The PAMPA Work Flow and Comparison of UV-Plate Reader Method vs. LC/MS Method Kevin Chen, Ph.D. BD Biosciences August 13, 2009
The Traditional PAMPA Workflow 96-well filter plate, un-coated Coat filter plate with lipids 96-well filter plate, coated with lipids Prepare compound solutions; Add solutions to plate Couple plates together for permeation Incubate for several hours Transfer solutions to UV Plates Transfer solutions, add internal standard UV Plate Reader Or LC/MS Analysis Calculate Permeability
The PAMPA Workflow Using BD Gentest Pre-coated PAMPA Plate System 96-well filter plate, un-coated Coat filter plate with lipids 96-well filter plate, coated with lipids Prepare compound solutions; Add solutions to plate Couple plates together for permeation Incubate for several hours Transfer solutions to UV Plates Transfer solutions, add internal standard UV Plate Reader Or LC/MS Analysis Calculate Permeability
Step 1: Prepare Compound Solutions Compound stock solution: usually prepared in DMSO Concentration ~10 mm Buffer PBS, ph 7.4 Ammonium acetate 10 mm, NaCl 200 mm, ph 7 Acetate 10 mm, NaCl 200 mm, add NaOH to desired ph (4 6) Compound solution: prepared by diluting DMSO stock solution into buffer For UV-plate reader, use compound concentration 100 200 μm For LC/MS, use compound concentration 10 50 μm
Step 2: Add Solutions to BD Gentest Pre-coated PAMPA Plates BD Gentest Pre-coated PAMPA Plates are stored at -20 C. Take out the plates from the freezer and let sit in room temp for at least half an hour before adding solutions. Plates can be taken out of the package before warming up to room temp. Add 0.3 ml/well of compound solutions in the bottom plate. Add 0.2 ml/well of buffer in the top plate. Alternatively, 0.2 ml compound solutions can be added in the top plate and 0.3 ml buffer added in the bottom plate 0.3 ml of compound solution 0.2 ml of buffer
Step 3: Couple Donor and Acceptor Plates Gently lower the top plate onto the bottom plate Use slow motion to avoid bubbles Coupling donor and acceptor plates manually Coupling donor and acceptor plates using a robotic arm
Step 4: Incubation Allow the coupled donor / acceptor plates to sit for 4 5 hours Stirring / shaking is not necessary Humidity control is not necessary Plates can be incubated at room temp or at 37 C Temperature control is recommended for reducing variations from day to day At the end of incubation, separate donor / acceptor plates and record actual incubation time Compounds permeate through membrane barrier (Incubation for 4 5 hours)
Analyze Compound Concentrations Using UV-Plate Reader (1) Transfer 150 μl / well from each plate to a BD Falcon 96-well UV-transparent plate (BD Cat. No. 353261). Add plain buffer to several un-used wells for background. In a third UV-transparent plate, add 150 μl / well of the original compound solution (initial donor solution) or a dilution series of the original compound solution in buffer. Scan three UV-transparent plates using a UV-plate reader. For each plate, scan at wavelength 250 nm and then at wavelength 280 nm. Save both sets of data.
Analyze Compound Concentrations Using UV-Plate Reader (2) For each compound, determine whether the UV absorption at 250 nm or UV absorption at 280 nm should be used for calculating compound concentrations: In general, choose the wavelength that has a larger value of UV absorption Record the UV absorption values of the initial donor solution (A 0 ), final donor solution (A D ), final acceptor solution (A A ), and plain buffer (A buffer ) at this chosen wavelength Calculate the final donor and acceptor concentrations: Initial donor concentration C 0 = 200 μm (or other actual values) Final donor concentration Final acceptor concentration C D AD Abuffer = C0 A0 Abuffer AA A CA = C0 A A 0 buffer buffer
Analyze Compound Concentrations Using LC/MS (1) Transfer 100 μl / well from each plate to a regular 96-well plate. Add equal amount of internal standard (for example, 10 μl of a 25 μm internal standard) to each well. In a third 96-well plate, add 100 μl / well of the original compound solution (initial donor solution) or a dilution series of the original compound solution in buffer. Add the same amount of internal standard to each well. Inject 10 μl of solution from each sample to the LC/MS. The solutions from each plate can be diluted, if needed (in the case that the MS signal is too high). Since the ratio of analyte concentration to internal standard concentration is calculated, the dilutions will not affect the results.
Analyze Compound Concentrations Using LC/MS (2) For each mass spectrum, calculate peak area of analyte (P analyte ) and peak area of the internal standard (P internal_standard ). Calculate the concentration of analyte: Panalyte Canalyte = Cinternal_s tandard P internal_standard For each compound, three concentrations are obtained: Initial donor concentration C 0 Final donor concentration C D Final acceptor concentration C A
Calculate the Permeability Permeability (cm/s): P e = ln[1 C S (1/ V D A / C + 1/ V equilibrium A ) t V D = donor volume (0.3 ml) V A = acceptor volume (0.2 ml) C equilibrium = [ CD VD + CA VA ]/( VD + VA ) ] Equilibrium concentration (compound concentration across donor and acceptor wells if the membrane is 100% permeable to the compound) S = membrane area (0.3 cm 2 ) t = incubation time (in seconds)
Calculate the Mass Retention Mass retention (percentage loss of compound due to non-specific binding to the plastic surfaces during the permeation assay): R = 1 [ CD VD + CA VA]/( C0 VD ) The permeability formula has taken mass retention into consideration. It assumes that the compounds bound to the surfaces do not contribute to the permeation, and it calculates the effective initial compound amount by adding the amount of compounds in the final donor and acceptor wells (the amount of compounds that are not bound and contribute to permeation).
