DE102005044071A1 - Method for measuring the permeation of a substance through a barrier - Google Patents

Abstract

The present invention relates to a method for measuring the permeation of at least one substance contained in a solvent through a permeation barrier and / or for determining permeation-influencing parameters, in particular for determining the permeability of the barrier for the at least one substance, using at least one luminescent probe comprising DOLLAR A - the provision of the at least one substance on one side of the permeation barrier, DOLLAR A - incubation of the permeation barrier with the at least one substance contained in the solvent and DOLLAR A - measurement of luminescence signals of the probe on at least one side of the permeation barrier, which depends on the respective concentration of the substance are modulated, DOLLAR A with the excitation and / or emission spectrum of the at least one probe overlapping the absorption spectrum of the at least one substance and a kit for use in this method.

Description

The The present invention relates to a method for measuring permeation at least one in a solvent contained substance through a barrier and / or for determination permeation-influencing parameter, in particular for determination the permeability the barrier for the at least one substance, as well as individual components and a Kit for use in this procedure.

In the pharmaceutical industry, potential active ingredients are tested, among other things, for their properties in the ADME area (absorption, distribution, metabolism, excretion). In particular, the inclusion of active substances in an organism such as the human body is the subject of intensive research. In the case of orally administered drugs, for example, the permeation of the active substance contained in the drug through the intestinal wall plays an important role as a natural permeation barrier. In recent years, a variety of methods have been developed to measure the uptake of an agent into the human body. In particular, the following procedures are described in the literature relevant:
In human experiments, direct measurements of permeation in the human body have been made. Three different clinical methods were used, namely the so-called open approach (ie without blockage of a bowel section), the so-called semi-open approach (ie with a blocking balloon on the proximal side of a bowel section) and the so-called closed approach (with two blocking balloons closing both sides a bowel section). In all cases, the active substance was administered via a drainage system, whereupon samples were taken at defined distances and examined for their active substance content (Lennernäs H., Human intestinal permeability, J. Pharm. Sci. 1998, 87, 403-410, Lennernäs H. Human Pharmacol., 1997, 49, 627-638).

In in vitro approaches use it frequently Permeation facilities, in which the permeation of a substance is observed through a barrier. Through the barrier is the Permeation device in two separate room units divided, the so-called compartments. To carry out a Permeation measurement becomes the substance to be examined usually on one side of the permeation barrier (in the donor compartment) submitted. With the onset of permeation, the concentration of the Substance in the donor compartment, while the substance in the donor compartment Enriches acceptor compartment. The progression of the permeation process can over appropriately Concentration measurements are observed, at least until a Concentration equilibrium between the two compartments adjusted Has.

Next special permeation chambers (eg the so-called Ussing chamber), among others with isolated membrane vesicles or with organ tissue preparations operated as a barrier layer, also find perfusion facilities a use with prepared Intestinal areas of dead animals work (Karlsson J. and Arthursson P., A new diffusion chamber system for the determination of drug permeability coefficients across the human intestinal epithelium that are independent of the unstirred water layer, Biochim. Biophys. Acta 1992, 1111, 204-210; Soderholm J.D., Hedman L., Arthursson P., Franzen L., Larsson J., Pantzar N., Permert J., Ol. G., Integrity and metabolism of human mucosa in vitro in the ussing chamber, Acta Physiol. Scand. 1998, 162, 47-56; Lennernäs H., Nylander S., Ungell A.L., Jejunal permeability: a comparison between the ussing chamber technique and the single-pass perfusion in humans, Pharm. Res. 1997, 14, 667-71; Arthursson P., Ungell A.L., Lofroth J.E., Selective paracellular permeability in two models of intestinal absorption: cultured monolayers human intestinal epithelial cells and rat intestinal segments, Pharm. Res. 1993, 10, 1123-1129).

These procedures are very cumbersome and time-consuming and beyond suitable only for the measurement of fewer substances. A simpler and more versatile way of determining or estimating the uptake of substances into an organism is to measure the permeation of substances through artificial permeation barriers. For example, in recent years, the use of cell monolayers as a permeation barrier has been extensively described in the literature (Ungell AL and Karlsson J. (2003) Cell Cultures in Drug Discovery: An Industrial Perspective.) In Drug Bioavailability; Estimation of Solubility, Permeability, Absorption and Bioavailability, Van de Waterbeemd H., Lennerrnas H., Arthursson P., eds., 90-131, Wiley-VCH). In particular, 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. Sci. 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. Commun. 1991, 175, 880-885; Cho MJ, Thompson DP, Cramer CT, Vidmar TJ, and Scieszka JF, The madin-Darby Canine Kidney (MDCK) epithelial cell monolayer as a model cellular transport Barrier, Pharm. Res. 1989, 6, 71-77).

