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WO2007028578A1 - Procede de mesure de la permeation d'une substance a travers une barriere - Google Patents

Procede de mesure de la permeation d'une substance a travers une barriere Download PDF

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Publication number
WO2007028578A1
WO2007028578A1 PCT/EP2006/008660 EP2006008660W WO2007028578A1 WO 2007028578 A1 WO2007028578 A1 WO 2007028578A1 EP 2006008660 W EP2006008660 W EP 2006008660W WO 2007028578 A1 WO2007028578 A1 WO 2007028578A1
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WIPO (PCT)
Prior art keywords
probe
permeation
substance
barrier
permeation barrier
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PCT/EP2006/008660
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German (de)
English (en)
Inventor
Johannes Schmitt
Antje Konrad
Joachim NÖLLER
Thorsten Hartmann
Thomas Müller
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Nimbus Biotechnologie GmbH
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Nimbus Biotechnologie GmbH
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Priority to EP06777169A priority Critical patent/EP1922538A1/fr
Publication of WO2007028578A1 publication Critical patent/WO2007028578A1/fr
Anticipated expiration legal-status Critical
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/08—Investigating permeability, pore-volume, or surface area of porous materials
    • G01N15/082—Investigating permeability by forcing a fluid through a sample
    • G01N15/0826—Investigating permeability by forcing a fluid through a sample and measuring fluid flow rate, i.e. permeation rate or pressure change
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/08—Investigating permeability, pore-volume, or surface area of porous materials
    • G01N2015/0846—Investigating permeability, pore-volume, or surface area of porous materials by use of radiation, e.g. transmitted or reflected light
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/08—Investigating permeability, pore-volume, or surface area of porous materials
    • G01N2015/0866—Sorption
    • G01N2015/0873—Dynamic sorption, e.g. with flow control means

