Pharmacogenomics: An Holistic Approach to. Drug - Organism Interaction. Michel
Eichelbaum. Me2450.ppt. Problems in Drug Therapy and Development. 1.
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
