The PAMPA Work Flow and Comparison of UV-Plate Reader Method vs. LC/MS Method

Kevin Chen, Ph.D.

BD Biosciences

August 13, 2009

Coat filter plate with lipids

Prepare compound solutions; Add solutions to plate

Couple plates together for permeation

96-well filter plate, un-coated 96-well filter plate, coated with lipids

Incubate for several hours

UV Plate Reader

LC/MS Analysis

Transfer solutions to UV Plates

Transfer solutions, add internal standard

Calculate PermeabilityOr

The Traditional PAMPA Workflow

Coat filter plate with lipids

Couple plates together for permeation

96-well filter plate, un-coated 96-well filter plate, coated with lipids

Incubate for several hours

UV Plate Reader

LC/MS Analysis

Transfer solutions to UV Plates

Transfer solutions, add internal standard

Calculate PermeabilityOr

Prepare compound solutions; Add solutions to plate

The PAMPA Workflow Using BD Gentest™

Pre-coated PAMPA Plate System

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

•

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.

0.3 mL of compound solution 0.2 mL of buffer

Alternatively, 0.2 mL compound solutions can be added in the top plate and 0.3 mL buffer added in the bottom plate

Step 2: Add Solutions to BD Gentest Pre-coated PAMPA Plates

Coupling donor and acceptor plates manually

Coupling donor and acceptor plates using a robotic arm

Step 3: Couple Donor and Acceptor Plates

•

Gently lower the top plate onto the bottom plate

•

Use slow motion to avoid bubbles

•

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

(Incubation for 4 – 5 hours)

Compounds permeate through membrane barrier

Step 4: Incubation

•

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 (1)

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 (A0

), final donor solution (AD

), final acceptor solution (AA

), and plain buffer (Abuffer

) at this chosen wavelength

•

Calculate the final donor and acceptor concentrations:–

Initial donor concentration C0

= 200 μM

(or other actual values)

–

Final donor concentration

–

Final acceptor concentration

buffer0

bufferD0D AA

AACC−−∗=

buffer0

bufferA0A AA

AACC−−∗=

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 (Panalyte

) and peak area of the internal standard (Pinternal_standard

). Calculate the concentration of analyte:

•

For each compound, three concentrations are obtained:

–

Initial donor concentration C0

–

Final donor concentration CD

–

Final acceptor concentration CA

tandardinternal_s

analytetandardinternal_sanalyte P

PCC ∗=

tVVSCC

P∗+∗

−−=

)/1/1(]/1ln[

AD

mequilibriuAe

)/(][ ADAADDmequilibriu VVVCVCC +∗+∗=

Calculate the Permeability

•

Permeability (cm/s):

–

VD

= donor volume (0.3 mL)

–

VA

= acceptor volume (0.2 mL)

–

Equilibrium concentration (compound concentration across donor and acceptor wells if the membrane is 100% permeable to the compound)

–

S = membrane area (0.3 cm2)

–

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):

•

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).

)/(][1 0 DAADD VCVCVCR ∗∗+∗−=

•

The permeability formula is derived from the bi-directional permeation model:

•

when t = 0, CA

= 0

•

when t ∞, CA = Cequilibrium

)1()( )/1/1(mequilibriuA

ADe tVVSPeCtC ∗+∗∗−−∗=

Derivation of the Permeability 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 calculationsAcceptor volume 0.2 mL

Donor volume 0.3 mL

Filter area 0.3 cm^2

Incubation time 4 hours = 14400 seconds

Input Data Calculation ResultsCompound

Acceptor concentration

Donor concentration

Initial concentration

Equilibrium concentration % Mass retention

Permeability (10^(-6)*cm/s)

Permeability (nm/s)

Example Cmp 1 25 170 200 112 6.67% 7.02 70.16Example Cmp 2 4 190 200 115.6 3.67% 0.98 9.78

Calculated using formula in previous slides

Data from Concentration Analysis

Calculation of Permeability Using a Worksheet Pre-loaded with Formula

Plate Map1 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 25B Cmp 2 Cmp 2 Cmp 2 Cmp 10 Cmp 10 Cmp 10 Cmp 18 Cmp 18 Cmp 18 Cmp 26 Cmp 26 Cmp 26C Cmp 3 Cmp 3 Cmp 3 Cmp 11 Cmp 11 Cmp 11 Cmp 19 Cmp 19 Cmp 19 Cmp 27 Cmp 27 Cmp 27D Cmp 4 Cmp 4 Cmp 4 Cmp 12 Cmp 12 Cmp 12 Cmp 20 Cmp 20 Cmp 20 Cmp 28 Cmp 28 Cmp 28E Cmp 5 Cmp 5 Cmp 5 Cmp 13 Cmp 13 Cmp 13 Cmp 21 Cmp 21 Cmp 21 Cmp 29 Cmp 29 Cmp 29F Cmp 6 Cmp 6 Cmp 6 Cmp 14 Cmp 14 Cmp 14 Cmp 22 Cmp 22 Cmp 22 Cmp 30 Cmp 30 Cmp 30G Cmp 7 Cmp 7 Cmp 7 Cmp 15 Cmp 15 Cmp 15 Cmp 23 Cmp 23 Cmp 23 Cmp 31 Cmp 31 Cmp 31H 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