Definitions

  • the present invention relates to a method for measuring the permeation of at least one substance contained in a solvent through a barrier and / or for determining permeation-influencing parameters, in particular for determining the permeability of the barrier for the at least one substance, as well as individual components and a kit for use in this procedure.
  • the permeation device is divided into two separate spatial units, the so-called Kompartimen- te.
  • Kompartimen- te the substance to be investigated is usually initially introduced on one side of the permeation barrier (in the donor compartment). With the onset of permeation, the concentration of the substance in the donor compartment gradually decreases while the substance accumulates in the acceptor compartment. The progression of the permeation process can be observed correspondingly via concentration measurements, at least until a concentration equilibrium has been established between the two compartments.
  • perfusion devices In addition to special permeation chambers (eg the so-called Ussing chamber), which are operated with isolated membrane vesicles or with organ tissue preparations as a barrier layer, among others, perfusion devices are also used which work with prepared intestinal areas of dead animals (Karlsson J.
  • Caco-2 cells and MDCK cells are used (Arthursson P., Epithelial Transport of Drugs in Cell Culture I: A Model for Studying the Passive Diffusion of Drugs over Intestinal Absorptive (Caco-2) Cells. J. Pharm. 1990; 79: 476-482; Arthursson P., Correlation between oral drug absorption in humans and apparent drug permeability coefficients in human intestinal epithelial (Caco-2) cells., Biochem., Biophys. Res.
  • the cells are cultured on permeable supports in special microtiter plates for a certain period of time. After formation of a monolayer cell layer, the cells are allowed to differentiate over a certain period of time and then take the natural permeation measurements. Depending on the cell type and cultivation conditions, this period is about 3 to 21 days. Subsequently, the cells are washed and submitted the actual measuring buffer on both sides of the cell layer. After addition of the substance to be measured on one side of the cell layer, samples are taken on the other side and the concentration of the substance to be examined is analyzed therein.
  • the schematic structure of a permeation measurement with a cell monolayer as a permeation barrier is shown in FIG.
  • the concentration measurements are usually carried out via an HPLC or LC / MS method.
  • the permeation can be measured both from the apical to the basolateral side and vice versa. Due to the morphology of the cells, this procedure allows conclusions to be drawn on the involvement of active transporter proteins in the permeation of the substance, since these transporter proteins are localized in the basolateral cell membrane. From these data, the so-called permeation coefficient can be calculated, which in turn correlates with in vivo data from the human body.
  • PAMPA Parallel Artificial Membrane Permeation Assay
  • the filter membranes are impregnated with mixtures of 1-20% lecithin in organic solvents (eg dodecane, hexadecane, 1, 9-decadiene), so that a lipophilic barrier layer is formed.
  • organic solvents eg dodecane, hexadecane, 1, 9-decadiene
  • a lipophilic barrier layer is formed.
  • This process reduces the diameter of the permeation barrier by using polycarbonate filter membranes with larger pore diameters (3 ⁇ m vs. 0.22 or 0.45 ⁇ m at Kansy) of 120 ⁇ m at Kansy to about 10 ⁇ m, thereby increasing the potential Throughput of substances per unit of time.
  • a method according to the invention is provided for investigating the permeation of a substance contained in a solvent through a barrier.
  • the simultaneous investigation of the permeation of several substances contained in a solvent is made possible by a method according to the invention.
  • a method according to the invention is eminently suitable for determining parameters influencing respiration, for example the permeability of the barrier for the at least one substance, which will be discussed later will be discussed in more detail. Permeation kinetics can also be determined with little effort by the method according to the invention.
  • An inventive method is characterized in particular by the fact that the permeation of the at least one substance is examined by using at least one luminescent probe.
  • luminescent is used as a generic term in particular for fluorescence and phosphorescence, but also for example for electro-, thermo- and chemiluminescence.
  • the at least one substance to be investigated is provided in a first step in a solvent on at least one side of a permeation barrier.
  • a permeation barrier This includes, inter alia, the possibility of providing different substances on both sides of the barrier and / or one or more identical substances on both sides of the barrier, in which latter case between the two sides of the barrier with respect to the substance or substance to be examined.
  • the substances to be examined must each have a concentration difference between the two sides of the barrier.
  • the at least one substance and the permeation barrier are allowed to interact for a certain period of time.
  • This incubation of the permeation barrier with the at least one substance contained in the solvent is followed in a further step by a measurement of luminescence signals of the at least one probe on at least one side of the permeation barrier.
  • the measured luminescence signals are modulated as a function of the respective concentration of the substance, it being absolutely necessary for the absorption spectra of the at least one substance to overlap with excitation and / or emission spectra of the at least one probe. In this way, statements about the concentration of the substance to be examined can be obtained on one or both sides of the permeation barrier, which in turn allows direct conclusions about the permeation of the substance through the barrier as well as on the various parameters influencing permeation.
