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OSAKA UNIVERSITY
1
Latest Achievement and Future Challenge on Pharmacogenomics
and Personalized Medicine
Clinical Application of Clinical Application of Pharmacogenomics (PGx) Pharmacogenomics (PGx)
Junichi Azuma, M.D.
Osaka University, Japan
OSAKA UNIVERSITY
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Alcohol drinks and Individuals
Many individuals of Asian descent are extremely sensitive to even small doses of alcohol drinks, principally because of the different activity of key enzymes in different activity of key enzymes in alcohol metabolismalcohol metabolism. Those that seem to predispose individuals to unpleasant effects after alcohol consumption are common in certain ethnic groups including Asians.
OSAKA UNIVERSITY
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Responses to drugs and Individuals
This phenomenon is not limited to the alcoholic beverages. It is well known that different people frequently show varied responses to drugsvaried responses to drugs. Even if a drug of the same amount is taken, an individual difference occurs in the effectivenesseffectiveness and degree of an adverse adverse drug reactiondrug reaction (ADR). Then unpredictable reactions that may result in significantly high morbidity and mortality may happen.
040104040104
OSAKA UNIVERSITY
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Non responder Disease Drug Class Non responder (%)
Asthma β2 adrenergic agonist 4~755-LO, LTD4
Cancer Various 70~100(breast,lung,brain) Depression SSRIs, Tricyclics, MAOs 20~40
Diabetes Sulfonylurea, Biguanides, 50~75Glitazones
Duodenal ulcer H2 antagonists, 20~70Proton pump inhibitors
Hyperlipidemia HMGCoA reductase, 30~75Resins, Niacin
Pharmacogenomics eds. Kalow, Tyndale, Meyer (p17, Marcel Dekker, May 2001)
OSAKA UNIVERSITY
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Paradox of DrugDevelopment and Therapy
● Clinical trials for drug development
provide evidence of efficacy and
safety at usual doses in populations. populations.
● Physicians treat individual individual patientwho can vary widely in their response
to drug therapy.
Need PGx Test?Need PGx Test?
OSAKA UNIVERSITY
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Internet survey on understanding and acceptance of pharmacogenetics in Japan
Purpose:
To identify the issues to solve in communication and education in pharmacogenetics in Japan
Study participants (number of expected responder):
500 from Yahoo internet monitors/panel (ca.200,000)
Date: 13-18 April 2005
Contents: 22 questions with 4 messages
Principal Investigators: Azuma J, Tsutani K
20-29 30-39 40-49 50-59 60-69 Totalmale 50 50 50 50 50 250female 50 50 50 50 50 250Total 100 100 100 100 100 500
OSAKA UNIVERSITY
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Q2: Have you ever heard about “Genome” ? あなたは、「ゲノム」という言葉を聞いたことがありますか。
84
75
84
80
82
16
25
16
20
18
20-29
30-39
40-49
50-59
60-69
(n=116)
(n=117)
(n=113)
(n=111)
(n=120)
Yes No
(c) Azuma J, Tsutani K
OSAKA UNIVERSITY
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Interindividual differences !
• An effective rate of most drugs is roughly 60-70%, and "a non responder" exists inevitably. One of a cause of this interindividual difference includes a hereditary factor.
• It is important to realize that human genetic variability can affect one or more of the stages in drug response:
① drug transporters ② drug metabolizing enzymes ③ drug target molecules
OSAKA UNIVERSITY
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Variation in Genes Encoding for Drug Metabolizing Enzyme Activity
GCCCG G CCTC
Patient A
Patient B
Concentration
Concentration
Time
Time
Same Dose
GCCCA A CCTC
Wild type
Mutation
Drug MetabolizingEnzyme
Drug MetabolizingEnzyme
however
High activity
Low activity
different plasma concentrations
OSAKA UNIVERSITY
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Many CYP450 Enzymes Are Polymorphic: Example CYP 2D6
Family: CYP 2Subfamily: CYP 2D6Gene: CYP 2D6*1
Relative contribution of CYP isozymes and other enzymes to the phase I metabolism of drugs.
