IMPACT OF EXPERIMENTAL CONDITIONS ON IN VITRO PERMEABILITY: (1) CORRELATIONS IN INTER-LABORATORY Caco-2
Y.H. Lee1, K.J. Lee1, N. Johnson1, J. Castelo1, R. Mower1, K. Click1, R. Christopher1, P. Gordon1, T. Hollenbeck1, D. Moylan1, V. Ereso1, K. Holme1, G. Grass1, P.J. Sinko2
1LION
ABSTRACT
bioscience, San Diego, CA; 2College of Pharmacy, Rutgers University, Piscataway, NJ.APS Annual Meeting, Oct. 21, 2001, Denver, CO
3. Adjusted Inter-Lab Variations
RESULTS
Propranolol
Verapamil
Inter-Lab Permeability (xE-6 cm/s)
Atenolol
Mannitol
Propranolol
Verapamil
Mean
SD
CV (%)
Range
N (inter-day data #)
28.5
5.49
19
18.5 - 36.8
12
21.6
2.55
12
18.7 - 23.6
3
Mean
SD
CV (%)
Range
N (Inter-lab data #)
1.50
1.36
91
0.38 - 4
10
34.8
25.2
72
11.5 - 110
11
38.0
27.6
73
9.2 - 69.4
4
Adjusted
Mean
SD
CV (%)
Range
N (Inter-lab data #)
1. Intra and Inter-Lab Variations
•
1.08
0.33
31
0.69 - 1.80
12
0.46
0.01
3
0.45 - 0.47
3
•
Intra-lab variation: low (CV < 50%)
2.02
1.68
83
0.1 - 4.5
9
Transport
Mechanism
Passive, paracellular
Passive, transcellular
Passive, paracellular
Passive, transcellular
Passive, paracellular
Passive, paracellular
Passive, paracellular
Passive, paracellular
Mixed (paracellular, efflux)
Passive, transcellular
Passive, transcellular
Passive, transcellular
Passive, transcellular
Passive, paracellular
Passive, transcellular
Passive, transcellular
Passive, transcellular
Passive, transcellular
Passive, paracellular
Passive, transcellular
Active, efflux
Oral Absorption
in Humans
16-30
100
50
100
13
62
60
3-8
74
80-91
100
92
95
15
90
100
90
100
50
100
100
1.50
1.36
91
0.38 - 4
34.8
25.2
72
11.5 - 110
38.0
27.6
73
9.2 - 69.4
0.55
0.27
49
0.17 - 1.01
10
28.2
10.9
39
6.7 - 51.4
11
39.6
33.9
86
5.0 - 82.4
4
Good correlations between Lion and other labs:
–
–
–
R2
R-1 to R-8: Good log-log correlations with > 0.8
R-5: Good correlation between pH 6.5 data (R-5) and pH 7.4 data (Lion)
R-7(3d), R-8(3d): Good correlations between 3d culture and 21d culture
(Lion)
•
•
–
–
•
Poor correlations between Lion and other labs:
–
–
R2
R-9 and R-10: Poor log-log correlations with < 0.8
R-9(3d): better correlation in 3d culture data (R-9, 3d) vs Lion
data than in 21d culture data (R-9) vs Lion data
5
3
2
1
0
R-2
R-3
R-4
R-7
R7(3d)
Different Labs
R-9
R9(3d)
R-10
Mannitol, propranolol: variations
decreased (CV < 50%)
Atenolol: variations not improved (CV >
50%) due to the outlier in R-7 and poor
correlations with R-9 and R-10 (R2 <
0.8)
Verapamil: variations not improved (CV
> 50%) due to the poor correlations with
R-9 and R-10 (R2 < 0.8)
Adjusted inter-lab permeability:
–
Adjusted Atenolol Permeability
4
Lion
Adjusted inter-lab variations:
–
5
–
4
3
Atenolol, mannitol, propranolol: very
similar values between Lion and other
labs
Verapamil: mean permeability not
improved due to the poor correlations
with R-9 and R-10 (R2 < 0.8)
2
1
0
Lion
R-2
R-3
R-4
R-7
R7(3d)
R-9
R9(3d)
R-10
Different Labs
Reported Verapamil Permeability
Permeability (x E-6 cm/s)
Solubility
Class (a)
High
High
High
High
Low
High
High
High
High
High
Low
Low
High
N/A
High
Low
Low
High
High
High
High
Verapamil
Reported Atenolol Permeability
•
Model Compounds for Correlation
Compounds
MW
Permeability
Class
Acyclovir
225
Low
Antipyrine
188
High
Atenolol
266
Low
Caffeine
194
High
Chlorothiazide
296
Low
Cimetidine
252
Low
Furosemide
331
Low
Ganciclovir
255
Low
Hydrochlorthiazide
298
Low
Hydrocortisone
363
High
Ibuprofen
206
High
Ketoprofen
254
High
Labetalol
328
High
Mannitol
182
Low
Metoprolol
267
High
Naproxen
230
High
Phenytoin
252
High
Propranolol
259
High
Ranitidine
314
Low
Theophylline
180
High
Verapamil
455
High
Propranolol
Inter-lab variation: high (CV > 50%)
2. Inter-Lab Correlations
The assessment of a compound’s intestinal permeability is one of the key factors for the
successful prediction of oral absorption in the drug discovery and development processes. In
vitro determination of permeability using a Caco-2 monolayer is a currently recognized
method to predict the extent of in vivo oral absorption. To evaluate chemical candidates for
their absorption potential faster and cheaper, in silico absorption models using in vitro Caco-2
permeability have been developed and used in drug discovery and development processes.
