-Mephenytoin clearance) Genotype (Null) - 1994

Clinical Phenotype - 1956 (Phenytoin toxicity) Cloning of CYP2C9 variant alleles and functional characterization - 1990s

Adapted from Evans and Relling, 1999

CYP3A?

Drug Gene PK/PD Phenotype Minor Allele

Altered Risk/Effect

6-Mercaptopurine Warfarin Codeine Abacavir Carbamazepine Statins Clopidogrel Interferon/ribavirin

TPMT CYP2C9/VKORC1 CYP2D6 HLA-B*5701 HLA-B*1502 SLCO1B1 CYP2C19 IL28B

Reduced clearance Reduced clearance/enzyme Reduced bioactivation Antigen presentation Antigen presentation Reduced hepatic uptake Reduced bioactivation Reduced anti-viral response

Leukopenia Anticoagulation Analgesia Multi-organ toxicity SJS - TEN Myopathy Anticoagulation Hepatitis C cure rate

Irinotecan Nortripylline Nicotine Vincristine Cisplatin Metformin Tacrolimus Omeprazole Isoniazid Succinylcholine

UGT1A1 CYP2D6 CYP2A6 CYP3A5 TPMT/COMT SLC22A1 CYP3A5 CYP2C19 NAT2 BuChE

Reduced metabolite CL Reduced clearance Reduced clearance Increased clearance Reduced clearance Reduced hepatic uptake Increased clearance Reduced clearance Reduced clearance Reduced clearance

Leukopenia/GI tox Antimuscarinic effects Addiction/Treatment Neurotoxicity Hearing Loss Hyperglycemia Immunosuppression Acid secretion Neuropathy Muscle paralysis

Candidate Drugs for Pharmacogenetic Testing

CYP3A4 Polymorphisms • ~22 unique alleles (many coding-region SNPs) identified for CYP3A4 gene – see the P450 allele web-site (www.cypalleles.ki.se/cyp3a4.htm)

• At least one of these (3A4*20 - a frameshift inducing a truncated protein) abolishes enzyme activity in vitro, but the allele frequency activity is extremely low (<0.06% in a white German population). CYP3A4*22 associated with low mRNA levels (allelic imbalance) and reduced simvastatin clearance (Elens et al, Pharmacogenet Genomics, 2011)

Ingelman-Sundberg et al., The Pharmacogenomics Journal (2013)

(n=75) (n = 59) (n=9)

Zhang et al, Eur J Clin Pharmacol 66:61-66, 2010

Common CYP3A4 Polymorphisms: *1G and Fentanyl PK/PD

•  A couple of common polymorphisms in the CYP3A4 gene (*1B and *1G) have been associated with altered drug clearance and effects. Both are in LD with the CYP3A5*1 allele in Caucasians. Data for *1G looks most promising.

Kuehl et al., 2001; Lin et al., 2002

1 2 3 4 5 6 7 8 9 10 11 12 13 3B

CYP3A5*3 (G) CYP3A5*1 (A)

CYP3A5 protein

1 2 3 4 5 6 7 8 9 10 11 12 13 5’ UTR 3’ UTR

(wt-CYP3A5 mRNA) (SV1-CYP3A5 mRNA)

A→G (intron-3)

truncated, inactive protein

1 2 3 4 5 6 7 8 9 10 11 12 13

CYP3A5 Genetic Polymorphism

Metabolic Fate of Tacrolimus

Tacrolimus

13-DMT

12-HT

31-DMT

12-OH-DMT CYP3A5>4

CYP3A5>4

CYP3A5>4

•  Tacrolimus is used to prevent grafted organ rejection (immune suppressant) •  CYP3A5 is one of 2 enzymes (also CYP3A4) that metabolically clear tacrolimus

from the body •  CYP3A5 makes all 4 primary metabolites, but preferentially the major one (13-

DMT), 12-HT and, 31-DMT

15-DMT CYP3A4>5

Contribution of CYP3A5 to Hepatic��� Tacrolimus Metabolism

13-DMT Formation

CYP3A4 Km (µM) Vmax (nmol/min/nmol) Clint (ml/min/nmol)

0.21 8.0 38

CYP3A5 Km (µM) Vmax (nmol/min/nmol) Clint (ml/min/nmol)

0.21 17.0 82

Human Liver Microsomes

Tacrolimus Disappearance (mL/min/mg)

CYP3A4

6.1

(3.6)

CYP3A4 +CYP3A5

15.9 (9.8)

Unbound 13-DMT formation clearance (rCYP3A) and unbound liver microsomal disappearance clearance

Dai et al., DMD, 2006

The CYP3A4 content for the 10 matched microsomal preparations represented in each group was equivalent. The nominal initial tacrolimus conc was 0.2 µM; unbound conc determined after measurement of nonspecific binding.