The cells are cultured on permeable supports in special microtiter plates for a certain period of time. After the formation of a monolayer cell layer, the cells are allowed to differentiate over a certain period of time and then the actual permeation measurements are carried out. 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 investigated is analyzed therein (s. 1 ).

The Concentration measurements are usually carried out via a HPLC or LC / MS method. The permeation can be from the apical to the basolateral side and vice versa. Due to the morphology The cells allow this approach to draw conclusions about an active participation Transporter proteins at the permeation of the substance, as these transporter proteins located in the basolateral cell membrane. From this data let yourself Calculate the so-called permeation coefficient, which in turn with In vivo data from the human body correlates.

Next Cell monolayers are increasingly becoming more and more in recent years artificial Permeation barriers such. B. artificial lipid systems used. So Kansy and co-workers in 1998 called the PAMPA process described (PAMPA = Parallel Artificial Membrane Permeation Assay), in which the permeation of chemical substances by means of porous filter membranes (Kansy M. et al. (1998), Physicochemical high throughput screening: parallel artificial membrane permeability assay in the description of passive absorption process, J.Med.Chem. 41, 1007-1010). The filter membranes are mixed with 1-20% lecithin in organic solvents (eg dodecane, hexadecane, 1,9-decadiene) impregnated so that a lipophilic Separation layer (barrier) is created.

2001 became a modification of the PAMPA procedure of Wohnsland and Faller (Residential 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) described in which a Hexadecan layer between two water compartments is used as a permeation barrier. This procedure reduces the diameter of the permeation barrier with simultaneous use of polycarbonate filter membranes with larger pore diameters (3 μm vs. 0.22 or 0.45 μm at Kansy) of 120 μm at Kansy to about 10 microns and increases thereby the possible Throughput of substances per unit of time.

In Another modification of Kansy's procedure will take place lecithin complex lipid mixtures (Sugano K. et al., Optimized conditions of biomimetic artificial membrane permeability assay, Int. J. Pharm. 2001, 228, 181-188). Such a special Lipid composition better simulates the gastrointestinal tract and take care of that a higher one biological relevance of the measuring method. This modification is as a biometric PAMPA or BAMPA method (Biometric Artificial Membrane Permeation Assay) in the literature (Miret S., Abrahamse L., De Groene E.M., Comparison of in vitro models for the prediction of compound absorption across the human instestinal mucosa, J. Biomolec. Screen 2004, 9, 598-606).

All have described procedures for studying the permeation common that the determination of concentration the substance in the sample are made only discontinuously can. To judge, whether and to what extent a substance by a Barrier permeates, is the at least one withdrawal of a Sample from the acceptor compartment behind the barrier required followed by a transfer to an external analyzer in which the determination of the substance concentration in the sample takes place. Especially Measurements of permeation kinetics are thus only at great expense perform. For use as part of an automated, modern high-throughput process, if appropriate, the study of the permeation of hundreds up to thousands of substances daily and in particular the measurement of the permeation kinetics enable should, the described investigation methods are completely unsuitable.

Accordingly, it is an object of the present invention to provide new and improved means and means for assaying and measuring the permeation of a substance through a barrier, which do not have the mentioned disadvantages of the prior art. The examinations or measurements should be feasible without great effort and as user-friendly as possible and, in particular, should also be able to be designed for a high sample throughput.

These Task is solved by the method having the features of claim 1 and a Kit to carry out a method according to the invention with the features of claim 34. Preferred embodiments the method according to the invention are in the dependent claims 2 to 25 shown. The requirements 26 to 33 relate to the uses of certain individual components in a method according to the present invention. The wording all claims is hereby incorporated by reference into the content of this specification.