Derivation of the Permeability Formula The permeability formula is derived from the bi-directional permeation model: C A ( t) = C equilibrium (1 e when t = 0, C A = 0 P S (1/ V when t, C A = C equilibrium e D + 1/ V A ) t )
Calculation of Permeability Using a Worksheet Pre-loaded with Formula Permeability Calculation Worksheet for BD Gentest Pre-coated PAMPA Plate System (P/N 353015) Instructions: 1. Follow the User's Guide of the product to conduct a permeability assay. 2. Measure the concentration of each compound in the acceptor well and donor well at the end of the assay, as well as the initial concentration of the compound at the beginning of the assay. Note: the unit of the concentrations does not affect the results as far as the same unit is used for all the concentration values. 3. Copy the three concentration values in the "Input Data" Section below. 4. The results can be found in the "Calculation Results" Section on the right side of the input data. Note: the formula used for the calculations can be found in the User's Guide. Permeability values are given in two units: 10^(-6)*cm/s and nm/s. Parameters used in calculations Acceptor volume Donor volume Filter area 0.2 ml 0.3 ml 0.3 cm^2 Incubation time 4 hours = 14400 seconds Compound Acceptor concentration Input Data Donor concentration Initial concentration Equilibrium concentration Calculation Results % Mass retention Permeability (10^(-6)*cm/s) Permeability (nm/s) Example Cmp 1 25 170 200 112 6.67% 7.02 70.16 Example Cmp 2 4 190 200 115.6 3.67% 0.98 9.78 Data from Concentration Analysis Calculated using formula in previous slides
Setting Up a Worksheet Based on the Experimental Design (1) Plate Map 1 2 3 4 5 6 7 8 9 10 11 12 A Cmp 1 Cmp 1 Cmp 1 Cmp 9 Cmp 9 Cmp 9 Cmp 17 Cmp 17 Cmp 17 Cmp 25 Cmp 25 Cmp 25 B Cmp 2 Cmp 2 Cmp 2 Cmp 10 Cmp 10 Cmp 10 Cmp 18 Cmp 18 Cmp 18 Cmp 26 Cmp 26 Cmp 26 C Cmp 3 Cmp 3 Cmp 3 Cmp 11 Cmp 11 Cmp 11 Cmp 19 Cmp 19 Cmp 19 Cmp 27 Cmp 27 Cmp 27 D Cmp 4 Cmp 4 Cmp 4 Cmp 12 Cmp 12 Cmp 12 Cmp 20 Cmp 20 Cmp 20 Cmp 28 Cmp 28 Cmp 28 E Cmp 5 Cmp 5 Cmp 5 Cmp 13 Cmp 13 Cmp 13 Cmp 21 Cmp 21 Cmp 21 Cmp 29 Cmp 29 Cmp 29 F Cmp 6 Cmp 6 Cmp 6 Cmp 14 Cmp 14 Cmp 14 Cmp 22 Cmp 22 Cmp 22 Cmp 30 Cmp 30 Cmp 30 G Cmp 7 Cmp 7 Cmp 7 Cmp 15 Cmp 15 Cmp 15 Cmp 23 Cmp 23 Cmp 23 Cmp 31 Cmp 31 Cmp 31 H Cmp 8 Cmp 8 Cmp 8 Cmp 16 Cmp 16 Cmp 16 Cmp 24 Cmp 24 Cmp 24 Cmp 32 Cmp 32 Cmp 32 Copy concentration data into the 3 highlighted regions below: Plate A Plate D Plate W A B C D E F G H A B C D E F G H A B C D E F G H 1 2 3 4 5 6 7 8 9 10 11 12 1 2 3 4 5 6 7 8 9 10 11 12 1 2 3 4 5 6 7 8 9 10 11 12
Setting Up a Worksheet Based on the Experimental Design (2) Permeability (10^(-6)*cm/s) 1 2 3 4 5 6 7 8 9 10 11 12 A 0.21 0.23 0.24 3.42 3.23 3.67 0.46 0.23 0.35 4.12 3.6 3.71 B 4.12 4.45 3.91 10.76 11.43 10.11 0.98 0.78 0.84 0.15 0.09 0.17 C 8.14 7.87 7.68 0.02 0.05 0.01 2.03 2.59 2.44 0.67 0.82 0.63 D 0.89 0.56 0.74 3.67 3.98 4.52 8.83 8.06 8.4 8.89 9.83 9.23 E 3.24 3.65 3.42 0.53 0.62 0.48 9.34 8.89 9.8 5.46 5.67 5.98 F 6.14 5.89 6.34 0.25 0.16 0.17 0.33 0.51 0.32 3.54 3.98 3.76 G 0.45 0.35 0.32 0.09 0.15 0.01 7.44 7.9 7.12 0.82 0.67 0.83 H 5.67 5.43 5.34 7.23 7.46 7.77 0.66 0.42 0.6 4.76 4.29 4.93 Permeability (10^(-6)*cm/s) Standard deviation Classification Cmp 1 0.23 0.02 Low permeability Cmp 2 4.16 0.27 High permeability Cmp 3 7.90 0.23 High permeability Cmp 4 0.73 0.17 Low permeability Cmp 5 3.44 0.21 High permeability Cmp 6 6.12 0.23 High permeability Cmp 7 0.37 0.07 Low permeability Cmp 8 5.48 