Pharmacogenomics: An Holistic Approach to Drug

Problems in Drug Therapy and Development
Dr. Margarete Fischer-Bosch-Institut
für Klinische Pharmakologie
Robert Bosch Stiftung
Stuttgart
IKP
STUTTGART
1. No effective drug treatment for many disease; limited
number of evaluated drug targets
Pharmacogenomics:
2. No or only insufficient response to drug treatment in
substantial proportion of patients - high attrition rate
of NCEs in clinical trial‘s
An Holistic Approach to
Drug - Organism Interaction
3. Selection of appropriate dose for individual patient;
fixed dose in clinical trials
4. Substantial morbidity and mortality by severe
adverse drug reactions
Michel Eichelbaum
Me2450.ppt
Drug associated morbidity and mortality
Pharmacogenomics: Variability in Drug Response
Same disease, same drug but different responses
Overall incidence of all severe adverse drug
reactions causing admission to hospital plus
ADR‘s occurring
no or insufficient
response
Class of Drug
ACE inhibitors
10- 30 %
Beta
- blocker
15- 25 %
Selective serotonin reuptake inhibitors
10- 25 %
Tricylic anti- depressants
20- 50 %
HMG
-
30- 70 %
CoA reductase inhibitors
Beta
- 2agonists
in the hospital:
6.7 % = 2.216.000 patients
Fatal ADR‘s:
0.32 % = 106.000 patients
Fourth to sixth leading cause of death
40- 70 %
Lindpaintner, 2000
ME2435.PPT
Lazarou et al., JAMA 1998
ME2048.PPT
Concentration – Effect Relationship
Dose of Drug is a Poor Predictor for
Response and Toxicity
Css of Desipramine and Nortriptyline at 25 mg 3x/day
75
20
20 to 40 fold difference
number of patients
[pmol/min/mg]
Drug effect
60
45
30
15
15
Nortriptyline
10
Desmethylimipramine
5
0
-11
-10
-9
-8
-7
-6
-5
-4
-3
0
log [Isoproterenol]
20
40
no response
(Mason et al. 1999)
ME3133
60
80 100 120 140
260 280 300
Plasma levels [ng/ml]
Drug concentration
ME3129
response
toxicity
(Hammer & Sjöqvist 1967)
Pharmacogenetics
Nongenetic Factors and Drug Response
Individual variability in the efficacy and toxicity
of drugs due to polymorphisms of genes
involved in their disposition and action
Age, body weight, sex, disease, diet, alcohol, smoking, drugs,
hepatic and renal function
ME3131
Me2818.ppt
Use of genomics to determine an individual‘s
drug response
First Description of a Pharmacogenetic Trait
PHARMACOGENE TICS
"Quod aliis cibus est, aliis
fuat acre venenum"
PHARMACOGENOMICS
What is food to some men
may be fierce poison to others
Use of genomics to design drugs and target
drugs to specific patient populations
ME2823.PPT
Human Genome
•
•
25 000 genes
100 000 – 300 000 proteins
•
•
99.9 % identical sequences
0.1 – 0.2 % different sequences
Same dose but different plasma concentrations
Drug
A
10
GCCCCGCCTC
Genetic variability
wild type
100 new mutations per individual
3 deleterious mutations per person per generation
5.4 Mio. SNPs (single nucleotide polymorphisms)
identified
P 450
ME3017.PPT
AUC 1
1
Time
Drug
B
10
Genetic polymorphisms
approximately every 3rd gene exists in the
population with frequent (>1 %) allelic variants. 47
to 61 % of all protein loci polymorphic
Concentration
3. 5 x 109 nucleotides
Mechanism of Genetic Variability in Drug Response
GCCCCACCTC
mutation
ME3012.PPT
P450
Concentration
•
Lucretius Caro
De Rerum Natura 4:641, 65 B.C.