  • the method according to the invention thus utilizes the so-called internal filter effect, in which the light absorption of substances can be determined indirectly via a weakening of luminescence intensities of suitable probes.
  • This phenomenon is based on the fact that the intensity of light rays, which are absorbed on the way to a suitable probe or on the way from the probe to a detector of other dissolved substances, that is, thus attenuated.
  • the overlap of excitation or emission wavelengths with the absorption band of the substance to be examined leads in the measurement at suitable wavelengths (ie in the range of the absorption band of the substance) to a modulation or attenuation of the luminescence intensity proportional to the concentration of the substances to be examined before and / or is behind the barrier.
  • the excitation light passes through the sample and is attenuated in the event of an overlap of the absorption spectrum of the substance to be examined with the excitation spectrum of the probe before it reaches the probe.
  • This is excited to luminesce and emits light, which in turn traverses the sample and is attenuated in the event of an overlap of the absorption spectrum of the substance with the emission spectrum of the probe.
  • the overall attenuation of the signal is proportional to the substance concentration in the sample.
  • WO 94/17388 describes the use of the internal filter effect for the determination of ions in liquids by means of a sensor membrane.
  • US 4,822,746 is concerned in general with the use of the internal filter effect for the determination of a ligand binding, whereby ligands here mean ions, substances or the like.
  • the ligand to be detected is made accessible to analysis by a suitable (color) reaction.
  • US Pat. No. 4,654,300 describes the use of such a quenching effect in immunoassays. In each of these cases, the internal filter effect is used for analytical purposes. However, this effect is in no case directly or exclusively caused by the substance to be investigated, as provided in a method according to the present invention.
  • a method according to the invention enables a continuous observation and measurement of a permeation process and is therefore particularly suitable for the measurement of permeation kinetics.
  • individual measurements can be made at a given time.
  • process steps such as sampling and sample transfer for the purpose of external concentration determinations can be dispensed with particular advantage.
  • Concentration changes with respect to a substance to be investigated as a result of a permeation process can thus be traced without difficulty on one side or on both sides of a permeation barrier at the same time.
  • a method according to the present invention is particularly suitable for use in the context of an automated, modern high throughput method.
  • a permeation barrier that can be used in a method according to the invention comprises, in a preferred embodiment, an artificial membrane.
  • the artificial membrane preferably comprises in a further development a porous carrier material, in particular a filter membrane.
  • the nature of the carrier material is basically not critical, so can be used, for example, porous plastics, silicates, glass, ceramics and metals.
  • the artificial membrane comprises organic material.
  • the organic material forms a layer on the surface of the porous support material and / or is present in the pores of the porous support material.
  • the organic material according to the invention is preferably lipophilic or hydrophobic.
  • the organic material is essentially at least one hydrocarbon having a preferred chain length of 8 or more carbons and / or at least one alcohol having a preferred chain length of 4 or more carbons. Hydrocarbons having chain lengths of more than 12 carbons, especially 14-16 carbons, are more preferred among those mentioned.
  • the organic material is essentially a lipid or a lipid mixture.
  • This may be a simple lipid layer, a so-called monolayer, or a double lipid layer, a so-called bilayer.
  • the composition of such lipid layers can be chosen freely depending on the desired application.
  • such lipid layers can be constructed to be substantially homogeneous, or different lipids, for example, can be used as constituents of the membrane layers, as a result of which, for example, the conditions of a native membrane are readjusted can.
  • the lipids or the lipid mixtures those which have membrane-bound, membrane-integral and / or trans-membrane proteins are more preferred.
  • Cells and / or cell fragments may also be preferred as the organic material, the cells then being present in particular in the form of a cell monolayer or cell monolayer. Particularly preferred is a monolayer of CACO-2 cells or of MDCK cells.
  • the permeation barrier comprises a non-artificial membrane instead of an artificial membrane, in particular a membrane of organ tissue. Preference is then given to barriers prepared from intestinal tissue, it also being possible in other cases for barriers prepared from skin tissue to be preferred.
  • the at least one luminescent probe is preferably part of the permeation barrier in preferred embodiments of the method according to the invention.
  • the porous support material of an artificial membrane, in particular the filter membrane is probe-marked.
  • the probe may be contained both in the carrier material and, in particular, also bound to it, preferably covalently.
  • the at least one probe may also be part of the organic material which, as already mentioned above, is present in particular as a layer on the surface of the porous carrier material and / or in the pores of the porous carrier material.
  • the at least one probe may also be preferred to apply the at least one probe to the barrier, in particular in FIG Shape of a segmenting, probe-labeled solid.
  • silicate beads provided with a probe are particularly well suited, but also sedimentable solids of glass, ceramic, silicate, metal and / or polymer material or of combinations of these materials are conceivable.
  • the at least one probe is contained in the solvent.
  • the probe is either used in dissolved form on one or both sides of the barrier or, which may be particularly preferred, in the form of a dispersible, non-sedimenting solid.
