Download Strategies for in vitro Transporter Testing and

Strategies for in vitro Transporter Testing
and Alignment with FDA Guidance
Elke S. Perloff, Ph.D.
BD Biosciences – Discovery Labware
www.bdbiosciences.com/admetox
Presentation Overview
• Guidance documents and position papers
• Transporter expression and function
– Role in drug-drug interactions
– In vitro models to study transporter interactions
• Focus on assessment of P-glycoprotein (P-gp)
Interactions
– Identification of P-gp substrates and inhibitors
– Technical considerations (cell lines, positive controls,
assay conditions, parameter calculations)
– Decision trees
Guidance Documents and
Position Papers
Guidance and Position Papers
•
FDA DRAFT Guidance for Industry (Sept 2006)
Drug Interaction Studies – Study Design, Data Analysis and Implications for Dosing and
Labeling (http://www.fda.gov/cder/guidance/index.htm)
•
Tucker, et al. Optimizing drug development: strategies to assess drug metabolism/
transporter interaction potential – toward a consensus. Basel Conference 2001,
sponsored by FDA, EUFEPS, AAPS:
Clin. Pharmacol. Ther. 70:103-14 (2001)
Br. J. Clin. Pharmacol. 52:107-17 (2001)
Eur. J. Pharm. Sci. 13:417-28 (2001)
Pharm. Res. 18:1071-80 (2001)
•
Zhang, et al. Scientific Perspectives on Drug Transporters and Their Role in Drug
Interactions. Mol. Pharm. 3:62-69 (2006)
•
Zhang, et al. A Regulatory Viewpoint on Transporter-based Drug Interactions.
Xenobiotica 38:709-724 (2008)
•
Huang, et al. New Era in Drug Interaction Evaluation: US FDA Update on CYP Enzymes,
Transporters, and the Guidance Process. J. Clin. Pharmacol. 48:662-670 (2008)
FDA Guidance Documents
•
Over 400 draft or final guidance documents
http://www.fda.gov/cder/guidance/index.htm
•
Represent the Agency's current thinking
•
Do not bind the FDA or the public, but provide pharmaceutical
companies with assurance
– An alternative approach may be used if it satisfies requirements of any
applicable statutes, or regulations
– If in doubt, contact the originating office (e.g. CDER)
Transporter Expression and
Function
Role in drug-drug interactions
In vitro models to study transporter interactions
Transporters
• Membrane-bound proteins with asymmetric distribution
in polarized cells of various tissues
– e.g., intestinal enterocytes, hepatocytes, proximal tubules,
blood-brain barrier capillary endothelial cells
– Function as uptake and efflux pumps
– Transport a variety of solutes: nutrients, cellular by-products,
environmental toxins and drugs into and out of cells
• Estimate: > 400 human transporters
• Active (ATP-dependent, Na+ or H+ gradient driven)
or passive (concentration gradient driven) transport
mechanisms
Major Human Transporters (ABC)
Gene
ABCB1
Aliases
P-gp, MDR1
Tissue
Drug Substrate
intestine, liver, kidney, brain, digoxin, fexofenadine,
placenta, adrenal, testes
indinavir, vincristine,
colchicine, topotecan,
paclitaxel
ABCB4
MDR3
liver
ABCB11
ABCC1
BSEP
MRP1
ABCC2
MRP2,
CMOAT
MRP3,
CMOAT2
MRP4
MRP5
MRP6
BCRP
ABCC3
ABCC4
ABCC5
ABCC6
ABCG2
Inhibitor
ritonavir, cyclosporine,
verapamil, erythromycin,
ketocoanzole, itraconazole, quinidine,
elacridar (GF120918)
LY335979, valspodar (PSC833)
digoxin, paclitaxel,
vinblastine
liver
vinblastine