Unbound Km and Clint calculated after correction for nonspecific binding.

CYP3A5 Genotype and In Vivo Tacrolimus Disposition

Haufroid et al. 2004

High CLint

Low CLint

Tacrolimus Metabolic Clearance In Vivo

•  Subjects carrying a functional CYP3A5*1 allele have a higher clearance than those with two variant alleles.

•  The importance of individual metabolic pathways and sensitivity to the CYP3A5 genotype matches the in vitro prediction. Zheng et al, CPT, 2012

CYP3A5*1 and Tacrolimus Clinical Outcomes

Ojo, NEJM, 2003

Zheng et al, Clin Pharmacol Ther, 2012

O

O OCH3

O O

H

H

O

OHGua-N7

AFB1 Guanine-N7 adduct

O

O OCH3

O O

H

H

O

OHGS

AFB1 GSH adduct

O

O

O

OCH3

O O

H

H

O

O OCH3

O O

H

H

O

O

O

O OCH3

O O

H

H

O

HOOH

AFB1 AFB1 exo-8,9-epoxide AFB1 dihydrodiol

CYP3A

GSH GST

DNA

H2O

EH

multiple monohydroxylation and keto reduction products

CYP3A5*1 allele associated with higher

aflatoxin-albumin adducts in Africans

(Wojnowski et al, 2004)

Aflatoxin Activation -

CYP3A5

Protein-S-aflatoxin adduct (e.g., albumin adduct)

Non-toxic

Primary elimination pathway

Non-toxic

Toxic DNA Adduct Non-toxic: BIOMARKER FOR EXPOSURE TO AFB

Other Representative CYP3A5 Substrates

Anti-cancer Drugs vincristine (neurotoxicity) irinotecan (efficacy response rate) etoposide (efficacy)

Immunosuppressants cyclosporine (neurotoxicity, renal toxicity) tacrolimus (neurotoxicity, renal toxicity)

Antimalarials quinine (cinchonism)

Sedative/Hypnotics alprazolam midazolam

Non-CYP, Polymorphic Phase I Enzymes

FMO TMA, nicotine, cimetidine

xanthine oxidase theophylline

cholinesterase succinylcholine

paroxonase organophosphates

ADH ethanol

ALDH2 acetaldehyde

•  ALDH2 catalyzes the elimination of acetaldehyde from the body (converts it to acetic acid).

•  Individuals homozygous and heterozygous for the ALDH2*2 allele have a pronounced impairment in their ability to clear acetaldehyde after ingestion of an acetaldehyde source (e.g., ethanol); 47x and 220x increase in the AUC, respectively.

•  Acetaldehyde accumulation results in profound cardiovascular changes; e.g., increase in arterial blood flow to the face (flushing).

Acetaldehyde – ALDH2 Genotypes

ALDH2*2/*2

ALDH2*1/*1

ALDH2*1/*2

•  The ALDH2*2 mutation involves a single coding SNP (Glu487Lys) that results in a 150-fold increase in the Km for NAD+ (i.e., reduced cofactor binding) and 10-fold decrease in Vmax. M=methoxyacetaldehyde; A = Acetaldehyde; B = benzaldehyde; P = propionaldehyde

•  Because ALDH2 is functional as a tetramer and heteroteramers exhibit reduced function, the *2 allele appears semi-dominant. Strongly associated with reduced risk of alcoholism (presumably an aversion to acetaldehyde). Other data suggests increased risk of MALD (ME) toxicity in *2 carriers (Ginsberg et al., Reg Toxicol Pharmacol 36:297-309, 2002).

