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Transporters 2015/11/04 Jun Min Jung

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Page 1: Transporters 2015/11/04 Jun Min Jung. Overview Membrane transporters increase the influx and efflux of substrate compounds Transporters are found in many

Transporters

2015/11/04

Jun Min Jung

Page 2: Transporters 2015/11/04 Jun Min Jung. Overview Membrane transporters increase the influx and efflux of substrate compounds Transporters are found in many

Overview

• Membrane transporters increase the influx and efflux of sub-

strate compounds

• Transporters are found in many tissues in vivo

• P-glycoprotein efflux in the blood-brain barrier, cancer cells,

and intestine is important for some compounds.

Page 3: Transporters 2015/11/04 Jun Min Jung. Overview Membrane transporters increase the influx and efflux of substrate compounds Transporters are found in many

Transporters as Drug Targets

• Membrane transporters (T) play roles in pharmacokinetic pathways (drug absorption, distribution, metabolism, and ex-cretion), thereby setting systemic drug levels.

Page 4: Transporters 2015/11/04 Jun Min Jung. Overview Membrane transporters increase the influx and efflux of substrate compounds Transporters are found in many

• Many important nutrients, including glucose and amino acids, are water soluble

• Transporter proteins enable water soluble nutrients to cross the lipid double membrane and enter the cell

• Transporters can be divided into pumps (ATP binding cassette) which consume energy by hydrolyzing ATP, and carriers which facil-itate the diffusion of solutes without use of ATP

Page 5: Transporters 2015/11/04 Jun Min Jung. Overview Membrane transporters increase the influx and efflux of substrate compounds Transporters are found in many

Transporter Fundamentals

Page 6: Transporters 2015/11/04 Jun Min Jung. Overview Membrane transporters increase the influx and efflux of substrate compounds Transporters are found in many

Basic Mechanisms of Transporter

• Light blue circles depict the substrate. Arrows show the direction of flux. • Dark blue ovals depict transport proteins

Page 7: Transporters 2015/11/04 Jun Min Jung. Overview Membrane transporters increase the influx and efflux of substrate compounds Transporters are found in many

Transporter Effects

• Uptake transporters enhance the absorption of some drug mol-

ecules in the intestine.

• Transporters assist the uptake of some molecules into hepato-

cytes to enhance metabolic ad biliary clearance.

• Elimination of many drugs and metabolites is enhanced by ac-

tive secretion in the nephrons of the kidney.

Page 8: Transporters 2015/11/04 Jun Min Jung. Overview Membrane transporters increase the influx and efflux of substrate compounds Transporters are found in many

Examples of Drugs with Active Uptake Transport

혈압약

고혈압 , 심부전

페니실린

페니실린

파킨스병

강압제 (고혈압 )

항암제

진통제 , 해열제

고지혈증항히스타민

Page 9: Transporters 2015/11/04 Jun Min Jung. Overview Membrane transporters increase the influx and efflux of substrate compounds Transporters are found in many

Kidney Basolateral Transport: OCT1, OCT2, OAT1, OAT2, OAT3, MRP1

Kidney Apical Transport: Secretion: P-gp, OAT4 Reabsorption: PEPT2

Blood-Brain BarrierP-gp (MDR1), OAT3, OATP-A, MRP1, MRP3

Liver Sinusoidal Transport Uptake:OCT1,OATP-C, OATP-B, OATP8, NTCP,OAT2Secretion: MRP1, MRP3

Liver Canalicular (Biliary) Transport Secretion: P-gp, MRP2, BCRP, BSEP, MDR3

Intestinal Lumen Ab-sorption: PEPT1 Secre-tion: P-gp, OATP3

Transporter Expression

Page 10: Transporters 2015/11/04 Jun Min Jung. Overview Membrane transporters increase the influx and efflux of substrate compounds Transporters are found in many

Gene Aliases Tissue Drug Substrate InhibitorABCB1 P-gp, MDR1 intestine, liver, kidney, brain,

placenta, adrenal, testesdigoxin, fexofenadine,

indinavir, vincristine, colchicine, topotecan,

paclitaxel

ritonavir, cyclosporine,verapamil, erythromycin, ketocoan-

zole, itraconazole, quinidine, elacridar (GF120918)