Me2824.ppt
AUC 20
1
Time
Mechanism of Genetic Variability in Drug Response
Same plasma concentrations but different concentrations at site
of action
Mechanism of Genetic Variability in Drug Response
Same concentration at site of action but differences in
response at drug target
Ser49Gly
concentration
concentration
Arg389
Arg389Gly
BBB
Gly389
BBB
(Hein 2001)
time [h]
(Mason et al. 1999)
time [h]
ME2290.PPT
ME2291.PPT
Drug Metabolism: First Line of Defense
Against Xenobiotics
Importance of Metabolism for Drug Elimination
OG
C H
N
H
C H
N
H
C H
N
H
GO
> 3000
> 500
∼ 3 million substances
Industry:
∼ 3000 chemicals/year
Drugs:
∼ 6000 (WHO)
Daily intake of xenobiotics:
ME3105
> 400
3000
3000
2000
2000
[ng/ml]
Biosphere:
[ng/ml]
Rifa
Biotransformation
Phase I and II
Enzymes
Biotransformation
Phase I and II
Enzymes
1000
∼ 10.000 substances; 1.5 grams of
natural pesticides (plant phenols,
flavonoids, saponines) carcinogenes
(aflatoxines, pyrrolizidine alkaloids ...)
1000
t½: 30 - 50 hours
0
0
10
20
t½: 800 - 1000 hours
0
0
30
200
Functional Alleles
1A1 1A2
1B1
2A6 2A7 2A13 2A18P
*1
3A4 3A5 3A5P 3A7 3A43
Conversion 2D6/2D7 (Intron 1)
2850C>T
*2
-1584C>G
1C1P
2C8 2C9 2C18 2C19
2D6 2D7P 2D8P
*35
genetic
polymorphisms
significant
R296C
2D6*1
2D8P
2D7P 2D6*2
2D6*2
2D8P
2D7P 2D6*35 2D6*35
138 ins T
1 2 3 4 5 6 7 8 9
100C>T
*4
N-1
N-1
*5
1 2 3 4 5 6 7 8 9
100C>T
2F1 F1P 2G1P 2G2P 2J2 2R1 2S1 2T2P 2T3P 2U1 2W1
4180G>C
1 2 3 4 5 6 7 8 9
1023C>T
S486T
2850C>T 4180G>C
1 2 3 4 5 6 7 8 9
T107I
100C>T
*36
K281del
R296C
S486T
Conversion 2D6/2D7
1 2 3 4 5 6 7 8 9
P34S
*41
1 2 3 4 5 6 7 8 9
R296C
S486T
*16
1 2 3 4 5 6 7 8 9
*18
1 2 3 4 5 6 7 8 9
*19
1 2 3 4 5 6 7 8 9
253 ter
4125-33 ins GTGCCCACT
chromosomal deletion of CYP2D6 gene
468-470 ins VPT
T1707 del
1 2 3 4 5 6 7 8 9
153 ter
2399 papers
dealing with
CYP2D6
*8
1 2 3 4 5 6 7 8 9
1758G>T
2850C>T
*20
1 2 3 4 5 6 7 8 9
*21
1 2 3 4 5 6 7 8 9
253 ter
2573 ins C2850C>T
4180G>C
1 2 3 4 5 6 7 8 9
2850C>T
*38
R296C
S486T
297 ter
1023C>T
1 2 3 4 5 6 7 8 9
*42
1 2 3 4 5 6 7 8 9
*14
T107I 172-74 ins FRPFRP
2850C>T
253 ter
1758G>A 2850C>T
4180G>C
1 2 3 4 5 6 7 8 9
P34S
G169R R296C
1863 ins (TTT CGC CCC)
2 4180G>C
*40
1 2 3 4 5 6 7 8 9
100C>T
1 2 3 4 5 6 7 8 9
4180G>C
2D7P/2D6-hybrid (138 ins T)
*13
4180G>C
2587-90 GACT del
1 2 3 4 5 6 7 8 9
G42R
4180G>C
273 ter
883G>C (splice site) 2850C>T
124G>A
1973 ins G 2850C>T
4180G>C
169 ter
*12
4180G>C
259 ter
*7
*11
2539-42 del AACT 2850C>T
2935A>C
1 2 3 4 5 6 7 8 9
12 amino acid changes
Conversion 2D6/2D7 (Intron 1) 2988G>A (Intron 6)
2850C>T 4180G>C
-1584C
2D7P/2D6-Hybrid (138 ins T)
1 2 3 4 5 6 7 8 9
P34S L91M 182 ter
H94R
*6
Since 1982
PubMed:
1 2 3 4 5 6 7 8 9