One inventive method is to study the permeation of one contained in a solvent Substance provided by a barrier. Also the simultaneous Investigation of the permeation of several substances contained in a solvent is achieved by a method according to the invention allows. In particular, a method according to the invention is outstandingly suitable for the determination of permeation-influencing parameters such as the permeability the barrier for the at least one substance, which will be discussed in more detail later. Also Permeation kinetics can be easily with the method of the invention determine.

One inventive method is characterized in particular by the fact that the permeation of at least a substance using at least one luminescent Probe is examined. The term "luminescent" is used as a generic term especially for Fluorescence and phosphorescence, but also for example for electric, Thermoluminescence and chemiluminescence used.

The At least one substance to be examined is in a first step in a solvent provided at least one side of a permeation barrier. This concludes including the possibility a, various substances on both sides of the barrier and / or one or more identical substances on both sides of the barrier in the latter case between the two sides of the Barrier regarding the substance or substances to be tested each a concentration difference between the two sides the barrier must exist.

After that you leave the at least one substance and the permeation barrier over one interact with each other for a certain period of time. On this incubation of Permeation barrier with the at least one in the solvent contained substance follows in a further step, 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 dependent modulated by the respective concentration of the substance, whereby mandatory it is necessary that the Absorption spectra of the at least one substance with excitation and / or emission spectra overlap at least one probe.

On In this way, statements about the concentration can be made examining substance on one or both sides of the permeation barrier win, which in turn directly draw conclusions the permeation of the substance through the barrier as well as on the various Permeationsbeeinflussenden parameter permits.

The inventive method exploits the so-called internal filter effect, in which the Light absorption of substances indirectly via a attenuation of luminescence intensities suitable probes can be determined. This phenomenon is based on the fact that the intensity of light rays, the on the way to a suitable probe or on the way from the probe absorbed to a detector of other dissolved substances be, that is so toned down become. The overlap of excitation or emission wavelengths with the absorption band leads to the substance to be examined when measuring at suitable wavelengths (ie in the range the absorption band of the substance) to a modulation or weakening of the luminescence, proportional to the concentration of the substances to be investigated before and / or behind the barrier. Crossed during a measurement the excitation light will be the sample and in case of overlap the absorption spectrum of the substance to be examined with the Stimulation spectrum of the probe attenuated before it reaches the probe. This is stimulated to luminesce and emits light, which in turn traverses the sample and in case of overlap of the absorption spectrum the substance is attenuated with the emission spectrum of the probe. The overall weakening of the signal is proportional to the substance concentration in the sample.

The internal filter effect has already been exploited for various analytical methods. Beispielswei WO 94/17388 describes the use of the internal filter effect for the determination of ions in liquids by means of a sensor membrane. The US 4,822,746 is generally concerned with the use of the internal filter effect to determine ligand binding, which is understood here to mean ligands ions, substances or the like. The ligand to be detected is made accessible to analysis by a suitable (color) reaction. Furthermore, the describes US 4,654,300 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.

One inventive method allows a continuous observation and measurement of a permeation process and is therefore particularly suitable for the measurement of permeation kinetics. Next the continuous measurement can Naturally Also, individual measurements can be made at a given time. On process steps such as sampling and sample transfer for the purpose of external concentration determinations may with special Advantage be waived. Due to a permeation process changes in concentration in terms of a substance to be examined can thus easily on one Side or on both sides of a permeation barrier at the same time be followed. Thus, a method according to the present invention is suitable Invention especially for the use in the context of an automated, modern high-throughput process.

The execution a method according to the invention is preferably carried out at a constant temperature, in particular in a temperature range between approx. 5 ° C and approx. 40 ° C. By doing temperature range can continue to a temperature of approx. 37 ° C (ie Body temperature) particularly preferred. In most cases, however, are measurements at about 20 ° C (Room temperature) is preferred. According to the invention, the method is preferably carried out in aqueous solution.

A in a method according to the invention usable permeation barrier comprises in a preferred embodiment an artificial membrane. The artificial membrane comprises in development preferably a porous one Support material in particular a filter membrane. The nature of the substrate is basically not critical, for example, porous plastics, Use silicates, glass, ceramics and metals.

Further it is preferred that the artificial membrane comprises organic material. In preferred embodiments The organic material forms a layer on the surface of the porous From support material and / or is present in the pores of the porous support material.

