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Drug Transporters in the Cornea PD Dr. Stephan Reichl Institute of Pharmaceutical Technology TU Braunschweig Germany Orlando October 29, 2015
AAPS Annual Meeting
Outline • The eye, the cornea and transcorneal drug transport
• Development and characterization of an organotypic, threedimensional in-vitro cell culture model of human cornea for drug absorption studies as replacement for animal testing
• Expression of ABC transporters in animal and human corneal models in-vitro and ex-vivo • Expression of phase I and II metabolizing enzymes in corneal tissue • SLC Transporter - short review from the literature
Orlando October 29, 2015 | S. Reichl | AAPS Annual Meeting
2
The Eye - Drug transport routes
Anterior
Posterior Barar J et al. (2008) Expert Opin Drug Deliv 5:567-581
Orlando October 29, 2015 | S. Reichl | AAPS Annual Meeting
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Human Cornea Epithelium
Tight junctions
Superficial cells Stroma
Wing cells Basal cells Bowman Membrane
Endothelium
Reichl S. et al. (2011) Expert Opin Drug Metab Toxicol 7:559-578 Orlando October 29, 2015 | S. Reichl | AAPS Annual Meeting
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Transepithelial drug absorption Influx transport
apical
Efflux transport
Orlando October 29, 2015 | S. Reichl | AAPS Annual Meeting
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• The eye, the cornea and transcorneal drug transport
• Development and characterization of an organotypic, threedimensional in-vitro cell culture model of human cornea for drug absorption studies as replacement for animal testing
• Expression of ABC transporters in animal and human corneal models in-vitro and ex-vivo • Expression of phase I and II metabolizing enzymes in corneal tissue • SLC Transporter - short review from the literature
Orlando October 29, 2015 | S. Reichl | AAPS Annual Meeting
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Experimental determination of eye-related pharmacokinetic parameters (in-vivo/ex-vivo) • mostly performed in rabbits • per formulation at least 36 animals are needed • enucleation at fixed time points, dissection and drug analysis in ocular tissues and fluids
• Ex-vivo studies using excised corneas (rabbit, pig, cow, …) • per formulation 6 animals are needed • enucleation, Ussing chamber system, determination of transcorneal drug transport Orlando October 29, 2015 | S. Reichl | AAPS Annual Meeting
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Disadvantages of animal experiments • Ethical concerns • High costs
• Poor standardization and high variability • Questionable extension of animal data to human
Are the cell culture models valid?
Orlando October 29, 2015 | S. Reichl | AAPS Annual Meeting
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Requirements for a valid corneal cell culture model for in-vitro drug absorption studies • KISS Principle „Keep it simple and significant“ • High equivalence of organotypic characteristics (epithelium plus stroma) • High expression of tight junctions (high transepithelial electrical resistance TEER) • Human cell lines • Serum-free cell cultivation • Equivalence of permeability to ex vivo tissue (marker) • Low variability, high intra- and interlaboratory reproducibility • Similar expression pattern of transport proteins (ABC, SLC transporters) and metabolizing enzymes (phase I and II) Orlando October 29, 2015 | S. Reichl | AAPS Annual Meeting
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Human corneal cells Tear film Superficial cells
HCE-T
Wing cells
cell line Basal cells Basement membrane
Epithelium
Epithelial cells
HCK
Basement membrane Bowman membrane
cell line Stromal cells
Stroma
HENC cell line
Descemet membrane Endothelium
Endothelial cells
Orlando October 29, 2015 | S. Reichl | AAPS Annual Meeting
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Tight junctions expression in HCE-T cells 40x
40x
40x
DAPI
ZO-1
Occludin
Immunohistological staining of HCE-T monolayer cultures. Detection and localization of tight junctions proteins ZO-1 (red) and occludin (green). Nucleus staining by DAPI (blue).
