transporters 2015/11/04 jun min jung. overview membrane transporters increase the influx and efflux...
TRANSCRIPT
Transporters
2015/11/04
Jun Min Jung
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.
Transporters as Drug Targets
• Membrane transporters (T) play roles in pharmacokinetic pathways (drug absorption, distribution, metabolism, and ex-cretion), thereby setting systemic drug levels.
• 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
Transporter Fundamentals
Basic Mechanisms of Transporter
• Light blue circles depict the substrate. Arrows show the direction of flux. • Dark blue ovals depict transport proteins
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.
Examples of Drugs with Active Uptake Transport
혈압약
고혈압 , 심부전
페니실린
페니실린
파킨스병
강압제 (고혈압 )
항암제
진통제 , 해열제
고지혈증항히스타민
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
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)
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)
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.
Various Transporters
Intestinal epithelium
Absorption direction = Drug Concentration IncreaseSecretion direction = Drug Concentration Decrease
Liver Hepatocytes
Uptake (Ex-cretion)
Efflux(Retention recirculation)
Clearance
Kidney Epithelial Cells
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)
Efflux Transporters
• Facilitate the export of compounds from the cell
• Belong to the ATP-binding cassette (ABC) family
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
• 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)
• 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.
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
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.
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
• 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
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)
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
Case Study of Pgp Efflux
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
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
Structure Modification Strategies to Reduce Pgp Efflux
Structure Modification Strategies to Reduce Pgp Efflux
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)
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
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)
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)
Q&A
A,B,C,D
B,D
A,B,C,D
Thank You