biopharmaceutics classification system: defining a ... · pdf filebiopharmaceutics...

© 2011 Absorption Systems

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Biopharmaceutics Classification System: Defining a Permeability Class

Blair Miezeiewski , M.S. Senior Scientist, In Vitro Permeability Lab


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Definition of Bioequivalence

The United States Food and Drug Administration (FDA) has defined bioequivalence as, "the absence of a significant difference in the rate and extent to which the active ingredient or active moiety in pharmaceutical equivalents or pharmaceutical alternatives becomes available at the site of drug action when administered at the same molar dose under similar conditions in an appropriately designed study.

http://en.wikipedia.org/wiki/United_States

http://en.wikipedia.org/wiki/Food_and_Drug_Administration

http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/ucm070124.pdf


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BCS Framework

1.If 2 drug products containing the same drug have the same concentration-time profile at the intestinal membrane surface, they will have the same rate and extent of absorption

2.If two drug products have the same in vivo dissolution profile under all luminal conditions, they will have the same rate and extent of drug absorption

Class I-High solubility + High Permeability

Class II-Low solubility + High Permeability

Class III-High solubility + Low Permeability

Class IV-Low Solubility + Low Permeability


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Guidance Recommendations for Determining Drug Substance Permeability Class

-Human studies :

-mass balance

-absolute BA

-intestinal perfusion

- Non-human studies :

-In vivo or in situ animal model intestinal perfusion

-In vitro permeability methods

-Excised intestinal tissues

-Monolayers of epithelial cells


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Caco-2 System

-human colon adenocarcinoma cell line

-forms polarized monolayers with an apical brush border

-morphologically homogeneous and comparable to human colon when seeded on dual chamber Transwell® system

- Considered an appropriate model for assessing the permeability of passively absorbed drugs


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Guidance Permeability Class Model Compounds

The FDA guidance states:

“To demonstrate suitability of a permeability method intended for application of the BCS, a rank-order relationship between test permeability values and the extent of drug absorption data in human subjects should be established using a sufficient number of model drugs.”

-minimizes inter-laboratory variability in permeability results


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Suitability of Caco-2 Monolayer Model for BCS Permeability Class

23 model compounds with high and low human absorption potential have well separated Papp values

Low High

0.01

0.03

0.05

0.07

0.10

0.25

0.50

0.75

1.00

2.50

5.00

7.50

10.00

25.00

50.00

75.00

100.00

Minoxidil

Human Absorption

Caco

-2 P

ap

p x

10^

6 c

m/s

ec


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Absorption Systems QC

-10 µM Atenolol

(paracellular monolayer integrity)

-10 µM Propranolol

(passive transcellular integrity)

-5 µM E3S (BCRP transport)

-10 µM Digoxin (P-gp transport)

-Qualifying properties of each batch ensures consistency between Caco-2 permeability and human intestinal absorption.

QC Criteria

(SOP 023)

ASI-4

Compound/Probe QC Measure Caco-2

TEER (pre-experimental) (Ω*cm2) 450 to 650

LY (0.5 mM) Papp(A→B) x 10-6 (cm/s) <0.40

Atenolol (10 μM) Papp(A→B) x 10-6 (cm/s) <0.50

Propranolol (10 μM) Papp(A→B) x 10-6 (cm/s)

10.0 - 30.0 (CPT) 15.0 - 25.0 (BCS)

Digoxin (10 μM ) Papp(A→B) x 10-6 (cm/s) N/A

Digoxin (10 μM ) Efflux Ratio (no units) >10

E3S (5 μM ) Efflux Ratio (no units) >15 (BCS)


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Guidance on in vitro cultured epithelial cells

-Presence of efflux transporters

-Lack of efflux transporters (compared to human levels) => potential misclassification of permeability class

-BCS guidance recommends “functional expression of efflux systems”

-Functional QC data showing asymmetric permeability of model chemicals

Cell Plating Notebook Ref AS1576-32

QC Notebook Ref AS1544-28

Cel l Line CACO2

Passage # 61

Date Seeded 04/09/2014

Assay Date 04/29/2014

Cel l Age 20 STDV

TEER (ohm-cm2) 471 16

Pass ive di ffus ion, Papp (x10-6, cm/s)

Atenolol AB 0.217 0.0214

Propranolol AB 15.5 0.952

Pgp transport, Papp (x10-6, cm/s)

Digoxin AB 0.619 0.0517

Digoxin BA 17.3 1.85

Net Flux 16.7

Efflux Ratio 27.9

BCRP transport, Papp (x10-6, cm/s)