Hahne M. et al. (2012) J Pharm Sci 101:2976-2988 Orlando October 29, 2015 | S. Reichl | AAPS Annual Meeting
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Hemicornea model Day Tag 11
Casting of Gießen des collagen solution Stromaäquivalents with HCK cells Verfestigung des Stromaäquivalents Gelling of im collagenLAF solution
Transwell® Insert Polycarbonate Filter
Stroma Biomatrix containing Keratocytes
min ca.ca. 1010 Minuten
Vollständige CompleteGelierung Gelling im Inkubator of collagen solution
Culture Medium
60 ca.Minuten 60 min
Orlando October 29, 2015 | S. Reichl | AAPS Annual Meeting
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Hemicornea model
Day Tag 1
Seeding ofder Aufbringen epithelial cells Epithelzellsuspension
Corneal Epithelial Cell Sheet
Metal Plate
Day Tag 7
Cultivation Anheben an dieatLuft Air-Liquid Interface Medium Grenze
until Day10 10 bis Tag
Formation of a multilayered epithelium
Corneal Epithelial Cells at Air-Liquid Interface
Metal Plate Multilayered Corneal Epithelium
Orlando October 29, 2015 | S. Reichl | AAPS Annual Meeting
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Histology
Epithelium
StromaBiomatrix
HE staining Orlando October 29, 2015 | S. Reichl | AAPS Annual Meeting
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SEM
Toluidine Staining
Histology
20 µm
Fibronectin
5 µm
Laminin
Collagen IV
Orlando October 29, 2015 | S. Reichl | AAPS Annual Meeting
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Drug transport studies
Day 10
Determination of TEER values
Incubation with Krebs-Ringer buffer
Buffer
ca. 30 min
Permeation Acceptor:
Orlando October 29, 2015 | S. Reichl | AAPS Annual Meeting
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Drug transport studies
Excised Cornea
Hemicornea Construct Acceptor:
Orlando October 29, 2015 | S. Reichl | AAPS Annual Meeting
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Drug transport studies 2,5 2.5 Transwell® insert
Permeated amount [µg/cm²]
2,0 2.0
Donor compartment Microporous membrane Acceptor compartment
1,5 1.5
Papp [cm / s]
1,0 1.0
Flux Donor concentration
0,5 0.5
0.0 0,0
0
50
100
150
200
Time [min] Orlando October 29, 2015 | S. Reichl | AAPS Annual Meeting
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Comparison between HC Model and excised Cornea Permeation coefficent Papp [cm/s]
Marker Substance
Hemicornea
Porcine Cornea
Rabbit Cornea
Sodium fluorescein
1.98±0.45 x 10-7
1.75±1.51 x 10-7
3.76±1.12 x 10-7
Rhodamine B
9.80±0.72 x 10-6
5.43±1.26 x 10-6
13.49±2.11 x 10-6
FD-4
4.96±1.46 x 10-8
1.67±0.43 x 10-8
9.08±4.88 x 10-8
Hahne M., Reichl S. (2011) Int J Pharm 416:268-279 Orlando October 29, 2015 | S. Reichl | AAPS Annual Meeting
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Prevalidation
Hahne M. et al. (2012) J Pharm Sci 101:2976-2988 Orlando October 29, 2015 | S. Reichl | AAPS Annual Meeting
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Interlaboratory variability and comparison with ex-vivo cornea
Hahne M. et al. (2012) J Pharm Sci 101:2976-2988 Orlando October 29, 2015 | S. Reichl | AAPS Annual Meeting
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Interlaboratory variability and comparison with ex-vivo cornea
Hahne M. et al. (2012) J Pharm Sci 101:2976-2988 Orlando October 29, 2015 | S. Reichl | AAPS Annual Meeting
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Equivalence test 1.8E-07
Maximum value of rabbits confidence intervals
1.6E-07
Maximal variation of rabbit confidence intervals
1.4E-07
95% confidence interval Mean of all rabbit batches
1.2E-07
Mean of all HC values
1.0E-07
Acceptance limits for HC batches
8.0E-08 6.0E-08 4.0E-08 2.0E-08 0.0E+00 rabbit/1 rabbit/2 rabbit/3 TUBS-A/1 TUBS-A/2 TUBS-A/3 TUBS-B/1 TUBS-B/2 AB/1 AB/2 UKE/1 UKE/2 UKE/7 UKE/8 UKE/10 UKE/11 UKE/12 UKE/13 UKE/14 UKE/15
Permeation coefficient Papp [cm/s] permeation coefficient [cm/s]
FD-4: 95% confidence intervals for each batch of Hemicornea and rabbit
Hahne M. et al. (2012) J Pharm Sci 101:2976-2988
Orlando October 29, 2015 | S. Reichl | AAPS Annual Meeting
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Equivalence test Intralaboratory Intralaboratory Intralaboratory TUBS AB UKE
equivalent
not equivalent
FD-4
Na-Fluorescein
Rhodamine B
Aciclovir
Bimatoprost
Dexamethasone
Timolol
Orlando October 29, 2015 | S. Reichl | AAPS Annual Meeting
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Equivalence test Intralaboratory Intralaboratory Intralaboratory Interlaboratory TUBS AB UKE equivalence
equivalent
not equivalent
FD-4
Na-Fluorescein
Rhodamine B
Aciclovir
Bimatoprost
Dexamethasone
Timolol
Orlando October 29, 2015 | S. Reichl | AAPS Annual Meeting
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• The eye, the cornea and transcorneal drug transport
• Development and characterization of an organotypic, threedimensional in-vitro cell culture model of human cornea for drug absorption studies as replacement for animal testing
• Expression of ABC transporters in animal and human corneal models in-vitro and ex-vivo • Expression of phase I and II metabolizing enzymes in corneal tissue • SLC Transporter - short review from the literature
Orlando October 29, 2015 | S. Reichl | AAPS Annual Meeting
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Membrane bound transport proteins Substrates of SLC transporters: amino acids, sugars, peptides, lactate, vitamins etc.