LY335979, valspodar (PSC833)

ABCB4 MDR3 liver digoxin, paclitaxel,Vinblastine

ABCB11 BSEP liver vinblastineABCC1 MRP1 intestine, liver, kidney, brain adefovir, indinavir

ABCC2 MRP2,CMOAT

intestine, liver, kidney, brain indinavir, cisplatin, cyclosporine

ABCC3 MRP3,CMOAT2

intestine, liver, kidney,placenta, adrenal

etoposide, methotrexate,tenoposide

ABCC4 MRP4ABCC5 MRP5ABCC6 MRP6 liver, kidney cisplatin, daunorubicinABCG2 BCRP intestine, liver, breast,

placentadaunorubicin, doxorubicin,

topotecan, rosuvastatin, sulfasalazine

elacridar (GF120918),gefitinib

Major Human Transporters (ABC)

Page 11: Transporters 2015/11/04 Jun Min Jung. Overview Membrane transporters increase the influx and efflux of substrate compounds Transporters are found in many

Gene Aliases Tissue Drug Substrate Inhibitor

SLCO1B1 OATP1B1, OATP-COATP2

liver rifampin, rosuvastatin, methotrexate,pravastatin, thyroxine

cyclosporine, rifampin

SLCO1B3 OATP1B3, OATP8, liver digoxin, methotrexate, rifampin,

SLCO2B1 SLC21A9, OATP-B intestine, liver, kidney, brain pravastatin

SLC10A1 NTCP liver, pancreas rosuvastatin

SLC10A2 ASBT ileum, kidney, biliary tract

SLC15A1 PEPT1 intestine, kidney ampicillin, amoxicillin, captopril,valacyclovir

SLC15A2 PEPT2 kidney ampicillin, amoxicillin, captopril,Valacyclovir

SLC22A1 OCT1 liver acyclovir, amantadine, desipramine,ganciclovir, metformin

disopyramide, midazolam,phenformin, phenoxy-benzamine

quinidine, ritonavir, verapamil

SLC22A2 OCT2 kidney, brain amantadine,cimetidine, memantine

desipramine, phenoxy-benzamine,quinine

SLC22A3 OCT3 skeletal muscle, liver, placenta,kidney, heart

cimetidine desipramine, prazosin, phenoxy-benzamine

SLC22A4 OCTN1 kidney, skeletal muscle,placenta, prostate, heart

quinidine, verapamil

SLC22A5 OCTN2 kidney, skeletal muscle,prostate, lung, pancreas, heart,

small intestine, liver

quinidine, verapamil

SLC22A6 OAT1 kidney, brain acyclovir, adefovir,methotrexate, zidovudine

probenecid, cefadroxil,cefamandole, cefazolin,

SLC22A7 OAT2 liver, kidney zidovudine

SLC22A8 OAT3 kidney, brain cimetidine, methotrexate, zidovudine probenecid, cefadroxil,cefamandole, cefazolin,

Major Human Transporters (SLC)

Page 12: Transporters 2015/11/04 Jun Min Jung. Overview Membrane transporters increase the influx and efflux of substrate compounds Transporters are found in many

Drug-Drug Interactions Due to Transporters

• Currently considered most important transporters for Drug-

Drug Interactions (DDI):

– ABC: P-gp, BCRP, BSEP

– SLC: OATP1B1, OATP1B3 , OATP2B1, OCT1, OCT3, OAT2

• Currently considered less important:

– MRP transporters

• Depending on the expression pattern of the affected transporter, DDI

can result in changes to absorption, tissue distribution (CNS,

tumors), or elimination of the victim drug.