The Organic material is preferably lipophilic or hydrophobic. In particular, it is the organic material essentially at least one hydrocarbon with a preferred chain length of 8 or more carbons and / or at least one alcohol with a preferred chain length of 4 or more carbons. Hydrocarbons with chain lengths of more than 12 carbons, especially with 14-16 carbons, are under the said more preferred.

In a further preferred embodiment If the organic material is substantially to a lipid or a lipid mixture. This can be a simple lipid layer, a so-called monolayer, or one double lipid layer, a so-called bilayer act. The composition Such lipid layers can be chosen freely depending on the desired application. For example, you can Such lipid layers be largely homogeneous, or it can for example, different lipids as constituents of the membrane layers be used, whereby, for example, the circumstances of a native membrane can be adjusted. Among the lipids or the lipid mixtures are further preferred, the membrane-bound, membranintegrale and / or transmembrane proteins.

Also Cells and / or cell fragments can be preferred as organic material, the cells then in particular in the form of a cell monolayer or a cell multilayer. Especially preferred is a monolayer of CACO-2 cells or of MDCK cells.

In certain embodiments the method according to the invention it may be preferred that the Permeation barrier in place of an artificial membrane a non-artificial Membrane comprises, in particular a membrane of organ tissue. Preferred are then off Intestinal tissue prepared Barriers, in other cases also prepared from skin tissue Barriers may be preferred.

The at least one luminescent probe is in preferred embodiments the method according to the invention preferably part of the permeation barrier. In these cases is it prefers that the porous support material an artificial membrane, in particular the filter membrane, is probe-marked. The probe may contain both in the carrier material and in particular also be bound to this, preferably covalently.

In further preferred embodiments the at least one probe may also be part of the organic Be material, which, as already mentioned above, in particular as a layer on the surface of the porous one support material and / or in the pores of the porous one support material is present.

Next The possibility, use the at least one probe as part of the barrier, however, it may also be preferred that the at least one probe applied to the barrier, in particular in the form of a sedimenting, probe-labeled solid. As such are suitable, for example silicate beads provided with a probe are particularly good, they are but also sedimentable solids of glass, ceramic, silicate, Metal and / or polymer material or combinations of these materials conceivable.

Farther it may also be preferred that the at least one probe in the solvent is included. In these cases The probe will be on either or both sides of the barrier either in dissolved Form used or, which may be particularly preferred in shape a dispersible, non-sedimenting solid. As not sedimenting solids are in particular probe-marked polymer beads used.

In further embodiments it may be preferred that the at least one probe is part of a gel, that too can be arranged on one or both sides of the barrier.

Of course they are the possibilities mentioned the probe assembly in a method according to the invention also with each other combined.

The Probe itself can be both inorganic and organic in nature be. Examples here are inorganic nanoparticles, so-called "quantum dots", dye-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 latexes. Organic dyes are preferred among the named probes. There are also probes but also luminescent proteins such as GFP or luminescent proteins whose luminescent properties are changes by binding substances, as is the case with HSA and AGP, for example.

One inventive method is particularly preferred in at least one permeation device carried out. Such includes preferably at least one donor compartment in which the at least a substance is provided, at least one barrier and at least one acceptor compartment into which the substance permeates can. The measurement of the luminescence signals can either nor in the Do nor or only in the acceptor compartment or in both compartments simultaneously.

Especially in a Permeationseinrichtung exists in addition to those already mentioned Variations of the probe assembly additionally or instead also the possibility, the at least one probe in the form of a probe-marked insert into the at least one donor and / or acceptor compartment. One such use is preferred in shape and size to a permeation device or a compartment of a permeation device tuned so that the at least one probe repeatable in a defined position can be placed in the respective compartment. Also this kind The probe assembly may optionally be provided with one or more the already mentioned possibilities the probe assembly are combined.

Basically 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 the progression continuously follow a dialysis procedure. Especially can be easily recognized when a dialysis process is completed is.