TPMT: thiopurine methyltransferase XO: xanthine oxidase HGPRT: hypoxanthine guanine phosphoribosyltransferase TIMP: 6-thioinosine monophosphate MTMP: 6-S-methylthioinosine monophosphate TGN: 6-thioguanine nucleotides 6-MP: 6-mercaptopurine MeMP: 6-S-methylmercaptopurine 6-TU: 6-thiouric acid

6-MP TGN

MeMP

TPMT

6-TU

XO

HGPRT DNA

TPMT

Bioactivation Pathway

Det

oxifi

catio

n Pa

thwa

y

TIMP

MTMP

TPMT

(multiple enzymatic steps)

(Purine salvage)

TPMT and 6-Mercaptopurine Disposition

Both TPMT and bioactivation enzymes found in hematopoietic cells; XO found only in the liver

Krynetski and Evans, Pharmacology 61:136-46, 2000

Common Impaired Function TPMT Alleles •  Although there are over

15 different mutant alleles, only a few account for the majority of PM activity throughout the world; *3A more common in Caucasians, *3C in Asians and Africans.

These mutations affect the stability of the enzyme (enhanced proteasomal

degradation), with reduced steady-state Vmax and Clint

Protein t1/2 ~ 18 hr

Protein t1/2 ~ 0.25 hr

PM: 1/300 IM: 1/10

Increased # base adducts in blood of PM

FDA Review: 6-MP Product Labeling Changes •  No mandatory requirement for genetic testing

•  Genetic information listed under Pharmacokinetics, Warnings and Precautions sections

•  Dose and Administration - “states tests are available”

Concerns with Mandatory Testing:  Extensive clinical experience  High cure rates with ”acceptable toxicity profile”  Fear of under-dosing and reduced cure rates (heterozygotes)  No standardized therapy (requires center-specific interpretation of genetic

results)  Delay in treatment initiation  Legal consequences

R. Padzur, Oncology Drug Products, FDA, 2004

Clinical Pharmacogenetics Implementation Consortium (CPIC)

Relling et al, CPT 89:387-91, 2011.

•  Reduce dose to 1/10 that of normal and frequency to 3 times/week (vs daily) for low activity phenotype.

•  For intermediate activity phenotype, reduced dose by 30-50%.

Glucuronosyl Transferases (UGTs)

•  18+ different genes in humans   UGT1A – 9 genes, 4 pseudogenes   UGT2A (2 genes), 2B (7 genes)   Membrane bound, ‘microsomal’   Catalyze the transfer of glucuronic acid to a ‘reactive’ heteroatom

(oxygen, nitrogen, sulfur) •  products - glucuronide conjugates- are readily excreted in urine and bile

•  UGT1 involved in endogenous bilirubin metabolism, and some xenobiotics;   rare variants – Crigler-Najar Syndrome   common variants Gilbert’s (idiopathic hyperbilirubinemia)

•  UGT2 involved in steroid metabolism, phenolic xenobiotics (at least 7 forms)

Glucuronosyl Transferases

Guillemete, Pharmacogenomics J

(2003): 3, 136-158

UGT1A1 Variants •  There are 60+ unique variants in the UGT1A1 gene

•  4 are associated with a ‘mild phenotype’ Gilbert’s Syndrome – idiopathic hyperbilirubinemia

3 nonsynonymous (rare); 1 common and 2 rare promoter tandem TA repeats

Moderate decrease in function

•  24+ SNPs associated with severe phenotype 18 with Criglar-Najar Syndrome Type I 6 with Criglar-Najar syndrome Type II

Nonsynonymous or truncating; loss of function

UGT1A1 Polymorphism

•  Variable tandem repeat, (TA)5-8TAA, in the UGT1A1 promoter modulates gene transcription, protein expression and catalytic activity; 6 repeats – wild type.

•  Homozygosity for the (TA)7 allele, UGT1A1*28, associated with ~30% normal bilirubin conjugating activity – Gilbert syndrome (also 7/8 or 8/8 genotypes).

•  PMs: 10% allele frequency in Whites; 29-47%, African; 3%, Asians

VNTR

•  Undergoes ester hydrolysis (not all that rapid) to active SN-38, which in turn is detoxified by glucuronidation. Multiple UGTs catalyze the reaction (1A1, 1A9, 1A10), but 1A1 seems to dominate.