253 ter
984A>G
1846G>A (splice site) 4180G>C
974C>A
H324P
*9
*15
260 ter
N-1
S486T
AGA 2613-5 del
P34S
ME2569.ppt
2D7P 2D6*1
1 2 3 4 5 6 7 8 9
Alleles with decreased function
*17
http://drnelson.utmem.edu/CytochromeP450.html
*2xN
*35xN
2D8P
A237S
Conversion 2D6/2D7 (Intron1)
G31A
2850C>T
4180G>C
V11M
*10
2E1
*1xN
S486T
1 2 3 4 5 6 7 8 9
*3
Alleles with increased function
4180G>C
1 2 3 4 5 6 7 8 9
R296C
2483G>T
*33
2B6 2B7
Nonfunctional Alleles
A2549 del
1 2 3 4 5 6 7 8 9
-1584C>G
ME3043
2D8P 2D7P 2D6
Alleles with normal function
CYP3
800
Genetics of CYP2D6
Chr. 22q13.1
CYP2
600
time [h]
ME2283
Human Drug-Metabolizing Cytochromes P450
CYP1
400
time [h]
S486T
3259 ins GT
4180G>C
R296C 375 ter
CYP2D6: Consequences of genotype for systemic drug exposure
Enzyme
60
chromosome
22
decreased
slow metabolism
PM
CYP2D6
5’
q13.1
2
3 - 13
13
0
24
48
normal
normal metabolism
60 - 70 %
normal
EM
UM
ca. 2 - 3%
side effects
1
10
drug response
1
2-4
10
no drug response
1
amplification
ME3016.PPT
Time [h]
Pharmacogenetics
Like most disease phenotypes, drug phenotypes
Nonresponse
PM
7%
UM
ultra rapid metabolism
(response, nonresponse, toxicity) are complex
UM
100
80
1
increased
The Impact of CYP2D6 Genotype on Adverse Drug Reaction
and Nonresponse During Treatment with Antidepressants
Adverse Effects
10
Time [h]
72
(Bertilsson et al., 1997)
1
Time [h]
EM
time [h]
ME2828.PPT
toxicity,
side effects
mutations
3’
1 (*9 *10 *17 *41) IM
0
Response
10
Concentration
phenotype
0
10
IM
0.2
genes
20
0.1
5 - 10 %
functional
30
PM
5 - 10 %
Concentration
Time [h]
50
40
Dose 1
Concentration
plasma concentration [nmol/l]
mutations
Phenotype
deficient
extreme slow
metabolism
Concentration
Gene
Nortriptyline plasma levels in relation to CYP2D6 genotype
Concentration
How predictive is the Genotype for the Phenotype
polygenic traits with nongenetic factors
29 %
contributing to the manifestation of phenotypes.
60
The extent to which genetic factors contribute to
40
phenotype will depend whether the candidate
20
expected observed
Rau et al., 2004
Kawanishi et al., 2004
ME3019
ME2848.ppt
amplification
time [h]
Concentration
time [h]
MDR1
Dose [mg]
T/T
97
C/T
97
Level [nmol/l]
Hypotension
MADRS
293
25 %
12.5
306
11 %
13.7
C/C
103
345
0%
12.7
10
normal
MAO
time [h]
SERT
10
TCA
BBB
time [h]
BBB
time [h]
BBB
time [h]
Mechanism: Autonomic and central α-receptor blockade
1
mutations
time [h]
TPH2
concentration
1
5-HT
ME3045
BBB
concentration
Concentration
BBB
conc.
1
time [h]
CYP2D6
MDR1 C3435T SNP and nortriptyline-induced postural
hypotension
10
conc.
Concentration
Polygenic Nature of Drug Response: Antidepressants
Drug
gene is a gene of major, moderate or minor effect.