• P-Glycoprotein • MRPs • BCRP Hediger M et al. (2004) Pflugers Arch - Eur J Physiol 447:465–468
Orlando October 29, 2015 | S. Reichl | AAPS Annual Meeting
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Expression of efflux transporters MDR1 bp
bp
500
500
300
300
100
100
PC
(1) Caco-2 Cells (2) HCE-T Cells (3) Hemicornea Construct (4) Porcine Cornea (5) Rabbit Cornea (6) Human Epithelial Cornea Cells
M PC 1
M
2 1
32
4 3
54
65
6
Verstraelen J., Reichl S. (2013) Int J Pharm 441:765-775 Orlando October 29, 2015 | S. Reichl | AAPS Annual Meeting
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Expression of efflux transporters MDR1 3
4
5
6
-
-
-
+
-
Caco-2 Cells
+
2
Rabbit Cornea
1
(1) Caco-2 Cells (2) HCE-T Cells (3) Hemicornea Construct (4) Porcine Cornea (5) Rabbit Cornea (6) Human Epithelial Cornea Cells
Verstraelen J., Reichl S. (2013) Int J Pharm 441:765-775 Orlando October 29, 2015 | S. Reichl | AAPS Annual Meeting
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Expression of efflux transporters • Bidirectional Transport Basolateral Apical
Apical Basolateral Transwell® Insert
Acceptor Krebs-Ringer Buffer
Cell Layer Donor specific substrate
• Substrates and Inhibitors MDR1 Substrate
Rhodamine123 (50-100 µg/mL)
Inhibitor
Verapamil (200 µM)
Localisation
Apical
Orlando October 29, 2015 | S. Reichl | AAPS Annual Meeting
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Expression of efflux transporters • Bidirectional permeation coefficients Papp (x 10-6 cm/s) Rhodamine123 Caco-2 Cells
HCE-T Cells
Hemicornea Construct
Porcine Cornea
Rabbit Cornea
AB
1.07 ± 0.13
0.90 ± 0.21
0.58 ± 0.05
0.25 ± 0.04
0.27 ± 0.17
BA
3.49 ± 0.41 *
0.96 ± 0.61
0.55 ± 0.06
0.16 ± 0.09
0.80 ± 0.09 *
• Efflux ratio: B A / A B > 2 3,5
MDR1
3
Efflux Ratio
2,5 2 1,5 1 0,5 * p < 0.05, t-test n=6, mean ± SD
0 1
2
3
4
5
Orlando October 29, 2015 | S. Reichl | AAPS Annual Meeting
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Expression of efflux transporters • Bidirectional permeation coefficients Papp (x 10-6 cm/s) Rhodamine123 Caco-2 Cells
HCE-T Cells
Hemicornea Construct
Porcine Cornea
Rabbit Cornea
AB
1.07 ± 0.13
0.90 ± 0.21
0.58 ± 0.05
0.25 ± 0.04
0.27 ± 0.17
BA
3.49 ± 0.41 *
0.96 ± 0.61
0.55 ± 0.06
0.16 ± 0.09
0.80 ± 0.09 *
• Efflux ratio: B A / A B > 2 3,5
MDR1
3
Efflux Ratio
2,5 2 1,5 1 0,5 * p < 0.05, t-test n=6, mean ± SD
0 1
2
3
4
5
Orlando October 29, 2015 | S. Reichl | AAPS Annual Meeting
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Expression of efflux transporters BCRP (1) Caco-2 Cells (2) HCE-T Cells (3) Hemicornea Construct (4) Porcine Cornea (5) Rabbit Cornea (6) Human Epithelial Cornea Cells
MRP3
Verstraelen J., Reichl S. (2013) Int J Pharm 441:765-775 Orlando October 29, 2015 | S. Reichl | AAPS Annual Meeting
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Expression of efflux transporters BCRP
+
+
-
-
-
-
+
+
+
-
+
-
MRP3
(1) Caco-2 Cells (2) HCE-T Cells (3) Hemicornea Construct (4) Porcine Cornea (5) Rabbit Cornea (6) Human Epithelial Cornea Cells Verstraelen J., Reichl S. (2013) Int J Pharm 441:765-775 Orlando October 29, 2015 | S. Reichl | AAPS Annual Meeting
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Caco-2 Cells
HCE-T Cells HCE-T Cells Porcine Cornea
MRP3
Caco-2 Cells
BCRP
Hemicornea
Expression of efflux transporters
Orlando October 29, 2015 | S. Reichl | AAPS Annual Meeting
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Expression of efflux transporters BCRP
Transport of methotrexate in the presence of verapamil
Verstraelen J., Reichl S. (2013) Int J Pharm 441:765-775 Orlando October 29, 2015 | S. Reichl | AAPS Annual Meeting
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Expression of efflux transporters MRP 3
Transport of methotrexate in the presence of verapamil and Ko143
