determinants of drug disposition - drug metabolismscdmdg.org/slides/20110419-kim.pdf · schroth et...

4/29/2011

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Drug Transporters: In Vitro and Knockout Model Systems Pharmacogenomics and Clinical Relevance

Richard B. Kim MD, FRCP(C)

Professor & Chair, Division of Clinical PharmacologyDirector, Centre for Clinical Investigation & Therapeutics

Systems, Pharmacogenomics, and Clinical Relevance

Department of Medicine, London Health Sciences Centre

Schulich School of Medicine & DentistryThe University of Western Ontario

[email protected]://www.uwoclinpharm.ca

Intestinal Lumen

UptakeEfflux

Determinants of Drug Disposition

metabolism

Efflux

Intestine

DrugSystemic Circulation

Uptake

Drug/Metabolite

Biliary and Renal Elimination

Liver,Kidney metabolism

Efflux

p

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Case Study

• 59 yr old female with type 2 diabetes on metformin presents with a 3 month history of vague abdominalpresents with a 3 month history of vague abdominal pain and diarrhea.

• Also 3 months ago, started on cimetidine for duodenal ulcer treatment.

• On examination, noted to be agitated, hypotensive (BP 85/40 mmHg). Arterial blood gas showed

f d b li id i ( H 6 5)profound metabolic acidosis (pH 6.5)

• Dx: Metformin induced metabolic acidosis

Dawson et al Diabetes Care 26:2471-2472, 2003

Case Study

• Why did she develop this?

• Metformin is not significantly metabolized. It is cleared by glomerular filtration and tubular secretion.

• Cimetidine is known to be capable of inhibiting renal tubular secretion of metformin.

Dawson et al Diabetes Care 26:2471-2472, 2003

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Renal Transporters

Ho RH and Kim RB Clin Pharmacol Ther, 78:260-77, 2005

OCT2 is the metformin transporter

Kimura N et al et al Pharm Res 22:255-259, 2005

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OCT2 can be inhibited by cimetidine

Kimura N et al et al Pharm Res 22:255-259, 2005

Portal blood flowOCT1 OCT3

OATP

MRP2

Sinusoidal membrane

OAT21B1 1B3 2B1 NTCP

Organic cations, drugs Organic anions, drugs and bile acids

humanhepatocyte bile

Canalicular membrane

ATP

MRP2

ATP BCRP

BSEP

MDR1ATP

ATP

MATE1

Sinusoidal membrane

MDR3ATP ATP

MRP3 MRP4

ATP

Portal blood flow

DeGorter MK et al Hepatology, 2009

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OATP1B1(OATP‐C, LST‐1, OATP2)

OATP1B3(OATP8, LST‐2)

Hepatic OATP Transporters

Endogenous Substrates:

Estrone Sulfate, PGE2, Bilirubin, thyroid hormone (T3, T4) Bilirubin‐glucuronides Estradiol 17β‐d‐glucuronide, bile acids

Drug Substrates:

Atorvastatin, Cerivastatin, Pravastatin

Rosuvastatin, Pitavastatin, Caspofungin,

Endogenous Substrates:

CCK‐8, PGE2 Thyroid hormone (T3, T4) Estradiol 17 β ‐d‐glucuronide, Bile acids, Deltophin, DPDPE,

Drug Substrates:

Pravastatin, Pitavastatin, Rosuvastatin,, Fexofenadine, BQ‐123, Oubain, Digoxin,

©Richard B. Kim M.D.

Troglitazone‐sulfate, Rifampin, Arsenic, Atrasentan, Valsartan, Olmesartan, Enalapril, MTX, Temocaprilat, SN‐38

Toxins:

Phalloidin, Microcystin‐LR

Doxotaxel, Paclitaxel,, Rifampin, MTX, Bilirubin, Repaglinide, Telmisartan, Valsartan,

Olmesartan, Enalapril, Temocaprilat, SN‐38

Toxins:

Phalloidin, Microcystin‐LR

mRNA and Protein Expression

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400400

Expression of Hepatic Statin Transporters

200200

300300

mR

NA

Co

py

Nu

mb

er

mR

NA

Co

py

Nu

mb

er

OATP1B1OATP1B1 OATP1B3OATP1B3 OATP2B1OATP2B1 NTCPNTCP00

100100

Human Liver SamplesHuman Liver Samples

mm

Ho et al., Gastroenterology 2006

Protein Expression of Hepatic OATPs

Ho et al., Gastroenterology 2006 Ho RH et al, Gastroenterology, 130:1793-1806, 2006