In particularly preferred embodiments of the method according to the invention, this is carried out in parallel, which is possible in particular in compartments of adjacent permeation devices. For carrying out a method according to the invention, in particular a method carried out in parallel, microtiter plates are particularly well suited. The wells of microtiter plates are suitable excellently as compartments of a permeation device. For example, microtiter plates with 24, 48, 96, 384 and 1536 cavities can be used. Preferably, a system of two coordinated microtiter plates is used, wherein the cavities of a first plate each have a filter membrane (which can be used as part of an artificial membrane as stated above in a method according to the invention) and the second plate with the first by sandwiching can be combined , In the combined state, the second plate is preferably the lower plate. Preferably, then 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 Permeationsrichtung, ie the direction in which the permeating Substance penetrates the permeation barrier, however, is in principle freely selectable (For example, the top plate with the donor or filter membranes may be used Be acceptor plate, but possibly both at the same time). In the following specified embodiments is basically of Permission from bottom to top, if not otherwise stated. In the corresponding pictures is the Permeation direction marked by black arrows.

at It is the measurement of luminescence signals in microtiter plates possible, the measurement in different measuring arrangements make. For one thing, it may be preferable to use the excitation light from the bottom of the plate to the probe introduce and to detect the emission light from the direction of the plate underside. This arrangement will hereinafter be referred to as "bottom-up" measurement, the other possibility consists in a measurement "of above ", at which the Excitation light passes from the top of the plate to the probe and the Emission light from the direction of the top plate is detected.

The thereby measurable Luminescence signal is proportional to the substance concentration. These can be used in further steps, if necessary using calibrations, be converted directly into the corresponding concentrations.

The basis for determining the concentration-proportional variable c 'is the Lambert-Beersche law c '= log I 0 / I = ε · c · d (1)

I ₀ is the luminescence intensity of a reference without substance (usually only with buffer solution) and I the luminescence intensity in the presence of the substance to be examined. The concentration-proportional quantity c 'for the concentration c can thus be determined at each instant t by measuring two luminescence intensities. This is schematically in 2 played.

The permeability P lets himself using equation (2) (Homes F. and Faller B., High-Throughput Permeability pH Profiles and High Throughput Alkanes / Water log P with Artificial Membranes, J. Med. Chem. 44, 2001, 923-930).

Here, V _D and V _A, the volumes of donor and acceptor, A is the accessible Permeationsoberfläche (calculated by multiplying the total surface of the barrier used with its porosity), t is the selected incubation time and c _acceptor and c are _balanced, the substance concentrations in Acceptor compartment after an incubation period 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, but in practice 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 _acceptor and c _equilibrium .

The quotient of the two concentrations c _acceptor and c _equilibrium In Equation (2), the quotient of the two concentration-proportional quantities determinable directly by the method according to the invention makes it possible to replace c ' _acceptor and c' _equilibrium , which leads to equation (3).

In order to determine the permeability, therefore, it is not necessary first to determine the actual substance _{concentrations} from the variables c ' _acceptor and c' _equilibrium , which (as already mentioned above) z. B. can be done with the help of calibration functions. The size c ' _acceptor to be determined can be easily determined at an arbitrarily determinable time t by measuring the luminescence activity. A number of approaches are available for determining the remaining unknown quantity c ' _equilibrium .

In one case, after determining the size c ' _{acceptor, one} simply waits until a concentration equilibrium between donor and acceptor cavity has been established, and then measures the luminescence intensity I and I ₀ . Equation (1) yields the sought-after quantity c ' _equilibrium analogous to the already determined quantity c' _acceptor .

Another possibility for determining c ' _equilibrium may be particularly preferred if the binding of the substance to the permeation barrier is negligible. In this case, the equilibrium concentration can be calculated at least approximately from the volume-averaged value of acceptor and donor concentration. For example, with equal volumes of solvent in the donor and acceptor compartment, a concentration of 0.5 M in the acceptor and donor compartments should have set each other after providing a substance in the donor compartment at 1 M concentration after a certain time. To determine c ' _equilibrium , the donor and / or acceptor compartment is filled with a solution having the calculated equilibrium concentration. From simple measurement of the luminescence intensity of the filled compartments and a reference value, the desired size c ' _equilibrium then again results according to equation (1).

A third, particularly preferred way to determine the concentration-proportional size c ' _equilibrium is to measure the concentration-proportional magnitudes c' _acceptor and c ' _donor by simultaneously measuring the fluorescence intensities of a reference and a sample in the acceptor and donor compartments both "from above" and " from below ", wherein at different path lengths of the excitation and emission light through the sample, if necessary, the concentration-proportional magnitudes must be normalized to this. With negligible binding of the substance to the permeation barrier, c ' _equilibrium results from the mass balance, ie the volume-averaged value of acceptor and donor concentration. This is schematically in 3 played.