Verstraelen J., Reichl S. (2013) Int J Pharm 441:765-775 Orlando October 29, 2015 | S. Reichl | AAPS Annual Meeting
37
Expression of efflux transporters MRP1, 2, 4 and 5 (1) Caco-2 Cells (2) HCE-T Cells (3) Hemicornea Construct (4) Porcine Cornea (5) Rabbit Cornea (6) Human Epithelial Cornea Cells
Verstraelen J., Reichl S. (2014) Mol Pharm 11:2160-2171 Orlando October 29, 2015 | S. Reichl | AAPS Annual Meeting
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Expression of efflux transporters MRP1, 2, 4 and 5
Verstraelen J., Reichl S. (2014) Mol Pharm 11:2160-2171 Orlando October 29, 2015 | S. Reichl | AAPS Annual Meeting
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Expression of efflux transporters
Hemicornea
Rabbit Cornea
Caco-2 Cells
HCE-T Cells
MRP1
Verstraelen J., Reichl S. (2014) Mol Pharm 11:2160-2171 Orlando October 29, 2015 | S. Reichl | AAPS Annual Meeting
40
Expression of efflux transporters
Rabbit Cornea
HCE-T Cells
Caco-2 Cells
MRP2
Verstraelen J., Reichl S. (2014) Mol Pharm 11:2160-2171 Orlando October 29, 2015 | S. Reichl | AAPS Annual Meeting
41
Expression of efflux transporters
Hemicornea
Porcine Cornea
HCE-T Cells
Caco-2 Cells
MRP4
Verstraelen J., Reichl S. (2014) Mol Pharm 11:2160-2171 Orlando October 29, 2015 | S. Reichl | AAPS Annual Meeting
42
Expression of efflux transporters
Hemicornea
Porcine Cornea
HCE-T Cells
Caco-2 Cells
MRP5
Verstraelen J., Reichl S. (2014) Mol Pharm 11:2160-2171 Orlando October 29, 2015 | S. Reichl | AAPS Annual Meeting
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Expression of efflux transporters MRP2
Transport of erythromycin in the presence of verapamil
Permeation coefficient [cm/s]
1.0E-05 9.0E-06
**
8.0E-06 7.0E-06 6.0E-06
*
5.0E-06 4.0E-06
*
3.0E-06
2.0E-06
*
1.0E-06 0.0E+00 Caco-2 cells
HCE-T epithelial model
Hemicornea construct
Apical - Basolateral
Porcine cornea
Rabbit cornea
Basolateral - Apical Verstraelen J., Reichl S. (2014) Mol Pharm 11:2160-2171
Orlando October 29, 2015 | S. Reichl | AAPS Annual Meeting
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Expression of efflux transporters Uptake Assay MRP1 and 5 CDCFDA
CDCF
Intracellular esterase activity
Cell
X Inhibitor (probenecid)
MRP4 [H3] Adefovir dipivoxil
Cell
X
scintillation counting
Inhibitor (indometacin) Orlando October 29, 2015 | S. Reichl | AAPS Annual Meeting
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Expression of efflux transporters MRP1 and 5
CDCF concentrations in the acceptor compartment
Concentration CDCF [µM]
2.25E+00 4.5E-01
4.0E-01 2.0E+00 3.5E-01 1.75E+00
3.0E-01 1.5E+00
**
2.5E-01 1.25E+00
2.0E-01 1.0E+00 1.5E-01 1.5E-01
1.0E-01 1.0E-01 5.0E-02 0.5E-01
***
0.0E+00 0.0E00 Caco-2 cells
*
* HCE-T epithelial model
Hemicornea construct
Without Inhibitor
Porcine cornea
Rabbit cornea
With Inhibitor Verstraelen J., Reichl S. (2014) Mol Pharm 11:2160-2171
Orlando October 29, 2015 | S. Reichl | AAPS Annual Meeting
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Expression of efflux transporters MRP4
Accumulated cellular concentrations of adefovir
Concentration Adefovir [pmol]
1.8E+02
*
1.6E+02
1.4E+02
*
1.2E+02
1.0E+02
*
8.0E+01
*
6.0E+01
*
4.0E+01 2.0E+01 0.0E+00 Caco-2 cells
HCE-T epithelial model
Without Inhibitor
Hemicornea construct
Porcine cornea
Rabbit cornea
With Inhibitor Verstraelen J., Reichl S. (2014) Mol Pharm 11:2160-2171
Orlando October 29, 2015 | S. Reichl | AAPS Annual Meeting
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Expression of efflux transporters - Summary MDR1 Hemicornea Construct
MRP1
MRP2
MRP3
MRP4
MRP5
BCRP
◊ -
- + + + + -
- + + - + + + + + + + ◊ +
Porcine Cornea
-
- + -
- + ◊ - + + + + + - ◊ -
Rabbit Cornea
+ + + + + + + + + + ◊ -
HEC
-
◊ -
- + -
/ + ◊ / + -
-
-
- ◊ ◊ + -
-
-
/ + ◊ / ◊ ◊ / + ◊ / + ◊ /
+: Detected -: Not Detected ◊: Weak Detection /: Not tested, Tissue not available Orlando October 29, 2015 | S. Reichl | AAPS Annual Meeting
