Post on 14-Aug-2020
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