Page 13: Transporters 2015/11/04 Jun Min Jung. Overview Membrane transporters increase the influx and efflux of substrate compounds Transporters are found in many

Various Transporters

Page 14: Transporters 2015/11/04 Jun Min Jung. Overview Membrane transporters increase the influx and efflux of substrate compounds Transporters are found in many

Intestinal epithelium

Absorption direction = Drug Concentration IncreaseSecretion direction = Drug Concentration Decrease

Page 15: Transporters 2015/11/04 Jun Min Jung. Overview Membrane transporters increase the influx and efflux of substrate compounds Transporters are found in many

Liver Hepatocytes

Uptake (Ex-cretion)

Efflux(Retention recirculation)

Clearance

Page 16: Transporters 2015/11/04 Jun Min Jung. Overview Membrane transporters increase the influx and efflux of substrate compounds Transporters are found in many

Kidney Epithelial Cells

Page 17: Transporters 2015/11/04 Jun Min Jung. Overview Membrane transporters increase the influx and efflux of substrate compounds Transporters are found in many

Blood-Brain Barrier Cells

Efflux (from the BBB endothelial cells back into the blood) Uptake (from the blood, through the BBB endothelial cells and into the brain)

Page 18: Transporters 2015/11/04 Jun Min Jung. Overview Membrane transporters increase the influx and efflux of substrate compounds Transporters are found in many

Efflux Transporters

• Facilitate the export of compounds from the cell

• Belong to the ATP-binding cassette (ABC) family

Page 19: Transporters 2015/11/04 Jun Min Jung. Overview Membrane transporters increase the influx and efflux of substrate compounds Transporters are found in many

P-glycoprotein (Pgp)

• 170KD protein with 1280 amino acids & 12 trans-membrane segments

• Member of the ATP Binding Cassette family of transporters

• Gene known as MDR1 or ABCB1

Page 20: Transporters 2015/11/04 Jun Min Jung. Overview Membrane transporters increase the influx and efflux of substrate compounds Transporters are found in many

• 170KD protein with 1280 amino acids

• Member of the ATP Binding Cassette family of transporters

• Gene known as MDR1 or ABCB1

Positions of mutations that alter the substrate speci-

ficity of P-gp

The N-linked glycosylation sites (N91, N94, and N99)

Phosphorylation sites (S661, S667, S671, and S683)

Page 21: Transporters 2015/11/04 Jun Min Jung. Overview Membrane transporters increase the influx and efflux of substrate compounds Transporters are found in many

• Initially identified as a major cause of resistance by cancer cells to multiple drugs (e.g., paclitaxel, etoposide) having a variety of structures.

• It is present in many tissues of the body.

• Pgp is abundant in cell barriers that have a protective function blood-brain barrier, small and large intestine liver, kidney, adrenal gland, pregnant uterus.

• In the liver and kidney, Pgp enhances drug and metabolite clearance to the bile and urine, respec-tively.

• Pgp attenuates penetration of some compounds into the brain, uterus, testes and other tissues.

Page 22: Transporters 2015/11/04 Jun Min Jung. Overview Membrane transporters increase the influx and efflux of substrate compounds Transporters are found in many

P-glycoprotein (Pgp)

• A drug molecule attaches to the binding domain of pgp

• 2 ATPs bound to the ATP binding regions, become hydrolyzed and induce

conformational change to open pathway for the drug molecule to pass

through into the extracellular fluid

Page 23: Transporters 2015/11/04 Jun Min Jung. Overview Membrane transporters increase the influx and efflux of substrate compounds Transporters are found in many

Rules for Pgp Efflux Substrates

More likely to be a Pgp substrate More likely to be a Pgp non-substrate

Increasing numbers of hydrogen bond acceptors (N+O) ap-

pear to confer increasing likelihood of Pgp efflux.

This may be because binding to Pgp occurs in the lipophilic

membrane region.

Page 24: Transporters 2015/11/04 Jun Min Jung. Overview Membrane transporters increase the influx and efflux of substrate compounds Transporters are found in many

Models to Study Pgp Interactions

Assay Type Tissues Parameters CommentsBi-DirectionalTransport

Caco-2 cellsMDCK-MDR1 cells LLC-PK1-MDR1cells

Net drug flux ratio ofB to A and A to B

Directly measure efflux across cellbarrier

Evaluation of P-gp transport and in-hibition

Allow for localization/identification of the transporters within the apical or ba-solateral side of the membrane

Uptake/efflux tumor cellscDNA transfected cells oocytes injected with cRNA of transporters

Inhibition of uptake or efflux offluorescent probe(Calcein-AM, rhodamine-123)

Cannot distinguish substrate frominhibitorTends to fail to identify substrate and/or inhibitor with low permeability

ATPase membrane vesicles fromtissues or cells expressing P gp, Reconstituted P-gp