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Expression of efflux transporters - Summary MDR1 Hemicornea Construct
MRP1
MRP2
MRP3
MRP4
MRP5
BCRP
◊ -
- + + + + -
- + + - + + + + + + + ◊ +
Porcine Cornea
-
- + -
- + ◊ - + + + + + - ◊ -
Rabbit Cornea
+ + + + + + + + + + ◊ -
HEC
-
◊ -
- + -
/ + ◊ / + -
-
-
- ◊ ◊ + -
-
-
/ + ◊ / ◊ ◊ / + ◊ / + ◊ /
+: Detected -: Not Detected ◊: Weak Detection /: Not tested, Tissue not available Orlando October 29, 2015 | S. Reichl | AAPS Annual Meeting
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Expression of efflux transporters - Summary MDR1 Hemicornea Construct
MRP1
MRP2
MRP3
MRP4
MRP5
BCRP
◊ -
- + + + + -
- + + - + + + + + + + ◊ +
Porcine Cornea
-
- + -
- + ◊ - + + + + + - ◊ -
Rabbit Cornea
+ + + + + + + + + + ◊ -
HEC
-
◊ -
- + -
/ + ◊ / + -
-
-
- ◊ ◊ + -
-
-
/ + ◊ / ◊ ◊ / + ◊ / + ◊ /
+: Detected -: Not Detected ◊: Weak Detection /: Not tested, Tissue not available Orlando October 29, 2015 | S. Reichl | AAPS Annual Meeting
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Expression of efflux transporters - Summary MDR1 Hemicornea Construct
MRP1
MRP2
MRP3
MRP4
MRP5
BCRP
◊ -
- + + + + -
- + + - + + + + + + + ◊ +
Porcine Cornea
-
- + -
- + ◊ - + + + + + - ◊ -
Rabbit Cornea
+ + + + + + + + + + ◊ -
HEC
-
◊ -
- + -
/ + ◊ / + -
-
-
- ◊ ◊ + -
-
-
/ + ◊ / ◊ ◊ / + ◊ / + ◊ /
+: Detected -: Not Detected ◊: Weak Detection /: Not tested, Tissue not available Orlando October 29, 2015 | S. Reichl | AAPS Annual Meeting
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Comparison with data from literature MDR1
Excised cornea mRNA level Human Rabbit
Protein level
Functionality
+++
References 1, 2, 3
--
--
4, 5, 6,
+
++++
7, 8, 9, 10
-
6
1
Dey S, Patel J, Anand BS, et al. Invest Ophthalmol Vis Sci 2003;44:2909-18
2
Xiang CD, Batugo M, Gale DC, et al. Drug Metab Dispos 2009;37:992-8
3
Zhang T, Xiang CD, Gale D, et al. Drug Metab Dispos 2008;36:1300-7
4
Vellonen KS, Mannermaa E, Turner H, et al. J Pharm Sci 2010;99:1087-98
5
Becker U, Ehrhardt C, Daum N, et al. J Ocul Pharmacol Ther 2007;23:172-81
6
Becker U, Ehrhardt C, Schneider M, et al. ATLA 2008;36:33-44
7
Dey S, Gunda S, Mitra AK. J Pharmacol Exp Ther 2004;311:246-55
8
Kawazu K, Oshita A, Nakamura T, et al. Biol Pharm Bull 2006;29:846-9
9
Hariharan S, Minocha M, Mishra GP, et al. J Ocul Pharmacol Ther 2009;25:487-97
10
Hariharan S, Gunda S, Mishra GP, et al. Pharm Res 2009;26:1270-82
Orlando October 29, 2015 | S. Reichl | AAPS Annual Meeting
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Comparison with data from literature MDR1
Corneal epithelial cells in culture
Human Rabbit
mRNA level
Protein level
Functionality
References
----
-
-----
1-4
++++
+++
+++++++
5 -11
-
1
1
Becker U, Ehrhardt C, Schneider M, et al. ATLA 2008;36:33-44
2
Becker U, Ehrhardt C, Daum N, et al. J Ocul Pharmacol Ther 2007;23:172-81
3
Xiang CD, Batugo M, Gale DC, et al. Drug Metab Dispos 2009;37:992-8
4
Vellonen KS, Mannermaa E, Turner H, et al. J Pharm Sci 2010;99:1087-98
5
Dey S, Patel J, Anand BS, et al. Invest Ophthalmol Vis Sci 2003;44:2909-18
6
Kawazu K, Yamada K, Nakamura M, Ota A Invest Ophthalmol Vis Sci 1999;40:1738-44
7
Kawazu K, Oshita A, Nakamura T, et al. Biol Pharm Bull 2006;29:846-9
8
Dey S, Gunda S, Mitra AK. J Pharmacol Exp Ther 2004;311:246-55
9
Hariharan S, Minocha M, Mishra GP, et al. J Ocul Pharmacol Ther 2009;25:487-97
10
Hariharan S, Gunda S, Mishra GP, et al. Pharm Res 2009;26:1270-82
11
Katragadda S, Talluri R, Mitra AK. J Ocul Pharmacol Ther 2006;22:110-20
Orlando October 29, 2015 | S. Reichl | AAPS Annual Meeting
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Comparison with data from literature
HCC
Human corneal cell culture models
Human cornea ex-vivo
Rabbit corneal cell culture models
Rabbit cornea ex-vivo
MDR1
-
-
-
+
+
MRP1
+
+
+
n.d.