  • probe-marked polymer beads can be used as non-sedimenting solid.
  • the at least one probe is part of a gel which may also be disposed on one or both sides of the barrier.
  • the probe itself can be both inorganic and organic in nature.
  • examples here are inorganic nanoparticles, so-called “quantum dots", colorant-containing, dispersible or sedimentable and / or luminescent silicate or ceramic particles, organic dyes such as rhodamine or fluorescein, dye-containing, dispersible or sedimentable and / or luminescent polymer latices, organic dyes are among the named probes
  • luminescent proteins such as, for example, GFP or luminescent proteins whose luminescence properties change as a result of binding of substances, as is the case, for example, with HSA and AGP, are also conceivable as probes.
  • a method according to the invention is particularly preferably carried out in at least one permeation device.
  • Such preferably comprises at least one donor compartment in which the at least one substance is provided, at least one barrier and at least one acceptor compartment into which the substance can permeate.
  • the measurement of the luminescence signals can take place either only in the donor or in the acceptor compartment or in both compartments at the same time.
  • the at least one probe in the form of a probe-labeled insert into the at least one donor and / or acceptor compartment.
  • a probe-labeled insert is preferably matched in shape and size to a permeation device or a compartment of a permeation device, so that the at least one probe can be placed repeatedly in a defined position in the respective compartment.
  • This type of probe arrangement can also optionally be combined with one or more of the possibilities of the probe arrangement already mentioned.
  • a method according to the invention can also be used in incomplete, compartmentalized systems. For example, it is possible to provide a dialysis membrane with a probe and to follow the progression of a dialysis process continuously. In particular, it can be easily recognized when a dialysis process has been completed.
  • microtiter plates are particularly well suited.
  • the wells of microtiter plates are outstandingly suitable as compartments of a permeation device.
  • microtiter plates with 24, 48, 96, 384 and 1536 cavities can be used.
  • a system of two coordinated microtiter plates is used, wherein the cavities of a first plate in each case have a filter membrane (which can be used as part of an artificial membrane as mentioned above in a method according to the invention) and the second plate with the first sandwiched by juxtaposing can be combined.
  • the second plate is preferably the lower plate.
  • each cavity of the upper plate is associated with a vertically located underneath cavity of the lower plate (or vice versa).
  • the system of the two plates thus forms a permeation device with a plurality of adjacent donor and acceptor compartments.
  • the permeation direction that is to say the direction in which the permeating substance penetrates the permeation barrier, is, however, in principle freely selectable (for example, the upper plate with the filter membranes may be a donor or acceptor plate, but possibly also both at the same time).
  • the upper plate with the filter membranes may be a donor or acceptor plate, but possibly also both at the same time.
  • the permeation direction is marked by black arrows.
  • the measurable luminescence signal is proportional to the substance concentration. This can be converted in further steps, if necessary using calibrations, directly into the corresponding concentrations.
  • I 0 is the intensity of luminescence of a reference without substance (usually so only with buffer solution), and I is the luminescence intensity in the presence of the test substance.
  • concentration-proportional quantity c 'for the concentration c can thus be determined at each instant t by measuring two luminescence intensities.
  • the construction of a measuring arrangement "from above" is shown schematically in Fig. 2.
  • the permeation direction is marked therein by the black arrow, in the left permeation means is the permeating substance while the measurement in the right permeation means serves as a reference.
  • the permeability P can be determined using equation (2) (Wohnland F. and Faller B., High-Throughput Permeability pH Profiles and High-Throughput Alkanes / Waterlog P with Artificial Membranes, J. Med. Chem. 44, 2001 , 923-930).
  • V 0 and V A the volumes of donor and acceptor compartment
  • A is the Permeationsober Structure accessible (calculated by multiplying the total surface of the barrier used with its porosity)
  • t the selected incubation time
  • C A kzepto r and h is CGieic Weight are the substance concentrations in the acceptor compartment after an incubation time t and after equilibration, ie after the substance has evenly distributed in donor and acceptor compartment.
  • V D , V A and A are basically freely selectable, in practice, however, usually fixed by the measurement setup, t results from the chosen duration of the incubation.
  • the quantities to be determined by the method according to the invention are thus the concentrations c A acceptor and CGieichong-
  • the equilibrium concentration can be calculated at least approximately from the volume-averaged value of acceptor and donor concentration. For example, for equal volumes of solvent in the donor and acceptor compartments, a concentration of 0.5 M in the acceptor and in the donor compartment should have been established after the provision of a substance in the donor compartment at 1 M concentration after a certain time.
  • c'Gieic h gewicw filled donor and / or acceptor compartment with a solution with the calculated equilibrium concentration. From simple measurement of the luminescence intensity of the filled compartments and a reference value, the sought-after result then again according to equation (1)
