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Dr. Margarete Fischer-Bosch-Institut für Klinische Pharmakologie Robert Bosch Stiftung Stuttgart Pharmacogenetics today: What we don‘t know STUTTGART IKP

Dr. Margarete Fischer-Bosch-Institut für Klinische Pharmakologie … · 2018-08-02 · ME3122. Polygenic Nature of Drug Response: Antidepressants ME3050 Drug Target Contribution

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Dr. Margarete Fischer-Bosch-Institut für Klinische Pharmakologie

Robert Bosch StiftungStuttgart

Pharmacogenetics today:

What we don‘t know

STUTTGART

IKP

Pharmacogenomics and Drug Therapy

ME3015.ppt

Selection of appropriate drug and dose for the individual patient in order to

• achieve optimal therapeutic response

• avoid therapeutic failure

• minimize side effects and toxicity

ME3290.ppt

Pharmacogenetics and Drug Therapy

Candidate genes associated with response and side effects/toxicity are known

Value of pharmacogenetic test in predicting response and selection of appropriate and dose has been established in prospective clinical trials in two independent study cohorts

Ideal:

ME3289.ppt

Pharmacogenetics and Drug Therapy

Reality: Focus on one gene, limited number of mutations tested

Predictions based on case reports only

Retrospective studies with poor descriptionof patient characteristics, clinical outcome & confounders

ME2849.ppt

Prerequisites for Pharmacogenetic Testing

• Clear definition of phenotype (confounders)

• Genotype-phenotype relationship

• Sufficient sample size to identify all relevant

mutations associated with phenotype

• Association studies: Plausible biological hypothesis

ME3292.ppt

Pharmacogenetics and Drug Therapy

How is the phenotype drug concentration

related to drug response, side effects?

How predictive is the drug effect for efficacy,

clinical endpoint?

Pitfalls in Pharmacogenetic Testing

ME2838.ppt

• Of the known functionally important mutations only a limited number are tested

• Presence of unknown mutations

• Penetrance of gene

• Phenotype studied is only in part caused by candidate gene

• Other genetic and nongenetic factors contribute to phenotype

Schaeffeler et al. 2003, 2004, 2005

TPMT*11

TPMT*12

TPMT*13

TPMT*14

TPMT*15

84A>T

1A>G

-1G>A

TPMT*16488G>A

TPMT*17124C>G

374C>T

TPMT*18211G>A

TPMT*19365A>C

TPMT*20712A>G

TPMT*21205C>G

395G>A

I II III IV V VI VII VIII IX X

TPMT*2

TPMT*3A

TPMT*3B

TPMT*3C

238G>C

460G>A

460G>A

719A>G

719A>G

TPMT*3D

TPMT*4

TPMT*5

TPMT*6

TPMT*7

TPMT*8

460G>A 719A>G292G>T

146T>C

539A>T

681T>G

644G>A

A G

TPMT*9356A>C

TPMT*10430G>C

TPMT*22488A>C

ME3277

ME3291.ppt

Requirements for Pharmacogenetic Testing

How many mutations should be tested?

Restricted to most common mutations or as complete

as possible?

Example of TPMT: 22 loss of function mutations identified

Cohort of ~ 15000 patients: 10 new mutations identified.

1 patient with complete deficiency was classified

as heterozygous based on genotyping which was restricted

to 3 most common SNPs

Polygenic Nature of Drug Response: Antidepressants

ME3045

BBB BBBconc

.

conc

.

time [h] time [h]

MAO

HT Receptors

5-HT

TCA

Co

nce

ntr

atio

n

10

1

time [h]

time [h]

10

1

Co

nce

ntr

atio

n

10

1

time [h]