+
MRP2
-
-/+
-
+
+
MRP3
-/+
-/+
-/+
n.d.
-/+
MRP4
+
+
-/+
n.d.
-
MRP5
+
+
+
+
+
BCRP
+
+
+
n.d.
-
Orlando October 29, 2015 | S. Reichl | AAPS Annual Meeting
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Comparison with data from literature
HCC
Human corneal cell culture models
Human cornea ex-vivo
Rabbit corneal cell culture models
Rabbit cornea ex-vivo
MDR1
-
-
-
+
+
MRP1
+
+
+
MRP2
-
-/+
-
MRP3
-/+
-/+
-/+
-/+
MRP4
+
+
-/+
-
MRP5
+
+
+
BCRP
+
+
+
+ +
+
+
+ -
Orlando October 29, 2015 | S. Reichl | AAPS Annual Meeting
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Overexpression of P-Glycoprotein MDR1 – Rabbit (SIRC cell culture model)
• SIRC cells were incubated with 10 µg/mL levofloxacin, ofloxacin and gatifloxacin for 3 weeks • Efflux activity was measured by uptake studies, flowcytometry and qRT-PCR • long-term exposure induced significant overexpression of P-gp in SIRC cells
Orlando October 29, 2015 | S. Reichl | AAPS Annual Meeting
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10 *
5
Upregulation of P-Glycoprotein in corneal models 0
Medium
Rhodamine123 Rhodamine123 Rhodamine123 4 hours 24 hours 72 hours
30 18
**
20
16 * Changes in the MDR1 mRNA level * 14 relative to control (medium) by real12 10 time PCR in the HCE-T cells following 8 treatment with rhodamine 123, * 6 4 levofloxacin and acebutolol
Upregulation
Upregulation
25
15 10 *
5
2
0 Medium
0
Rhodamine123 Rhodamine123 Rhodamine123 4 hours 24 hours 72 hours
Medium
Levof loxacine 4 hours
Levof loxacine 24 hours
Levof loxacine 72 hours
12 18 16
*
*
Upregulation
Upregulation
14
12 10 8 *
6
**
10
4
8 6
*
4
*
2
2
0
0 Medium
Levof loxacine 4 hours
Levof loxacine 24 hours
Levof loxacine 72 hours
lation
8
Acebutolol 4 hours
Acebutolol 24 hours
Acebutolol 72 hours
Verstraelen J., Reichl S. (2015) J Pharm Pharmacol 67:605-615
12 10
Medium
Orlando October 29, 2015 | S. Reichl | AAPS Annual Meeting **
57
Upregulation of P-Glycoprotein in corneal models Caco-2 cells
HCE-T model
Hemicornea
Medium
4 hours
24 hours
72 hours
Rhodamine 123
3.04 ± 0.33
3.32 ± 0.51
5.08 ± 0.73*
5.03 ± 1.33*
Levofloxacin
2.40 ± 0.06
2.56 ± 0.29
2.83 ± 0.51*
2.19 ± 0.69*
Acebutolol
2.99 ± 0.60
2.98 ± 0.14
3.05 ± 0.14*
4.44 ± 1.13*
Rhodamine 123
0.58 ± 0.07
0.55 ± 0.02
0.52 ± 0.01*
3.42 ± 0.26**
Levofloxacin
0.71 ± 0.12
2.44 ± 1.03
1.54 ± 0.31*
1.63 ± 0.06**
Acebutolol
0.19 ± 0.02
0.22 ± 0.02
0.56 ± 0.18*
1.05 ± 0.69**
Rhodamine 123
0.53 ± 0.04
1.54 ± 0.31*
0.81 ± 0.07*
2.23 ± 0.24*
Levofloxacin
0.74 ± 0.15
0.70 ± 1.01*
1.65 ± 0.08*
1.72 ± 0.05*
Acebutolol
0.12 ± 0.01
0.18 ± 0.02*
0.30 ± 0.05*
0.38 ± 0.12*
The amount of digoxin (μg) transported from the basolateral to the apical side tested for the in-vitro cornea models prior and after stimulation Verstraelen J., Reichl S. (2015) J Pharm Pharmacol 67:605-615 Orlando October 29, 2015 | S. Reichl | AAPS Annual Meeting
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Inhibition of P-Glycoprotein by drug interaction MDR1 – Rabbit (primary cell cultures and in-vivo model)
6α-methyl prednisolone, prednisolone, prednisone • increased intracellular accumulation of erythromycin in rabbit corneal epithelial cells in-vitro
• 2 h single-dose continuous infusion of erythromycin to the rabbit eye in absence and presence of corticosteroids resulted in 2-3 fold higher ka, cmax and AUC
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• The eye, the cornea and transcorneal drug transport
• Development and characterization of an organotypic, threedimensional in-vitro cell culture model of human cornea for drug absorption studies as replacement for animal testing
• Expression of ABC transporters in animal and human corneal models in-vitro and ex-vivo • Expression of phase I and II metabolizing enzymes in corneal tissue • SLC Transporter - short review from the literature
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mRNA Expression of phase I und II enzymes CYP1A2 CYP2A6 CYP2B6 CYP2C8 CYP2C9 CYP2C19 CYP2D6 CYP2E1 CYP3A4 CYP3A5 GSTA4-4 GSTO1-1 GSTP1-1 NAT1 NAT2 SULT1A1 UGT1A1
Human Liver
Caco-2
Human Corneal Epithelium
HCE-T
HCK
HENC
Hemicornea Construct
+ + + + + + + + + + ◊ + ◊ ◊ ◊ ◊ +
◊ + + + + + ◊ + + + + + ◊ ◊ +
