Transcript Transport

Transporters
2015/11/04
Jun Min Jung
Overview
• Membrane transporters increase the influx and efflux of
substrate 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
excretion), 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 facilitate 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
molecules in the intestine.
• Transporters assist the uptake of some molecules into
hepatocytes to enhance metabolic ad biliary clearance.
• Elimination of many drugs and metabolites is enhanced by
active secretion in the nephrons of the kidney.
Examples of Drugs with Active Uptake Transport
혈압약
파킨스병
고혈압, 심부전
강압제(고혈압)
페니실린
페니실린
항암제
진통제, 해열제
고지혈증
항히스타민
Transporter Expression
Liver Sinusoidal
Transport Uptake:
OCT1,
OATP-C, OATP-B, OATP8,
NTCP,
OAT2
Secretion: MRP1, MRP3
Intestinal Lumen Abs
orption: PEPT1 Secreti
on: P-gp, OATP3
Blood-Brain Barrier
P-gp (MDR1), OAT3, OATP-A, MRP1
, MRP3
Liver Canalicular (Biliary) Transport
Secretion: P-gp, MRP2, BCRP, BSEP,
MDR3
Kidney Basolateral Transport: O
CT1, OCT2, OAT1, OAT2, OAT3,
MRP1
Kidney Apical Transport:
Secretion: P-gp, OAT4 Re
absorption: PEPT2
Major Human Transporters (ABC)
Gene
ABCB1
Aliases
P-gp, MDR1
ABCB4
MDR3
liver
ABCB11
ABCC1
BSEP
MRP1
liver
intestine, liver, kidney, brain
digoxin, paclitaxel,
Vinblastine
vinblastine
adefovir, indinavir
ABCC2
MRP2,
CMOAT
MRP3,
CMOAT2
MRP4
MRP5
MRP6
BCRP
intestine, liver, kidney, brain
indinavir, cisplatin,
intestine, liver, kidney,
placenta, adrenal
etoposide, methotrexate,
tenoposide
liver, kidney
intestine, liver, breast,
placenta
cisplatin, daunorubicin
daunorubicin, doxorubicin,
topotecan, rosuvastatin,
sulfasalazine
ABCC3
ABCC4
ABCC5
ABCC6
ABCG2
Tissue
Drug Substrate
intestine, liver, kidney, brain,
digoxin, fexofenadine,
indinavir, vincristine,
placenta, adrenal, testes
colchicine, topotecan,
paclitaxel
Inhibitor
ritonavir, cyclosporine,
verapamil, erythromycin, ketocoanzole
, itraconazole, quinidine, elacridar (GF
120918)
LY335979, valspodar (PSC833)
cyclosporine
elacridar (GF120918),
gefitinib
Major Human Transporters (SLC)
Gene
SLCO1B1
Aliases
OATP1B1, OATP-C
OATP2
Tissue
liver
Drug Substrate
rifampin, rosuvastatin, methotrexate,
pravastatin, thyroxine
SLCO1B3
SLCO2B1
SLC10A1
SLC10A2
SLC15A1
OATP1B3, OATP8,
SLC21A9, OATP-B
NTCP
ASBT
PEPT1
liver
intestine, liver, kidney, brain
liver, pancreas
ileum, kidney, biliary tract
intestine, kidney
digoxin, methotrexate, rifampin,
pravastatin
rosuvastatin
SLC15A2
PEPT2
kidney
ampicillin, amoxicillin, captopril,
Valacyclovir
SLC22A1
OCT1
liver
acyclovir, amantadine, desipramine,
ganciclovir, metformin
SLC22A2
OCT2
kidney, brain
amantadine,
cimetidine, memantine
desipramine, phenoxy-benzamine,
quinine
SLC22A3
OCT3
skeletal muscle, liver, placenta,
kidney, heart
cimetidine
desipramine, prazosin, phenoxybenzamine
SLC22A4
OCTN1
kidney, skeletal muscle,
placenta, prostate, heart
quinidine, verapamil
SLC22A5
OCTN2
SLC22A6
OAT1
kidney, brain
acyclovir, adefovir,
methotrexate, zidovudine
SLC22A7
SLC22A8
OAT2
OAT3
liver, kidney
kidney, brain
zidovudine
cimetidine, methotrexate, zidovudine
kidney, skeletal muscle,
prostate, lung, pancreas, heart, sm
all intestine, liver
Inhibitor
cyclosporine, rifampin
ampicillin, amoxicillin, captopril,
valacyclovir
disopyramide, midazolam,
phenformin, phenoxy-benzamine qu
inidine, ritonavir, verapamil
quinidine, verapamil
probenecid, cefadroxil,
cefamandole, cefazolin,
probenecid, cefadroxil,
cefamandole, cefazolin,
Drug-Drug Interactions Due to Transporters
•
Currently considered most important transporters for DrugDrug 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 Increase
Secretion direction = Drug Concentration Decrease
Liver Hepatocytes
Uptake
(Excretion)
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
The N-linked glycosylation
sites (N91, N94, and N99)
Positions of mutations that
alter the substrate
specificity of P-gp
Phosphorylation
sites (S661, S667,
S671, and S683)
• 170KD protein with 1280 amino acids
• Member of the ATP Binding Cassette family of transporters
• Gene known as MDR1 or ABCB1
• 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,
respectively.