0.17 High permeability Cmp 9 3.44 0.22 High permeability Cmp 10 10.77 0.66 High permeability Cmp 11 0.03 0.02 Low permeability Cmp 12 4.06 0.43 High permeability Cmp 13 0.54 0.07 Low permeability Cmp 14 0.19 0.05 Low permeability Cmp 15 0.08 0.07 Low permeability Cmp 16 7.49 0.27 High permeability Cmp 17 0.35 0.12 Low permeability Cmp 18 0.87 0.10 Low permeability Cmp 19 2.35 0.29 High permeability Cmp 20 8.43 0.39 High permeability Cmp 21 9.34 0.46 High permeability Cmp 22 0.39 0.11 Low permeability Cmp 23 7.49 0.39 High permeability Cmp 24 0.56 0.12 Low permeability Cmp 25 3.81 0.27 High permeability Cmp 26 0.14 0.04 Low permeability Cmp 27 0.71 0.10 Low permeability Cmp 28 9.32 0.48 High permeability Cmp 29 5.70 0.26 High permeability Cmp 30 3.76 0.22 High permeability Cmp 31 0.77 0.09 Low permeability Cmp 32 4.66 0.33 High permeability
Classification Criteria In the PAMPA set-up, if the compound solution is added in the bottom plate and buffer is added in the top plate, then the classification criteria is P e > 1.5 * 10-6 cm/s P e < 1.5 * 10-6 cm/s High Permeability Low Permeability In the PAMPA set-up, if the compound solution is added in the top plate and buffer is added in the bottom plate, then the classification criteria is P e > 4.0 * 10-6 cm/s P e < 4.0 * 10-6 cm/s High Permeability Low Permeability
BD Gentest Pre-coated PAMPA Plates have been Validated using both UV-Plate Reader and LC/MS Analysis Human absorption values (Fa%) Permeability by LC/MS Low Permeability Compounds Nadolol 30% 0.028 0.16 Sulpiride 35% 0.04 0.18 Famotidine 40% 0.03 0.04 Acebutalol 50% 0.07 0.15 Atenolol 54% 0.03 0.1 Furosemide 61% 0.73 0.46 High Permeability Compounds Timolol 90% 3.3 4.45 Pindolol 92% 2.28 2.64 Metoprolol 95% 4.28 4.34 Warfarin 98% 5.0 5.28 Diclofenac 99% 7.46 6.3 Antipyrine 100% 9.66 7.33 Caffeine 100% 8.8 9.58 Propranolol 100% 11.8 8.6 Ketoprofen 100% 2.6 4.13 Permeability by UV Plate Reader In these PAMPA experiments, the compound solutions were added in the bottom plate.
BD Gentest Pre-coated PAMPA Plates have been Validated using both UV-Plate Reader and LC/MS Analysis Correlation plots using the same group of compounds Results obtained using UV-Plate Reader Results obtained using LC/MS 100 100 Human Absorption (%FA) 90 80 70 60 50 40 30 False negative Low permeability High permeability False positive Human Absorption (%FA) 90 80 70 60 50 40 30 False negative Low permeability High permeability False positive 20-8 -7-6 -5-4 PAMPA Permeability Log P (cm/s) e 20-8 -7-6 -5-4 PAMPA Permeability Log P (cm/s) e Both methods produced correct predictions for high and low permeability compounds.
Summary The PAMPA assay consists of set-up, incubation, compound concentration analysis, and permeability calculations The UV-plate reader method provides a high throughput way to obtain data The LC/MS method provides a more sensitive way to detect compounds BD Gentest pre-coated PAMPA plates have been validated using both UV-plate reader analysis and LC/MS analysis
BD Gentest Pre-coated PAMPA Plate System
Contact Us Questions? Contact information: Kevin Chen e-mail: kevin_chen@bd.com Technical Support: In the U.S. tel: 877.232.8995 e-mail: labware@bd.com Outside the U.S. Contact your local distributor or visit bdbiosciences.com/offices to locate your nearest BD Biosciences office. For research use only. Not intended for use in diagnostic or therapeutic procedures. BD, BD Logo, and all other trademarks are the property of Becton, Dickinson and Company. 2009 BD