Prof. Alberto Corsini Università degli Studi di Milano IUSS/29-4... · unrelated substrates • Species differences. Human: MDR1 Rodents: mdr1a (or mdr3) and mdr1b (or mdr1) •

Farmacogenetica e malattiecardiovascolari

Prof. Alberto CorsiniUniversità degli Studi di Milano

• Interazione tra farmaci

• Presenza di una patologia concomitante

• Genetica

• Età del paziente

• Polifarmacia

Variabili interindividuali che influenzano la Variabili interindividuali che influenzano la risposta ai farmacirisposta ai farmaci

• Mendelian genetics– Autosomal Dominant Hypercholesterolemia

• LDLR, APOB, PCSK9

– Familial Thoracic Aortic Aneurysm and Dissection syndromes:• FBN1, TGFBR1, TGFBR2, ACTA2, MYH11, NOTCH

• Genetic markers for CAD– Multiple genes contribute to small percentage of phenotype

• Chromosome 9p21/ANRIL• KIF6

• Pharmacogenetics– Controversial, but gathering speed

• Clopidogrel • Warfarin sensitivity• Statin efficacy

Recent Developments in CV Genetics

FARMACOGENETICA

Scienza che studia il ruolo dell’ereditarietà nella risposta individuale al farmacoStudia i polimorfismi dei singoli geni coinvolti nel metabolismo di un farmacoAnalizza le differenze individuali in termini di risposta al farmaco

Obiettivi finali di uno studio di farmacogenetica:

1. Personalizzare la terapia del paziente in cura

2. Aumentare l’efficacia del farmaco

3. Ridurre la possibilità di comparsa di effetti collaterali

POLIMORFISMOPOLIMORFISMO

Un POLIMORFISMOPOLIMORFISMO e’ una variazione della sequenza del DNA che avviene con una elevata frequenza (ogni 300-500 bp). In alcuni casi puo’ determinare una variabile FENOTIPICAFENOTIPICA (es. colore degli occhi)

Tempo(le frecce indicano somministrazioni ripetute)

Metabolizzatori lenti (PM)

Metabolizzatori veloci (EM, normali)

Rilevanza di un metabolismo insolitamente lento di alcuni farmaci da parte del CYP450 o di altri enzimi

L’andamento normale è riportato nel pannello superiore, dove i livelli plasmatici di farmaco si mantengono entro un certo intervallo quando viene assunta una particolare dose ripetuta

Un metabolismo insolitamente lento (pannello inferiore) porta ad un aumento dei livelli plasmatici del farmaco

Live

lli p

lasm

atic

i di f

arm

aco

Delezione del gene Singolo geneGeni duplicati o moltiplicati

No enzima Enzima instabile Enzimanormale

Alterata specificità di substrato

Livelli più alti di enzima

Nessun metabolismo

Metabolismo ridotto

Metabolismo normale

Metabolismoaumentato

Possibile formazionedi altri metaboliti

mRNA-AAAA mRNA-AAAA mRNA-AAAA mRNA-AAAA

CYP2D6,*4,*5CYP2C19,*2,*3

CYP2D6,*4,*10 CYP2D6,*1CYP2C19,*1CYP2C9,*1

CYP2D6,*17CYP2C9,*3

CYP2D6,*2xN

Principali meccanismi molecolari che possono causare un alterato metabolismo dei farmaci

Human polymorphic cytochrome P450 enzymes and the Human polymorphic cytochrome P450 enzymes and the global distribution of their major variant allelesglobal distribution of their major variant alleles

Pharmacogenetics of Phase I Drug MetabolismPharmacogenetics of Phase I Drug Metabolism

N Engl J Med 2003; Vol 348(6), Febr 6:529-537

Ruolo dei trasportatori di membrana nel percorso farmacocinetico

Giacomini KM, et al. Goodman and Gilman’s. The Pharmacological Basis of Therapeutics, 11/E, 2006

Le proteine di trasportoCaptazione: facilitano

l’entrata di sostanze (farmaci) all’interno delle cellule.

• OATP (organic aniontransporting polypeptide)

• OAT (organic aniontransporter)

• PEPT (peptide transporter)

Efflusso: esportano sostanze (farmaci) all’esterno delle cellule, anche contro un gradiente di concentrazione.