E3S AB 0.341 0.0103

E3S BA 41.0 2.52

Net Flux 40.6

Efflux Ratio 120


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Internal Reference Standards

Guidance indicates “a low and high permeability model drug should be used as internal standards”

High permeability reference: Minoxidil

-fraction absorbed reported > 90% in humans

-validated acceptance criteria: Papp of 2.33-7.91

Low permeability reference: Atenolol

-fraction absorbed < 50% in humans

-validated acceptance criteria: Papp of <1

-Caco-2 monolayers clearly discriminate low and high permeability compounds


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Absorption Systems BCS Approach

Phase 1A:

Pre-qualification and determination of the eligibility of test article for BCS biowaiver (a go/no go decision point)

-Bidirectional with co-dosed controls

Phase 1B:

Protocol optimization and conduct of FDA-required experiments to establish protocol for pivotal studies

- NSB, pH verification/tolerability, and bidirectional in absence of co-dosed controls

Phase 2:

GLP BCS classification of permeability pivotal study


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Amlodepine Phase 1A Plan

-Brand name: Norvasc

-Calcium channel blocker

-used to treat hypertension and other coronary artery diseases

-BCS Phase 1A protocol:

Treatment Dose Direction Replicates Sample Donor Sample Receiver Post-

Experiment

1 Amlodipine, minoxidil (10 µM)

and atenolol (100 µM) AB 3 0 and 45 min 15, 30 and 45 min N/A


and atenolol (100 µM) BA 3 0 and 45 min 15, 30 and 45 min

Lucifer Yellow


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Phase 1B: Non-specific Binding

-used to determine if test article is lost due to binding of the apparatus

Treatment Dose Direction Replicates Donor Sampling Receiver Sampling

1 Test article only AB 3 0 and 45 min 45 min


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Phase 1B: Monolayer Tolerability and pH Verification

- Used to measure effects of test article on Caco-2 monolayer integrity (tolerability) and to elucidate any pH shift

Treatment Dose Direction Replicates*

Sample Donor

Sample Receiver

Donor and Receiver pH Measurement

Post-Experiment

1 Test article, atenolol,

and minoxidil AB 4

0 and 45 min**

15, 30 and 45 min**

45 min N/A

2 Test article only BA 4 0 and

45 min*** 15, 30 and 45 min***

45 min PEDS run AB

for 30 min

3 Atenolol and minoxidil AB 4 0 and

45 min** 15, 30 and 45 min**

45 min N/A

4 Buffer only N/A 4 0 and

45 min*** 15, 30 and 45 min***

45 min PEDS run AB

for 30 min


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Phase 1B: Bi-directional Permeability

-re-run bidirectional permeability in the absence of controls to confirm Papp and that controls don’t impact permeabiilty

Treatment Dose Direction Replicates Sample Donor Sample Receiver Post-Experiment

1 Test article (1% HDS) only

AB 4 or 6* 0 and 45 min 15, 30 and 45 min PEDS or Clysate


BA 4 or 6* 0 and 45 min 15, 30 and 45 min PEDS or Clysate










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Thank You!

Please feel free to ask questions!


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Data Processing and Interpretation


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QC: TEER Measurements

= (measured TEER – Rb)*insert area (1.13 cm2)

Caco-2 TEER acceptance: 450-650 ohm cm2

Measured TEER TEER Adjusted AVE TEER STDEV

Repeat (ohm) (ohm-cm2) (ohm-cm2) (ohm-cm2)

R1 448 504 518 43

R2 475 534

R3 517 582

R4 480 540

R5 419 471

R6 422 475

TEER


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Apparent Permeability

Papp = (dCr/dt) x Vr / (A x 60 x DNC) x 106 (unit: 10

-6 cm/s)

Where,

dCr/dt is the slope of the cumulative concentration in the receiver compartment versus time;

Vr is the volume of the receiver compartment in cm3;

A is the area of the cell monolayer (1.13 cm2 for 12-well Transwell

®);

DNC is the nominal dosing concentration.

L.Y. Conc. L.Y. Papp Ave LY Papp STDEV

(uM) (cm/s) E-6 (cm/s) E-6 (cm/s) E-6

0.660 0.24 0.22 0.02

0.552 0.20

0.590 0.22

LY


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LY apparent permeability

data points.