1 2 3 4 5 6 7 8 9 10 11 12ABCDEFGH

Plate D1 2 3 4 5 6 7 8 9 10 11 12

ABCDEFGH

Plate W1 2 3 4 5 6 7 8 9 10 11 12

ABCDEFGH

Setting Up a Worksheet Based on the Experimental Design (1)

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 3.71B 4.12 4.45 3.91 10.76 11.43 10.11 0.98 0.78 0.84 0.15 0.09 0.17C 8.14 7.87 7.68 0.02 0.05 0.01 2.03 2.59 2.44 0.67 0.82 0.63D 0.89 0.56 0.74 3.67 3.98 4.52 8.83 8.06 8.4 8.89 9.83 9.23E 3.24 3.65 3.42 0.53 0.62 0.48 9.34 8.89 9.8 5.46 5.67 5.98F 6.14 5.89 6.34 0.25 0.16 0.17 0.33 0.51 0.32 3.54 3.98 3.76G 0.45 0.35 0.32 0.09 0.15 0.01 7.44 7.9 7.12 0.82 0.67 0.83H 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 ClassificationCmp 1 0.23 0.02 Low permeabilityCmp 2 4.16 0.27 High permeabilityCmp 3 7.90 0.23 High permeabilityCmp 4 0.73 0.17 Low permeabilityCmp 5 3.44 0.21 High permeabilityCmp 6 6.12 0.23 High permeabilityCmp 7 0.37 0.07 Low permeabilityCmp 8 5.48 0.17 High permeabilityCmp 9 3.44 0.22 High permeabilityCmp 10 10.77 0.66 High permeabilityCmp 11 0.03 0.02 Low permeabilityCmp 12 4.06 0.43 High permeabilityCmp 13 0.54 0.07 Low permeabilityCmp 14 0.19 0.05 Low permeabilityCmp 15 0.08 0.07 Low permeabilityCmp 16 7.49 0.27 High permeabilityCmp 17 0.35 0.12 Low permeabilityCmp 18 0.87 0.10 Low permeabilityCmp 19 2.35 0.29 High permeabilityCmp 20 8.43 0.39 High permeabilityCmp 21 9.34 0.46 High permeabilityCmp 22 0.39 0.11 Low permeabilityCmp 23 7.49 0.39 High permeabilityCmp 24 0.56 0.12 Low permeabilityCmp 25 3.81 0.27 High permeabilityCmp 26 0.14 0.04 Low permeabilityCmp 27 0.71 0.10 Low permeabilityCmp 28 9.32 0.48 High permeabilityCmp 29 5.70 0.26 High permeabilityCmp 30 3.76 0.22 High permeabilityCmp 31 0.77 0.09 Low permeabilityCmp 32 4.66 0.33 High permeability

Setting Up a Worksheet Based on the Experimental Design (2)

•

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 isPe

> 1.5 * 10-6

cm/s High Permeability

Pe

< 1.5 * 10-6

cm/s 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 isPe

> 4.0 * 10-6

cm/s High Permeability

Pe

< 4.0 * 10-6

cm/s Low Permeability

Classification Criteria

Human absorption values (Fa%)

Permeability by LC/MS

Permeability by UV Plate Reader

Low Permeability CompoundsNadolol 30% 0.028 0.16Sulpiride 35% 0.04 0.18Famotidine 40% 0.03 0.04Acebutalol 50% 0.07 0.15Atenolol 54% 0.03 0.1Furosemide 61% 0.73 0.46High Permeability CompoundsTimolol 90% 3.3 4.45Pindolol 92% 2.28 2.64Metoprolol 95% 4.28 4.34Warfarin 98% 5.0 5.28Diclofenac 99% 7.46 6.3Antipyrine 100% 9.66 7.33Caffeine 100% 8.8 9.58Propranolol 100% 11.8 8.6Ketoprofen 100% 2.6 4.13

•

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

20

30

40

50

60

70

80

90

100

-8 -7 -6 -5 -420

30

40

50

60

70

80

90

100

-8 -7 -6 -5 -4

False negativeFalse positiveHigh permeability

Low permeability

Hum

an A

bsor

ptio

n (%

FA)

PAMPA PermeabilityLog Pe (cm/s)

Results obtained using UV-Plate Reader

Correlation plots using the same group of compounds

Results obtained using LC/MS

False negativeFalse positiveHigh permeability

Low permeability

Hum

an A

bsor

ptio

n (%

FA)

PAMPA PermeabilityLog Pe (cm/s)

Both methods produced correct predictions for high and low permeability compounds.

BD Gentest Pre-coated PAMPA Plates have been Validated using both UV-Plate Reader and LC/MS Analysis

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 Chene-mail: kevin_chen@bd.com

Technical Support:In the U.S.tel: 877.232.8995e-mail: labware@bd.comOutside the U.S.Contact your local distributor or visitbdbiosciences.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