However, it is well known that in vitro Caco-2 permeability can vary lab-to-lab due to the
differences in culture and transport conditions. As cited in 58 references, in vitro mannitol
permeability values vary 345 fold (0.019 – 6.55 x 10-6 cm/s)1. Therefore, an external
validation is needed to compare data between labs. The key issue is the quantitative
comparison of Caco-2 permeability between labs, but there is no standardized method to
evaluate inter-lab variations.
In this report, in vitro permeability of 21 marker compounds representing various transport
processes was studied in a standardized Caco-2 protocol at Lion to investigate inter-lab
variations of Caco-2 permeability. In vitro permeability values of marker compounds were
also collected from 10 references with various culture conditions (passage #, serum, filter
type/size, seeding density, monolayer age) and various transport conditions (agitation, pH,
buffer), and they were compared and correlated to the data studied at Lion. Finally, the
adjusted Caco-2 permeability of 4 marker compounds was back-calculated using a log linear
correlation curve. Atenolol, mannitol, propranolol, and verapamil were chosen as 4 marker
compounds as they were most frequently used in 10 references.
1.2
1.08
90
0.01 - 3.69
9
Mannitol
Perm eab ility (xE-6 cm/s)
INTRODUCTION
Intra-Lab (Lion) Permeability (xE-6 cm/s)
Atenolol
Mannitol
Inter-Lab Permeability (xE-6 cm/s)
Atenolol
Reported
Mean
2.02
SD
1.68
CV (%)
83
Range
0.1 - 4.5
Perm eability (xE-6 cm /s)
Purpose. To investigate inter-laboratory correlation of Caco-2 permeability using marker compounds. Methods. In vitro permeability of 21 marker compounds
representing various transport processes was studied in a standardized Caco-2 monolayer (24-well transwell format/ 20-24 day culture/ passage 30-40). A  B
permeability was studied at 37C, 50 opm, 95% humidity, and 5% CO2 using a 100 M donor concentration with 1% DMSO. In vitro permeability values of marker
compounds were also collected from 10 references with various culture conditions (passage #, serum, filter type/size, seeding density, monolayer age) and various
transport conditions (agitation, pH, buffer), and they were correlated to the data studied at Lion. Results. The study results at Lion showed reproducible permeability
for 4 transport markers at different Caco-2 monolayer batches (Mannitol: 0.45 – 0.47 x 10-6 cm/s (n=3); atenolol: 0.69 – 1.80 x 10-6 cm/s (n=12); propranolol: 18.5 –
36.8 x 10-6 cm/s (n=12); and verapamil: 18.7 – 23.6 x 10-6 cm/s (n=3)). However, the in vitro permeability values varied 11 fold (0.38 – 4.0 x 10-6 cm/s) for mannitol,
45 fold (0.1 – 4.5 x 10-6 cm/s) for atenolol, 10 fold (11.5 – 110 x 10-6 cm/s) for propranolol, and 8 fold (9.2 – 69.4 x 10-6 cm/s) for verapamil in inter-laboratory Caco2. It is also known that in vitro mannitol Papp values vary 345 fold (0.019 – 6.55 x 10-6 cm/s) from 58 published references1. By contrast, the correlation of Caco-2
permeability between Lion and each of 10 references was good (r2 > 0.8, 8 of 10). Conclusions. The in vitro permeability of marker compounds is reproducible under
a standardized experimental condition, but can vary significantly between laboratories due to the differences in culture and experimental conditions. This work
suggests that correlating inter-laboratory Caco-2 permeability for certain transport markers may be a valuable standardization method to normalize inter-laboratory
differences of Caco-2 permeability.
