Biopharmaceutics Classification System: Defining a Permeability Class Blair Miezeiewski, M.S. Senior Scientist, In Vitro Permeability Lab
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.
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
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
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
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
Caco-2 Papp x 10^6 cm/sec 2011 Absorption Systems Suitability of Caco-2 Monolayer Model for BCS Permeability Class 100.00 75.00 50.00 25.00 23 model compounds with high and low human absorption potential have well separated P app values 10.00 7.50 5.00 2.50 1.00 0.75 0.50 Minoxidil 0.25 0.10 0.07 0.05 0.03 0.01 Low High Human Absorption
-10 µm Atenolol Absorption Systems QC (paracellular monolayer integrity) QC Criteria (SOP 023) -10 µm Propranolol (passive transcellular integrity) -5 µm E3S (BCRP transport) ASI-4 Compound/Probe QC Measure Caco-2 TEER (pre-experimental) (Ω*cm 2 ) 450 to 650 LY (0.5 mm) P app(a B) x 10-6 (cm/s) <0.40 Atenolol (10 μm) P app(a B) x 10-6 (cm/s) <0.50-10 µm Digoxin (P-gp transport) Propranolol (10 μm) P app(a B) x 10-6 (cm/s) 10.0-30.0 (CPT) 15.0-25.0 (BCS) -Qualifying properties of each batch ensures consistency between Caco-2 permeability and human intestinal absorption. Digoxin (10 μm ) P app(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)
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 QC Notebook Ref Cell Line AS1576-32 AS1544-28 CACO2 Passage # 61 Da te Seeded 04/09/2014 Assay Date 04/29/2014 Cel l Age 20 STDV TEER (ohm-cm 2 ) 471 16 Passive diffusion, P app (x10-6, cm/s) Atenolol A B 0.217 0.0214 Propranolol A B 15.5 0.952 Pgp transport, P app (x10-6, cm/s) Digoxin A B 0.619 0.0517 Digoxin B A 17.3 1.85 Net Fl ux 16.7 Effl ux Ra tio 27.9 BCRP transport, P app (x10-6, cm/s) E3S A B 0.341 0.0103 E3S B A 41.0 2.52 Net Fl ux 40.6 Effl ux Ra tio 120
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: P app of 2.33-7.91 Low permeability reference: Atenolol -fraction absorbed < 50% in humans -validated acceptance criteria: P app of <1 -Caco-2 monolayers clearly discriminate low and high permeability compounds
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
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 2 Amlodipine, minoxidil (10 µm) and atenolol (100 µm) Amlodipine, minoxidil (10 µm) and atenolol (100 µm) A B 3 0 and 45 min 15, 30 and 45 min N/A B A 3 0 and 45 min 15, 30 and 45 min Lucifer Yellow
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 A B 3 0 and 45 min 45 min
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 A B 4 0 and 45 min** 15, 30 and 45 min** 45 min N/A 2 Test article only B A 4 0 and 45 min*** 15, 30 and 45 min*** 45 min PEDS run A B for 30 min 3 Atenolol and minoxidil A B 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 A B for 30 min
Phase 1B: Bi-directional Permeability -re-run bidirectional permeability in the absence of controls to confirm P app and that controls don t impact permeabiilty Treatment Dose Direction Replicates Sample Donor Sample Receiver Post-Experiment 1 Test article (1% HDS) only 2 Test article (1% HDS) only 3 Test article (10% HDS) only 4 Test article (10% HDS) only 5 Test article (100% HDS) only 6 Test article (100% HDS) only A B 4 or 6* 0 and 45 min 15, 30 and 45 min PEDS or C lysate B A 4 or 6* 0 and 45 min 15, 30 and 45 min PEDS or C lysate A B 4 or 6* 0 and 45 min 15, 30 and 45 min PEDS or C lysate B A 4 or 6* 0 and 45 min 15, 30 and 45 min PEDS or C lysate A B 4 or 6* 0 and 45 min 15, 30 and 45 min PEDS or C lysate B A 4 or 6* 0 and 45 min 15, 30 and 45 min PEDS or C lysate
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Data Processing and Interpretation
QC: TEER Measurements = (measured TEER Rb)*insert area (1.13 cm 2 ) Caco-2 TEER acceptance: 450-650 ohm cm 2 TEER Measured TEER TEER Adjusted AVE TEER STDEV Repeat (ohm) (ohm-cm 2 ) (ohm-cm 2 ) (ohm-cm 2 ) R1 448 504 518 43 R2 475 534 R3 517 582 R4 480 540 R5 419 471 R6 422 475
Apparent Permeability P app = (dc r /dt) x V r / (A x 60 x D NC ) x 10 6 (unit: 10-6 cm/s) Where, dc r /dt is the slope of the cumulative concentration in the receiver compartment versus time; V r is the volume of the receiver compartment in cm 3 ; A is the area of the cell monolayer (1.13 cm 2 for 12-well Transwell ); D NC is the nominal dosing concentration. LY L.Y. Conc. L.Y. P app Ave LY P app 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 apparent permeability oints.