• 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)
appear 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
Bi-Directional
Transport
Tissues
Caco-2 cells
MDCK-MDR1 cells L
LC-PK1-MDR1cells
Parameters
Net drug flux ratio of
B to A and A to B
Comments
Directly measure efflux across cell
barrier
Evaluation of P-gp transport and inh
ibition
Allow for localization/identification of t
he transporters within the apical or basol
ateral side of the membrane
Uptake/efflux
tumor cells
Inhibition of uptake or efflux of
cDNA transfected cells oocyte fluorescent probe
s injected with cRNA of transp (Calcein-AM, rhodamine-123)
orters
ATPase
membrane vesicles from
ATPase stimulation
tissues or cells expressing P gp,
Reconstituted P-gp
Cannot distinguish substrate from
inhibitor
Tends to fail to identify substrate and/or
inhibitor with low permeability
Same comments as uptake/efflux
assay
Bi-directional Pgp Transport Assays
• 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
1 = P-gp-mediated transport
2 = Passive diffusion
A
(apical)
B
(basolateral)
1
ATP
2
Cell monolayer
Filter membrane
Cell Lines for Pgp Transport Assay
•
Cells used for bi-directional transport studies should form a functionally polar
ized cell monolayer, complete with tight junctions (verified by pre-experiment
al 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
Calculation of Papp and Efflux Ratios
Apparent Permeability (Papp)
Papp [cm/sec] = Vr/C0 x 1/S x dC/dt
Vr
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
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
bilayer
Structure Modification Strategies to Reduce Pgp Efflux
Structure Modification Strategies to Reduce Pgp Efflux
Decision Tree for Pgp Substrates
Bidirectional
Bidirectional transport
transportassay
assay
IsIsefflux
effluxratio
ratio≥≥22??**
IsIsefflux
effluxratio
ratio<<22??**
IsIsefflux
effluxinhibited
inhibitedby
byPgp
P-gpinhibitors?
inhibitors?****
YES
Likely
Likelyto
tobe
bePgp
P-gpsubstrate
substrate
In
Invivo
vivodrug
druginteraction
interaction study
studywith
withaa
Pgp
P-gpinhibitor
inhibitormay
maybebewarranted
warranted
Unlikely
Unlikelyto
tobe
bePgp
P-gpsubstrate
substrate
NO
Transporters
Transporters other
otherthan
thanPgp
P-gpmim
ght
be
involved
ight be involved
Further
Further in
invitro
vitrostudies
studiesto
toidentify
identify tt
ransporters
ransportersmay
may be
bewarranted
warranted
FDA DRAFT Guidance for Industry (Sept 2006)
Decision Tree for Pgp Inhibitors
Bidirectional
Bidirectional transport
transport assay
assay wit
wit
hh P-gp
P-gp probe
probesubstrate
substrate
Net
Netflux
fluxratio
ratioof
of probe
probe substrate
substrate de
de
creases
with
increasing
concentrati
creases with increasing concentrati
ons
ons of
of test
testcompound
compound
Pgp
P-gpinhibitor
inhibitor
Net
Net flux
flux ratio
ratio of
of probe
probe substrate
substrate isis nn
ot
affected
by
increasing
ot affected by increasing concentrati
concentrati
ons
of
test
compound
ons of test compound
Poor
Pooror
ornon-inhibitor
non-inhibitor of
ofPgp
P-gp
Determine
DetermineIC
IC5050(or
(or KKi)i)
[I]/IC
[I]/IC5050>> 0.1
0.1
[I]/IC
[I]/IC5050<< 0.1
0.1
In
In vivo
vivodrug
drug interaction
interaction study
studywith
withaa
Pgp
P-gpprobe
probesubstrate
substrate(e.g.
(e.g.digoxin)
digoxin)isisr
ecommended
recommended
In
In vivo
vivodrug
drug interaction
interaction study
study w
w
ith
ith aa Pgp
P-gpprobe
probesubstrate
substrateisis not
not
needed
needed
FDA DRAFT Guidance for Industry (Sept 2006)
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