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Hepatic OATP Expression

Ho et al., Gastroenterology 2006

Recombinant Transporter Expression Systems

Rat Human

HeLa Cells

Transporters

Labeled DrugAdded

Overnight co‐incubationwith virus (vtf7)


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40004000

50005000

60006000

70007000

******

***

***

n U

pta

ken

Up

take

y C

on

tro

l)y

Co

ntr

ol)

Rosuvastatin Transporters

Con

trol

Con

trol

OA

TP

OA

TP

--A)

A)

OA

TP

OA

TP

--B)

B)

OA

TP

OA

TP

--C)

C)

OA

TP

8)O

AT

P8)

(N

TC

P)

(N

TC

P)

2 (A

SB

T)

2 (A

SB

T)

(O

CT

1) (

OC

T1)

(O

atp1

) (

Oat

p1)

4 (O

atp2

)4

(Oat

p2)

5 (O

atp3

)5

(Oat

p3)

2 (O

atp4

)2

(Oat

p4)

a1 (

Ntc

p)a1

(N

tcp)

a2 (

Asb

t)a2

(A

sbt)

00

10001000

20002000

30003000

40004000

******

***

******

Ro

suva

stat

inR

osu

vast

atin

(% V

ecto

r(%

Vec

tor--

on

lyo

nly

OA

TP

1A2

(OO

AT

P1A

2 (O

OA

TP

2B1

(OO

AT

P2B

1 (O

OA

TP

1B1

(OO

AT

P1B

1 (O

OA

TP

1B3

(O

AT

P1B

3 (

SLC

10A

1S

LC10

A1

SLC

10A

2S

LC10

A2

SLC

22A

1S

LC22

A1

Slc

o1a1

Slc

o1a1

Slc

o1a4

Slc

o1a4

Slc

o1a5

Slc

o1a5

Slc

o1b2

Slc

o1b2

Slc

10a

Slc

10a

Slc

10a

Slc

10a

HumanHuman RatRat

Ho et al., Gastroenterology 2006.

Ho RH et al, Gastroenterology, 130:1793‐1806, 2006

OATP1B3/MRP2 Double Transfected MDCK Cells

©Richard B. Kim M.D.Cui Y et al Mol Pharm 60:934, 2002

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So what should you do about OATPs??

AppetizerHepatocytes (could use rat). Q. Uptake transport relevant?

Salad

AppetizerConsider labeled estradiol 17-B glucuronide as a model substrate

Salad

Substrates Galore Lots of Inhibition

Salad

Express OATP1B1, 1B3, 2B1 (consider 1A2).Consider expressing rat or mouse Oatps.

EntréeIs uptake 1.5-fold or > than control?If no, OATPs may not be the relevant transporter(s)

Express 1B1 and 1B3 (might consider other OATPs).

EntréeDetermine IC50. Include known positive control inhibitors (e.g. RIF).

Dessert

Consider I/IC50 values.Dessert

Determine Km and Vmax(use a standardized transporter model system. i.e. do not mix oocytes + Cell lines + vesicles etc)

Beverages

OATP1B1- Consider PGEN

Difficult to know when a clinical interaction study should be carried out.

Beverages

If a clinical study is necessaryRosuvastatin or pravastatin as substratesOATP1B3 (mostly) telmisartanPK + and - your drug could be done


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OATP substrates: What to do step‐by‐step

Step 1:Hepatocytes (could use rat). Q. Uptake transport relevant? Step 4:

D t i K d VQ. Uptake transport relevant?

Step 2:

Express OATP1B1, 1B3, 2B1 (consider 1A2).Consider expressing rat or mouse Oatps (why? Species differences).

Determine Km and Vmax(use a standardized transporter model system. i.e. do not mix oocytes + Cell lines + vesicles etc)

Step 5:OATP1B1 C id PGEN i )

Step 3:Is uptake 1.5-fold or > than control?If no, OATPs may not be the relevant transporter(s)

OATP1B1- Consider PGEN in vivo study

OATPs: What to do if you are worried that your compound is an inhibitor

Step 1Consider labeled estradiol 17 B glucuronide as a

Step 4

Consider I/IC50 values.17-B glucuronide as a model substrate

Step 2

Express 1B1 and 1B3 (might consider other OATPs).

Step 3

Difficult to know when a clinical interaction study should be carried out.