intestine, liver, kidney, brain adefovir, indinavir
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
FDA DRAFT Guidance for Industry (Sept 2006)
cyclosporine
elacridar (GF120918),
gefitinib
Major Human Transporters (SLC)
Gene
SLCO1B1
SLCO1B3
SLCO2B1
SLC10A1
SLC10A2
SLC15A1
Aliases
OATP1B1, OATP-C
OATP2
OATP1B3, OATP8,
SLC21A9, OATP-B
NTCP
ASBT
PEPT1
Tissue
liver
intestine, liver, kidney, brain
liver, pancreas
ileum, kidney, biliary tract
intestine, kidney
SLC15A2
PEPT2
kidney
SLC22A1
OCT1
liver
SLC22A2
OCT2
kidney, brain
SLC22A3
OCT3
SLC22A4
OCTN1
SLC22A5
OCTN2
SLC22A6
OAT1
SLC22A7
SLC22A8
OAT2
OAT3
liver
Drug Substrate
rifampin, rosuvastatin, methotrexate,
pravastatin, thyroxine
digoxin, methotrexate, rifampin,
pravastatin
rosuvastatin
ampicillin, amoxicillin, captopril,
valacyclovir
ampicillin, amoxicillin, captopril,
valacyclovir
acyclovir, amantadine, desipramine,
ganciclovir, metformin
amantadine,
cimetidine, memantine
skeletal muscle, liver, placenta, cimetidine
kidney, heart
kidney, skeletal muscle,
quinidine, verapamil
placenta, prostate, heart
kidney, skeletal muscle,
quinidine, verapamil
prostate, lung, pancreas, heart,
small intestine, liver
acyclovir, adefovir,
kidney, brain
methotrexate, zidovudine
liver, kidney
zidovudine
kidney, brain
cimetidine, methotrexate, zidovudine
FDA DRAFT Guidance for Industry (Sept 2006)
Inhibitor
cyclosporine, rifampin
disopyramide, midazolam,
phenformin, phenoxy-benzamine
quinidine, ritonavir, verapamil
desipramine, phenoxy-benzamine,
quinine
desipramine, prazosin, phenoxybenzamine
probenecid, cefadroxil,
cefamandole, cefazolin,
probenecid, cefadroxil,
cefamandole, cefazolin,
Transporter Expression
Liver Sinusoidal
Transport
Uptake:
OCT1,
OATP-C, OATP-B, OATP8,
NTCP,
OAT2
Secretion: MRP1, MRP3
Intestinal Lumen
Absorption: PEPT1
Secretion: 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:
OCT1, OCT2, OAT1, OAT2, OAT3,
MRP1
Kidney Apical Transport:
Secretion: P-gp, OAT4
Reabsorption: PEPT2
From: Zhang, L. et al, Mol Pharm. 3(1):62-69 (2006).
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
•
Most critical for drugs with a narrow therapeutic index (e.g., digoxin)
where co-administration of a transporter inhibitor might require dose
adjustment.
•
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.
Clinical Examples for Transporter-Related
Drug-Drug Interactions
• Cerivastatin (HMG-CoA reductase inhibitor)
– Substrate of hepatic uptake transporter OATP1B1
– Increased plasma concentrations with inhibition of OATP1B1 by co-administered
cyclosporine (JPET 304:610 [2003])
– (Withdrawn from market in 2001 after 52 deaths due to kidney failure)
• Digoxin (cardiac glycoside)
– Substrate of efflux transporter P-glycoprotein
– Elimination of unchanged digoxin is by renal and biliary excretion.
– AUC increased and renal clearance decreased with co-administration of P-gp inhibitor
itraconazole (Ther. Drug Monit. 10:609 [1997])
– AUC decreased with co-administration of P-gp inducer rifampin (J. Clin. Invest. 104:147
[1999]).
• Cephalosporin antibiotics
– Substrates of uptake transporter OAT
– Co-administration of OAT inhibitor probenecid resulted in decreased renal clearance,
increased Cmax and AUC (Clin. Pharmacokinet. 24:289 [1993]).