If, in equation (3), the size c ' _{equilibrium is} replaced by the volume-averaged value of acceptor and donor concentration, equation (4) is obtained, with the aid of which the permeability of a barrier can be conveniently determined.

These Approach possible also a quasi-simultaneous determination of the enrichment in the acceptor and the impoverishment in the Donorkavität and thus also allows statements about the actual permeation coefficient addition. So can too Processes such as the permeation of a substance through a barrier occurring membrane retention are tracked time-resolved.

This is also possible in accordance with a further preferred embodiment of the method according to the invention, according to which the measurement of the luminescence takes place exclusively "from above." Measurements are carried out in 3 permeation devices (using a system of microtiter plates in three cavities) 4 is illustrated schematically. A reference cavity serves to determine the luminescence intensity I _{0 of} a reference without substance (the reference cavity in FIG 4 top right). The reference cavity contains only buffer solution. A second cavity (the acceptor cavity in 4 top left) is used to determine the luminescence intensity I _acceptor In it substance accumulates, which permeates from below through a barrier. A third cavity (the donor cavity in 4 Middle top) is used to determine the luminescence intensity I _donor . In it substance was provided, which permeates down through the barrier.

The concentration-proportional quantities are in this arrangement as usual according to c ' _acceptor = log I ₀ / I _acceptor and c ' _donor = log I ₀ / I _donor determined. It is assumed that the accumulation in the acceptor and the depletion in the donor cavity are proportional to each other.

In a method according to the invention it is generally preferred that the Measurement of the luminescence signals at one or more discrete Wavelengths, in particular excitation and emission wavelengths takes place. on the other hand can However, entire spectra are measured and from this conclusions pulled the modulation of the respective signals of certain wavelengths become.

The at least one luminescent probe preferably has fluorescent and / or phosphorescent properties. Here, the Excitation of the respective fluorescence or phosphorescence either with one or more defined wavelengths, or it will be excitation spectra added. Furthermore, as a probe and a luminescent Probe can be used, which is a signal without prior light excitation radiates. For example, a probe with electric, Thermo- and / or chemiluminescence can be used. The usage however, probes with fluorescence and phosphorescence are particular preferred, since this is established in laboratory practice procedures which are very easy to handle.

In a further preferred embodiment the method according to the invention a single probe or a single probe type is used. on the other hand it may also be preferred that a Combination of different probes is used. Such a combination of probes can in a preferred embodiment via the so-called Förster energy transfer be coupled with each other.

• – die Verwendung von mindestens einer Mikrotiterplatte, insbesondere als Bestandteil mindestens einer Permeationseinrichtung,
• – die Verwendung von mindestens einem Farbstoff, insbesondere als mindestens eine lumineszierende Sonde,
• – die Verwendung eines Einsatzes zum Einbringen einer lumineszierenden Sonde in ein Kompartiment einer Permeationseinrichtung,
• – die Verwendung mindestens eines porösen Trägermaterials, bevorzugt mindestens einer Filtermembran, insbesondere als Bestandteil mindestens einer Permeationsbarriere,
• – die Verwendung mindestens eines Lipids oder einer Lipidmischung, insbesondere als Bestandteil mindestens einer Permeationsbarriere,
• – die Verwendung mindestens eines Kohlenwasserstoffs mit einer bevorzugten Kettenlänge von 8 oder mehr Kohlenstoffen und/oder mindestens eines Alkohols mit einer bevorzugten Kettenlänge von 4 oder mehr Kohlenstoffen, insbesondere als Bestandteil mindestens einer Permeationsbarriere,
• – die Verwendung von Zellen, vorzugsweise in Form einer Zellmono- oder einer Zellmultischicht, insbesondere als Bestandteil mindestens einer Permeationsbarriere,
• – die Verwendung von Zellfragmenten, insbesondere als Bestandteil mindestens einer Permeationsbarriere und
• – die Verwendung mindestens einer Membran aus Organgewebe, insbesondere als Permeationsbarriere.

