US 7,022,528 B2Granted

Method and apparatus for improving in vitro measurement of membrane permeability of chemical compounds

AssigneePion, Inc.

InventorsAlex Avdeef, Per E. Nielsen, Chau M. Du

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Abstract

This invention improves the PAMPA (parallel artificial membrane permeability assay) method used in pharmaceutical, biotechnological, and agrochemical R&D. This new high-throughput method and apparatus for measurement of permeability and membrane retention of compounds overcomes shortcomings of prior art and includes sensitivity and speed enhancing reagents. The phospholipid membranes used here consist of 10–74% wt/vol soybean lecithin extract dissolved in dodecane. Concentrations are measured by direct UV spectroscopy. To reduce membrane retention, surfactants, cyclodextrins, or water-soluble lipophilic polymers with low UV absorption are used in the acceptor comparment of the permeation cells and create an artificial sink state. A pH gradient established between donor and acceptor solutions creates a secondary sink state. This “double-sink” makes successful modeling of passive-diffusion transport of molecules possible. By accelerating transport of certain molecules, it shortens measurement time and increases assay throughput. A new permeability equation accounts for the “double-sink” condition as well as membrane retention.

An assay method for high-throughput spectroscopic measurement of the membrane permeability and membrane retention values for a compound, said method not requiring knowledge or measurement of the molar absorptivity of said compound, and not requiring a calibration curve relating known concentrations of said compound to a spectroscopic property of said compound, said method comprising the steps of:

a. preparing a sample solution of said compound in an aqueous buffer of known pH and separating said sample solution from any precipitate, said separated solution constituting a reference solution,
b. preparing an initial donor solution of said compound, by placing an aliquot of said reference solution in a donor compartment, said donor compartment being on one side of a membrane barrier,
c. placing an initial acceptor solution in an acceptor compartment, said acceptor compartment being on the second side of said membrane barrier, wherein said acceptor solution comprises a buffer of known pH and one or more sink-forming additives, wherein said sink-forming additives possess one or more of the properties selected from the group consisting of (1) high capacity to bind said compound, (2) low UV absorption, (3) high water solubility, and (4) low vapor pressure,
d. preparing a donor-blank solution free of said compound, but otherwise of the same composition as said reference solution,
e. preparing an acceptor-blank solution of the same composition as said initial acceptor solution,
f. measuring a spectroscopic property of said compound in said reference, donor-blank, and acceptor-blank solutions at the start of the assay,
g. measuring said spectroscopic property of said compound in each of said final donor and final acceptor solutions after known permeation time from the start of the assay,
h. determining the relative concentration of said final donor and acceptor solutions by comparing the measured spectroscopic property of said final acceptor, final donor, reference, acceptor-blank and donor-blank solutions, and
i. calculating from said determination the membrane permeability of said compound using the equation
P
e
(
D
)
=
-
2.303
⁢
⁢
V
D
A
⁡
(
t
-
τ
SS
)
⁢
(
1
1
+
r
a
)
⁢
log
10
⁡
[
-
r
a
+
(
1
+
r
a
1
-
R
)
⁢
C
D
⁡
(
t
)
C
D
⁡
(
0
)
]
where R is membrane retention, calculated from the equation
R=1−[CD(t)+CA(t)·VA/VD]/CD(0)
and where
ra=(VD/VA)(Pe(A)/Pe(D))
and where Pe(D) and Pe(A) are the membrane permeabilities in the donor-to-acceptor and in the acceptor-to-donor directions, respectively;
VA and VD are volumes of the acceptor and donor compartments, respectively;
A is the area of the membrane barrier;
t is time;
τSS is steady-state time;
CA(t) and CD(t) are the acceptor and donor sample concentrations at time t, respectively;
and CD(0) is the concentration of the initial donor solution.

77 more elements

Facts

Application

10/351,263Filed 23 Jan 2003 · us provisional application

Priority

31 Jan 2002

Published

US 2003/0219716 A127 Nov 2003

Granted

4 Apr 2006

Term ends

Not estimatedThe source records don’t support an estimate.

Classification11 codes

G01N 13/04
Investigating surface or boundary effects, e.g. wetting power; Investigating diffusion effects; Analysing materials by determining surface,…:…
G01N 13/00
Investigating surface or boundary effects
G01N 15/0826
Investigating permeability by forcing a fluid through a sample: and measuring fluid flow rate, i.e. permeation rate or pressure change
B01L 3/50255
For parallel transport of multiple samples: multi-well filtration

Abstract

Claims26