• ABC (ATP bindingcassette):– ABCB (P-glicoproteina)– MDR1 (multidrug

resistance protein 1)

• BCRP (breast cancerresistance protein)

P-gp TISSUE DISTRIBUTION

C. Marzolini et al., Clin. Pharmacol. Ther., 75: 13-33, 2004

P-glycoprotein

• P-glycoprotein (170 kDa ATPase): Complex mechanism of drug transport.

• At least 3 substrate binding sites• Two-stage process: (membrane partitioning (rate limiting) and P-gp interaction)

• Found in many tissues and organisms

• Structurally diverse and unrelated substrates

• Species differences. Human: MDR1Rodents: mdr1a (or mdr3) and mdr1b (or mdr1)

• Polymorphism and induction of P-gp

So far no truly general and conclusive SAR available

Need for different types of P-gp assays

HUMAN DRUG TRANSPORTERS

Ho R.H. et al., Clin. Pharmacol. Ther. 78: 260-77, 2005

Additional points to consider: PolymorphismAdditional points to consider: Polymorphism

• - C3435T: correlation with functional activity. C/C wildtype allelecorrelates with 2-fold higher activity [Hoffmeyer, PNAS, 2000]

• - Interethnic differences (African population with high C/C frequencies)

• But: Very limited impact:• - No association between C3435T SNP and cyclosporine PK

(106 renal transplant patients [Anglicheau, Clin Pharm Ther, 2004])• - > 20 single nucleotide polymorphisms in MDR1

Hypothesis:

Compensatory protective mechanisms in P-gp deficient individuals

Marzolini et al., Clin. Pharmacol. & Ther., 2004; 75:13-33

Genetic polymorphism of MDR1Genetic polymorphism of MDR1

N Engl J Med 2009;360.

AIMS

• 2208 patients presenting with an acute myocardial infarctionin a nationwide French registry and receiving clopidogreltherapy

• The relation of allelic variants of genes modulatingclopidogrel absorption (ABCB1), metabolic activation(CYP3A5 and CYP2C19), and biologic activity (P2RY12 and ITGB3) to the risk of death from any cause, nonfatalstroke, or myocardial infarction during 1 year of follow-up.

Simon T et al. N Engl J Med 2009;360

Roles in ClopidogrelActivity of Proteinswith Known Genetic

Polymorphisms

Simon T et al. N Engl J Med 2009;360

Simon T et alN Engl J Med2009;360

Distribution of Cmax and AUC of clopidogrel and itsactive metabolite according to MDR1 C3435T genotype in

PCI patients after clopidogrel

Taubert D et al. Clin Pharmacol Ther 2006;80:486-501

Simon T et alN Engl J Med2009;360

Relationship between CYP2C19 Genetic Classification and Pharmacokinetic and Pharmacodynamic Responses

of Clopidogrel in Healthy Subjects

Mega JL et al. N Engl J Med 2009;360

Rates of Death from Any Cause, Nonfatal MyocardialInfarction, or Stroke, According to ABCB1Variant-Allele

Polymorphisms

Simon T et al N Engl J Med 2009;360

Rates of Death from Any Cause, Nonfatal MyocardialInfarction, or Stroke, According to CYP2C19 Variant-

Allele Polymorphisms

Simon T et al N Engl J Med 2009;360

N Engl J Med 2009;360

Aims

The association between functional genetic variants in CYP genes, plasma concentrations of active drug metabolite, and platelet inhibition in response to clopidogrel in 162 subjects

The association between these genetic variants and cardiovascular outcomes in a separate cohort of 1477 patientswith acute coronary syndromes treated with clopidogrel


Genetic Effects on Pharmacokinetic and Pharmacodynamic Responses to Clopidogrel


Association between a Carrier of a CYP2C19 Reduced-Function Allele and the Primary Efficacy Outcome

in Subjects Receiving Clopidogrel


Association between a Carrier of a CYP2C19 Reduced-Function Allele and the Stent Thrombosis

in Subjects Receiving Clopidogrel


Efficacy and Safety Outcomes at 15 Months in Subjects Treated with Clopidogrel, According to

Genotype Status


Warfarin PGx• Warfarin is commonly prescribed drug• Variants in CYP2C9 and VKORC1 account for high

percentage of variability of warfarin response• FDA relabeled warfarin in Aug. 2007 to encourage

pharmacogenetic testing• Utility still remains low

– Controversial topic– Unanswered questions

Warfarin• Widely prescribed anticoagulant• 12th most commonly prescribed drug

Wysowski, D. K. et al. Arch Intern Med 2007;167:1414-1419.