  • a third, particularly preferred possibility for determining the concentration-proportional variable c'cieichites is the concentration-proportional magnitudes c ⁇ k z eptor and c ' D ⁇ nor by simultaneous measurement of the fluorescence intensities of a reference and a sample in the acceptor and donor compartment both "from above” and For different path lengths of the excitation and emission light through the sample, where appropriate, the concentration-proportional quantities must be normalized to this "from below”. With negligible binding of the substance to the permeation barrier, the caloric weight results from the mass balance, ie the volume-averaged value of acceptor and donor concentration.
  • the arrangement of a measuring arrangement for the simultaneous measurement "from above” and “from below” for determining the concentration-proportional magnitudes c ⁇ kzeptor and c'oonor is shown schematically in Fig. 3.
  • equation (4) is obtained, with the aid of which the permeability of a barrier can be conveniently determined.
  • This procedure also allows a quasi-simultaneous determination of the enrichment in the acceptor and the depletion in the donor cavity and thus also allows statements beyond the actual permeation coefficient. In this way, processes such as membrane retention during the permeation of a substance through a barrier can be monitored in a time-resolved manner.
  • the measurement of the luminescence signals takes place at one or more discrete wavelengths, in particular excitation and emission wavelengths.
  • discrete wavelengths in particular excitation and emission wavelengths.
  • entire spectra can be measured and conclusions can be drawn on the modulation of the respective signals of specific wavelengths.
  • the at least one luminescent probe preferably has fluorescent and / or phosphorescent properties.
  • the excitation of the respective fluorescence or phosphorescence either takes place with one or more defined wavelengths, or excitation spectra are recorded.
  • a luminescent probe can be used, which emits a signal without prior excitation of light.
  • a probe with electro-, thermo- and / or chemiluminescence can be used.
  • the use of probes with fluorescence and phosphorescence is particularly preferred, since this is established in the laboratory practice procedures that are very easy to handle.
  • a single probe or a single probe type is used.
  • such a combination of probes can be coupled to one another via the so-called Förster energy transfer.
  • the present invention also includes, in addition to the method already described, the use of various individual components in a method according to the present invention. These include
  • At least one microtiter plate in particular as a component of at least one permeation device, the use of at least one dye, in particular as at least one luminescent probe,
  • At least one porous carrier material preferably at least one filter membrane, in particular as a constituent of at least one permeation barrier
  • cells preferably in the form of a cell monolayer or a cell multilayer, in particular as a constituent of at least one permeation barrier,
  • cell fragments in particular as part of at least one permeation barrier and the use of at least one membrane of organ tissue, in particular as a permeation barrier.
  • kits for carrying out a method according to the invention comprises at least 2 members from the group with
  • At least one insert for introducing a luminescent probe into at least one compartment of a permeation device, in particular into the wells of a microtiter plate,
  • At least one porous carrier material in particular at least one filter membrane
  • At least one solvent at least one suitable buffer system
  • Cells in particular in the form of a cell monolayer or a cell multi-layer,
  • At least one non-artificial membrane in particular at least one membrane of organ tissue.
  • the individual components of the kit have already been explained in detail above. The relevant parts of the description are hereby incorporated by reference.
  • kits according to the invention may contain individual components separately as a "kit” in order to allow the user to combine them specifically for a very specific embodiment of a method according to the invention
  • the kit may be preferred for the kit to have a filter membrane which is already probe-marked and / or provided with an organic layer, for example composed of hydrocarbons, and directly in a novel membrane Method can be used.
  • the permeation of substances from any substance classes can be examined.
  • potential active substances in particular potential pharmaceutical and / or biological active substances, which can be used for therapy and / or diagnosis of various diseases can be investigated with the aid of the method according to the invention. It may also be, for example, peptides or proteins.
  • low molecular weight substances as frequently occurring in the case of potential pharmaceutical active substances, can also be investigated.
  • Fig. 1 Schematic structure of a permeation measurement with a
  • Fig. 4 Schematic representation of a luminescence measurement exclusively "from above” for the simultaneous determination of the concentration-proportional magnitudes c ⁇ kzeptor and cOonor- The permeation directions are marked by the black arrows.
  • Example 1 Preparation of a passivated, probe-labeled, fluorescence-active carrier
  • a porous silicate material (Nucleoprep 300-12 from Macherey-Nagel, Düren) are concentrated in a silane solution consisting of 9.2 ml of N- (2-aminoethyl) -3-aminopropyltrimethoxysilane (EDA) and 243 ⁇ l Acetic acid in 450 ml of deionized water and rotated slowly for three hours. Thereafter, the silicate material is sedimented, washed three times with deionized water and dried at 80 0 C.
  • a porous silicate material Nucleoprep 300-12 from Macherey-Nagel, Düren
  • the support is dispersed in 18 ml of carbonate buffer (pH 9.5) and mixed with 2 ml of a solution of rhodamine isothiocyanate (RITC) in carbonate buffer (final concentration 40 ⁇ mol / l) and rotated overnight. To remove unreacted RITC, it is first washed twice with carbonate buffer and finally three times with PBS (phosphate buffered saline, 10 mM sodium phosphate, 0.9% sodium chloride, pH 7.4).
  • PBS phosphate buffered saline, 10 mM sodium phosphate, 0.9% sodium chloride, pH 7.4