Step 5If a clinical study is necessary•Rosuvastatin or pravastatin Step 3

Determine IC50. Include known positive control inhibitors (e.g. RIF).

pas substrates•OATP1B3 (mostly) telmisartan•PK of substrate +/- your drug could be done

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In vivo relevance

Focus on OATPs

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Loss of hepatic Oatp1b2

Rifampin continuous infusion

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Pravastatin continuous infusion

Low dose: 8 µg/hr

High dose: 32 µg/hr

Oatp KO mice data

DeGorter M and Kim RB, Clin Pharmacol Ther 2011

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Oat, Oct, and Mate1 KO mice

DeGorter M and Kim RB, Clin Pharmacol Ther 2011

OATPs and CNS Drug Entry?

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OATP1A2 is expressed at the level of the BBB

©Richard B. Kim M.D.Lee et al, J.Biol.Chem. 2005

OATP1A2

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OATPs and Breast Cancer

OATP1A2 is Expressed in Breast Cancer

Meyer zu Schwabedissen et al Cancer Res 2008

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OATP1A2 and PXR go together


Modulation of PXR alters OATP1A2 Expression


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PXR antagonist A792611 reduces PXR effect on OATP1A2 expression


OATP1A2 is regulated by PXR

0 100 200 300 400

pGL3 basic

-5120 to -6600

DMSO

A-792611

Rifampin

Rifampin & A-792611

Luciferase activity [%-of control]

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PXR, OATP and Breast Cancer Proliferation


OATPs and Colon Cancer

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Expression of OATP1B3 in Colon Cancer

Lee et al, Cancer Res., 2008

OATPs and Irinotecan (CPT‐11) Chemotherapy

• Topoisomerase I inhibitor used to treat pcolorectal cancer.

• Active drug, SN‐38 undergoes hepatic transport and metabolism.

• Emerging evidence suggest hepatic OATP1B1 d ff k dand 1B3 transporters affect SN‐38 uptake and

disposition.

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IrinotecanIrinotecan SNSN--38 (active drug)38 (active drug)Carboxy esterases

Irinotecan Disposition Pathway

BILEBILE

BLOODBLOOD

BCRPBCRP**

BILEBILE

OATP1B1OATP1B1** OATP1B3OATP1B3**

IrinotecanIrinotecan

SNSN--38 (active drug)38 (active drug)

UGT1A1UGT1A1**

HEPATOCYTEHEPATOCYTE

BLOODBLOOD

BILEBILEBILEBILEMDR1MDR1**

MRP2*MRP2*

MRP2MRP2**

UGT1A1UGT1A1**

SNSN--38G38G

CYP3A4, CYP3A4, CYP3A5CYP3A5**

MetabolitesMetabolites

OATPs and Statin Therapy

30 million Americans take them deveryday…..

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Case Study

• 52 yr old female, renal failure secondary to polycystic kidney disease, underwent renal transplant in 1996transplant in 1996.

• Presents in May 1999 complaining of myalgia and muscle weakness.

• From Dec 1996 to Nov 1998, had been on simvastatin (10 mg/day) without any side effects.

• April 1999, switched to cerivastatin 0.1 mg/day.April 1999, switched to cerivastatin 0.1 mg/day. Also on cyclosporine, mycophenolate, prednisone, and ranitidine.

Rodriguez et al Ann Int Med 132:598, 2000

Case Study

• On admission, CK 12,615 U/L.

• Cerivastatin discontinued.

• CK level normalizes in 10 days.

Rodriguez et al Ann Int Med 132:598, 2000

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In vivo human relevance

Functional Polymorphisms in OATP1B1 (OATP‐C)


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200200

mL)

mL) 2

OATP1B1 GenotypeOATP1B1 Genotype

Impact of OATP1B1 Haplotypes to Pravastatin Pharmacokinetics

100100

150150

vast

atin

AU

C (

ng•h

r/m

vast

atin

AU

C (

ng•h

r/m

25

34 29

1

8

8

AA TT

GG TT

AA CC

388388 521521

*1b*1b

*1a*1a

*5*5GG CC

*15*15

00

5050

Pra

vP

rav 1

AUCAUC 6767 9090 8787 5959 100100 121*121* 167*167*

Ho RH et al., Pharmacogenetics Genomics 17:647-656, 2007

Genetics and Statin‐Induced Muscle Injury


Aug, 2008

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The genetic defect turned out to be one my group had first identified in 2001


Aug, 2008

What about clinical relevance?