In Vitro Models for Transporter Interaction
In vitro Model
Description
Membranes from insect cells expressing
transporter cDNA
ATPase assay; Discovery screen; Indirect assay; does not
differentiate substrates/inhibitors
“Inside-out” Vesicles from insect cells
expressing transporter cDNA
Direct, functional uptake assay with vesicles to identify
substrates and inhibitors
Polarized cell monolayers
Caco-2
Direct, functional efflux assay; Substrate and/or inhibitor
testing; IC50, Ki determination
Transfected cell lines (LLC-PK1, MDCK)
Transfection of single (e.g. MDR1) or multiple transporters
Xenopus leavis oocytes expressing SLC
transporters (e.g. OAT, OATP, OCT,
NTCP, PEPT)
Direct, functional uptake assay; Substrate and/or inhibitor
screening; Affinity determination Km/Vmax, IC50, Ki
Hepatocytes
Fresh hepatocytes plated or in suspension
Transporter Characterized BD Gentest™
Cryopreserved Human Hepatocytes
(OATP, NTCP, OCT1)
Sandwich cultured human hepatocytes
Assess inter-individual variability
Direct, functional uptake assay; Substrate and/or inhibitor
screening; Affinity determination Km/Vmax, IC50, Ki
Assess biliary excretion
Assessment of P-gp Interactions
Identification of P-gp substrates and inhibitors
Technical considerations
Decision trees
FDA’s Current Thinking
•
“…in vitro studies can frequently serve as a screening mechanism
to rule out the importance of a metabolic pathway and the drugdrug interactions that occur through this pathway so that
subsequent in vivo testing is unnecessary.”
•
“...not every drug-drug interaction is metabolism-based, but may
arise from changes in pharmacokinetics caused by absorption,
distribution, and excretion interactions. Drug-drug interactions
related to transporters are being documented with increasing
frequency and are important to consider in drug development. “
•
“Of the various transporters, P-gp is the most well understood and
may be appropriate to evaluate during drug development.”
FDA DRAFT Guidance for Industry (Sept 2006)
P-gp
•
Apical side
(e.g. intestinal lumen)
P-glycoprotein (P-gp) commonly
refers to MDR1
– P-glycoprotein (MDR1) is encoded
by the ABCB1 gene.
– MDR3 (encoded by ABCB4) is
present in various human tissues,
but there is little evidence of a
major role in drug transport.
•
170 kDa glycoprotein
•
ATP-binding cassette transporter
Filaria Journal 2(Suppl 1):S8 (2003).
P-gp consists of 2 sections, each containing 6 transmembrane
domains and a large cytoplasmic domain with an ATP-binding site
P-gp (MDR1)
•
Mainly present on the apical side of epithelial cells:
–
–
–
–
brush border membrane of small intestine enterocytes
proximal tubule cells in the kidney
canalicular membrane of hepatocytes
blood brain barrier capillary endothelial cells
•
Efflux pump with broad substrate specificity. Substrates tend to be
large, lipophilic, positively charged or neutral molecules
•
Likely evolved as a defense mechanism against harmful substances
•
Modulation of P-gp activity and/or expression may affect oral
bioavailability, biliary and renal clearance, and tissue distribution
(e.g. tumors, placenta, CNS) of drugs potentially resulting in DDIs
after co-administration of drugs that interact with P-gp
Models to Study P-gp Interactions
Assay Type
Bi-Directional
Transport
Tissues
Caco-2 cells
MDCK-MDR1 cells
LLC-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
inhibition
Uptake/efflux
ATPase
Allow for localization/identification of
the transporters within the apical or
basolateral side of the membrane
Inhibition of uptake or efflux of Cannot distinguish substrate from
tumor cells
fluorescent probe
inhibitor
cDNA transfected cells
oocytes injected with cRNA (Calcein-AM, rhodamine-123) Tends to fail to identify substrate
and/or inhibitor with low permeability
of transporters
membrane vesicles from
ATPase stimulation
Same comments as uptake/efflux
tissues or cells expressing Passay
gp, Reconstituted P-gp
“The bi-directional transport assay is regarded as the definitive assay
for identifying P-gp substrates and inhibitors because it measures drug
efflux in a more direct manner than other methods.”