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Atenolol

Acceptance: Papp <0.50

Papp = (dCr/dt) x Vr / (A x 60 x DNC) x 106 (unit: 10

-6 cm/s)

Atenolol Conc. Atenolol Papp Ave Atenolol PappSTDEV

Repeat (uM) (cm/s) E-6 (cm/s) E-6 (cm/s) E-6

R1 0.0129 0.238 0.229 0.00804

R2 0.0122 0.225

R3 0.0121 0.223

Atenolol Receiver

AB


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Propranolol

Acceptance criteria: Papp 15.0 - 25.0

Propranolol Conc.Propranolol Papp Ave Propranolol PappSTDEV


R1 0.835 15.4 16.0 1.48

R2 0.812 15.0

R3 0.961 17.7

Propranolol Receiver

AB


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Digoxin Efflux: Functionality of P-gp Transporter

Digoxin ER ≥ 10

The efflux ratio (ER) is defined as Papp (B-to-A) / Papp (A-to-B)

Digoxin Conc. Digoxin Papp Ave Digoxin Papp STDEV


R1 0.0286 0.527 0.536 0.0105

R2 0.0289 0.533

R3 0.0297 0.548

R1 1.86 11.4 13.4 2.33

R2 2.09 12.8

R3 2.60 16.0

Net Flux Papp(B-A)-Papp(A-B) 12.9

Efflux Ratio Papp(B-A)-Papp(A-B) 25.0

Digoxin Receiver

AB

BA


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Digoxin Historical Data


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E3S Efflux: Functionality of BCRP Efflux Transporter

ER ≥ 15

E3S Conc. E3S Papp Ave. E3S Papp STDEV

Repeat (µM) (cm/s) E-6 (cm/s) E-6 (cm/s) E-6

R1 0.010 0.35 0.37 0.02

R2 0.011 0.39

R3 0.010 0.38

R1 1.550 19.05 20.03 1.40

R2 1.580 19.42

R3 1.760 21.63

Net Flux Papp(B-A)-Papp(A-B) 19.66

Efflux Ratio Papp(B-A)/Papp(A-B) 53.80

E3S Receiver

A-B

B-A


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E3S Historical Data


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Amlodipine Experimental Plan: Phase 1A

Treatment Dose Direction Replicates Sample Donor Sample Receiver Post-

Experiment


and atenolol (100 µM) AB 3 0 and 45 min 15, 30 and 45 min N/A


and atenolol (100 µM) BA 3 0 and 45 min 15, 30 and 45 min

Lucifer Yellow


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Amlodipine Data Analysis: Part 1

For A-to-B receiver samples, Cc15 = Cm15

Cc30 = Cm30 + Cm15 × 2/15 Cc45 = Cm45 + Cm15 × 2/15 + Cm30 × 2/15

For B-to-A receiver samples, Cc15 = Cm15

Cc30 = Cm30 + Cm15 × 2/5

Cc45 = Cm45 + Cm15 × 2/5 + Cm30 × 2/5

Cumulative Concentration (nM)

15 min. 30 min. 45 min.

R1 1550 3127 4986

R2 2370 3786 6379

R3 2220 3916 5559

Raw data (nM)

15 min. 30 min. 45 min.

R1 1550 2920 4390

R2 2370 3470 5600

R3 2220 3620 4780


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Amlodipine A-B Data


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Amlodipine B-A Data

B to A 15 30 45 0 45 Papp X 1e6 *

R1 4030 8672 12266 76800 72700 20.7

R2 2930 6622 10012 82100 87600 17.8

R3 5630 9452 14892 77200 86100 23.3

Mean 4197 8249 12390 78700 82133 20.6

STD 1358 1462 2442 2951 8204 2.7413

Receiver conc (nM) Donor conc (nM)

Replicate slope R2

R1 274.533 0.99

R2 236.067 1.00

R3 308.733 0.99 Efflux Ratio = 0.747


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Recovery Calculation

RecoveryNC (%) = 100 x ((Vr x Cr

final) + (0.05 x Cini) + (Vd x Cd

final))/(Vd x DNC)

Where,

Vr is the volume of the receiver compartment in cm

3;

Vd is the volume of the donor compartment cm3;

Cr final

is the cumulative receiver concentration at the end of the incubation period; Cd

final is the concentration of the donor at the end of the incubation period;


Cini is the initial donor concentration;


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Amlodepine Recovery Calculations

Nominal Initial Donor

Recovery Recovery

74.7 92.6

71.5 88.8

75.4 93.3

73.8 91.6

2.09 2.41

Nominal Initial Donor

Recovery Recovery

78.5 100

92.7 110

92.7 117

88.0 109

8.17 8.75

A-B B-A

Initial donor concentration (Dini) may be used in certain occasions (e.g. when the measured concentration is much lower than nominal due to significant non-specific

binding to the apparatus or cell accumulation) if properly justified by the Study Director.