•  Although SN-38 is 1000x more potent Topo-I inhibitor than irinotecan, it circulates at levels 2-3% that of irinotecan and is more highly plasma protein bound (95% v 50%).

•  Parallel intestinal/hepatic CYP3A4 pathway and P-gp mediated efflux also significant.

•  Conversion of APC to SN-38 does not seem to occur (steric/polarity hindrance), although not tested rigorously.

Innocenti, DMD, 2001; PDR, 2004; Pauluzzi, J Clin Pharmacol, 2004’ Ohuri, Anticancer Res, 2004

P-gp

Association between UGT1A1*28 Genotype��� and Serum Bilirubin

Marcuello et al., Br J Cancer, 2004

Association Between UGT1A1*28 Genotype and��� Post-Irinotecan ANC Nadir

•  All grade 4 neutropenia (shaded area) occurred in patients carrying the UGT1A1*28 allele; ANC = absolute neutrophil count.

•  Exclusion of 7/7 genotype (10% of population) from standard dose would reduce frequency of grade 4 toxicity by 50%. Exclusion of anyone carrying a TA7 allele would eliminate such toxicity, but have poor positive predictive value.

Innocenti et al, J Clin Oncol, 2004

N-Acetyltransferases

•  NAT1 and NAT2 are on chromosome 8   2 active genes separated by a pseudogene

•  Both are expressed in liver   Only NAT1 expressed in human leukocytes

•  Both have distinct but overlapping substrates   N- and O-acetylation of aromatic amines - both   N,O-acetyltransferase activity by NAT1 only   PABA, PASA - NAT1 (monomorphic)   Sulfametazine, isoniazid, hydralazine- NAT2 (clear polymorphic

phenotype)

NAT2 ‘Slow Acetylator’ Phenotype

•  NAT2 polymorphism first identified as the ‘slow acetylator’ phenotype; isoniazid

•  Trimodal phenotype distribution   55-60% in Caucasians / Northern Europeans   8-10% Japanese   20% Chinese   90% North Africans

•  SA due largely to polymorphisms in NAT2 gene

Role of NAT1 and NAT2 in Aromatic Amine Metabolism

NAT1 appears to function as an O-acetyltransferase (OAT) and an N,O-acetyltransferase (N,O-AT) when using acetyl coenzyme A or hydroxamic acids, respectively, as acetyl donors. NAT2 appears to act preferentially as an OAT and NAT.

Decomposes to reactive arylnitrenium ion (DNA binding)

(immune toxicity)

Redox cycling

Sulfation (DNA adduction)

Aromatic amine

Polymorphisms in NAT2 ���(multiple variants)

50

Gln64 (191A) Tyr94

(282T)

Thr114 (341C)

100 150 200 250

Pro145 (434C)

Leu161 (481T)

Gln197 (590A)

Arg268 (803G)

Thr282 Gln286 (857A) 2*5 A,B,C A,B B,C

2*6 A A,B 2*7 B A,B 2*12 B A 2*13 (A) 2*14 A B 2*17 (A) 2*18 (A)

Note, haplotypes for *5, *6, *7 and *14; confer SA phenotype.

A

B

C

NAT2 ‘Slow Acetylator’ PM and Cancer Risk���

•  SA-Elevated risk of differentiated thyroid cancer   Modulates response to ionizing radiation

•  SA-Elevated risk for bladder cancer   In aromatic-amine exposed workers  In smokers (candidate gene and GWAS studies)

•  SA-Decreased risk for colon cancer   Acetylation of N-hydroxy heterocyclic amines

•  Transported to bile as glucuronide •  Hydrolyzed by β-glucuronidase •  Increased formation or reactive O-acetyl-

Population Study •  Multi-center study of

bladder cancer reproduced association with loci containing NAT2, GSTM1 and UGT1A.

•  Gene products involved in detoxication of reactive metabolites or pro-carcinogens

•  GxE (smoking)

Nature Genet 42:978-84, 2010

Genetic Variation and Rare ADRs

Characteristic Type A Type B

Dose dependent generally yes no clear relationship

Predictable from known pharmacology

yes no

Animal models usually predictable none known

Frequency/severity common/variable rare/severe

First detected Phase I-III Phase IV (post approval)

Pirmohamed, AAPS Journal 8:E20-26, 2006

Hepatic and Idiosyncratic Drug Toxicities

•  Because the liver is the dominant site of biotransformation, it is also highly susceptible to metabolically-based toxicity, including immune-based toxicity.