Co

nce

ntr

atio

n

CYP2D6

amplification

normal

mutations

MDR1

TPH2

SERT

Drug

Pharmacogenetics and Prediction of Response and Toxicity

ME3010.ppt

The example of TPMT

• Of the known functionally important mutations only a limited number are tested

• Presence of unknown mutations

• Penetrance of gene

• Phenotype studied is only in part caused by candidate gene

• Other genetic and nongenetic factors contribute to phenotype

ME3273

Dose dependent ADRs

Dose independent ADRs

• hematotoxicity (leukopenia, pancytopenia)• hepatotoxicity (most cases)

• pancreatitis• gastrointestinal disturbancies* (eg. nausea, vomiting, diarrhoea)• flu-like symptoms (such as fever*, headache)• rash*• arthralgia*, myalgia*

* commonly termed also as Azathioprine intolerance

Patients (n= 41) with Crohn‘s Disease and SevereMyelosuppression during Azathioprine Therapy

Colombel et al.Gastroenterology 2000

homozygousmutant

wildtype0

10

20

30

heterozygous

num

ber

n=4 n=8 n=299.8%

19.5%

70.7%TPMT genotype

29.3%

ME3278

13 (14%) comedicationallopurinol

391CMV Infection (36% received

ASA �12 mo )

53 wildtype (56%)

Schwab et al. unpublished data (Mai 2005)

12homozygous

28heterozygous

2het+allopurinol

42 mutant (44%)

onset within 3 - 8 weeks

95 patients with severe myelosuppression(pancytopenia)

Spontaneous reporting of cases (n=14 066); 92 % IBD

Patients on thiopurine therapy (n=4525)

Results of TPMT diagnostics in Stuttgart

ME3280

� DPD catalyzes 1st and rate limiting step

� commonly expressed Fe-S protein (predominately in human liver)

� cytosolic enzyme

� endogenous substrates known

� association to inborn error (familial pyrimidinemia) and

� severe 5-FU toxicity(Diasio et al., 1988)

�-alanine �-F-�-alanine �-amino-isobutyrate

Dihydropyrimidine Dehydrogenase (DPD)

dihydropyrimidinedehydrogenase (DPD)

ME2755.ppt

ME2843.ppt

798patients included

78 (9.7 %)concomitant chemotherapy

30 (3.8 %)incomplete documentation

5 other causes (0.6 %)

Reasons for exclusion

German Study-Groupon 5-FU Toxicity

GI tumors 96 %, breast cancer 2.4 %, CUP 1.6 %

685study patients

Pitfalls in Pharmacogenetic Testing:DPYD Mutations and 5-FU Toxicity

DPYD Exon 14 Skipping Mutation explains only ~ 15 % of 5-FU toxicity

patients 685

*2 / *2 0wt / *2 13

German Study-Groupon 5-FU Toxicity

2.2%

WHO grade IV III II I-0

number of pts 35 91 80 479

11.4%3.8% 0.8%

n=4 n=2 n=3 n=4Tox 4+3 vs 0-2:

95%CI 1.3-12, P = 0.019Odds Ratio 3.91.9%

8/2003

ME2844.ppt

Phenotype is only in part caused by candidate gene

Female Sex is a Risk Factor for 5FU Toxicity

0

10

20

30

40

50

60

70

WHO° 0-I476 patients

WHO° II81 patients

WHO° III92 patients

WHO° IV34 patients

p = 0.0015

mal

e

fem

ale

ME3030

Sex ratio [%] : 61:39 47:53 42:58 47:53

Total 56:44

Sex and DPYD*2 Allele associated 5-FU Toxicity

0

2

4

6

8

10

12

14

16

18

20

WHO° 0-I476 (4)

WHO° II81 (3)

WHO° III92 (2)

WHO° IV34 (4)

Dis

t rib

uti

on

of

DP

YD

*2 (

%)

male: p < 0.0001

female: n. s.