Mean RecoveryNC must be > 80% to be considered acceptable.

If recovery of test and/or control compounds is < 80%, mass balance needs to be

established by measuring the residual concentration associated with the insert and in

certain occasions, rinsing the receiver side to recover compound.


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Internal References: minoxidil

Nominal

A to B 15 30 45 0 45 Papp X 1e6 * Recovery

R1 19.2 51.0 115 12600 9190 7.07 98

R2 16.5 55.2 98.3 7830 9020 6.03 92

R3 18.7 66.3 134 10100 10400 8.50 107

Mean 18.1 57.5 116 10177 9537 6.55 99

STD 1.44 7.92 17.88 2386 752 0.732 7.84

Cum. Receiver Conc (nM) Donor Conc (nM)

slope R2

3.194 0.96

2.726 1.00

3.843 0.99


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Internal reference: atenolol

A to B 15 30 45 0 45 Papp X 1e6 * Recovery

R1 10.5 25.1 54.2 79000 81000 0.322 81.0

R2 10.0 39.0 62.3 92800 103000 0.385 102

R3 11.2 33.0 62.2 85700 81900 0.376 82.4

Mean 10.57 32.38 59.54 85833 88633 0.361 88.5

STD 0.60 6.99 4.66 6901 12450 0.0342 11.9

Cum. Receiver Conc (nM) Donor Conc (nM)

slope R2

1.455 0.96

1.742 1.00

1.700 0.99


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Amlodipine Phase 1A TEER and LY

Rb 14

LY.Papp Ave LY Papp

(cm/sec) (cm/sec)

1 434 474.6

2 451 493.81

3 419 457.65

4 434 474.6 0.6700 0.5

5 470 515.28 0.4160 0.3

6 426 465.56 0.8770 0.6

AVE. TEER 480.25

STDEV 20.98

97.8 µM amlodepine, +100 µM

atenolol, +10 µM minoxidil A →BNO PELY

97.8 µM amlodepine, +100 µM

atenolol, +10 µM minoxidil B→A0.5

Well# LY, µM PASS/FAILTEER

(ohm-cm2)SampleRt


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Amlodepine: Non-Specific Binding

A to B

Receiver Papp Recovery

Time Point (min) 0 45 0 45 (cm/sec, 10-6) (%)

R1 0 44.4 876 535 22.3 68.3

R2 0 39.6 911 609 19.9 74.4

R3 0 49.1 822 581 24.7 74.5

Mean 22.3 72.4

SD 2.388 3.53

Donor


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Special Case: Mass Balance

(2) Mass Balance

Mass Balance = ((Vr x Cr final

) + (0.05 x Cini) + (Vd x Cd final

) + (Vlysate x Clysate))/( Vd x Dnc) x 100 %

Where,

Vr is the volume of the receiver in mL (1.5 mL for A-to-B, 0.5 mL for B-to-A); Vd is the volume of the donor in mL (0.5 mL for A-to-B, 1.5 mL for B-to-A);

Cr final

is the cumulative receiver concentration at the end of the incubation period;

Cd final

is the concentration of the donor at the end of the incubation period;

Vlysate is the volume of cell lysate, 0.5 mL; Clysateis the measured concentration in cell lysis homogenate;


Cini is the initial donor concentration;


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Additional Considerations: Case Study

Table 4. Caco-2 Permeability and Recovery of Test Article in Ca2+

/Mg2+

-free HBSSg

With and Without a pH Gradient

Compound

Dosing Conc.

(µM)

A-to-B

(mean , n=3)

pH 6.5/7.4 pH 7.4/7.4

Nominal

Measured Papp

(10-6

cm/s)*

Recovery

(%)

Papp

(10-6

cm/s)*

Recovery

(%) pH

6.5/7.4

pH

7.4/7.4

Test Article 13.5 13.9 12.5 NR 108 ± 8.65 NR 101 ± 9.99

Atenolol 100 99.8 98.6 9.06a ± 1.08 101 ± 4.28 9.36

a ± 0.997 90.6 ± 7.00

Minoxidil 10 11.0 9.90 NR 100 ± 0.899 NR 97.0 ± 9.02

Two possible alternative approaches were considered: A

pH gradient (apical 6.5, basolateral 7.4) and Ca2/Mg2+-

free HBSSg (to reduce the buffer-specific chelation of

test article with divalent cations and possibly overcome

solubility limitations).

biopharmaceutics classification system: defining a ... · pdf filebiopharmaceutics...

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