Responsible for metabolism of 40% of all Rx drugs
OSAKA UNIVERSITY
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Metabolic pathway of Venlafaxine
O-desmethylvenlafaxine
HOOH
CH 3
CH 3
CH 2 N
CH 3 OOH
H
CH 3
CH 2 N
OH
CH 3
CH 3
CH 2 N
CH 3 O
CYP2D6Venlafaxine
N-desmethylvenlafaxine
(ODV)CYP3A
CYP2C19
OSAKA UNIVERSITY
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Plasma concentration of venlafaxine
time(hr)
Venlafaxine
Plasma concentration (ng/ml)
0
50
100
150
200
0 5 10 15 20 25
Main metabolic enzyme likely to be CYP2D6, but
OSAKA UNIVERSITY
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Main CYP2D6 polymorphism in Japanese population
CYP2D6*5
CYP2D6*4
CYP2D6*2
genotype activity
CYP2D6*10 Decreased
None
None
(≒Normal)
A deletion of the entire CYP2D6 gene
G1934AAberrant 3' splice recognition site
G4268CC2938T296Arg→Cys 486Ser→Thr
gene structure
34Pro→Ser
G4268CC188T486Ser→Thr
Exon with a mutation affecting the activityExon at least with one mutation
CYP2D6*1 Nomal
(%)
43.0
12.3
0.2
4.5
36.3
CYP2D6*36 Decreased
34Pro→Ser 6 AA changesC188T 1.8
OSAKA UNIVERSITY
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Relationship between CYP2D6*10 andthe plasma concentration of venlafaxine
Venlafaxine
121110987654321 *1/*2*10/*10
*1/*1
*1/*10
*2/*10
*1/*2
*5/*10
*10/*10
*10/*10
*1/*10*1/*2
*2/*10
Plasma concentration (ng/ml)
200
150
100
50
0
time(hr)
0 5 10 15 20 25
OSAKA UNIVERSITY
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Pharmacogenetics of CYP2D6
0
20
40
60
80
100
120
No. of subjects
Ultra-rapidmetabolism
Extensive metabolism
PoormetabolismCutoff
PM = 5-10 % Cauc Americans>1 % Japanese
Intermediatemetabolism
0 0.01 0.10 1 10 100
Debrisoquin:4-Hydroxydebrisoquin Metabolic Ratio
Adapted from: Bertilsson L et al, Clin Pharmacol Ther 51:388-97,1992
OSAKA UNIVERSITY
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Treatment of Pulmonary Tuberculosis in Japan
• Approximately 20% of the patients treated with Rifampicin and INH develop hepatotoxicity.
• Metabolic enzyme of INH is NAT2.• Rifampicin strongly induces many drug metabolizing enzymes.
OSAKA UNIVERSITY
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Metabolic Pathways of IsoniazidMetabolic Pathways of Isoniazid
N-acetyltransferase2(NAT2)
Hydrolysis
amidase
Acetylation
Diacetylhydrazine(excretion)
Hydrazine
Acetylisoniazid
CONHNHCOCH3
NIsoniazid
(INH)
Isonicotinic acid
COOH
N
AcetylhydrazineNH2 NH2
N
CONHNH2
Hepatotoxicmajoralternative
OSAKA UNIVERSITY
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Genetic polymorphism of NAT2
803481C→T
590G→A
857G→A
845341
191G→A
282C →T
T→C
434A→C
A→G
A→C
190C→T
499G→A
759C→T
* * *** * * **** * *
NAT2*5
859T→CDel
411
364
* * *111T→C
G→A
A→T
NAT2*6 NAT2*7
Active allele(wild): NAT2*4
Low-active alleles(mutant): NAT2* NAT2* NAT2*5 6 7
The 4 genotypes can explain almost all Japanese phenotypes.
Mutant alleles 0 → Rapid acetylator type (RA-type)
Mutant alleles 1 → Intermediate acetylator type (IA-type)
Mutant alleles 2 → Slow acetylator type (SA-type)*
OSAKA UNIVERSITY
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INHINH--RFP induced hepatotoxicityRFP induced hepatotoxicity
How to Rationalize Dosing ?
