Pharmacogenetic and Genomic Applications for Safety and ... · PDF file2e-NADPH+H+ NADP ++2H+...

Pharmacogenetic and Genomic Applications for Safety and

Therapeutic Efficacy Assessment in Drug Development Programs

[email protected]

- Topics of my presentation –

Centre for Pharmacology and Toxicology

• Translational genomics(HUGO, disease diagnostics, clinical trialsand patient stratification)

• How SNPs affect drug safety andtherapeutic efficacy (PK, ADME genes,treatment „responder“, clinical outcome..)

• Genetic factors linked to DILI

Human Genome Project• At the DNA sequence level all humans are >99% identical

(initial draft 2001; the 1000 Genomes Project in 2010)

• Mice and chimpanzee genomes are very similar to humans; even protein coding regions are ~ 90% identical

• “Housekeeping” genes are highly conserved (human versus yeast 26% or fruit fly ~ 44%)

• Individual genomes carry ∼3 million SNPs but most do not impact protein function.

• SNPs in non-coding regions affect gene splicing, transcription factor binding, messenger RNA degradation, or the sequence of non-coding RNAs.

• Each person carries ~ 250 to 300 loss-of-function and 50 to 100 variant genes previously implicated in inherited disorders


.


N=149 pharmacogenomic studies listedin EU Clinical Trials Register (03.2017)


149

60

21 2011 9 6

22

0

20

40

60

80

100

120

140

160

Pharmacogenetics of metoprolol drug disposition

2 4 6 8 12 24Time (h)

200

400

600

Deficient metabolism of drugs is referred to as POOR METABOLIZERas these individuals exert a dramatic change in pharmacokinetic parameters.

METABOLIC RATIO EM

METABOLIC RATIO EM PM

3.5 billion years

today

Cytochrome P450

• There is no life without CYP monooxygenases; all livingsystems (plants, bacteria, fungi, animals) carry CYP genes.

• In animals original functions lie within chlosterolmetabolism/ membrane biology and host defense againstplant „secondary synthesis products“ such as alkaloids


Humans have 57 CYP genes and ~ 59 pseudogenes.CYP genes are divided among 18 families & 43 subfamilies.Mice have ~ 100 CYP genes

Examples of CYP monoxygenase functions

P450 family FunctionCYP1-CYP3 xenobiotic/steroid metabolismCYP4 fatty acid hydroxylation, foreign compound metabolismCYP5 thromboxane synthesisCYP7 cholesterol 7-hydroxylaseCYP11 cholesterol side chain cleavage

steroid 11 hydroxylaseCYP17 steroid 17 hydroxylaseCYP19 steroid aromataseCYP21 steroid 21-hydroxylaseCYP24 vitamin D metabolismCYP27 cholesterol 27-hydroxylase



OOH

N

O NO

N

N

O

NS

O ON

O

O

N

O

PROPANOLOL

BUFURALOL

TIMOLOL

METOPROLOL

O

O

ON

N N

NN

N

HH

HH

N

O

NO

O

ONH

N H 2

N H

NH

N

O

NO

H

N

O H

NO H

H

P R O P A F E N O N

D E B R IS O Q U IN E

E N C A IN ID E

G U A N O X A N

S P A R T E IN E

P E R H E X IL IN E

N -P R O P Y L A JM A L IN E

ß-adrenergic blocking agents and antiarrhythmics are substrates for polymorphic CYP monooxygenase genes


Reaction cycle of cytochrome P450

HP-Fe3+

HP-Fe3+

RH

RH

HP-Fe2+

RH

1e-

HP-Fe2+ -O2

RHHP-Fe3+ -O2

-

RH

HP-Fe3+ -O22-

RH

HP-Fe3+

RH

O21e-

O

H2O

2H+

ROH

Flavoprotein2e-

NADPH+H+ NADP++2H+


Most drug metabolising enzymes exhibit clinically relevant genetic polymorphism

CYP1A1/2CYP1B1

CYP2A6CYP2B6

CYP2C8

CYP2C9CYP2C19

CYP2D6

CYP2E1

CYP3A4/5/7

ALDH

ADH

DPD

NQO1

esterases

epoxidehydrolase

others NAT1NAT2

GST-M

GST-T

GST-P

GST-A

STs

HMT

COMT

TPMT

UGTs

others

Phase 1 Phase 2


Discovery of the first genetic polymorphism of human cytochrome P450 (CYP2D6)

Melatonin (MT), N-acetyl-5-methoxytryptamine, is a major hormone of the pineal gland, is a multitasking molecule and exerts anti-excitatory and sedating effects. MT is an anticonvulsant and functions through two MT-receptor (GPCR).