0

5000000

10000000

15000000

20000000

25000000

30000000

35000000

Annual Number of

Prescriptions

1998 1999 2000 2001 2002 2003 2004

Year

Annual Number of Outpatient Warfarin Prescriptions, 1998-2004 45% increase from

1998 to 2004

Warfarin• Challenges in regulating warfarin dosing• Prothrombin time (INR) must remain in narrow

therapeutic range• Elevated INR: risk for major bleeding complications

– Risk for serious bleeding increases with INR > 4.0– Most likely to occur within first few weeks of initiating

treatment

• Subtherapeutic INR: thrombotic complications

Sources of variability in Warfarin dosing

VKORC1 25%

CYP2C9 17%

CYP4F2 2%

Weight 9%

Age 7%

Other 28%

Drugs 12%

Warfarin Pharmacology• Racemic mixture of R- and S-enantiomers

– S-Warfarin approx. 7-10X as potent as R-– Majority of in vivo activity of warfarin resides in S-

warfarin

• Metabolized mainly through CYP2C9• Targets VKORC1 (Vitamin K epoxide reductase

complex subunit 1)– Interferes with recycling of Vit K in the liver– Reduced activation of clotting factors

Pharmacokinetics Pharmacodynamics

CYP2C9

CYP2C19CYP3A4CYP1A1CYP1A2CYP2C8CYP2C9

4-hydroxywarfarin

6-hydroxywarfarin

7-hydroxywarfarin

8-hydroxywarfarin

10-hydroxywarfarin

VKOR

(hepatocyte) (hepatocyte)

S-WarfarinR-Warfarin

6-hydroxywarfarin

7-hydroxywarfarinVitamin K (oxidized)

Inactive Vitamin K-dependent clotting

factors

Active Vitamin K-dependent clotting

factors

Vitamin K (reduced)

S-WarfarinR-Warfarin

inactive

GGCX

Elimination

Warfarin PGx• CYP2C9

– CYP2C9*2 variant• Arg144Cys• 30% decrease in enzymatic activity

– CYP2C9*3 variant• Ile359Leu• 70%-95% decreased enzymatic activity

• VKORC1– Promoter SNP -1639G>A – 44% decrease in promoter activity

Allele Frequencies

8%89%42%

VKORC1-

1639G>A6-10%8-13%Caucasian

1-4%

AIMS

• To developed and used an algorithm for estimating the appropriate warfarin dose that is based on both clinical and genetic data from a broad population base

• Clinical and genetic data from 4043 patients were used tocreate a dose algorithm that was based on clinical variablesonly and an algorithm in which genetic information wasadded to the clinical variables

N Engl J Med, 360: 753-764, 2009

Demographic and clinical characteristics of the derivation and validation cohorts

N Engl J Med, 360: 753-764, 2009

N Engl J Med, 360: 753-764, 2009

Comparison of warfarin doses predicted according to

the clinical and the pharmacogneics algorithm

N Engl J Med, 360: 753-764, 2009

N Engl J Med, 360: 753-764, 2009

Conclusions

• The use of a pharmacogenetic algorithm forestimating the appropriate initial dose of warfarinproduces recommendations that are significantlycloser to the required stable therapeutic dose thanthose derived from a clinical algorithm or a fixed-dose approach

Warfarin PGx: Summary

• Some studies suggest that warfarin PGx testing is beneficial– Reduced time to stable INR– Reduced adverse events

• Large multi-center studies currently underway– Clinical outcomes– Validated dosing algorithms

• New technology allowing for more rapid results• Genetic variants explain 40-45% of variability in response to

warfarin– Important to realize the impact of the compound effects of

• polymorphisms in CYP2C9 AND VKORC1• Drug-drug interactions, co-morbidities, age, BMI

Structural Formulas of HMG-CoA and of Statins

ES Istvan, J Deisenhofer Science 2001; 292: 1160-1164

Regulation of Cholesterol Metabolismin the Liver Cell

Modified from D. Rader Nat Med 2001, 7: 1282-1284

Individual LDL-C % Response to Atorvastatin 10mg/day

Pedro-Botet J et al. Atherosclerosis 158 (2001) 183-193

Inter-individual variability in response to statins

poor compliancebackground diet

dose and uptitration of drugconcomitant drug therapy

Extrinsic factors(extraneous influences)