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Patients

Our Group’s Approach

Blood sample

Drugs known to have narrow

Therapeutic index or variable efficacy

DetermineDrug level

DetermineGenotype

Individualize Drug selection and dosing


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We have a Personalized Statin Clinic


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Tamoxifen

CYP2D6 has a major impact to tamoxifen therapy

Schroth et al JAMA 302:1429‐1436, 2009

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Drug level and genetic testing at our clinic

Collect blood Extract DNAObtain PlasmaCollect blood Extract DNAObtain Plasma

GenotypingDrug Level Analysis

CYP2D6 and Clinical Relevance

Breast Cancer Therapy

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Case

• 60 year old female, dx (R) breast cancer Aug 2008.

ER/PR iti• ER/PR positive

• Chemo: FEC‐D

• Tried aromatase inhibitor but discontinued due to side effects (arthralgia)

• Tamoxifen started April 2010

• Referred to Personalized Tamoxifen Clinic at UH

Current medications

• insulin 30/70 46 units BID

• furosemide 40 mg once a dayg y

• enalapril 5 mg once a day

• bupropion 150 mg once a day

• Celebrex 200 mg once a day

• potassium supplement 600 mg once a day

• metformin 500 mg BID

• aspirin 81 mg once a day

• Lipitor 10 mg once a day

• calcium and vitamin D

• Ativan 1 mg QHS

• Mirapex 0.25 mg in the evening for restless leg syndrome

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Genotype and drug levels

• Date of Blood Sample: August 4, 2010

– CYP2D6 genotype: *1/*1– CYP2D6 genotype: 1/ 1

– Tamoxifen level: 247 ng/ml (667 nM)

– Endoxifen level: 3.85 ng/ml (10.3 nM)

• How do you interpret the genotype?

• Should you stay on tamoxifen?

CYP2D6 genotype and Endoxifen levelsCYP2D6 inhibitors:

Minimal:SSRI

venlafaxinevenlafaxine(Effexor)

Weak:SSRI

sertraline(Zoloft)

citalopram(Celexa)

Strong:SSRI

paroxetine(Paxil)

fluoxetine(Prozac)

buproprion (Wellbutrin, Zyban)

Borges et al. 2006. Clin. Pharm. Ther. 80: 61

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What have we learned from our clinic?

Tamo ifenTamoxifen Endoxifen

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How is endoxifen eliminated from the body??

Drug Transporters

P‐Glycoprotein

• ATP‐dependent efflux pump encoded by MDR1 gene in humanshumans.

• Can actively extrude various anticancer agents from intracellular to extracellular compartment, thus preventing cell death.

• Expression in some cancer cells• Expression in some cancer cells result in the phenomenon of multidrug resistance (MDR).

Kartner N and Ling V, Sci Am1989;260(3):44-51

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P‐glycoprotein (MDR1) Expression

PGP

Blood-brainbarrier

PGP

PGP

Liver bileducts

Small

Kidneytubules

OvariesPGP

PGP

PGP

PGP

PGP

Smallintestine

Testes

Placenta

P‐glycoprotein and Drug Interactions

• Substrates • Inhibitors– Digoxin

– Fexofenadine (Allegra)

– Cyclosporine

– Tacrolimus

– Quinidine

– Verapamil (but not

Inhibitors– Quinidine

– Verapamil

– Ketoconazole

– Itraconazole

– Cyclosporine

– HIV protease inhibitors– Verapamil (but not nifedipine or felodipine)

– Taxol

– HIV protease inhibitors

– Erythromycin

– Erythrmycin

– Clarithromycin

• Inducers

– Rifampin– St John’s wort

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P-gp: Identifying Drug SubstratesL-MDR1 Cells Top view (XY Plane)

Apical compartment

Side view (XZ Plane)

Cells

} P-gp {Basal Compartment

Exdoxifen is a P‐gp substrate

A B

CD

Teft W et al, Drug Metab Disp (2011)

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Endoxifen Plasma Level in P‐gp wildtype and deficient mice

60

20

40

60 WT

Mdr1adefo

xife

n p

lasm

a co

nc

(ng

/ml)

0 1 2 3 40

Time (h)

En

do


Brain endoxifen levels are far higher in P‐gp deficient mice

300)

100

200

300***

en b

rain

co

nc

(ng

/g)

WT Mdr1adef0E

nd

oxi

fe


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Conclusion• Certain transporters have very broad substrate specificity.

• Drug uptake transporters should be considered• Drug uptake transporters should be considered earlier in the drug development process

• Many drug transporters exhibit tissue specific expression.

• Genetic variation in transporters exist.• Drug transporters may be important to drug disposition and diseasedisposition and disease.

• Drug transporters could be targeted to enhance drug delivery

• Drug transporters are relevant to optimal drug therapy

determinants of drug disposition - drug metabolismscdmdg.org/slides/20110419-kim.pdf · schroth et...

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