FDA DRAFT Guidance for Industry (Sept 2006)
Bi-directional P-gp 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
Materials and Equipment Needed
•
Filter plate system (available in 6-, 12-, 24-, 48-, and 96-well formats)
Example:
BD Falcon™ 24-Multiwell HTS Insert Plate
(1 µm PET) with feeder tray (for culture),
24-well cluster plate (for assay), and lid
•
37°C incubator
•
TEER (Trans-Epithelial Electrical Resistance) meter
to confirm presence of functionally polarized monolayers
prior to the experiment
– TEER [Ω·cm2] = resistance [Ω] x filter surface area [cm2]
– varies with cell line, 100-800 Ω·cm2
•
Liquid scintillation counter or LC/MS/MS for sample analysis
•
Pipettors (manual or automated) for liquid transfers
Cell Lines for P-gp Transport Assays
•
Cells used for bi-directional transport studies should form a functionally
polarized cell monolayer, complete with tight junctions (verified by preexperimental 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)
•
A paracellular marker such as [14C] mannitol may be used as an additional
integrity marker (typical permeability: < 0.2-2 x 10-6 cm/sec)
Cell Lines for P-gp Transport Assays
•
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 P-gp transport of drugs
– Various uptake and efflux transporters are expressed in Caco-2 cells, however,
P-gp 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 P-gp
•
MDCK-MDR1
– Transfected canine kidney cell line
– High background dog P-gp activity
Bi-Directional P-gp Transport Assays
• The transcellular transport assay should be used as a
definitive method for identifying P-gp substrates and
inhibitors
• Bi-directional transport assays may fail to identify highly
permeable compounds as P-gp substrates, however
for such compounds, P-gp is not likely to be a significant
barrier to cross membranes
• These experiments require the use of known
P-gp probe substrates and P-gp inhibitors
P-gp Probe Substrate Characteristics
•
Ideally
–
–
–
–
–
•
Selective for P-gp
Low to moderate passive permeability (2-30x10-6 cm/sec)
May be used as an in vivo P-gp probe substrate (optional)
No significant metabolism of the substrate (optional)
Commercially available (optional)
Realistically
– A P-gp substrate that meets all of the above criteria has not been
identified, due to overlapping substrate selectivity between different
transporters as well as transporters and enzymes
– Acceptable P-gp substrates meet the majority of the above mentioned
criteria. These P-gp substrates serve as positive controls to ensure the
cell systems have functional P-gp expression.
FDA DRAFT Guidance for Industry (Sept 2006)
Acceptable P-gp Probe Substrates
Efflux Ratio
Drug
Concentration [µM]
Caco-2
MDR1-MDCK
MDR1-LLCPK1
Digoxin
0.01-10
4-14
4
4
Loperamide
1-10
2-5
3.4
Quinidine
0.05
3
5
Vinblastine
0.004 -10
2-18
Talinolol
30
26
>9
3
•
Acceptable P-gp substrates are not limited to compounds listed
•
Selection of other compounds as probe P-gp substrates may be
appropriate based on scientific justification
FDA DRAFT Guidance for Industry (Sept 2006)
P-gp Inhibitor Characteristics
•
Ideally
–
–
–
–
–
•
Selective for P-gp
Inhibit P-gp with low Ki or IC50 values (e.g., IC50 < 10 µM)
No significant metabolism of the inhibitor occurs in the cells (optional)
Commercially available (optional)
May be used as an in vivo P-gp inhibitor (optional)
Realistically
– A P-gp inhibitor that meets all of the above criteria has not been
identified, due to substantial overlap between different transporters
(e.g., cyclosporine is also a potent MRP2 and OATP-C inhibitor;
quinidine and verapamil also inhibits various organic cation transporters)
– Because of the lack of specificity, the use of multiple inhibitors is
recommended to determine whether the efflux activity observed in vitro
is related to P-gp
FDA DRAFT Guidance for Industry (Sept 2006)
Acceptable P-gp Probe Inhibitors
IC50
(µM)
Ki (µM)
Caco-2
Caco-2
MDCKMDR1
LLC-PK1MDR1
Cyclosporine A
1.3
0.5
2.2
1.3
Ketoconazole
1.2
Inhibitor
LY335979
0.024
Nelfinavira
1.4
Quinidine
2.2
Ritonavir
3.8
Saquinavir
6.5
Tacrolimus
0.74
Valspodar (PSC833)
0.11
Verapamil
2.1
5.3
3.2
8.6
• Acceptable P-gp
inhibitors are not limited
to compounds listed
• Selection of other
compounds as probe
P-gp inhibitors may be
appropriate based on
scientific justification
• Use of multiple
inhibitors is
recommended
8
15
Elacridar (GF120918)
(GG 918)
0.4
0.4
Reserpine
1.4
11.5
FDA DRAFT Guidance for Industry (Sept 2006)
23
Assay Design – P-gp Substrate Testing
•
Test a range of concentrations (e.g., 1, 10, and 100 µM)
•
Test multiple time points (30 min up to 4 hours)
•
Include a P-gp substrate as a positive control
– Acceptable cell systems produce net flux ratios of probe substrates similar
to values reported in the literature (net flux ratios of ≥ 2 are recommended)
•
Include parental LLC-PK1 and MDCK cells, respectively, side-by-side
as negative controls
•
Test in presence and absence of 2-3 potent P-gp probe inhibitors to
determine if efflux can be inhibited
•
Triplicate monolayers for each condition
•
Assess recovery of substrate, to assess metabolism and non-specific
binding
Time- and Concentration-Dependence of
Digoxin Transport in MDR1-LLC-PK1 cells
1200
1400
digoxin 5.0 uM
digoxin 50 uM
digoxin 30 min
digoxin 60 min
1200
P-gp transport [pmol]
1000
P-gp transport [pmol]
P-gp facilitated Transport
Concentration dependence
P-gp facilitated Transport
Time dependence
digoxin 0.5 uM
800
600
400
200
digoxin 90 min
digoxin 120 min
1000
800
600
400
200
0
0
0
50
100
min
150
0
10
20
30
40
50
60
uM
Transport of P-gp probe substrate digoxin is linear over the concentration
range of 0.5 - 50 µM and incubation times of 30 -120 min.