Drug Indication Active Metabolite ToxicityChloramphenicol Antimicrobial - restricted Acyl chloride Hematopoietic ToxicityAcetaminophen Analgesia Quinoneimine HepatotoxicityNSAIDS (zomepirac) Inflammation Acyl glucuronides AnaphylaxisPhenytoin Epilepsy Arene oxides Stevens-Johnson, SLEHalothane Anesthesia Trifluoroacetic acid Mild & Severe HepatitisFelbamate Epilepsy - restricted Conjugated alkene HepatotoxicityValproic Acid Epilepsy Acyl CoA HepatotoxicityTiclopidine Antiplatelet - monitored Oxidized thiophene Blood dyscrasiasClozapine Antipsychotic Conjugated imminium ion AgranulocytosisFlutamide Prostate CA - monitored Nitroaromatic HepatotoxicityTolcapone Parkinson’s-monitored Nitroaromatic HepatotoxicityTrovofloxacin Antibiotic - restricted Aminopiperidine oxidation HepatotoxicityTroglitazone Antidiabetic - withdrawn Quinone methide HepatotoxicityZileuton Antiasthmatic-monitored ? HepatotoxicityAbacavir HIV Infection -restricted Carbovir triphosphate Hypersensitivity

Representative drugs that cause severe toxicities

Biological Basis for Immune-Based ADR

•  MHC - Major Histocompatability Complex  Located on chromosome 17  Multiple tandem genes; Class I, II and III regions  Class I (A, B, C) and class II (D, DR) genes - involved in antigen

presentation, tolerance, self/non-self recognition  Class I and II gene products can bind intracellularly processed antigenic

peptides (ligand modified self or neoantigen) within antigen presenting cells (APCs; e.g., Langerhan cells in skin) and the complex is presented to the cell surface where it causes specific recruitment of T-helper cells, which then activate killer T-cells and precipitate autoimmune reaction

 There is also evidence that direct binding of drug to the HLA molecule can alter its recognition of self proteins and thereby precipitate the autoimmune reaction

 HLA allele frequencies vary throughout the world populations  Genetic variation in HLA genes (protein products) affects peptide

binding and recognition of the complex by T-cells

Abacavir Hypersensitivity

•  Nucleoside analog reverse transcriptase inhibitor used in combination therapy to treat HIV-1 infection

•  About 5-8% of treated patients will develop a multi-system hypersensitivity reaction, which can be fatal.

•  Indications of MHC Gene Involvement:  Develops within 6 wks or rarely at all  Racial differences in risk (decreased risk in black population)  Familial predisposition  Evidence for neoantigen formation (haptenation) or direct binding

to MHC

Association of Abacavir Hypersensitivity with���HLA Genotype

•  Lancet 359:727-32, 2002

•  Lancet 359:1121-2, 2002 (GSK Group)

Loci AbacavirHypersensitive

(n = 18)

AbacavirTolerant(n = 167)

Odds Ratio(95% CI)

Pc

HLA-B*5701 14 (78%) 4 (2%) 117 (29-481) < 0.0001

HLA-DR7, HLA-DQ3

13 (72%) 6 (3%) 73 (20-268) < 0.0001

HLA-B*5701, HLA-DR7, HLA-DQ3

13 (72%) 0 (0%) 822 (43-15,675) < 0.0001

Caucasoids

Loci Cases Controls Odds Ratio

HLA-B57 36/65 (55%) 2/80 (3%) 23.6 (8.0-70.0)

HLA-B*5701 36/65 (55%) 1/80 (1%)

HLA-B*5701 and HLA-DR7 15/46 (33%) 1/69 (1%)

GSK group also, reported a significant association with CYP1A2 and TNFα SNPs

Prospective HLA B*5701 Genotyping

•  Prospective testing with exclusion of HLA B*5701 positive patients from receiving abacavir reduced the incidence of confirmed hypersensitivity rxns (diamond) and all related symptoms (circles). Those 2004-05 subjects with a reaction requested drug despite risk.