ATPase stimulation Same comments as uptake/effluxassay

Page 25: Transporters 2015/11/04 Jun Min Jung. Overview Membrane transporters increase the influx and efflux of substrate compounds Transporters are found in many

• Cell monolayers grown on filters and placed in cluster plates

• Filters are typically PET or PC membranes with 0.4-1 μm pores

• Transport is measured in two directions:

–Apical (A) to Basolateral (B), i.e. test compound added to apical side

–Basolateral (B) to Apical (A), i.e. test compound added to basolateral side

Drug

= P-gp-mediated transport

= Passive diffusion

1

2

Filter membrane

Cell monolayer

ATP

A(apical)

B(basolateral)

1 2

Bi-directional Pgp Transport Assays

Page 26: Transporters 2015/11/04 Jun Min Jung. Overview Membrane transporters increase the influx and efflux of substrate compounds Transporters are found in many

Cell Lines for Pgp Transport Assay

• Cells used for bi-directional transport studies should form a functionally po-larized cell monolayer, complete with tight junctions (verified by pre-experi-mental TEER of 100-800 Ω·cm2)

• At present, preferred cells lines include

– Caco-2 cells

– MDR1 transfected LLC-PK1 cells

– MDR1 transfected MDCK cells

• LLC-PK1 and MDCK wild type cells should be used as negative controls

• Cells should be allowed to grow to confluence (typically 3-7 days for LLC-PK1

or MDCK, 18-21 days for Caco-2, however accelerated 3-5 day Caco-2 models are available and produce similar results)

Page 27: Transporters 2015/11/04 Jun Min Jung. Overview Membrane transporters increase the influx and efflux of substrate compounds Transporters are found in many

Cell Lines for Pgp Transport Assay

• Caco-2– Human colon carcinoma cell line

– Morphologically similar to small intestinal epithelial cells

– Most extensively characterized human cell-based model for investigating permeability and Pgp transport of drugs

– Various uptake and efflux transporters are expressed in Caco-2 cells, however, Pgp is functionally the most predominant

– No wild-type cells to run alongside

• LLC-PK1-MDR1

– Transfected porcine kidney cell line

– Low transporter background, especially for Pgp

• MDCK-MDR1– Transfected canine kidney cell line

– High background dog Pgp activity

Page 28: Transporters 2015/11/04 Jun Min Jung. Overview Membrane transporters increase the influx and efflux of substrate compounds Transporters are found in many

Case Study of Pgp Efflux

Page 29: Transporters 2015/11/04 Jun Min Jung. Overview Membrane transporters increase the influx and efflux of substrate compounds Transporters are found in many

Apparent Permeability (Papp)

Papp [cm/sec] = Vr/C0 x 1/S x dC/dtVr

C0

S

dC/dt

is the volume in the receiver chamber [cm3]

is the concentration in the donor chamber at t=0

is the filter surface area [cm2]

is the is the linear slope of the drug concentration in the receiver chamber with time after correcting for dilution [mM/sec]

Efflux Ratio (RE)

RE = Papp (B to A) / Papp (A to B)

Papp (B-A) is the Papp value measured in the B to A direction

Papp (A-B) is the Papp value measured in the A to B direction

Calculation of Papp and Efflux Ratios

Page 30: Transporters 2015/11/04 Jun Min Jung. Overview Membrane transporters increase the influx and efflux of substrate compounds Transporters are found in many

Structure Modification Strategies to Reduce Pgp Efflux

• Introduce steric hindrance to the hydrogen bond donating

atoms

• Attach a bulky group

• Methylate the nitrogen

• Decrease H-bond acceptor potential

• Add an adjacent electron withdrawing group

• Replace or remove the hydrogen bonding group

• Modify the overall structure’s Log P to reduce penetration into the lipid bi-

layer

Page 31: Transporters 2015/11/04 Jun Min Jung. Overview Membrane transporters increase the influx and efflux of substrate compounds Transporters are found in many

Structure Modification Strategies to Reduce Pgp Efflux

Page 32: Transporters 2015/11/04 Jun Min Jung. Overview Membrane transporters increase the influx and efflux of substrate compounds Transporters are found in many