INH+RFP
slow acetylator (SA type)
intermediate acetylator (IA type)
rapid acetylator (RA type)
Continue
Change regimen
DelayedCured
Liver Toxicity Increase dose
OSAKA UNIVERSITY
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Trough plasma concentration of INH in relation to NAT2 genotype
RA-type
Trough conc. of INH (mg/mL)Standard dose of INH
MIC0.05-0.2 mg/mL
0 2 4 6 8
Dose (mg/kg)
10
1
0.1
0.01
0.001
n=60 IA-type
IA-type + HTJapan 400 mg/daySA-type + HT(8 mg/kg/day)
Europe& USA
300 mg/day
(5 mg/kg/day)
Frequency of slow acetylator(SA-type)
Japanese 10 %
Caucasians 50 %
HT: hepatotoxicity
OSAKA UNIVERSITY
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Next clinical trial for genotype based chemotherapy against pulmonary tuberculosis
Collectively, it could be proposed that the dose adjustment of INH based on NAT2 genotypes is a promising strategy for tuberculosis therapy. Based on the obtained results, optimum INH dose for appropriate plasma concentration was calculated once daily as follows, 450 mg for RA-type, 300 mg for IA-type and 150 mg for SA-type, respectively.
IA-type300mg
RA-type450mg
SA-type150mg
(Once a day)
Trial groupINH dose
Control groupall
300mg
Prof. Dr. med. Uwe FuhrUniversity of Cologne
NAT2Gene chip
OSAKA UNIVERSITY
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THE PRINCIPAL RESEARCH QUESTION
We want to examine whether a dose individualization based on NAT2 genotype may improve the risk/benefit ratio of isoniazid treatment in tuberculosis by reducing variation in drug concentrations. Specifically, our major hypotheses are that :
● The incidence of drug-induced hepatotoxicity in subjects with no high activity NAT2 allele (SA)can be reduced.
● The incidence of early treatment failure in subjects with two high activity NAT2 alleles (RA)can be reduced by a corresponding doseadjustment of isoniazid within the standard multi-drug therapy of tuberculosis.
● As an additional research question, we want to quantitatively assess role of NAT2 genotype and other covariates (e.g. age, sex, body weight) in pharmaco-kinetics and –dynamics of isoniazid.
● Finally, the impact of a NAT2-based isoniazid dose adjustment on the costs of healthcare will be evaluated.
OSAKA UNIVERSITY
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Nicotine and CYP2A670ー80% of nicotine metabolized by CYP2A6
CYP2A6 polymorphism
decreased
null
normal
Activity
21%
20%
49%
Frequency
CYP2A6 *9
CYP2A6 *4
Genotype
CYP2A6 *1
3’5’
3’5’
3’
T- 48G
5’
Blood concentration of nicotine varied depending on CYP2A6 polymorphism; Clin Pharmacol Ther, 2003,74,69-76
OSAKA UNIVERSITY
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CPT-11 and UGT1A1 genepolymorphism
The patients with UGT1A1*28 are at higher risk for
severe toxicity by CPT-11.
SN-38
SN-38 G
UGT1A1(&1A7)
UGT1A1UGT1A1**2828(TA)6(TA)6→→(TA)7(TA)7Promoter regionPromoter region
Intestinal lumenBone marrow
Carboxylesterase
β-Glucuronidase
IrinotecanIrinotecanCPTCPT--1111
OSAKA UNIVERSITY
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WARNINGS
Patients with Reduced UGT1A1 Activity
Individuals who are homozygous for the UGT1A1*28allele are at increased risk for neutropenia following initiation of CAMPTOSAR treatment. A reduced initial dose should be considered for patients known to be homozygous for the UGT1A1*28 allele (see DOSAGE AND ADMINISTRATION).
Heterozygous patients (carriers of one variant allele and one wild-type allele which results in intermediate UGT1A1 activity) may be at increased risk for neutropenia; however, clinical results have been variable and such patients have been shown to tolerate normal starting doses.
OSAKA UNIVERSITY
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DOSAGE AND ADMINISTRATION
Dosing for patients with bilirubin >2 mg/dLcannot be recommended since such patients were not included in clinical studies.