  • Acceptor plate are first filled for one hour with 150 .mu.l of a polyethyleneimine solution (1 mg / ml in deionized water). The two plates are put together to incubate the filter. The cavities are then washed 3 times with deionized water and poured into
  • Rebuilt carbonate buffer In a further step, the wells of donor and acceptor plate are filled with 150 .mu.l of a 10 .mu.mol / l solution of rhodamine isothiocyanate in carbonate buffer (pH 9.5) and placed in each other for two hours. It is then washed once with carbonate buffer and then with deionized water. Finally, the cavities are dried.
  • Example 3 Production of probe-marked filter plates with a hexadecan membrane
  • the filter plate (acceptor plate in this example) from Example 3 are each 150 ul PBS buffer pH 7.4, which contains 1% DMSO, pipetted.
  • the corresponding wells of the donor plate are each filled with 135 .mu.l of PBS buffer and 15 .mu.l of a 1, 0 mM substance solution in PBS buffer / DMSO (90:10 v: v).
  • Further cavities of the donor plate and the acceptor plate are filled only with a 1% solution of DMSO in PBS and serve as a reference for determining the fluorescence intensity I 0 .
  • the permeation of the substances is started by assembling the two plates. After 4 hours, the fluorescence intensities are measured in a measuring arrangement "from above" in the cavities of the acceptor plate, and the concentration-proportional magnitudes c ⁇ cceptor and c'cieichmati of the permeated substances were determined from the measured intensities according to the above-described procedure.
  • the investigated substances in the experiment were warfarin and carbamazepine, for which the following permeabilities could be determined by means of equation (3):
  • Example 5 Preparation of a microtiter plate with hexadecane membranes
  • Example 6 Determination of the Substance Permeation by the Hexadecane Membranes of a Microtiter Plate from Example 5, Read by UV Detection
  • Cavities of the donor plate as well as the acceptor plate are filled only with a 1% solution of DMSO in PBS buffer and serve as a reference.
  • the permeation of the substances is started by assembling the two plates. After 4 hours donor and acceptor plates are separated and an aliquot of 50 ⁇ l is transferred from the wells of the two plates to a UV transparent microtiter plate. The concentration of the substances is determined in a UV plate reader (SpektraMax plus 384 from Molecular Devices).
  • the investigated substances were desipramine, quinidine,
  • Example 7 Determination of the Substance Permeation by the Hexadecane Membranes of a Microtiter Plate from Example 5, Read through Probe-Labeled Silicate Beads
  • 115 ⁇ l of PBS reagent are added to several of the wells of the microtiter plate from Example 5 which are provided with filter membranes.
  • Buffer 20 .mu.l of the carrier suspension prepared in Example 1 (with 200 mg of solids per 1 ml of suspension) and 15 .mu.l of a 10% solution of DMSO in PBS buffer pipetted.
  • the corresponding wells of another microtiter plate (without filter membranes) are filled with 135 ⁇ l of PBS buffer and 15 ⁇ l of a 1.0 mM substance solution in PBS buffer / DMSO (90:10 v: v).
  • Some cavities of the microtiter plate from Example 5 act here as acceptor cavities, other than donor cavities.
  • microtiter plate from example 5 is filled with a 1% solution of DMSO in PBS as a reference, as are their corresponding cavities in the further microtiter plate.
  • the permeation of the substances is started by assembling the two plates. After 4 hours, the fluorescence intensities in the measuring arrangement "from above" are determined in all filled wells of the microtiter plate from Example 5. From these intensities, the concentration-proportional sizes of the permeated substance were determined.
  • the investigated substances were desipramine, quinidine,
  • the determined logarithmic permeabilities of the substances are shown in correlation to the measured values of the measurement from Example 6 in FIG. 5.
  • Example 8 Determination of the Substance Permeation by a Hexadecan Membrane on a Microtiter Plate from Example 5, continuously read out by dye-coated silicate beads
  • Example 8 two permeation directions are to be considered. In case 1, the substance permeates from “bottom to top” into the microtiter plate from example 5 (acceptor), in case 2 the substance permeates from the microtiter plate from example 5 (donor) from “top to bottom”.
  • the permeation of the substances is started by combining the two plates. At discrete time intervals of 5 min over a total time of 4 hours, the fluorescence intensities in the measurement geometry were measured "from above” in all filled wells of the microtiter plate from Example 5. From these intensities, the concentration-proportional sizes of the permeated substance were determined for each measured discrete time ,
  • the investigated substances were warfarin, carbamazepine and propranolol.
  • the obtained mean permeabilities of the substances were:
  • Example 9 Determination of the Substance Permeation by a Hexadecan Membrane on a Microtiter Plate from Example 5, Read Out by Dye-loaded Dispersible Polymer Particles.
  • 120 ⁇ l of PBS buffer, 15 ⁇ l of a 10% strength solution of DMSO in PBS buffer and 15 ⁇ l of a suspension of dye-loaded polystyrene particles (1 ⁇ m, 0.05 ⁇ m) are introduced into several wells of an acceptor plate prepared according to Example 5 %, Manufacturer
  • the corresponding wells of another microtiter plate serving as a donor plate are filled with 135 ⁇ l of PBS buffer and 15 ⁇ l of a 1.0 mM substance solution in PBS buffer / DMSO (90:10 v: v). Other cavities of the donor plate (and the corresponding ones on the
  • Acceptor plate are filled only with a 1% solution of DMSO in PBS and serve as a reference for determining the fluorescence intensity I 0 .
  • the permeation of the substances is started by assembling the two plates. After 4 hours, the fluorescence intensities of the dye-loaded polystyrene particles in the measurement geometry "from above" are measured in all filled wells of the acceptor plate.
  • warfarin was investigated u.a. the substance warfarin.