◊ ◊ ◊ ◊ + ◊ ◊ ◊ ◊ + + ◊ ◊ ◊
◊ ◊ ◊ ◊ + ◊ ◊ ◊ ◊ + + + ◊ + ◊
◊ ◊ + ◊ ◊ ◊ + + + + ◊
◊ ◊ + ◊ ◊ ◊ + + + ◊ +
◊ ◊ ◊ + ◊ ◊ ◊ ◊ + + + + ◊
Kölln C., Reichl S. (2012) J Ocul Pharm Ther 28:271-277 Orlando October 29, 2015 | S. Reichl | AAPS Annual Meeting
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Cytochrome P450 Expression CYP2D6 Hemicornea Construct
Porcine Cornea
Rabbit Cornea
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Cytochrome P450 expression Cytochrome P450 Total Activity
CYP
NADPH 7-Ethoxycoumarin
7-Hydroxycoumarin (7HC)
CYP2D6 Activity
CYP2D6
NADPH Luciferin-ME EGE
D-Luciferin
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Cytochrome P450 Expression CYP 450 Total Activity
CYP2D6 Activity
(Substrate: 7-ethoxycoumarin)
(Substrate: Luciferin-ME EGE) 45 CYP2D6 activity per cornea or construct [fmol D-Luciferin ∙ min-1]
P450 activity activity per cornea or or construct construct [pmol 7HC 7HC ∙ min-1]
3 2.5 2,5 . 2 1.5 1,5 . 1 0.5 . 0,5 0
Hemicornea
Porcine Cornea
Rabbit Cornea
***
40 35 30 25 20 15 10 5 0
Hemicornea
Porcine Cornea
Rabbit Cornea
*** p < 0.001, t-test n=3, mean ± SD
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Glutathione Transferases Expression Hemicornea Construct
Porcine Cornea
Rabbit Cornea
GSTO1
GSTP1
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Glutathione Transferases Expression Glutathione Transferase Total Activity GST
-HCl
Glutathione (GSH)
1-chloro-2,4-dinitrobenzene
GSTP1 Activity GSTP1
Glutathione (GSH)
ethacrynic acid
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Glutathione Transferases Expression GSTO1 Activity GSTO1 GSTO1
H+ S-(4-nitrophenacyl)glutathione (4-NPG)
4-nitroacetophenone
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GST Total Activity (Substrate: 1-chloro-2,4-dinitrobenzene ) 200 160
140
50
GSTO1 Activity
45 50
(Substrate: 4-NPG)
40 45 35 40 30 35 25 30 20 25 15 20 10 15
5 10
120
0 5
100
0
**
80
60
14
40 20
0
Hemicornea
Porcine Cornea
Rabbit Cornea
** p < 0.01, t-test n≥3, mean ± SD
GSTP1 GSTP1 activity activity per per cornea cornea or construct or construct -1] -1] [nmol [nmol ∙ min ∙ min
GST activity per cornea or construct [nmol ∙ min-1]
180
GSTO1 GSTO1 activity activity per per cornea cornea or construct or construct -1] -1] [nmol [nmol ∙ min ∙ min
Glutathione Transferases Expression
14 12
Hemicornea Hemicornea
Porcine Cornea Porcine Cornea
Rabbit Cornea Rabbit Cornea
GSTP1 Activity (Substrate: ethacrynic acid)
** **
12 10 10 8 8 6 6 4
4 2 2 0 0
Hemicornea Hemicornea
Porcine Cornea Porcine Cornea
Rabbit Cornea Rabbit Cornea
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Summary – Part I What do we know? • organotypic and valid cell culture models of human cornea for drug absorption studies have been successfully established • expression patterns of efflux transporters seem to be quite different for human and animal cornea models (ex-vivo and in-vivo) • transfer of data obtained with rabbit corneas to human situation remains difficult • expression of ABC transporters could be induced in corneal cells by long-term exposure of transporter subtrates
But, what don’t we know yet? • Are efflux transporters functionally expressed in human cornea in-vivo? → Problem: Availability of sufficient human donor tissue • Could efflux transporters play a significant role for drug absorption and transcorneal transport after topical application to human eye? → Problem: In vivo data from human subjects are needed High drug concentration in ophthalmics (saturation?) Dynamic conditions on eye surface (fast dilution, drainage … resulting in short precorneal residence time) Orlando October 29, 2015 | S. Reichl | AAPS Annual Meeting
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Dynamic Micro Tissue Engineering System (DynaMiTES) 5A
5B Inverse HC
Drive for the stirrer Upper TEER electrode Sampling point Top level Transwell®/ insert level Bottom level Lower TEER electrode Inlet and outlet
Figure 5: Engineering DynaMiTES; 5A: Engineering design;design 5B: First prototype
First prototype
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• The eye, the cornea and transcorneal drug transport