A reduction in the starting dose by at least one level of CAMPTOSAR should be considered for patients known to be homozygous for the UGT1A1*28 allele (See CLINICAL PHARMACOLOGY and WARNINGS). The appropriate dose reduction in this patient population is not known.
OSAKA UNIVERSITY
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Metabolism of bilirubin
Hemoglobin
Unconjugated bilirubin
spleen
Unconjugated bilirubin + albumin
Direct (conjugated) bilirubin
RBC
UDPUDP--glucuronosyl transferase (UGT)1A1glucuronosyl transferase (UGT)1A1
polymorphism
PhenobarbitalLow calorie
into bile acid
into blood stream
OATP
MRP2MRP3
OSAKA UNIVERSITY
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UGT1A1 Molecular Assay UGT1A1 GENE
TATA box exon1 2 3 4 5
*28 *6 *27
5‘ 3‘
*29 *7mutation method
*28 (TA)6 → (TA)7 Melting Curve
*6 211G → A
*27 686C → A
*29 1099C →G
*7 1456T →G
PCR-RFLP
OSAKA UNIVERSITY
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FDA Clears Genetic Test That Advances Personalized Medicine Test Helps
Determine Safety of Drug TherapyFDA News August 22, 2005 Today, FDA cleared for marketing a new blood test that will help doctors make personalized drug treatment decisions for some patients. The Invader UGT1A1 Molecular Assay detects variations in a gene that affects how certain drugs are broken down and cleared by the body. Doctors can use this information to help determine the right drug dosage for individual patients, and minimize harmful drug reactions.“This test represents the power of DNA-based testing to provide individualized medical care,” said Daniel Schultz, MD, Director of FDA’s Center for Devices and Radiological Health. “These technologies can significantly improve patient management and reduce the risk of ineffective or even harmful drug therapy by telling doctors how to individualize drug dosing."The Invader assay joins a growing list of genetic tests used by physicians to personalize treatment decisions, including the Roche AmpliChip, used to individualize dosage of antidepressants, antipsychotics, beta-blockers, and some chemotherapy drugs, and TRUGENE HIV-1 Genotyping Kit, used to detect variations in the genome of the human immunodeficiency virus that make the virus resistant to some anti-retroviral drugs.
OSAKA UNIVERSITY
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UGT1A1 gene and allele frequency
Frequency
Allele Control (n=230) High indirect Bil (n=22)
*1 52.2% 4.5% *
*6 22.2% 31.8%
*28 10.9% 45.5% *
*60 14.8% 18.2%
n: number of allele*60 :-3279T>G
* :p<0.001 cross test
Simultaneous assay system : UGT1A1 *6、*28、*60 etc
OSAKA UNIVERSITY
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Candidate Genes for a genetic factor related to SSRI response
Polymorphism in pharmacogenetic factors analyzed in the present study
Gene/Polymorphism
PK1: Metabolic enzyme CYP2D6 IM / EM
Drug Transporter MDR-1 C3435T
5HT transporter 5HT transporter LPR/VNTR
5HTR1A Gly272Asp
5HTR2A A-1438G, T102C
TPH(1) A218C
MAO-A VNTR
Target molecule
5HT receptors
5HT synthase
5HT oxidase
PK2:CNS (BBB)
PD
cooperated with Kansai Medical University
OSAKA UNIVERSITY
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FDA Approves BiDil Heart Failure Drug for Black Patients (1)
The Food and Drug Administration (FDA) approved BiDil (bye-DILL), a drug for the treatment of heart failure in self-identified black patients, representing a step toward the promise of personalized medicine. (June 23, 2005 )
Heart failure is a condition in which the heart is weakened and does not pump enough blood. It can be caused by a variety of damage to the heart, including heart attacks, high blood pressure, and infections.
The approval of BiDil was based in part on the results of the African-American Heart Failure Trial (A-HeFT). The study, which involved 1,050 self-identified black patients with severe heart failure who had already been treated with the best available therapy, was conducted because two previous trials in the general population of severe heart failure patients found no benefit, but suggested a benefit of BiDil in black patients. Patients on BiDil experienced a 43% reduction in death and a 39%decrease in hospitalization for heart failure compared to placebo, and a decrease of their symptoms of heart failure.
is marketed by NitroMed, Inc. of Lexington, MA.