a. High: Solubility at pH 1.5-7.5 > dosage/250 mL; Low: Solubility at pH 1.5-7.5 < dosage/250 mL
120
100
80
60
40
20
0
Lion
R-9
R-9(3d)
R-10
Different Labs
Adjusted Verapamil Permeability
METHODS
Perm eability (xE-6 cm /s)
120
Lab to Lab Difference of Culture and Experimental Conditions
Experimental
Lion
R-1
R-2
R-3
R-4
R-5
R-6
R-7
Source
ATCC#37-HTB
ATCC
ATCC
ATCC
ATCC
Pfizer
SKB
ATCC
R-7(3d)
R-8
R-8(3d)
Culture media
DMEM, 10% FBS
1% NEAA, w L-glutamine
DMEM, 10% FBS
1% NEAA, 2mM L-glutamine
DMEM, 10% FBS
1% NEAA, 1% L-glutamine
DMEM, 10% FBS
1% NEAA
DMEM w MITO serum
DMEM, 10% FBS
1% NEAA
DMEM/F12
DMEM, 10% FBS
6 well (Coster)
3um
4.71
24 well (Coster)
PTEE, 0.4um
0.33
6 well (Coster)
Polycarbonate, 0.3um
4.71
6 well (Coster)
Polycarbonate, 3um
4.71
6 well
3um
4.71
6 well BIEGE
1um
4.19
6 well
Polycarbonate, 3um
Surface area (cm2)
24 well (BD)
PET membrane, 1um
0.31
DMEM w 25mM Hepes
10% FBS, 1% NEAA
1% L-glutamine
12 well
Polycarbonate, 0.4um
1.13
MEM, 10% FBS
Filter type
DMEM w high glucose
20% FBS, 0.1mM NEAA
2mM L-glutamine
12 well (Felcon)
PETP, 0.45um
0.83
6 well BIEGE
1um
4.71
24 well
PET, 1um
0.31
Cell passage
30-40
35-41
31-42
23-50
52-80
30-35
40-50
35-37
ATCC
R-9
74k
63k
64k
3x per week
3x per week
Every 2days for first 6 days
Everyday thereafter
Cell age
20-23 days
21-25 days
18-21 days
TEER (ohm cm2)
>200
Buffer used
Ringers
w glucose
PBS/ 15 mM Hepes
w glucose
HBSS/ 10mM Hepes
HBSS/ Hepes
HBSS
HBSS
Ringers
w glucose
HBSS
HBSS
HBSS
pH at A & B
7.4
7.2
7.4
7.4
7.4
7.4
7.4
7.4
A:0.3/ B:1.2
Transwell
A:0.3/ B:1
Ussing: 11mL
A:1.5
A:6.5 20mM Mes
B:7.4 20mM Hepes
A:0.5/B:1.5
7.4
Volume (mL) at A & B
Ussing: 6mL
A:1.5/B:2.6
A:1.5
A:0.31/B:1.0
Incubator
37C, 5% CO2
37C, 5% CO2
37C, 95% humidity
37C heating
37C, 5% CO2
37C water bath
37C heating
37C
37C, 5% CO2
37C, 5% CO2
Use of any cosolvent
1% DMSO
Type of agitation
50 rpm orbital
30 rpm reciprocal
Stirred by 5% CO2-95% O2 100 rpm orbital
70 rpm
Stirred by 5% CO2-95% O2
50 rpm orbital
100 rpm orbital
50 rpm
Transport time
90 min
120 min
60 min
60 min
120 min
240 min
120 min
120min
Sampling method
100uL/ 4 points
6 points
3 points
3-6 points
Drug concentration
100 uM
Bioanalytical method
LSC, LC/UV, LC/MS
Reference
15 days
25 days
63k
2-3x per week
every 2 days
21 days
20-26 days
>230
477k
200k
40
20
0
65k
R-9
R-9(3d)
R-10
806k
Every 2 days
21-25days
3days
21-25days
3days
300
100-150
840
440
21days
CONCLUSION
Correlating inter-lab Caco-2 permeability using certain transport markers may be a valuable standardization method to
normalize inter-lab differences of Caco-2 permeability.
Reference:
1. D.A. Volpe and A.S. Hussain. Impact of experimental conditions on the in vitro permeability of mannitol: An artificial neural network analysis.
4 points
100uM
100uM
10-100uM
<14uM
200uM
10-100uM
200uM
LSC
HPLC
LSC, HPLC
LSC, HPLC
LSC
LSC, HPLC
LSC, HPLC
HPLC
Pade et al. (1997)
Pharm. Res. 14:1210-
Irvine et al. (1999)
J. Pharm. Sci. 88: 28-
Yamashita et al.(2000)
EJPS. 10: 195-
Yazdanian et al. (1998) Yee. (1997)
Pharm. Res. 15: 1490- Pharm. Res. 14: 763-
Rubas et al. (1993)
Pharm. Res. 10: 113-
Chong et al. (1997)
Chong et al. (1997)
Pharm. Res. 14: 1835- Pharm. Res. 14: 1835-
Liang et al. (2000)
J. Pharm. Sci. 89: 336-
R-10: Lennernas (culture and experimental conditions are unavailable)
60
Different Labs
1% DMSO
120 min
80
Lion
Feeding schedule
120k
DMEM w MITO serum
20% FBS
30-50
Seeding density/cm2
80k
R-9(3d)
ATCC
100
Liang et al. (2000)
J. Pharm. Sci. 89: 336-
Withington et al. (1999)
AAPS. BD ViaSante
*The number in parenthesis: number of markers employed in correlation
Withington et al. (1999)
AAPS. BD ViaSante
2000 AAPS