concentration
0
19 %
2%
1%
10 %
expected observed expected observed
HT Receptors
Roberts et al, The Pharmacogenomics Journal, 2002
ME2648.ppt
Polygenic Nature of Drug Response: Antidepressants
Polygenic Nature of Drug Response: Antidepressants
Drug Target
Drug Target
SERT: 5‘-upstream regulatory region: 44 base pair insertion / deletion with
1. Concentration of serotonin in
synaptic cleft is influenced by
biosynthesis (TPH2), re-uptake
(SERT) and catabolism (MAOA)
Gene
MAOA
2. Inhibition of serotonin re-uptake
depends on drug concentration in
synaptic cleft
3. Mutations of receptors and
signalling pathways affect
neurotransmitter and drug effects
long (L) and short (S) variant
Short variant: Two fold decreased expression and transport activity
MDR1
5-HT
SERT
Response predictive genotype
SERT
L/L
VNTR
D/D
ACE
DRD 2/3/4
MAOA
TPH1 (2)
ß1AR
GNB3
HTR2A
HTR6
TPH2
TCA
HT Receptors
ME3122
OR
1.3 – 5.6
inconsistent
~ 2.0
n.s.
n.s.
n.s. – 5.3
< 0.05
1.8
< 0.02
n.s.
promoter
218A>G
Gly389Arg
825C>T
102T>C
267C>T
Kirchheimer et al., 2004
ME3051
5-Fluorouracil: Therapeutic Use and Toxicity
RNA-turnovergenes
colorectal Cancer:
RNA
cell alteration
FUTP
NDP-kinase
300.000 new cases
200.000 deaths
FUDP
each year in Europe
and the USA
FUMP
FUrd
5-Fluorouracil: drug of first choice
in adjuvant and metastatic setting
transporter (?)
response rate: 10- 30%
toxicity: 3- 30% (severe 3
- 5%)
Application type: Bolus > Continuous Infusion
(Meta Analysis Group in Cancer 1998)
orotatephosphoribosyltransferase
dTMP
FdUDP
FdUMP
DHF
DHFR
NMP-kinase
thymidylate synthase
thymidinkinase
FdUrd
dUMP 5,10-MeTHF
thymidine phosphorylase
5-fluorouracil
anabolism
catabolism
dihydropyrimidine dehydrogenase (DPD)
dihydrofluorouracil
neutropenia, anemia
mucositis, diarrhea
neurological symptoms
ME2717.ppt
Candidate Genes: 5-FU Toxicity and Response
Dihydropyrimidine dehydrogenase
DPYD
Methylene tetrahydrofolate reductase
MTHFR
Orotate phosphoribosyl transferase
OPRT
Thymidine phosphorylase
TP
Thymidylate synthase
TS
Uridine kinase
UK
Uridine phosphorylase
UP
Fluoropyrimidines
MRP8 (ABC C11)
Uracil transporter
Fur4p
Concentrative nucleoside transporter
CNT1&2
ME3026
apoptosis genes
cellcycle genes
NDP-kinase
dUTPhydrolase
uridinphosphorylase
uptake
apoptosis
FdUTP
ribonucleotidreductase
NMP-kinase
uridine kinase
DNA
DNA-repairenzyme
elimination
transporter (?)
dihydropyrimidinase
fluoroureidopropionic acid
patient
ß-ureidopropionase
fluoro-ß-alanine
versus
tumor
ME2774.ppt
Dihydropyrimidine Dehydrogenase (DPD)
• DPD catalyzes 1st and rate
limiting step
• Commonly expressed Fe
- Sprotein
(predominately in human liver)
dihydropyrimidine
dehydrogenase (DPD)
• Cytosolic enzyme
• Endogenous substrates known
• Association to inborn error
(familial pyrimidinemia) and
β-alanine
α-F-β-alanine
β-aminoisobutyrate
• Severe 5-FU toxicity
(Diasio et al., 1988)
ME2755.ppt
Age of Patient and 5-FU Toxicity
German Study-Group on 5-FU Toxicity
798
patients included
Reasons for exclusion
80
80 (10.0 %)
concomitant chemotherapy
70
60
30 (3.8 %)
incomplete documentation
< 70 years
50
not significant
40
≥ 70 years
WHO
0-II
30
5 other causes (0.6 %)
20
WHO
0-II
10
683
0
125 patients
study patients
GI tumors 95.6 %, breast cancer 2.3 %, CUP 2.0 %
ME3039.ppt
Female Sex is a Risk Factor for 5-FU Toxicity
70
WHO
III-IV
5-FU Toxicity in Relation to Continuous Infusion
versus Bolus Administration
WHO° toxicity
male
50
555 patients
ME3028
p = 0.0015
60
WHO
III-IV
IV
III
II
I-0
female
40
30
20
DYPD wt/*2A
10
0
Sex ratio [%]
Total
WHO° 0-I
476 patients
♂:♀
WHO° II
81 patients
61:39
WHO° III
92 patients
WHO° IV
34 patients
42:58
47:53
47:53
4 bolus
N
n=4
2 infusion
34
56:44
2 bolus
1 infusion
92
bolus infusion
26
8
ME3030
n=2
n=3
81
bolus infusion
55
37
3 bolus
1 both
both
3
bolus infusion
51
27
n=4
476
both
4
bolus infusion
269
203
ME3033.ppt
Sex and DPYD*2 Allele associated 5-FU Toxicity
Phenotype is only in part caused by candidate gene
DPYD Exon 14 Skipping Mutation explains only ~ 15 % of 5
- FU toxicity
20
Distribution of DPYD*2 ( %)
18
no difference in allele *2 frequency between
females (2.3%) and males (1.6%)
14
male:
12
female:
10
p < 0.0001
n. s.
