Pharmacogenetics g

Prof. dr. Henk-Jan GuchelaarClinical Pharmacy & ToxicologyLeiden University Medical Centre

25th A t 200725th August 2007

Variability in drug response

• “more than 90% of the currently used drugs is effective in only 30-50% of the patients”

(Collins – The Independent 8 dec 2003)

2

‘Most drugs don’t work’

Effective (%)…..• Alzheimer 30• Depression (SSRI) 62• Asthma 60Asthma 60• Diabetes mellitus 57• Incontinence 40• Migraine (acute) 52• Migraine (profyl.) 50

Cradiac dysrhythmia 60• Cradiac dysrhythmia 60• Tumors 25• Schizophrenia 60p• Reumatoid arthritis 50• Reumat. art. (Cox-2) 80

3

• Hepatitis C 47The Independent, 8 dec 2003

Variability in humans

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Heritability of common conditions

• Freckles 90%• Short sight 90%

• Arthritis 60%• Cataract 60%Short sight 90%

• Autism 80+%• Acne 80%

Cataract 60%• Motion sickness 60%• Migraine 50%

• Height 80%• Osteoporosis 75%

g• Varicose vein 50%• Menopause 50%

• Diabetes 70%• Obesitas 70%

• Blood pressure 50%• Parkinson’s disease 0-10%

• Blood clotting 70%• Backpain 65%

Q %

• Irritable bowel syndr 10%• Left handedness <10%

• IQ 65%• Asthma, allergic 60% • Drug response ??

5 Tim Spector, CBG meeting, Apr 2007

Gene-Environment interaction

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Pharmacogenetics

• The heritability of drug response

• Individual pharmacotherapy

• Tailor made pharmacotherapyp py

• “Personal Pills”

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“Book of Life”

• Complete sequence human genome is knowng

• “for everybody similar”

• Typographic failures:• Letter mis.ing• Letter toomuuch • Interhcange• Typing erlor• Dupliplipliplicationssss

Doubledouble paragraph• Doubledouble paragraph• Missing parts ……..• Tnorf ot kcab

8

Tnorf ot kcab

Check, Nature 2005:1084

Sequence variation in DNA

• Deletion• DNA

Wild type Mutant• GAA AAG CCT GGT GAA GCC TGG TGADNA

• Protein • Glu Lys Pro Gly Glu Ala Trp Stop

• SNPs• DNA • ATG AAC CCG ATG AAC TGG DNA• Protein • Met Asn Arg Met Asn Trp

• Microsatellieten • ATGAATATATATATATAGGCMicrosatellieten

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Genetic variation may lead to a different protein

…and to a different enzyme activity of a metabolic … y v y enzyme

variant aminoacid enzyme

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Single Nucleotide Polymorphisms - SNPs