• Development and characterization of an organotypic, threedimensional in-vitro cell culture model of human cornea for drug absorption studies as replacement for animal testing
• Expression of ABC transporters in animal and human corneal models in-vitro and ex-vivo • Expression of phase I and II metabolizing enzymes in corneal tissue • SLC Transporter - short review from the literature
Orlando October 29, 2015 | S. Reichl | AAPS Annual Meeting
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Oligopeptide Transporters PEPT1, 2 – Human (excised cornea and cell culture model), Rabbit mRNA level (+)
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Oligopeptide Transporters – PEPT1
Acyclovir
L-Valine Prodrug of Acyclovir
• In-vitro test using rabbit cornea • threefold higher permeability for L-Val-ACV than ACV • dipeptides, beta-lactam antibiotics and ACE inhibitors strongly inhibited the transport of L-Val-ACV Orlando October 29, 2015 | S. Reichl | AAPS Annual Meeting
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Amino Acid Transporters ASCT1 – Rabbit (excised cornea and cell culture), mRNA level (+), functionality level (+)
LAT1 – Rabbit (excised cornea and cell culture), mRNA level (+), functionality level (+); Human cornea, mRNA level (+)
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Amino Acid Transporters CAT1, 2 – Human (excised cornea, cell culture), mRNA level (+), protein level (+)
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Amino Acid Transporters
Acyclovir
γ-Glutamate Prodrug of Acyclovir
L- Tyrosine Prodrug of Acyclovir
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Amino Acid Transporters
Acyclovir (ACV)
γ-Glutamate Prodrug of Acyclovir (γ-Glu-ACV)
L- Tyrosine Prodrug of Acyclovir (Tyr-ACV)
• In-vitro test using rabbit cornea and primary rabbit cells
• γ-Glu-ACV transport was concentration-, energy- and sodium-dependent, and independent of pH; inhibited by neutral and cationic amino acids • Tyr-ACV was not recognized by this amino acid transporters
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Monocarboxylate transporters MCT1, 3, 4 – Human (excised cornea and cell culture), Rabbit cornea mRNA level (+), protein level (+), functionality level (+)
MCT1, 2, 4, 5 – Rabbit (primary cell culture), protein level (+), functionality level (+)
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Monocarboxylate transporters Rabbit (primary cell culture)
• ketoprofen uptake was time, temperature and pH dependent • ketoprofen uptake was significantly inhibited by various monocarboxylates and other NSAIDs and by MCT and/or organic anion transporter (OAT) inhibitors probenecid and p-aminohippurate
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Organic anion and cation transporters OCT1, 3; OCTN1, 2; OATP2B1 – Human (excised cornea and cell culture model), mRNA level (+)
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Vitamin transporters SMVT – Rabbit (excised cornea and cell culture), mRNA level (+), functionality level (+)
Riboflavin transport – Rabbit (excised cornea and cell culture), functionality level (+)
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Summary • ABC and SLC transporters are expressed in epithelium of human and animal corneas • Recent studies suggest that the expression patterns are species dependent • The impact of transport proteins on transcorneal drug absorption and disposition has to be further investigated under dynamic in vivo conditions
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Acknowledgment TU Braunschweig
Universität Düsseldorf
Matthias Hahne Gesa Grobe Jessica Verstraelen Christian Kölln Lucia Albrecht Anne Dolberg Nicole Beißner
Prof. Dr. Gerd Geerling Prof. Dr. Stefan Schrader Dr. Maria Borrelli
LU Hannover Dr. Nicola Hofmann
Universität Bremen
UKE Hamburg
PD Dr. Maria Engelke
Dr. Michaela Zorn-Kruppa Prof. Dr. Johanna Brandner
Across Barriers GmbH Dr. Eleonore HaltnerUkomadu Gustavo Guzman
TU Braunschweig Institut für Mikrotechnik Prof. Dr. Andreas Dietzel Dipl. Ing. Thomas Lorenz
Medizin. Hochschule Hannover Dr. Michael Meyer Prof. Dr. Rolf Winter
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