13α helices5ß-sheets

Ma et al. 2005;DMD 33(4):489-94.

(A) Melatonin 6-hydroxylation (A) and (B) O-demethylation.

Examples of variant CYP2D6 alleles but NOT all of them affect activity

Allele1Trivialname

Nucleotide differencesfrom wild-type CYP2D6

Enzymeactivity

CYP2D6*1 Wild-type none normalCYP2D6*2A CYP2D6L1 G1749C; C2938T; G4268C normal

CYP2D6N G4268C normalCYP2D6L2 G1749C; C2938T; G4268C increased

CYP2D6L2 x 2 G1749C; C2938T; G4268C increasedCYP2D6L2 x 12 G1749C; C2938T; G4268C increased

CYP2D6*3 CYP2D6A A2367 deletion inactiveCYP2D6*4 CYP2D6B C188T; C1062A; A1072G;C1085G;

G1749C; G1934A; G4268Cinactive

CYP2D6*5 CYP2D6D CYP2D6 deleted inactiveCYP2D6*6 CYP2D6T T1795 deleted; G2064A inactive



There are about 100 known SNPs within the CYP2D6 gene. However, only four of them, i.e. CYP2D6*3, *4, *5

and *6 account for 93% to 97% of PMs

(Source:http://www.genome.utah.edu/genesnps/genes/CYP2D6/)

flanking, 620,G/A

flanking, 660,G/C

flanking, 780,A/T

flanking, 801,C/G

flanking, 880,C/T

flanking, 883,A/G

flanking, 942,G/A

flanking, 1032,G/A

flanking, 1255,G/A

flanking, 1444,G/A

nonsyn, 1650,G/A

nonsyn, 1696,G/A

nonsyn, 1719,T/C

nonsyn, 1719,C/T

intronic, 1833,G/C

intronic, 1840,C/A

intronic, 1842,C/G

intronic, 1846,T/C

intronic, 1851,GA/CC

intronic, 1864,A/G

nonsyn, 4194,C/A

nonsyn 4469,T/C

nonsyn, 4482,C/G

nonsyn 4484,G/A

nonsyn, 4515,C/T

nonsyn, 4884,G/A

nonsyn, 4910,A/G

intronic, 5003,A/C

intronic, 5201,A/G

nonsyn, 5472,G/A

3'UTR, 5862,C/T

flanking, 7895,G/A

intronic, 1889,C/T

intronic, 1929,G/T

intronic 2348,C/A

intronic 2365,C/G

intronic, 2462,T/G

synon, 2658,C/T

intronic, 2789,G/A

nonsyn, 3280,C/G

nonsyn, 4085,T/C

synon, 4089,T/C


CYP2D6 metabolizes about 20–25% of clinically used drugs; the enzyme also catalyzes oxidation of hydroxytryptamines

4378 bp

CYP2D6

i1 i2 i3 i4 i5 i6 i7 i8

i3 intron 4

1846 1934 2005 27052528Exon 4 Exon 5

Ex1 Ex2 Ex3 Ex4 Ex5 Ex6 Ex7 Ex8 Ex9

1934 : G to A substitution*4 Allel

2637 : A deletion*3 Allel

Allan Gough et al., Nature 347, 773 – 776; Kagimoto M et al., J Biol Chem. 1990 Oct 5;265(28):17209-14

The case of tramadol and the challenge: Analysis of the most common SNPs within 60 minutes of blood with-drawl.