A A A A A A A AATC

AGC

AGC

AGC

AGC

AGC

AGC

AGC

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T T T T T T T TTGA

TGA

TCA

TGA

TCA

TGA

TGA

TGA

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G G G G G GG G

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GC

GC

GC

GA

GA

GA

GA

GC2000

Cox, Perlegen, 2006

Linkage of SNPs

A A A A A A A A AATC

AGC

AGC

AGC

AGC

AGC

AGC

AGC

AGC

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T T T T T T T T TTGA

TGA

TCA

TGA

TCA

TGA

TGA

TGA

TGA

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G G G G G G GG G

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GC

GA

GC

GA

GA

GC

GC

GA

GC2000

Cox, Perlegen, 2006

Grouping of frequent haplotypes

A A A A A A A A AAGC

AGC

AGC

AGC

AGC

AGC

AGC

AGC

ATC

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T T T T T T T T TTGA

TGA

TGA

TGA

TCA

TCA

TGA

TGA

TGA

1000

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G G G G G G GG G

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GA

GA

GC

GC

GC

GC

GC

GA

GA2000

Cox, Perlegen, 2006

Pharmacogenetics into clinical practice

• FDA approved PGx tests

• Amplichip® – CYP2D6 and CYP2C19

• Invader® – UGT1A1 *28

14

Cytochrome P450 enzymes and drugs

3% 3% 4%12%

CYP1A2CYP2E1

53%

CYP2C19CYP2C9CYP2D6

25%CYP2D6CYP3A4

15

Genetic variants CYP2D6allele mutation Enzyme-

activityallele-frequency %

Europe China ZimbabweEurope China Zimbabwe*1 wild type normal 33-40 43 54

*2[G] -1584C>G normal/high? 19[ ] g

*2xn*1xn

duplicationmultiplication

(very) high 1-5

*3 2549delA absent 2 0 0

*4 G1846A absent 22 0-1 2

*5 gen deletion absent 4 6 4

*9 2613-delAGA low 2 3 0

*10 C100T low 2 51 610 C100T low 2 51 6

*17 C1023T C2850T laag 0.1 34

*35 G31A normal/high? 4-8

16

*41 (*2[C]) 2850C>T 4180G>C low 10

http://www.cypalleles.ki.se/

CYP2D6 phenotype

17

debrisoquine/4-OH-debrisoquine

Case 1: codeine

• Mature baby• day 7: problems with breastfeeding and lethargic• day 11: birth weight• day 12: grey skin; no milk intake• day 13: died• day 13: died

• postmortem analysis• no anatomic abnormalities• Morphine plasmalevel 70 ng/ml (normal: 0-2.2 ng/ml with a breastfeeding

mother using codeine)• Mother: paracod initially 2 dd 1000/60 mg because of somnolence

decreased to 2 dd 500/30 mg for 2 weeks• Morphine concentration milk: 87 ng/ml (normal: 1.9-20.5 ng/ml with 4 dd 60

mg codeine)• Genotyping

• Mother: CYP2D6 *2A with gene-duplication UM high codeine morphine metabolism

18

• Kind: CYP2D6 *1/*2 EM

Koren, Lancet 2006

CYP2D6/2C19 variants and Chip technology

• 33 CYP2D6 variants

• 3 CYP2C19 variants

19

Affymetrix fluidic station 450Dx and Scanner 3000Dx

20

From genotype to phenotype

E

21 Roche, AmpliChip CYP450 Test, manual

Workingparty Pharmacogenetics WINAP

22 http://farmacogenetica.knmp.nl

Workingparty Pharmacogenetics WINAP

23

Workingparty Pharmacogenetics WINAP

24

Scientific evidence

• CYP2D6 and nortriptyline• search terms• search terms

• nortriptyline + CYP2D6 (‘only human’)• nortriptyline + debrisoquine• nortriptyline + debrisoquine• nortriptyline + sparteine• nortriptyline + dextromethorphan• nortriptyline + dextromethorphan• nortriptyline + metabolizer• nortriptyline + polymorph*• nortriptyline + polymorph• nortriptyline + IID6

• cross references• cross references

25 Swen, Guchelaar, Plos Med Aug 2007; e209

Scientific evidence

• Quality score:

• 4 controlled study; clinical relevant endpoint

• 3 controlled study; relevant surrogate endpoint

• 2 well documented case-report

• 1 incomplete case-report

• 0 animal or in vitro study or data on file

26 Swen, Guchelaar, Plos Med Aug 2007; e209

Scientific evidence

source Q n description

J Korean Med Sci 2004:750 2 1 at 1 dd 150 mg high level and side effects; 50 mg is effectiveJ Korean Med Sci 2004:750 2 1 at 1 dd 150 mg high level and side effects; 50 mg is effective

Neuropsychopharm 2001:737 3 36 geriatric; pt; dosed to targetCss; D IM at mean 30% lower vs EM

Br J Clin Pharmacol 2003;630 3 10 10 healthy vol; once 25 mg; heterozyg PM vs EM; no diff Cm, Tm, T1/2 AUC NT d 10OH NTT1/2, AUC NT and 10OH-NT