OSAKA UNIVERSITY
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β1AR Ser49Gly and Risk in CHF
Eur Heart J 2000;21:1853-8.
0 1 2 3 4 5
20
40
60
0
p = 0.016
p = 0.12
Follow-up (years)
Risk of end-point (%)
△
▲
☆
Ser49 homozygotes without β-blockers (n=63)
Gly49 variant without β-blockers (n=28)
Ser49 homozygotes with β-blockers (n=59)
Gly49 variant with β-blockers (n=33)
△
▲
☆
★
★
Increased down regulation
β-blocker more effective in patients with Gly allele
OSAKA UNIVERSITY
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Heart failure and Polymorphism
α2c AR Del322-325
Allele frequency
CHF healthy
0.62
0.11
???
Black 0.41
White 0.04
Yellow ???
(2002,10 reported)
α2A
α2C
Norepinephrine
β1 β3β2
Cardiac-cellmembrane
Sympatheticnerve
α2CDel322-325Decreased Function in vitro
β1Arg389GlyIncreased
Function in vitro
These two polymorphism of receptors act
synergistically to increase the risk
of heart failure in black.
Japanese ?
N Engl J Med (2002) 347, 1135-42
OSAKA UNIVERSITY
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Allelic frequency of adrenergic receptor polymorphisms in healthy and CHF
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
Ser49Gly Arg389Gly Arg16Gly Gln27Glu α2c Del
CHF (n = 66)
Healthy (n = 119)
β1 AR α2c AR
Allele frequency
β2 AR
• Allelic frequency of α2c AR Del322-325 in healthy Japanese is 0.14
• CHF>Healthy : Black and White
OSAKA UNIVERSITY
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Clinical significances of polymorphismsin β-blocker therapy against
Chronic Heart Failure
Mega trial of β-Blocker Treatment in Japanese Patients with Chronic Heart Failure (J-CHF)
Patient: NYHA II or III
EF<40%, 1500 patients (multicenter, 300 hospitals)
Drug:Carvedilol (2.5mg、5mg、20mg/day)
Endpoint:Mortality, Cardiovascular death, Mobility
Surrogate Marker : Cardiac function
Sub analysis : Genotyping
Directed by A Kitabatake MD, PhD (Hokkaido University)
Supported by Japanese Circulation Society
OSAKA UNIVERSITY
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Chronic Heart Failure
ββblokerbloker
responderresponder nonnon--responderresponder
CAUSEPlasma Concentration
of β blockerPolymorphisms
Drug Metabolizing Enzyme
Function of Target Molecules of β blocker
PolymorphismsAR and Target Molecules
OSAKA UNIVERSITY
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Individual Difference in βblocker Effect
0
5
10
15
20
25
Before
After
(%)
(33例:41.8%) (41例:51.9%) (5例:6.3%)
%FS ±3%
Responder Non-responder Bad-responder
11.8
21.7
14.9 15.0
19.3
12.2
Responder ; improvement of
3 % in the fractional shortening
%FS
79patients
For Individualized Medication
Genotyping
OSAKA UNIVERSITY
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Allelic frequency of adrenergic receptor polymorphisms in responder and non-responder
0.3
0.4
0.5
0.6
Arg16Responder (n=31)
Non-responder (n=15)
Allele frequency
Gly49 Gly389 C-470.2
Glu270.1
0
Ser49Gly Arg389Gly T-47C Arg16Gly Gln27Glu α2c Del
β2ARβ1AR α2c AR
OSAKA UNIVERSITY
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Polymorphisms associated with response to β-blocker
Ratio of responder (%)
0 20 40 60 80 100
C/C(n=10)
T+ (n=34)
G/G(n=35)
T+ (n=38)
C/C (n=31)
G+ (n=3)
A/A(n=66)
G+ (n=64)
T+ (n=30)C/C(n=39)
C+ (n=13)
T/T(n=56)
p=0.017
T+ (n=59)SNP A p=0.001
p=0.008SNP B
SNP C p=0.011
No responderSNP D p=0.029
C/C (n=5)SNP E p=0.033
SNP F
SNP Gp=0.004
OSAKA UNIVERSITY
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遺伝子多型のスコア化によるβ遮断薬の反応性予測
Score = 2x SNP A + SNP B + SNP C + SNP D + SNP E + SNP F + 2x SNP G
SNP A: T alleleSNP B: G alleleSNP C: C alleleSNP D: A alleleSNP E: G alleleSNP F: T alleleSNP G: C allele
各SNPを判定し、左記のアレルの数を上の式に当てはめて多型スコアを計算した。
SNPのA及びGは有意性が特に大きいため2を乗じた。
≧8points 7points≧
Responder(n=47) 46 1
Non responder(n=22) 9 13
OSAKA UNIVERSITY
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Individualized Medicine (1)
Patient a
X
Patient b
Gene A
Gene B×
×
Responder
Non responder
Drug X
Rationalize
Dosing
ADR?