2

3

ME3035

patients (n)

4 1

31

0 0

no difference in allele *2 frequency betweenfemales (2.3%) and males (1.6%)

Nomogram for the Prediction of 5-FU Toxicity

DPD: Dihydropyrimidine dehydrogenase

Points

Age

Sex (DPD wt)

Sex (DPD*2)

Thymidylatesynthase

Folinic acid

Mode

Methylenetetrahydro-folate reductase

Total Points

0 10 20 30 40 50 60 70 80 90 100

0 20 40 60 80 100 120 140 160 180 200 220

90 80 70

25 60

26 100

Infusion

yes

no

wt

mt

mt

0.10.2

0.30.4

0.50.6

0.70.8

0.9Probability (WHO�3) [%]

ME3152

50

17

27

11

8

16

Bolus

wt

Predictive model for 5-FU WHO4-toxicity

Factor female malesex (DPD wt) 17 0sex (DPD*2) 9 (26) 100TS (wt) 16 16MTFR (wt) 8 8Folinic acid 27 27Bolus 11 11age 50 50total points 138 212Probability 0.48 0.95

Contribution of DPD*2 is negligible (4 %) in female but substantial (45 %) in male patients.

ME3151

What is the information content of a genetic test ?

ME2748.ppt

Sensitivity likelihood that a patient with a given phenotype (ADR)

will test positive

Specifity likelihood that a patient without the phenotype (ADR)

will test negative (i.e. not test false positive)

Positive likelihood that a patient with a positive test will have the

predictive value phenotype (ADR)

Negative likelihood that a patient with a negative test will not

predictive value have the phenotype (ADR)

ME3185.ppt

Genotype and Phenotype

Limitations of present approach: Usually focus on one gene

Like most disease phenotypes, drug phenotypes

(response, nonresponse, toxicity) are complex polygenic

traits with nongenetic factors contributing to the

manifestation of phenotypes.

The extent to which genetic factors contribute to phenotype

will depend whether the candidate gene is a gene of major,

moderate or minor effect.

ME3211

41 Candidate genes withpotential relevance for

statin response(Zineh 2005)

Contribution of polymorphism of

most genes in lowering cholesterol

will be moderate.

ME3225.ppt

LimitationsLimitations to to currentcurrent statinstatin pharmacopharmaco--geneticsgenetics studiesstudies ((ZinehZineh et al. 2005et al. 2005))

• Generally not multi-gene studies (or studiesconsidering combinations of several genes)

• Statistically significant results are not necessarilyclinically meaningful

• Many studies - few results replicated• Gene-environment, gene-disease and dietary

factors not contolled• Candidate polymorphisms often associated with

baseline cholesterol

ME3228.ppt

Pharmacogenomics of Statins: Outlook

• Genotype groups with diminished lipidresponse may still show clinically usefuleffects!

• How predictive is lipid lowering efficacy forclinical endpoints?

• Will genetic testing to predict response and toxicity be feasible and cost-effective?- Maybe, but expectations are probably too high

• Large studies with many genes are needed

ME3126Can Genotype-based Dose Selecetion Reduce Toxicityand Increase Response ?

Genotype Dose Concentration Response Dose Concentration Responseadjusted

0.1

0.2

1

4

PM

IM

UM

EM

Co

nce

ntr

atio

n 10

1

Time [h]

Time [h]

10

1

Co

nce

ntr

atio

n

10

1

Time [h]

Co

nce

ntr

atio

n

toxicity,

side effects

10

1

Time [h]

Co

nce

ntr

atio

n

side effects

drug response

no drug response

1

1

1

1

10

1

Time [h]

Time [h]

10

1

Time [h]

no toxicity,

no side effects

10

1

Time [h]

no side effects

drug response

drug response

Co

nce

ntr

atio

nC

on

cen

trat

ion

Co

nce

ntr

atio

n

10

1

Co

nce

ntr

atio

n

The Impact of CYP2D6 Genotype on Adverse Drug Reactionand Nonresponse During Treatment with Antidepressants

0

20

40

60

80

100

Adverse Effects Nonresponse

PM UM

expected observed expected observed expected observed

ME3019Kawanishi et al., 2004Rau et al., 2004

7 % 29 %

2 % 19 % 1 % 10 %

ME3287.ppt0.1 100101 MRS

Indi

vidu

als

10

20

40

30

IM: intermediatemetabolizer~ 10-15 %

PMpoor metabolizer

~5-10 %

UM:ultrarapid

metabolizer~ 5-10 %

EM: extensive metabolizer (~70-80 %)