Rauch et al, Clinical Infectious Diseases 43:99-102, 2006

Reproducibility of Association Result

•  Test Sensitivity = 0.51; proportion of individuals with a positive test result who will get hypersensitivity (true positives + false negatives); positive predictive value = 82%

•  Test Specificity = 0.96; proportion of individuals with a negative test result who will not get hypersensitivity (true negatives + false positives); negative predictive value = 88%

Strength of the association is ethnicity-dependent

White males (n = 293): p = 5 x 10-18

White females (n = 56): p = 7 x 10-6

Hispanics (n = 104): p = 2 x 10-4

Blacks (n = 78): p = 0.27

Hughes et al, Pharmacogenomics, 2004

Hughes et al, Pharmacogenetics 14:335-42, 2004

HLA B*5701 Genotype - Abacavir Hypersensitivity

Screening for HLA-B*5701 Eliminates the Risk for Abacavir Hypersensitivity (Diagnosis Confirmed by Patch Testing)

Screened Control

Total Population Caucasians

Inci

denc

e of H

yper

sens

itivi

ty

Rea

ctio

n (%

)

0

1

2

3

4

5

2.7% 3.1%

Mallal et al. NEJM 358:568-579, 2008

0% 0%

Negative predictive value = 100% Positive predictive value = 48%

Critical Factors for Translation of Abacavir Pharmacogenetic Findings into Clinical Practice

•  Strong molecular and clinical evidence for association of HLA-B*5701 and hypersensitivity

•  Widely available genetic testing

•  Evidence for beneficial application of genetic data in clinical practice

•  Clear guidelines for the use of HLA-B*5701 genotype data

•  Willing and educated clinicians

•  Positive cost-benefit analysis

Carbamazepine and Phenytoin Immunotoxicity

•  Established drugs used to treat epilepsy (CBZ also used for other disorders) •  Associated with adverse reactions, many of which occur in the skin:

Mild maculopapular exanthema (16% of patients) Severe cutaneous reactions (Stevens-Johnson syndrome; SJS); 1:10,000 - 90% cases

occur within first 2 mo of therapy Toxic epidermal necrolysis (TEN, 40% fatal)

•  Multiple studies have found a strong association between the HLA-B*1502 gene and SJS/TEN with CBZ use in Han Chinese

Chung et al, Nature 428:486, 2004 Man et al, Epilepsia 48:1015-18, 2007

Hung et al., Pharmacogenetics and Genomics 16:297-306, 2006

•  A similar association between the HLA-B*1502 gene and SJS/TEN with DPH use in Han Chinese and Thai

Locharernkul et al, Epilepsia 49:2087-91, 2008 Man et al, Epilepsia 48:1015-18, 2007

Anti-Epileptic Drug Immunotoxicity

•  FDA has relabeled Carbamazepine (Tegretol) and Phenytoin (Dilantin) to recommend HLA testing in high risk populations

Warning Serious Dermatological Reactions and HLA-B*1502 Allele

… Patients with ancestry in genetically at-risk populations should be screened for the presence of HLA-B*1502 prior to initiating treatment

with Tegretol. Patients testing positive for the allele should not be treated with Tegretol unless the benefit clearly outweighs the risk

http://www.fda.gov/Drugs/ScienceResearch/ResearchAreas/Pharmacogenetics/ucm083378.htm

FDA – Rationale for Pharmacogenetic Testing

•  Drug exposure and clinical response variability •  Risk for adverse events •  Genotype-specific dosing •  Mechanisms of drug action •  Polymorphic drug target and disposition genes

Barriers to Clinical Implementation of Pharmacogenetic Tests

•  Logistics of genetic testing - Point-of-care testing, CLIA certification, etc.

•  Reimbursement concerns •  Lack of prospective randomized clinical trials (RCT)

- Does PGx improve clinical outcomes; alternative to RCT? -  What is the optimal clinical algorithm for its application? -  Is it cost effective?

•  Questions around the preparedness of health care providers to integrate genomic-based technologies with clinical practice - Educational needs are significant!

- How will the electronic medical record cope??

Alan Shuldiner