Genotyping in clinical trials

wildtype

hetero-zygote

homo-zygote

CYP2D6*3

CYP2D6*4 wildtype

heterozygote

homo-zygote


Pharmacokinetics of tramadol and M1


Results Standard deviations

Phenotype PM EM PM EM

Cmax(tramadol) [ng/ml] 303,75 340,23 90,76 48,40

Cmax(M1) [ng/ml] 34,43 87,00 23,24 20,03

AUC(tramadol) [ng/ml*h] 3482,57 2283,54 1534,79 572,19

AUC(M1) [ng/ml*h] 431,00 829,62 215,72 166,43

Tramadol

CYP2D6

M1

-opioidreceptor

NMDA, 5HT, nAChR, mAChRs, SRI, NRI...

CYP2D6

CYP2D6

CYP2D6

Tramadol receptors


The metabolite (+)-M1 showsthe highest affinity (Ki=3.4 nM)to the human mu-opioid receptor

Qiang Ma, and Anthony Y. H. Lu Pharmacol Rev 2011;63:437-459

(S)-warfarin blocks Vitamin K epoxide Reductase Complex VKORC1 activity resulting in the depletion of reduced vitamin K.This results in decreased γ-carboxylation of coagulation factors II, VII, IX, and X by gamma-glutamylcarboxylase GGCX and prolonged blood coagulation. CYP2C9 catalyzes the 7-hydroxylation of (S)-warfarin to inactivate the drug, whereas, calumenin modulates GGCX function to affect blood clotting. Variant CYP2C9 and VKORC1 genotypes require dose adjustment

Centre for Pharmacologyand Toxicology

Thiopurine methyltransferase (TPMT) and Dehydro-pyrimidine dehydrogenase (DPD) polymorphism –- implications for drug therapy -

Typical substrates for TPMT and DPD are mercaptopurine, ACE inhibitors such as captopril, D-penicilamine given to Morbus Wilson and arthritis patients, antidiuretic spironolactone, 5-flurouracil…


TPMT9ß-sheets6 α-helices

DPDHomodimer of2 x 111kDa prot.


Thiopurine methyltransferase

N NH

NN

SH

N NH

NN

SCH3

Ado-Met Ado-Hcy

6-Methylmercaptopurine

45 112 184 93 133 53 75 86 45 2075TPMT*1(wild type)

ATG

TPMT*2( activity)

ATG G238CAla Pro

TPMT*3(no activity)

ATG G460CAla Pro

A719GTyr Cys

• TPMT*3 is of great importance in cancer therapy. Drugs such as mercaptopurine, azathiopurine and thioguanine are prodrugs and must be converted to thiopurine nucleotides.

• TPMT competes with HPRT (hypoxanthine phosphoribosyl transferase) and inactivates drugs by S-methylation.

• TPMT deficient patients require dose adjustment because they experience severe toxicity to drug treatment due to increased levels of thioguanine nucleotides (TGN).

Thiopurine methyltransferase


N

NH2

O NH

NH

O NH

O

NH

O NH

O

N

NH2

O N

F

NH3H2O

NADPH + H

NADP+

H2O

-ureidopropionate-alanine

DihydropyrimidineDehydrogenase

fluoro--alanine

5-fluorouracil

DPD catalyzes the NADPH-dependent reduction of uracil and thymine to 5,6-dihydropyrimidines

cytosine uracil

dehydro-uracil


R Duschinsky, R Schnitzer, Hoffman-La Roche, C. Heidelberger US

Expression of DPD activity in human leucocytes

subjects

Patient number 8 is a cancer patient that experienced high toxicity when administered 5-FU.



Human NAT1 alleles (8p23.1-21.3); 26 SNPs identified

1 1150

1095 C > ANAT1*3NAT1*10NAT1*11A,CNAT1*14ANAT1*16NAT1*18ANAT1*26ANAT1*29

350, 351 G > CNAT1*5

NAT1*5497-499 G > C

884 A > GNAT1*5

NAT1*29NAT1*18ANAT1*14ANAT1*10

1088 T > A

445 G > ANAT1*11ANAT1*11B

459 G > ANAT1*11ANAT1*11BNAT1*11C

640 T > GNAT1*11ANAT1*11BNAT1*11C

NAT1 and NAT2 acetylate endogenes substrates and arylamine carcinogens and drugs.Slow acetylator show increased susceptibility to hydralazine (vasodilator) and isoniazidtoxicity and to occupational bladder cancer.