patients
685
wt / *2
*2 / *2
13
0
8
4
0
patients (n)
III
II
I-0
n= 2
n= 3
n=4
Tox 4+3 vs 0-2:
1.9%
n= 4
Odds Ratio 3.9
95%CI 1.3-12, P = 0.019
8/2003
6
2
ME3035
WHO grade IV
16
3
0
4
WHO° 0-I
476 (4)
1
WHO° II
81 (3)
3
1
11.4%
2
0
WHO° III
92 (2)
WHO° IV
34 (4)
number of pts
ME2844.ppt
35
2.2% 3.8% 0.8%
91 80 479
German Study-Group
on 5-FU Toxicity
Functional Consequences of the TS-promoter
Polymorphism
promoter
5´
repeats
0
Points
0
Sex (DPD wt)
TSER*2
TSER*3
10
90
Age
3´
Sex (DPD*2)
response
2 tandem
repeats
TS activity
toxicity
thymidylate
thymidylate
synthase
synthase (TS)
(TS)
FdUMP
Nomogram for the Prediction of 5-FU Toxicity
response
3 tandem
repeats
TS activity
2.6 fold
toxicity
ME2775.ppt
30
40
80
50
75
60
70
25 60
17
17/17
26
70
80
90
100
50/50
100
DPD: Dihydropyrimidine dehydrogenase
wt
Thymidylate
synthase
mt
Folinic acid
no
yes
Bolus
11
Mode
Infusion
wt
Methylenetetrahydro8
folate reductase
mt
Total Points
0
20 40
27/27
139
94
77
60
80
0.1
0.07
ME3128
196
100 120 140 160 180 200 220
0.2
Probability (WHO≥3)
[%]
Kaneda et al., Nucleic Acids Res 1987
Kawakami et al., Anticancer Res 1999
20
85
0.4
0.3
0.14
0.6
0.8
0.5
0.7
0.48
0.9
0.88
Pharmacogenetics and Drug Therapy
Pharmacogenetics and Drug Therapy
Selection of drug
Avoidance of severe adverse drug reactions
Section of SNP genotype profile
Patients with efficacy
with efficacy
SNP profile for toxcicity
without efficacy
without toxicity
Predictability of efficacy
efficacy
with toxicity
no efficacy
Roses, 2000
Roses, 2000
ME2474
ME2476
Pharmacogenetics and Drug Therapy
IKP
Selection of dose
STUTTGART
Patients with efficacy
Dr. Margarete Fischer-Bosch-Institut
für Klinische Pharmakologie
Robert Bosch Stiftung
Stuttgart
Acknowledgement
% average dose
250
200
150
100
50
25
PM
ME2475
IM
EM
UM
IKP:
O. Burk
M. Fromm
U. Hofmann
K. Kivistö
U. Klotz
T. Mürdter
M. Niemi
E. Schaeffeler
M. Schwab
U. Zanger
U. Brinkmann
I. Cascorbi
Robert Bosch Stiftung
I. Hauser
DFG (FR 1298/2-1)
H. Kroemer
BMBF (BEO/310311782)
U. Meyer
M. Oskarsson
Alexander von Humboldt Stiftung
I. Roots
G. Treiber
M. Stanulla, ALL Study Group Hannover
M. Kostrzewa, Bruker Leipzig
ME3062.ppt