Calculation of Papp and Efflux Ratios
Apparent Permeability (Papp)
Papp [cm/sec] = Vr/C0 x 1/S x dC/dt
Vr
is the volume in the receiver chamber [cm3]
C0
is the concentration in the donor chamber at t=0 [mM]
S
is the filter surface area [cm2]
dC/dt
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
Net Efflux Ratio (R) – for use with transfected cell models only
NER = RET / REW
RET
is the efflux ratio obtained in the P-gp transfected cells
REW
is the efflux ratio obtained in the wild-type control cells
FDA DRAFT Guidance for Industry (Sept 2006)
Decision Tree for P-gp Substrates
Bidirectional
Bidirectional transport
transport assay
assay
Is
Is efflux
efflux ratio
ratio ≥≥ 22 ?? **
Is
Is efflux
efflux ratio
ratio << 22 ?? **
Is
Is efflux
efflux inhibited
inhibited by
by P-gp
P-gp inhibitors?
inhibitors? **
**
Unlikely
Unlikely to
to be
be P-gp
P-gp substrate
substrate
YES
NO
Likely
Likely to
to be
be P-gp
P-gp substrate
substrate
Transporters
Transporters other
other than
than P-gp
P-gp
might
might be
be involved
involved
In
In vivo
vivo drug
drug interaction
interaction study
study with
with aa
P-gp
P-gp inhibitor
inhibitor may
may be
be warranted
warranted
Further
Further in
in vitro
vitro studies
studies to
to identify
identify
transporters
transporters may
may be
be warranted
warranted
* There is concern that this value is too liberal and will lead to too may positive results.
Alternatively, use a % value relative to a probe substrate, such as digoxin
** Reduces the efflux ratio significantly (> 50% or to unity)
FDA DRAFT Guidance for Industry (Sept 2006)
Example: Identification of a Test Article
as a P-gp Substrate in Caco-2 Cells
1.1
3.6
2.8
2.3
1.3
1.4
1.3
1.3
1.1
1.2
TA 10 uM
TA 1.0 uM +
keto 25 uM
TA 3.0 uM +
keto 25 uM
TA 10 uM +
keto 25 uM
TA 1.0 uM +
csa 10 uM
TA 3.0 uM +
csa 10 uM
TA 10 uM +
csa 10 uM
test article
with 10 uM cyclosporine
TA 3.0 uM
Efflux Ratios:
14
1.4
test article
with 25 uM ketoconazole
TA 1.0 uM
25
test article
without inhibitors
digoxin 5 uM +
csa 10 uM
positive control
+/- inhibitors
Papp [10-6 cm/s]
20
15
10
5
B-A Papp
digoxin 5 uM +
keto 25 uM
A-B Papp
digoxin 5 uM
0
Data are for 120 min incubation time; similar results were obtained at 30 and 60 min
Test article shows active efflux, which is inhibited by P-gp inhibitors
Æ likely a P-gp substrate
Assay Design – P-gp Inhibition Testing
•
Use a P-gp probe substrate that has been confirmed as a positive
control in the test system
– Acceptable cell systems produce net flux ratios of probe substrates
similar to values reported in the literature (net flux ratios of ≥ 2 are
recommended)
•
Initially, test a high concentration (e.g. >100 µM, solubility
permitting) of the compound
– If inhibition is observed, follow up with an IC50 determination
•
Include parental LLC-PK1 and MDCK cells, respectively, side-byside as negative controls
•
Use 2-3 potent P-gp probe inhibitors as positive controls
•
Triplicate monolayers for each condition
Calculations for P-gp Inhibition
IC50 Value
REi/REa = 1 – [(Imax* Ic) / ( Ic + IC50c )]
REi/REa is the efflux ratio of the probe P-gp substrate in the presence of inhibitor
relative to that without inhibitor
I
is the inhibitor concentration [µM]
Imax
is the maximal inhibitory effect [fraction of control activity]
c
is the Hill exponent
IC50
is the inhibitor concentration achieving half maximal inhibition effect [µM]
• There is considerable discussion in the scientific community regarding
the most appropriate way to calculate P-gp inhibition