Cured
NotCured
DIAGNOSISDIAGNOSIS
Gene A
Gene B
OSAKA UNIVERSITY
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Individualized Medicine (2)
×
Cured
Gene analysis
×
DrugX
Drug SelectionRationalize DosingClass of Drugs
Gene A
Gene B
Responder
Responder
Cured
Patient a
Patient b
DIAGNOSISDIAGNOSIS
Gene A
Gene B
DrugY
OSAKA UNIVERSITY
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OSAKA UNIVERSITY
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Next generation-full automatic systemConventional method
<PCR>
<GenelyzerTM>
Toshiba
<Full automatic system>
≪Future≫
Electrochemical method
<PCR>
<Hybri. apparatus>
<Detector>
<Labeling>
<DNA extraction> <DNA extraction>
≪Present≫
<Image processing>
Only inject blood sample into cassette
○ scene of hospital
○ rapid testing
Point of Care
OSAKA UNIVERSITY
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Pharmacogenomics Clinical Trial Management SystemPharmacogenomics Clinical Trial Management System
with high speed, high security, high quality & efficiency using IT net work infrastructure
Osaka Univ.
M/C M/C M/C
IT infrastructures
M/C
M/C
Privacy protection
PGx DB
1) PKI card for certification & coding
3) Coding & scrambling on inputting patient’s clinical data at clinical sites
2) Fiber & high-speed broad bands to share the information of clinical trials
PKI card゙
IP-VPN(MPLS net work)
PGx analysis center
Pgtt Inc
Security measures: Clinical trial DB
C
Osaka Medical CenterRespiratory & Allergic disease
Toneyama Hospital Kinki-CyuoChest Medical Center
Osaka Hospital JATA-Osaka
OSAKA UNIVERSITY
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IP-VPN network with secured security Internet Protocol ‒Virtual Private Network (IP-VPN) runs over the Multi-Protocol Label Suitching (MPLS) public IT network, which lets users set up a fully meshed IP-VPN.
Security
MPLSMPLSNetworkNetwork
RouterRouter
RouterRouter
Reliability
Flexibility
Economical
Performance
RouterRouter
RouterRouter
CROCRO
CompanyCompany
×
network intrusions and hackers
HospitalsHospitals
HospitalsHospitals
OSAKA UNIVERSITY
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Application of PGx providing individualized medicines
Bood samples(I.D.)
Clinical trials/Treatmentsbased on
genetic variation
Patients(I.D.)
DoctorsPharmacists
Published Evidence
New Evidencefrom
research institute
IT Network
GenotypingData & Gene Bank
Genetic Information Center(I.D.)
・Process information・Bioinformatics・Expect pharmacokinetics/ adverse effects in vivo
・Simulate response in silico
Personalized Medicationright treatment for the right
patient at the right time.
Patent obtaining
New Drug Discovery & Development
PG-TipTop Inc.
Creation of database for pharmacogenomic s
薬効ゲノム情報
OSAKA UNIVERSITY
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TTPPTTGGTTPPTTGG
TTPPTTGG TTPPTTGGPPGGTT
TT
Optimize medication most suitable for each patient
http://www.pgtiptop.com