Intrinsic factors(genetically-determined)

LDL-receptor gene mutationsapo-B-100 gene mutations

CYP/transporter polymorphism apoE polymorphismCETP polymorphism

rate of cholesterol biosynthesisrate of cholesterol absorption

Kajinami K et al. Atherosclerosis 2004, 177:219-234

Candidate Genes Involved in the Pharmacogenetics of Statins Responsiveness

Pharmacological effects of genetic variations associated with statin therapy

Schmitz et al, Curr opin Lipidol 18:164-173. 2007

Lipid metabolism







CYP/transporter polymorphism apoE polymorphismCETP polymorphism


Responsiveness to fluvastatin in patients with molecularly defined heterozygous familial

hypercholesterolemia

Patients characteristic according to the five genetic groups

Leitersdorf E. et al, Circulation, 1993;87[suppl. III]:III-35–III-44

Leitersdorf E. et al, Circulation, 1993;87[suppl. III]:III-35–III-44

Responsiveness to fluvastatin in patients with molecularly defined heterozygous familial

hypercholesterolemia LDL and HDL response in the five genetic groups

p46a ZL PD PF PW BP MMMP PAt PE PVp43 PAI PG PC

A

B

C

InheritanceInheritance of the of the mutation mutation in the codon for aa 3500 of in the codon for aa 3500 of apoapo B geneB gene

Corsini A et al.1991. Eur J Clin Invest 21: 389-397

FamilialFamilial defectivedefective ApoApo BB--100: 100: reducedreducedresponseresponse toto HMGHMG--CoA reductase CoA reductase inhibitorinhibitor

Patient (n) Initial LDLcholesterol

mg/dl

Percent decrease inLDL cholesterol

P.V. 274 15

*FH (48) 298 35

SIMVASTATIN (20 mg/die) FOR 4 WEEKS

* Typical response to simvastatin treatment of heterozygous

Corsini A et al, Lancet 1991, Vol 337, Feb 2:305







apoE polymorphismCETP polymorphism

CYP3A4 polymorphism


Determinants of Variable Response to Statin Treatment in Patients With Refractory Familial

Hypercholesterolemia

Frans H O’Neill et al, Arterioscler Thromb Vasc Biol 2001;21:832-837


LDL-C reductions of subjects according to the different mutations detected


Plasma MVA and 7-α-OHC in Good and Poor Responders After Each of the 4 Treatment

Regimens of the Srudy MVA, µmol/L 7-α-OHC, µmol/L

Good responders (n=11)

Placebo 34.4±6.1 87.4±69.9 A10 22.3±7.4 72.4±39.9

BAS 58.1±22.9 217.2±259.6

A10+BAS 29.0±14.2 159.7±177.2

Poor responders (n=8)

Placebo 26.3±6.1 87.4±52.4

A10 22.3±5.4 112.3±97.3

BAS 54.7±13.5 497.7±376.9

A10+BAS 25.0±8.8 169.7±192.2 Because the values were not normally distributed, the values given are the geometric

mean±approximate SDs


Reduction in LDL-C after 1 month of atorvastatin (10 mg/d) according to apoE phenotype

Poor responders to statins have a low basal rate of cholesterol synthesis due to an

increase in cholesterol absorption, possibly mediated by apolipoprotein E4








CYP/transporter polymorphismapoE polymorphismCETP polymorphism


Tissue localization and role of transport proteins in drug disposition

A. Ayrton et al., Xenobiotica 2001;31:469-497

Richard B. Kim, Clin Pharnacol Ther 2004;75:381-385

Uptake and Efflux Transporters Expressed

on Human Hepatocytes

Summary of the major human liver sinusoidal canalicular membrane transporters involved in transport of therapeutic drugs

A. Ayrton et al., Xenobiotica 2001;31:469-497

Atorvastatin, simvastatin, lovastatin, pravastatin, ezetimibe, cyclosporine,taxol, vinblastine, doxorubicin, digoxin,talinolol, loperamide, erythromycin

CABCB1MDR1

pravastatin, atorvastatin,methotrexate,cefodizime ezetimibeirinotecan, conjugates of drugs

CABCC2MRP2

Anticancer agents, anionic conjugates with glutathione, sulfate or glucuronideCerivastatin, pitavastatin, fluvastatin, pravastatin, rosuvastatin