Atenolol A B P app = (dc r /dt) x V r / (A x 60 x D NC ) x 10 6 (unit: 10-6 cm/s) Acceptance: P app <0.50 Atenolol Receiver Atenolol Conc. Atenolol P app Ave Atenolol P ap STDEV 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
Propranolol Acceptance criteria: P app 15.0-25.0 A B Propranolol Receiver Propranolol ConcPropranolol P app Ave Propranolol STDEV Repeat (um) (cm/s) E -6 (cm/s) E -6 (cm/s) E -6 R1 0.835 15.4 16.0 1.48 R2 0.812 15.0 R3 0.961 17.7
Digoxin Efflux: Functionality of P-gp Transporter The efflux ratio (ER) is defined as Papp (B-to-A) / Papp (A-to-B) Digoxin ER 10 A B B A Digoxin Receiver Digoxin Conc. Digoxin P app Ave Digoxin P app STDEV Repeat (um) (cm/s) E -6 (cm/s) E -6 (cm/s) E -6 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 P app(b-a) -P app(a-b) 12.9 Efflux Ratio P app(b-a) -P app(a-b) 25.0
Digoxin Historical Data
E3S Efflux: Functionality of BCRP Efflux Transporter ER 15 A-B B-A E3S Receiver E3S Conc. E3S P app Ave. E3S P app 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 P app(b-a) -P app(a-b) 19.66 Efflux Ratio P app(b-a)/ P app(a-b) 53.80
E3S Historical Data
Amlodipine Experimental Plan: Phase 1A Treatment Dose Direction Replicates Sample Donor Sample Receiver Post- Experiment 1 2 Amlodipine, minoxidil (10 µm) and atenolol (100 µm) Amlodipine, minoxidil (10 µm) and atenolol (100 µm) A B 3 0 and 45 min 15, 30 and 45 min N/A B A 3 0 and 45 min 15, 30 and 45 min Lucifer Yellow
Amlodipine Data Analysis: Part 1 Raw data (nm) 15 min. 30 min. 45 min. R1 1550 2920 4390 R2 2370 3470 5600 R3 2220 3620 4780 Cumulative Concentration (nm) 15 min. 30 min. 45 min. R1 1550 3127 4986 R2 2370 3786 6379 R3 2220 3916 5559 For A-to-B receiver samples, C c15 = C m15 C c30 = C m30 + C m15 2/15 C c45 = C m45 + C m15 2/15 + C m30 2/15 For B-to-A receiver samples, C c15 = C m15 C c30 = C m30 + C m15 2/5 C c45 = C m45 + C m15 2/5 + C m30 2/5
Amlodipine A-B Data
Amlodipine B-A Data Receiver conc (nm) Donor conc (nm) 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 Replicate slope R2 R1 274.533 0.99 R2 236.067 1.00 R3 308.733 0.99 Efflux Ratio = 0.747
Recovery Calculation Where, Recovery NC (%) = 100 x ((V r x C r final ) + (0.05 x C ini ) + (V d x C d final ))/(V d x D NC ) V r is the volume of the receiver compartment in cm 3 ; V d is the volume of the donor compartment cm 3 ; C r final is the cumulative receiver concentration at the end of the incubation period; C d final is the concentration of the donor at the end of the incubation period; D NC is the nominal dosing concentration. C ini is the initial donor concentration;
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 (D ini ) 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 Recovery NC 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.
Internal References: minoxidil Cum. Receiver Conc (nm) Donor Conc (nm) 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 slope R2 3.194 0.96 2.726 1.00 3.843 0.99
Internal reference: atenolol Cum. Receiver Conc (nm) Donor Conc (nm) 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 slope R2 1.455 0.96 1.742 1.00 1.700 0.99
Amlodipine Phase 1A TEER and LY Rb 14 TEER LY.Papp Ave LY Papp Well# Rt LY, µm PASS/FAIL (ohm-cm 2 Sample ) (cm/sec) (cm/sec) 1 434 474.6 97.8 µm amlodepine, +100 µm 2 451 493.81 atenolol, +10 µm minoxidil A B 3 419 457.65 NO PELY 4 434 474.6 0.6700 0.5 97.8 µm amlodepine, +100 µm 5 470 515.28 0.4160 0.3 0.5 atenolol, +10 µm minoxidil B A 6 426 465.56 0.8770 0.6 AVE. TEER 480.25 STDEV 20.98
Amlodepine: Non-Specific Binding A to B Receiver Donor 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
Special Case: Mass Balance (2) Mass Balance Mass Balance = ((V r x C r final ) + (0.05 x C ini ) + (V d x C d final ) + (V lysate x C lysate ))/( V d x D nc ) x 100 % Where, V r is the volume of the receiver in ml (1.5 ml for A-to-B, 0.5 ml for B-to-A); V d is the volume of the donor in ml (0.5 ml for A-to-B, 1.5 ml for B-to-A); C r final is the cumulative receiver concentration at the end of the incubation period; C d final is the concentration of the donor at the end of the incubation period; V lysate is the volume of cell lysate, 0.5 ml; C lysate is the measured concentration in cell lysis homogenate; D NC is the nominal dosing concentration. C ini is the initial donor concentration;
Additional Considerations: Case Study Table 4. Caco-2 Permeability and Recovery of Test Article in Ca 2+ /Mg 2+ -free HBSSg With and Without a ph Gradient Compound Nominal Dosing Conc. (µm) Measured ph 6.5/7.4 ph 7.4/7.4 A-to-B (mean, n=3) ph 6.5/7.4 ph 7.4/7.4 P app Recovery (10-6 cm/s) * (%) P app Recovery (10-6 cm/s) * (%) Test Article 13.5 13.9 12.5 NR 108 ± 8.65 NR 101 ± 9.99 Atenolol 100 99.8 98.6 9.06 a ± 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).