Structure Modification Strategies to Reduce Pgp Efflux

Page 33: Transporters 2015/11/04 Jun Min Jung. Overview Membrane transporters increase the influx and efflux of substrate compounds Transporters are found in many

Bidirectional transport assayBidirectional transport assay

In vivo drug interaction study with a P-gp inhibitor may be warranted

In vivo drug interaction study with a Pgp inhibitor may be warranted

Is efflux ratio ≥ 2 ? *Is efflux ratio ≥ 2 ? *

Likely to be P-gp substrateLikely to be Pgp substrate

Unlikely to be P-gp substrateUnlikely to be Pgp substrate

YES NO

Is efflux inhibited by P-gp inhibitors? **Is efflux inhibited by Pgp inhibitors? **

Is efflux ratio < 2 ? *Is efflux ratio < 2 ? *

Transporters other than P-gp might be involved

Transporters other than Pgp might be involved

Further in vitro studies to identify transporters may be warranted

Further in vitro studies to identify transporters may be warranted

Decision Tree for Pgp Substrates

FDA DRAFT Guidance for Industry (Sept 2006)

Page 34: Transporters 2015/11/04 Jun Min Jung. Overview Membrane transporters increase the influx and efflux of substrate compounds Transporters are found in many

FDA DRAFT Guidance for Industry (Sept 2006)

Bidirectional transport assay with P-gp probe substrate

Bidirectional transport assay with P-gp probe substrate

In vivo drug interaction study with a P-gp probe substrate (e.g. digoxin) is

recommended

In vivo drug interaction study with a Pgp probe substrate (e.g. digoxin) is

recommended

Net flux ratio of probe substrate decreases with increasing concen-

trations of test compound

Net flux ratio of probe substrate decreases with increasing concen-

trations of test compound

[I]/IC50 > 0.1[I]/IC50 > 0.1

Poor or non-inhibitor of P-gpPoor or non-inhibitor of Pgp

Determine IC50 (or Ki)Determine IC50 (or Ki)

Net flux ratio of probe substrate is not affected by increasing concen-

trations of test compound

Net flux ratio of probe substrate is not affected by increasing concen-

trations of test compound

[I]/IC50 < 0.1[I]/IC50 < 0.1

In vivo drug interaction study with a P-gp probe substrate is

not needed

In vivo drug interaction study with a Pgp probe substrate is

not needed

P-gp inhibitorPgp inhibitor

Decision Tree for Pgp Inhibitors

Page 35: Transporters 2015/11/04 Jun Min Jung. Overview Membrane transporters increase the influx and efflux of substrate compounds Transporters are found in many

Uptake Transporters

• Organic Anion Transporting Polypeptides (OATPs, SLCOs)

• OATP1B1

• Expressed in liver

• Rifampin, Pravastatin, Rosuvastatin, Cerivastatin, Benzylpenicillin

• OATP1A2

• Found in BBB, Hepatocytes, Renal epithelium

• Fexofenadine, Enalapril, Temocaprilat, N-methyl quinidine

• Di/Tri Peptide Transporters (PEPT1, PEPT2)

• Organic Anion Transporters (OATs)

• Organic Cation Transporters (OCT)

• Large Neutral Amino Acid Transporter (LAT1)

• Monocarboxylic Acid Transporter (MCT1)

Page 36: Transporters 2015/11/04 Jun Min Jung. Overview Membrane transporters increase the influx and efflux of substrate compounds Transporters are found in many

Other Uptake Transporters

• Glucose Transporter (GLUT1)

• Bile Salt Export Pump (BSEP, ABCB11)

• Sodium Dependent Taurocholate Co-transporting Polypeptide (NCTP)

• Uptake transporters in the BBB

• CAT1 (Cationic amino acids)

• CNT2 (Nucleosides)

• CHT (Choline)

• NBT (Nucleobase)

Page 37: Transporters 2015/11/04 Jun Min Jung. Overview Membrane transporters increase the influx and efflux of substrate compounds Transporters are found in many

Q&A

A,B,C,D

B,D

A,B,C,D

Page 38: Transporters 2015/11/04 Jun Min Jung. Overview Membrane transporters increase the influx and efflux of substrate compounds Transporters are found in many

Thank You