L

L

ABCC1

ABCP

MRP1

BCRP

S, sinusoidal; C, canalicular; L, lateral membranes.

fexofenadine, UK-191,005SSLC21A9OATP-B

BSP, rosuvastatinSSLC21A8OATP-8

Fluvastatin, pravastatin, cimetidineS?SLC22A8OAT3

atorvastatin, cerivastatin, simvastatin, rosuvastatin, pravastatin, pitavastatinfluvastatin, gemfibrozil, lovastatin, ezetimibe, BSP, eicosanoids, coniugated steroids, cyclosporine

SSLC21A6OATP-C/2/1B1

fexofenadine, rosuvastatin, UK-191,005SSLC21A3OATP-A

Known drug substratesSubcellular location

Gene family name

Common name of transporter

Yes

+nanana+na+na

Yes

++++na+-

na

Yes

++na+++++

Yes

+nanananana+na

Yes

++na+++-na

Yes

++na+++-

na

Yes

+nana+na+++

Transporter

OATP1B1OATP1B3OATP1A2OATP2B1OAT3BCRPMDR1MRP2

Enzymatic pathways involved in the pharmacokinetics of Statins

SimvaRosuvaPravaLovaFluva XL

FluvaAtorvaStatins

Corsini A, Bellosta S. Exp Rev Clin Pharmacol 2008; 1 (1): 105-113

G Schmitz Clin Chem Lab Med 2003, 41:581-589

Statin Metabolism and Elimination

Richard B. Kim, Clin Pharnacol Ther 2004;75:381-385

Single-nucleotide polymorphisms in OATP-C (SLCO1B1)

PHARMACOKINETIC CONSEQUENCES OF REDUCED OATP1B1 ACTYIVITY FOR PRAVASTATIN

Kivistö KT and Niemi M. Pharmaceut Res, 24: 239-247, 2007

Polymorphisms of OATP-C (SLC21A6) and OATP3 (SLC22A8) genes: Consequences for

pravastatin pharmacokinetics

Yohei Nishizato et al., Clin Pharmacol Ther 2003;73:554-65

Mikko Niemi et al., Pharmacogenetics 2004;Vol14 (7):429-440

Pravastatin Pharmacokinetics after a single 40mg oral in relation to the OATP-C single nucleotide polymorphisms and the haplotypes

PLASMA PRAVASTATIN CONCENTRATION-TIME AND LIPID RESPONSE IN SUBJECTS WITH A SLCO1B1 VARIANT AFTER 40 MG PRAVASTATIN

Kivistö KT and NiemiM. Pharmaceut Res,

24: 239-247, 2007

A NOVEL VARIANT ALLELE OFOATP-C (SLCO1B1) FOUND IN A JAPANESE PATIENT WITH PRAVASTATIN-INDUCED

MYOPATHY

K. Morimoto et al., Drug Metab. Pharmacokinet., 19 (6): 453-5, 2004

The SEARCH Collaborative Group, N Engl J Med, 359: 789-799, 2008

ODDS RATIOS FOR MYOPATHY ASSOCIATED WITH THE SLCO1B1 GENOTYPE AMONG SUBGROUPS OF PATIENTS TAKING 80 mg OF

SIMVASTATIN DAILY


ESTIMATED CUMULATIVE RISK OF MYOPATHY ASSOCIATED WITH TAKING 80 mg OF SIMVASTATIN ACCORDING TO SLCO1B1 GENOTYPE


In vivo studies of the association between statin elimination and SLCO1B1 polymorphism


Conclusions

Altogether, the pharmacogenetic must be considered in our efforts to understand the pharmacological properties of cardiovascular drugs and in our selection of intervention strategies to lower CVD risk

FARMACOGENOMICA

Scienza che studia l’intero genoma individualeIdentifica i profili genetici individuali di ogni singolo paziente

Obiettivi finali di uno studio di farmacogenomica:

1. Identificare nuovi geni bersaglio per terapie innovative

2. Prevedere la risposta ai principi attivi somministrati

3. Personalizzare la terapia del paziente in cura

Documents

Prof. Alberto Corsini Università degli Studi di Milano IUSS/29-4... · unrelated substrates • Species differences. Human: MDR1 Rodents: mdr1a (or mdr3) and mdr1b (or mdr1) •