N = 308

Sparteine

N

N

CYP2D6 Oxidation Phenotypes in Caucasians

ME3285.ppt

Limited Predictivity of CYP2D6 Gene Duplicationfor UM Phenotype

Indi

vidu

als

10

20

40

30

0.1 100101 MRS

N = 308

Carriers of 3 gene copies

6/16 UM (38%)

ME3286.ppt

CYP2D6 Protein and Propafenone EnzymeActivity in Human Liver

Correlation PPF-Protein

r=0.77

*4*4

*41*0

Dupl

0 25 50 75 100 125 1500

100

200

300

400

2D6 Protein pmol/mg

PP

F p

mo

l/mg

/min

N=125

Polygenic Nature of Drug Response: Antidepressants

ME3125

Co

nce

ntr

atio

n

10

1

time [h]

time [h]

10

1

Co

nce

ntr

atio

n

10

1

time [h]

Co

nce

ntr

atio

n

CYP2D6

amplification

normal

mutations

Drug

Drug Metabolism

Poor predictive value of CYP2D6 and CYP2C19 genotype for severe adverse drugreactions and non-response leading to discontinuation of treatment

• Comparable doses used; compliance

• Measurement of drug levels

• Coadministration of drugs: Phenocopying

• Coexisting diseases; age; gender

• Predictive value of genotype for phenotype:UM genotype predicts only 20 – 30 % of UM phenotype

Polygenic Nature of Drug Response: Antidepressants

Drug Transport

MDR1 at blood brain barrierComparable plasma concentration,yet different concentration at siteof action:

• Poor predictive value of drug concentration

• net concentration = influx (diffusion) – efflux (transport)

• Contribution of MDR1 polymorphism to

response only at comparable concentrations

BBB BBBconc

.

conc

.

time [h] time [h]

ME3124

Polygenic Nature of Drug Response: Antidepressants

Drug Target

1. Concentration of serotonin in synaptic cleft is influenced bybiosynthesis (TPH2), re-uptake(SERT) and catabolism (MAOA)

2. Inhibition of serotonin re-uptakedepends on drug concentration in synaptic cleft

3. Mutations of receptors and signalling pathways affectneurotransmitter and drug effects

MAOA

MDR1

HT Receptors

5-HT

TCA

TPH2

SERT

ME3122

Polygenic Nature of Drug Response: Antidepressants

ME3050

Drug Target

Contribution of receptor / signallingpathway to drug response shouldbe assessed at comparable plasmalevels

BBB: Stratification of patient groupsfor transporter polymorphisms

Comparable effect at receptor, but10 fold difference in concentrationrequired between genotypes

(Mason et al. 1999)

Gly389

Arg389

-11 -10 -9 -8 -7 -6 -5 -4 -3

log [Isoproterenol]A

den

ylyl

cycl

ase

acti

vity

[pm

ol/m

in/m

g]

0

15

30

45

60

75

ME3225.ppt

LimitationsLimitations to to currentcurrent statinstatin pharmacopharmaco--geneticsgenetics studiesstudies ((ZinehZineh et al. 2005et al. 2005))

• Generally not multi-gene studies (or studiesconsidering combinations of several genes)

• Statistically significant results are not necessarilyclinically meaningful

• Many studies - few results replicated• Gene-environment, gene-disease and dietary

factors not contolled• Candidate polymorphisms often associated with

baseline cholesterol

ME3228.ppt

Pharmacogenomics of Statins: Outlook

• Genotype groups with diminished lipidresponse may still show clinically usefuleffects!

• How predictive is lipid lowering efficacy forclinical endpoints?

• Will genetic testing to predict response and toxicity be feasible and cost-effective?- Maybe, but expectations are probably too high

• Large studies with many genes are needed