J Clin Psychopharmacol 2000:141

3/4 41 pt; 1 dd 25-120 mg; relation (10-OH)NT and NT levels with genotype

Clin Pharmacol Ther 1998:384 3 15 healthy vol; once 25 mg NT; relation (10-OH)NT and NT levels with genotype

Psychopharmacol 1996:315 3 21 pt; 1 dd 150 mg; relation (10-OH)NT and NT levels with genotypey p p ; g; ( ) g yp

Clin Pharmacol Ther 1996:522 1 8 pt with side effects; normal dose;

Clin Pharmacol Ther 1998:444 3 21 healthy vol; 1x25(50mg); relation (10-OH)NT and NT levels with genotypegenotype

Clin Pharmacokin 2001:869 3 40 pt and healthy vol; 2-3 dd 25-50mg; relation Cl NT and genotype

27 Swen, Guchelaar, Plos Med Aug 2007; e209

Clinical relevant application?

28

Tamoxifen label change

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CYP450 2D6 and tamoxifen

30 Goetz, J Clin Oncol 2005

Pharmacogenetics into clinical practice

• FDA approved PGx tests

• Amplichip® – CYP2D6 and CYP2C19

• Invader® – UGT1A1 *28

31

UGT1A1 polymorphism and Irinotecan

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Irinotecan: UGT1A1 and Invader® test

Carboxylesterase-2 UGT1A1

Irinotecan SN-38 SN-38-glucuronide

ABCB1?

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UGT1A1 genotype – shared exons

ATATATATATAT

RNA

34 Mackenzie, Pharmacogenet 1997

(TA)6 (TA)7= UGT1A1*28

UGT1A1 *28 genotype distribution

80%90%

100%

60%70%80%

TA7TA7

30%40%50% TA6TA7

TA6TA6

0%10%20%

Europ Asian African

35 Beutler, PNAS 1998

UGT1A1 and SN38-glucuronidation

36 Iyer, Clin Pharmacol Ther 1999

Risk for neutropenia

TA7/TA7 TA6/TA6+

TA6/TA7

Odds Ratio

Unadj

95% CI

TA6/TA7 Unadj.

Innocentic 2004 50% 6% 16.7 2.3-120.6

Roultsa 2004 57% 15% 7.5 1.4-38.5

Marcuelloa 2004 40% 21% 2.5 0.6-9.7

Andob 2000 57% 20% 5.4 1.1-25.9

a grade 3+ neutropeniab grade 4 leukopenia and/or grade 3+ diarrhoeac grade 4 neutropenia

37 Parodi, FDA Nov 2004

g p

UGT1A1 genotype and neutropenia

FDA: dosisreduction

Response?Response?

38 McLeod, Cancer Invest 2003

•

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Irinotecan dose adaptation

• 220 patients CAIRO1 studyM t t ti l t l• Metastatic colorectalcancer

• 1e line• Capecitabine 2 dd 1000 mg/m2 on days 1 14 every 3 weeks• Capecitabine 2 dd 1000 mg/m2 on days 1-14 every 3 weeks• Irinotecan 250 mg/m2 every 3 weeks

• 2e line (following capecitabine)2 line (following capecitabine)• Irinotecan 350 mg/m2 every 3 weeks

• Genotyping UGT1A1 *28 yp g

• Association with grade 3 of 4 febrile neutropenia

• Tumor response for each genotypep g yp

• Irinotecan dose for each genotype

40 Kweekel, submitted

Irinotecan dose adaptation• Grade 3 or 4 febrile neutropenia

TA7/TA7 TA6/TA7 TA6/TA6 p1e line 18 2% 5 5% 1 7% 0 0321e line 18.2% 5.5% 1.7% 0.0322e line 0 16.7 2.6 0.044

• Response• Response1e line 63% 42% 49% 0.9592e line 0 7 8 0.711

• Irinotecan dose; No differences per genotype in% dose reduction (entire treatment; first 3 cycles)% patients having received < 90% irinotecan dose% of nominal dosenumber of cycles

• Termination of treatment due to toxicity (first 3 cycles)82% grade 3 diarrhoea; 18% febrile neutropenia

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82% grade 3 diarrhoea; 18% febrile neutropeniaAssociation with ABCB1 polymorphisms C1236T, G2677T, T3435C

Kweekel, submitted

UGT1A1 *28 genotyping

numbers/week indication roundabout time

Univ. Pens. 20 100% Gilbert ?