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

• The use of 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,
• The use of an insert for introducing a luminescent probe into a compartment of a permeation device,
• The use of at least one porous carrier material, preferably at least one filter membrane, in particular as a constituent of at least one permeation barrier,
• The use of at least one lipid or a lipid mixture, in particular as a constituent of at least one permeation barrier,
• The use of 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, in particular as a constituent of at least one permeation barrier,
• The use of cells, preferably in the form of a cell monolayer or a cell multilayer, in particular as part of at least one permeation barrier,
• The use of 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.

All stated, preferably usable components have already been explained in detail above. On the relevant parts of the description are hereby incorporated by reference and reference.

• – mindestens einer Mikrotiterplatte,
• – mindestens einer lumineszierenden Sonde,
• – mindestens einem Einsatz zum Einbringen einer lumineszierenden Sonde in mindestens ein Kompartiment einer Permeationseinrichtung, insbesondere in die Kavitäten einer Mikrotiterplatte,
• – mindestens einem porösen Trägermaterial, insbesondere mindestens einer Filtermembran,
• – mindestens einem Lösungsmittel,
• – mindestens einem geeigneten Puffersystem,
• – mindestens einem Kohlenwasserstoff mit einer bevorzugten Kettenlänge von 8 oder mehr Kohlenstoffen und/oder mindestens einem Alkohol mit einer bevorzugten Kettenlänge von 4 oder mehr Kohlenstoffen,
• – mindestens einem Lipid oder einer Lipidmischung,
• – Zellen, insbesondere in Form einer Zellmono- oder einer Zellmultischicht,
• – Zellfragmenten und
• – mindestens einer nicht-artifiziellen Membran, insbesondere mindestens einer Membran aus Organgewebe.

Furthermore, the invention also includes a kit for carrying out a method according to the invention. A kit according to the invention comprises at least 2 members from the group with

• At least one microtiter plate,
• At least one luminescent probe,
• 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,
• 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,
• At least one lipid or a lipid mixture,
• Cells, in particular in the form of a cell monolayer or a cell multilayer,
• - cell fragments and
• - At least one non-artificial membrane, in particular at least one membrane of organ tissue.

Also The individual components of the kit have already been explained in detail above. On the corresponding parts of the description will be hereby accordingly referred and referred.

One Inventive kit may contain individual components separately as a "kit" to give the user the option to give this targeted for a very specific embodiment a method according to the invention to combine with each other. In a particularly preferred embodiment of the kit according to the invention However, individual components of the kit are already for one or more several specific embodiments the method according to the invention ready to use. For example, it may be preferred be that the Kit has a filter membrane already probe-marked and / or with an organic layer z. B. from hydrocarbons provided is and can be used directly in a method according to the invention can.

With a method and / or a kit according to the present invention can basically studied the permeation of substances from any substance classes become. In particular, you can with the aid of the method according to the invention potential active ingredients, in particular potential pharmaceutical and / or biological agents to be investigated for therapy and / or diagnostics of various diseases. It This may also be, for example, peptides or proteins. In particular, you can Also low molecular weight substances, as common in potential pharmaceutical Occurring agents are examined.

Further Features of the invention will become apparent from the drawings and from the following description of preferred embodiments in conjunction with the subclaims. Here you can the individual features for each one or more in combination with each other in one embodiment of the Invention be realized. The particular embodiments described are for explanation only and for better understanding of the invention and are in no way limiting. Also the The drawings described below are part of the present invention Description, which is hereby confirmed by express reference.

In show the drawings:

1 : Correlation of the Measured Values from Examples 6 and 7

2 : Course of the determined according to Example 8, concentration-proportional variables for the substances warfarin, propranolol and carbamazepine

Example 1: Preparation a passivated, probe-labeled, fluorescence-active carrier

2 g of a porous Silicate material (Nucleoprep 300-12 from Macherey-Nagel, Düren) in a silane solution, consisting of 9.2 ml of N- (2-aminoethyl) -3-aminopropyltrimethoxysilane (EDA) and 243 μl concentrated acetic acid in 450 ml of deionized water and slowly for three hours rotates. Thereafter, the silicate material is sedimented, with three times washed with deionized water and dried at 80 ° C.

Of the carrier 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 μmol / l) staggered and over Night is rotating. To remove unreacted RITC is first twice with carbonate buffer and finally three times with PBS (phosphate buffered sa line, 10 mM sodium phosphate, 0.9% sodium chloride, pH 7.4).

to Passivation becomes the carrier with 20 ml of a solution of polyethylene glycol bisglycidyl ether (Mm ~ 526 g / mol) in PBS (3 % (w / v)) and over Night on an overhead cutter rotated, whereupon washed three times with PBS. The suspension will be final to a 20% solids content (200 mg of solids per 1 ml of suspension).