Schematic representation of the NAT2 RFLP multiplex PCR

KapI digest DdeI digest

BamHI digest TaqI digest

A

547

433

114

B

345

137

114

65

C

547490

57

D

392

222170155

Kk kk KK Dd dd DD

Bb bb BB Tt tt TT

Constant

Constant

Constant


Association between NAT phenotypes and genotypes

Common TB Treatments:• Isoniazid (inhibition of cell wall lipid synthesis, depletion of• nucleic acid pools and metabolic enzymes

• Rifampin (interchelates DNA, suppresses RNA polymerases)• Ethambutol (inhibition of arabinosyl transferase)• Pyrazinamide (inhibition of growth,

pyrazinoic acid accumulation)

Portal fibrosis with early bridging on trichrome. Portal areas with inflammationon H&EAnnals of Clinical & Laboratory Science, 44, 1, 2014


• Significant differences in the allelic frequencies of the pNAT and CYP2D6 genes among epileptic patients (role of melatonin…)

• Actylation of melatonin is extremely important and protects 5HT against MAO degradation and thus permanent loss of function

• Melatonin increases in epileptic seizures• There was a significant increase of PMs

amongst epileptic patients (2=4.01, p<0.05), i.e. 9.1 vs 4.3%

• There was a significant increase of slow acetylators among epileptic patients and many anticonvulsive drugs require N-acetylation for metabolic clearance

Critical Care Clinics 1997;13[4]:763-83


Clinically relevant human UGT gene polymorphism

Human UGT Gene Family

UGT1*2P

UGT1*3

UGT1*5

UGT1*4

UGT1*1

UGT1*6

UGT1*02

UGT2B11

UGT2B4

UGT2B9

UGT2B7

UGT2B15

UGT2B10

UGT2B8

HugBr2 / HP2

HugBr1 / HP2

HP1

HP4

Hlug25 / UDPGTh-1

Hlug6

UDPGTh-2

Hlug7

Hlug4 / UDPGTh-3

human olfactory UGT

UGT1

UGT2

UGT2B

Percentage protein similarity 100%30%

Exon 1 2 3 4 5

A C

(TA)6 TAA

39 53

D B98-100 bp

253-255 bp

3’5’


Structure of UGT1*1 gene highlighting promotor region

Molecular probes to detect variant alleles of the TATA-insertion polymorphism of the UGT1A1 gene

http://onlinelibrary.wiley.com/doi/10.1053/jhep.2000.18193/abstrac


Box plots of serum total bilirubin for individual genotypes incohort of 266 unrelated individuals from Southern Germany.


Molecular diagnosis of a familial nonhaemolytic hyperbilirubinemia

clonedcontrols

individual genotypes

negative control (water)wild type (homozygote)mutation (heterozygote)mutation (homozygote)

(TA)6TAA (TA)7TAA

(TA)6TAA (TA)7TAA

SF2

Folie 32

SF2 Histologic image from the liver biopsy of a 54-year-old patient with drug-related hepatitis and Gilbert’s syndrome. (A) Presence of a significant granulocyte population in the portal tracts with a lymphocytic spill-over and morphology alterations to small and medium bile canaliculi (hematoxylin and eosin stain, original magnification ×10). (B) Hepatocellular intracytoplasmatic edema with few microthrombi inside canalicular bile ducts (hematoxylin and eosin stain, original magnification ×10)Schoppmeier, Frauke; 15.03.2017


Mean Total Serum Bilirubin Levels in Individual UGT1A1 Genotypes

Metabolism of irinotecan to APC


Centre for Pharmacologyand Toxicology

Pharmacokinetic parameter estimates of AWD21-360 after multiple oral doses of 200mg retigabine b.i.d.