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  • Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)

Abstract

L'invention concerne un procédé pour mesurer la perméation d'au moins une substance contenue dans un solvant à travers une barrière de perméation et/ou pour déterminer des paramètres influençant la perméation, en particulier pour déterminer la perméabilité de la barrière à cette substance, au moyen d'au moins une sonde luminescente. Ce procédé consiste à placer la ou les substances sur un côté de la barrière de perméation, à incuber la barrière de perméation et la ou les substances contenues dans le solvant, puis à mesurer des signaux de luminescence de la sonde sur au moins un côté de la barrière de perméation, ces signaux étant modulés en fonction de la concentration de la substance. Le spectre d'absorption de la ou des substances est superposé au spectre d'excitation et/ou d'émission de la ou des sondes. L'invention concerne en outre un nécessaire à utiliser dans ce procédé.
PCT/EP2006/008660 2005-09-07 2006-09-06 Procede de mesure de la permeation d'une substance a travers une barriere Ceased WO2007028578A1 (fr)

Priority Applications (1)

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EP06777169A EP1922538A1 (fr) 2005-09-07 2006-09-06 Procede de mesure de la permeation d'une substance a travers une barriere

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE102005044071.1 2005-09-07
DE200510044071 DE102005044071A1 (de) 2005-09-07 2005-09-07 Verfahren zur Messung der Permeation einer Substanz durch eine Barriere

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WO2007028578A1 true WO2007028578A1 (fr) 2007-03-15

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EP (1) EP1922538A1 (fr)
DE (1) DE102005044071A1 (fr)
WO (1) WO2007028578A1 (fr)

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DE102022132085B4 (de) * 2022-12-02 2024-12-24 Lts Lohmann Therapie-Systeme Ag. 3D-in-vitro-Permeationsmodell

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DE102005044071A1 (de) 2007-03-08

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