• A DIA initiative to assess inter-laboratory variability in P-gp inhibition
testing and to evaluate various calculation methods is ongoing
• Stay tuned for more information (white paper, final guidance)
FDA DRAFT Guidance for Industry (Sept 2006)
Decision Tree for P-gp Inhibitors
Bidirectional
Bidirectional transport
transport assay
assay
with
with P-gp
P-gp probe
probe substrate
substrate
Net
Net flux
flux ratio
ratio of
of probe
probe substrate
substrate
decreases
with
increasing
decreases with increasing
concentrations
concentrations of
of test
test compound
compound
Net
Net flux
flux ratio
ratio of
of probe
probe substrate
substrate is
is
not
affected
by
increasing
not affected by increasing
concentrations
concentrations of
of test
test compound
compound
P-gp
P-gp inhibitor
inhibitor
Poor
Poor or
or non-inhibitor
non-inhibitor of
of P-gp
P-gp
Determine
Determine IC
IC5050 (or
(or KKi)i)
[I]/IC
[I]/IC5050 >> 0.1
0.1
In
In vivo
vivo drug
drug interaction
interaction study
study with
with aa
P-gp
P-gp probe
probe substrate
substrate (e.g.
(e.g. digoxin)
digoxin)
is
is recommended
recommended
[I]/IC
[I]/IC5050 << 0.1
0.1
In
In vivo
vivo drug
drug interaction
interaction study
study
with
a
P-gp
probe
substrate
with a P-gp probe substrate is
is
not
needed
not needed
FDA DRAFT Guidance for Industry (Sept 2006)
[I] = total steady-state
Cmax at highest dose
Example: Identification of a Test Article
as a P-gp Inhibitor in Caco-2 Cells
1.2
1.1
12.2
8.4
4.9
3.5
1.6
digoxin 5 uM
+ TA 0.5 uM
digoxin 5 uM
+ TA 1 uM
digoxin 5 uM
+ TA 2.5 uM
digoxin 5 uM
+ TA 5 uM
digoxin 5 uM
+ TA 10 uM
Efflux Ratios:
14.8
digoxin 5 uM
+ csa 10 uM
14
increasing concentation of the test article
digoxin 5 uM
+ keto 25 uM
no inhibition
probe P-gp inhibitors
as positive controls
Papp [10-6 cm/s]
12
10
8
6
4
2
A-B Papp
B-A Papp
digoxin 5 uM
0
Test article shows concentration dependent inhibition
of digoxin efflux Æ P-gp inhibitor Æ determine IC50
Example: Identification of a Test Article
as a P-gp Inhibitor in Caco-2 Cells
Calculations performed:
Decrease in Efflux Ratio (or Net Efflux Ratio, respectively)
% Inhibition = (1-(REi – 1) / (REa – 1))*100
REi
is the efflux ratio of the probe P-gp substrate in the presence of inhibitor
REa
is the efflux ratio of the probe P-gp substrate in the absence of inhibitor
IC50 Value (SigmaPlot v8.0)
f = 1 – [(Imax* Ic) / ( Ic + IC50c )]
f
is the fraction of control activity ((REi – 1) / (REa – 1))
I
is the inhibitor concentration [uM]
Imax
is the maximal inhibitory effect [fraction of control activity]
c
is the Hill exponent
IC50
is the inhibitor concentration achieving half maximal inhibition effect [µM]
Example: Identification of a Test Article
as a P-gp Inhibitor in Caco-2 Cells
% Inhibition of digoxin efflux
120
[I] / IC50 > 0.1
is the recommended
cut-off for in vivo testing
100
80
60
Æ If [I] (steady-state
total Cmax after highest
dose) is >0.12 uM, an
in vivo P-gp interaction
study might be warranted
40
20
IC50 = 1.2 µM
0
0
2
4
6
8
Test article concentration [uM]
10
12
Inhibition of Digoxin Efflux by Ketoconazole
in Caco-2 and LLC-PK1 Cells
MDR1-LLC-PK1 cells
% Inhibition of digoxin efflux
100
80
60
40
20
IC50 = 2.6 uM
0
-20
0
10
20
Ketoconazole [uM]
30
% Inhibition of digoxin net efflux
Caco-2 cells
120
(using both P-gp expressing and
control cells)
100
80
60
40
20
IC50 = 1.8 uM
0
0
20
40
60
80
Ketoconazole [uM]
100
120
Non-Specific Binding / Recovery
•
Amount of compound recovered in the donor and receiver chambers at the