Polymorphisms in other phase II enzymes(GSTs, COMT, esterases, aldehyd (ALDH) /

alcohol (ADH) dehydrogenases andsulfotransferases)


SAM, 3,5-Dinitrocatechol


GSTs are of vital importance in maintaining cellular defenceagainst ROS. SNPs contribute to risk for carcinogenesis andindividual variation in drug response

position, allele

9363, A/G9425, A/T9616, C/T9685, C/T9920, A/G

implication

intronic, intron 7nonsyn, exon 83‘UTR3‘UTRflanking

function

unknownunknownunknownunknownunknown

submitter population ID: NIHPDRCollins et al (1998), Genome Research 8:1229-1231160 to 190 probes analysed

Source: http://www.genome.utah.edu/genesnps/genes/GSTM1

implication

exon 7whole gene

position, allele

534 G/Cnull allele

function

unchangedno activity

Brockmoller et al (1993), Cancer Research 53:1004-1011272 probes analysed

nomenclature

GSTM1*AGSTM1*BGSTM1*0

Human cytosolic GSTs involve alpha, zeta, theta, mu, pi, sigma, and omega classes

• Catechol-O-methyl-transferase playsan essential role in the metabolism ofneurotransmitter such as dopamine, epinephrine, norepinephrine, catecholestrogens, and drugs

• Polymorphism may be related to neuropsychiatricconditions involving catecholamine neurotransmission.

• COMT*2 encodes for a missense val 158 Met thatcauses lower activity and protein instability. The allelicfrequecies is about 0.46.

COMT polymorphisms



AIMS: A 23-year-old Caucasian woman, with cystic fibrosis, bilateral lung-transplantation and immunosuppressive therapy with prednisolone, tacrolimus and sirolimus, presented with clinical symptoms of a chronic transplant rejection.METHODS: Since constant sufficient blood level of tacrolimus and sirolimus had never been achieved, a genetic analysis was carried out to clarify drug metabolism.RESULTS: The genetic analysis for polymorphisms of cytochrome P450 (CYP) 3A4,5,7 revealed no sequence alterations in the CYP 3A4,5,7 gene. Thus, drug intake was scrutinized in detail, disclosing a missing interval between the intake of both immunosuppressiveagents. After a correct drug intake the woman's condition ameliorated and the blood levels reached normal range.CONCLUSIONS: This case report highlights the crucial importance of basic medical skills like an accurate and dainty drug anamnesis before high tech approaches were applied.

.

Centre for Pharmacology andToxicology

Fig. 1 - Current mechanistic understanding in the initiation and progression events relevant to idiosyncratic drug-induced liver injury.

Lazarou et al., 1998 estimate 6.7% ADR‘s [95%CI, 5.2 - 8.2%] for the US alone with 0.32% [95% CI, 0.23 – 0. 41%] fatal outcome ( > 100.000,00 patient die of ADRs, i.e. third leading cause of death!)



• HLA-B*5701 allele was associated with an 80-fold increased risk of developing DILI (Dale et al., 2009 Nature Genetics; DILIGEN & Serious Adverse Event consortium)

• This HLA allele is also associated with abacavir-hypersensitivity in HIV patients. • The HLA-B*5701 in the general population (6 to 8%) and the low incidence of DILI in

treated patients (1 in 10,000) leads to a low positive predictive value (0.12%).• Testing for HLA-B*5701 may help diagnose flucloxacillin cholestasis in patients that

develop jaundice but use of the drug should not be withheld in HLA-B*5701 positive individuals.

Fontana Gastroenterology. 2014 April ; 146(4): 914–928.

Genetic studies of Flucloxacillin hepatotoxicity


Genetic studies in pooled idiosyncratic DILI patients

• DILI susceptibility may be shared across multiple drugs but a GWA study in 783 Caucasian individuals who experienced DILI from over 200 different drugs did not reveal significant associations.

• Stratification of cases according to injury pattern, latency, severity, drug class and clinical phenotypes/medical history did not reveal any association

• Susceptibility to Amoxicillin-Clavulanate-induced liver injury is influenced by multiple HLA class I and II alleles

• Pharmacogenetic polymorphisms influence the metabolism and therefore PK of drugs.

• This can impact safety, particularly with drugs of narrow therapeutic index.

• Pharmacogenetics permits “personalized” dose selection to ensure safety and therapeutic efficacy.

• The pharmacogenetics of drug targets (receptors, ion channels etc.) will be key for defining “drug responders” and a sine qua non for precision medicine

• Pharmacogentics and translational genomics will bea major breakthrough in precision medicine

Summary


Pharmacogenetic and Genomic Applications for Safety and ... · PDF file2e-NADPH+H+ NADP ++2H+...

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