Univ. Chicago 6 50% Irinotecan 7 d

Wash. Univ.

St L i

1-3 100% Irinotecan 7 d

St. Louis

Dartmouth 5 100% irinotecan 2 d

Detroit Med Ctr 1-2 75% Irinotecan 2 d

42 Feldman, ACCP meeting sept 2006

Pharmacogenetic research

• Aimed at solving importantAimed at solving important clinical problems

• Providing scientific evidence for added value of PGt testing

• Aimed at providing clinically applicable and clear cutapplicable and clear cut guideline following a test result

• Combine PGt and non-genetic determinants for drug response

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Predictive model MTX response in RA patients

• 205 RA patients• Best-cohort (rheumatology LUMC)

• MTX monotherapy• 15 mg/wk + folic acid 1 mg/dag• Increased to 25 mg/wk if DAS>2.4

• Response: 47% • Adverse drug events: 30%Adverse drug events: 30%

• 17 polymorphisms in 13 candidate genes• folate and adenosine

• Non-genetic factors

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Non-genetic factors

Wessels, A&R 2005, 2006, 2007

Predictive model: weighted score

Baseline Variable Score

premenopausal1Gender Female

postmenopausal 1

Baseline Variable Score

premenopausal1Gender Female

postmenopausal 11Male 0

Disease activity DAS at baseline 3.8 0DAS at baseline >3.8, but 5.1 3

1Male 0

Disease activity DAS at baseline 3.8 0DAS at baseline >3.8, but 5.1 3

≤≤DAS at baseline 3.8, but 5.1 3

DAS at baseline >5.1 3.5

Immunological factors Rheumatoid factor negative and non -smoker 0Rheumatoid factor negative and smoker 1

DAS at baseline 3.8, but 5.1 3DAS at baseline >5.1 3.5

Immunological factors Rheumatoid factor negative and non -smoker 0Rheumatoid factor negative and smoker 1

≤

Rheumatoid factor negative and smoker 1Rheumatoid factor positive and non -smoker 1Rheumatoid factor positive a nd smoker 2

Genetic factors MTHFD1 1958 AA genotype 1

Rheumatoid factor negative and smoker 1Rheumatoid factor positive and non -smoker 1Rheumatoid factor positive a nd smoker 2

Genetic factors MTHFD1 1958 AA genotype 1Genetic factors MTHFD1 1958 AA genotype 1AMPD1 34 CC genotype 1ITPA 94 A - allele carrier 2ATIC 347 G -allele carrier 1Other genotypes 0

Genetic factors MTHFD1 1958 AA genotype 1AMPD1 34 CC genotype 1ITPA 94 A - allele carrier 2ATIC 347 G -allele carrier 1Other genotypes 0

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Other genotypes 0Other genotypes 0

Good clinical response rates

>80%10080%>80%

80

90>80%

good response % 60

70Good clinical response % at 6 months

<20%30

40

50

10

20 <20%

00,0 1,0 2,0 3,0 4,0 5,0 6,0 7,0 8,0 9,0

score index (points)

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( )

Conclusions

• Drug response is –at least in part- a heritable trait (‘proof of principle’)heritable trait ( proof of principle )

• PGt has the potential to solvePGt has the potential to solve important clinical problems and to improve pharmacotherapy

• The (added) value of PGt tests needs to be proven

• The approval of two PGt tests has introduced PGt into the clinic

47