end of the assay relative to the amount added to the donor chamber at t=0
•
Low recovery may significantly underestimate Papp values
•
•
–
Always consider recovery when interpreting Papp values and efflux ratios
–
Typical cut-offs used in the industry are 50-80%, depending on the stage of the
compound in the development process
Factors contributing to low recovery include:
–
Non-specific binding to the outside of the cells
–
Uptake into the cells
–
Non-specific binding to the plastic plate
–
Metabolism or non-metabolic instability
Addition of protein may improve recovery, however, the impact on Papp and
efflux ratios is not fully understood
Evaluation of P-gp Induction
•
•
Expression of P-gp is inducible (e.g. by rifampin, hypericin, ritonavir)
Methods for in vitro evaluation of P-gp induction are not well established
– Caco-2 cells are not suitable for P-gp induction testing (lack of PXR expression)
– As for CYPs, species differences in inductive response limit the use of animal
models
– Models that have been used successfully include:
• Human hepatocytes (mRNA induction)
• Human colon adenocarcinoma cells (LS180), and their adriamycin or vinblastine
resistant sublines (mRNA, protein, activity assessment)
•
Co-induction of P-gp and CYP3A (both are regulated by PXR)
– Because of similar mechanisms of CYP3A and P-gp induction, data on CYP3A
inducibility can inform decisions about P-gp
• If no CYP3A induction is observed in vitro,
no further tests of CYP3A and P-gp induction in vivo are necessary
• If CYP3A induction is observed in vitro, but not in vivo,
no further tests of P-gp induction in vivo is necessary
• If CYP3A induction is observed in vitro and in vivo,
an additional study of the drug’s effect on a P-gp probe substrate
in vivo is recommended
Comments on Draft Guidance Document
•
Draft guidance has been commented on extensively, especially the
transporter section:
http://www.fda.gov/ohrms/dockets/dockets/06d0344/06d0344.htm
•
Main issues raised:
– Degree of experimental detail prescribed
– Use of control cells with transfected cell lines
– Efflux ratio >2 as criteria for P-gp substrates
– Method of calculating inhibition and IC50 values
– Trigger for in vivo study and definition of [I]
Expectations for Future Guidance
•
Our expectations:
– Experimental detail for P-gp interaction testing may come into line with
other sections
– Clarification on calculation methods for P-gp inhibition
– More detail around triggers for in vivo studies, and requirements for those
– Number of transporters which require testing expected to increase
(e.g. BCRP, BSEP, OAPT2, NTCP)
• Recommendation on experimental systems and probes for transporters other
than P-gp
• Decision trees for other transporters
– Standardized language for labeling regarding transporter interactions
Recommendations
•
Try to generally follow recommendations in the draft FDA guidance
– Until document is final, consider the docket comments
– Use your scientific judgment to decide how to approach controversial
issues
– Consider “consensus” documents / White Papers published by
representatives from industry and/or government
•
In vitro transport studies are not required to be GLP compliant
– However, using validated methods and test systems, positive control
tracking processes, SOPs, etc. are good practice
•
Use well-accepted reagents from reputable suppliers
– Radiochemical purity of probe substrates and test compounds is critical
Contact Information
Elke S. Perloff
e-mail: [email protected]
tel: 781-935-5115 x2273