Example 2: Preparation probe-marked filter plates

The individual cavities of a commercially available system of two coordinated Microtiter plates (96well permeation of Millipore with a donor and an 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. Subsequently, the wells are washed 3 times with deionized water and rebuffered in 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 probe-marked filter plates with a hexadecane membrane

In the individual cavities a dye-coated filter plate of Example 2 are 15 μl of a 5% solution hexadecane in heptane is added to the membranes. Leave the filter plate so long under constant Air vent until the heptane solution has evaporated. The result is a probe-marked filter plate with a layer of hexadecane on the membranes (here also as hexadecane membrane designated).

Example 4: Determination the substance permeation through a hexadecane membrane of a probe-labeled filter plate

In several wells of the filter plate (acceptor plate in this example) from Example 3 are each pipetted 150 ul PBS buffer pH 7.4, which contains 1% DMSO. The corresponding wells of the donor plate are each filled with 135 μl of PBS buffer and 15 μl of a 1.0 mM solution of substance 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 .

To determine the concentration-proportional size c ' _equilibrium , both some cavities of the donor and the corresponding cavities of the acceptor plate are filled with 5E-5 molar solutions (the calculated concentration in equilibrium) of the substances to be investigated.

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 ' _acceptor and c' _{equilibrium of} the permeated substances were determined from the measured intensities according to the procedure described above.

The investigated substances in the experiment were warfarin and carbamazepine, for which the following permeabilities could be determined by means of equation (3): logP warfarin = -4,3 and logP carbamazepine = -3.9.

Example 5: Preparation a microtiter plate with hexadecane membranes

In the cavities of a microtiter plate provided with filter membranes a commercially available system made of two coordinated microtiter plates, as is it was used in Example 2, 15 ul of a 5% hexadecane solution in Heptane is added to the membranes and the filter plate under so long constant Leave air vent until the heptane solution has evaporated. You get one Microtiter plate with a layer of hexadecane on the membranes.

Example 6: Determination the substance permeation through the hexadecane membranes of a microtiter plate from Example 5, read by UV detection

In the cavities provided with filter membranes of the microtiter plate (acts here as acceptor plate) from Example 5 are each 135 ul PBS buffer pH 7.4 and 15 μl a 10% solution of DMSO in PBS buffer pipetted. The cavities of the corresponding Donor plate are each with 135 ul PBS buffer and 15 ul of a 1.0 mM substance solution in PBS buffer / DMSO (90:10 v: v). Another 4 cavities of Donor plate as well as the acceptor plate are only used with a 1% solution filled by 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 a 50 μl aliquot is added from the wells transferred the plates into a UV-transparent microtiter plate. The concentration of the substances is determined in a UV plate reader (SpektraMax plus 384 from Molecular Devices).

The substances studied were desipramine, quinidine, testosterone, furosemide, terbutaline, acyclovir, ketoprofen, carbamazepine, amiloride, metoprolol, imipramine, chloramphenicol, propranolol, corticosterone, naproxen, sulfasalazine, verapamil, progesterone and warfarin. The logarithmic permeabilities of the substances determined in accordance with equation (2) are correlated to the measured values of the measurement from example 7 in FIG 1 shown.

Example 7: Determination the substance permeation through the hexadecane membranes of a microtiter plate from Example 5, read by probe-marked silicate beads

Analogous to the in 4 Here, an arrangement is chosen in which the concentration-proportional quantities are determined by simultaneous measurement of the fluorescence intensities in acceptor and donor cavities "from above".

To be in several of the provided with filter membranes cavities of the Microtiter plate from Example 5 115 μl of PBS buffer, 20 μl of the preparation prepared in Example 1 support suspension (with 200 mg of solids per 1 ml of suspension) and 15 μl a 10% solution of DMSO in PBS buffer pipetted. The corresponding cavities of a another microtiter plate (without filter membranes) with 135 ul PBS buffer and 15 ul of 1.0 mM substance solution in PBS buffer / DMSO (90:10 v: v) filled.

In more cavities The microtiter plate from Example 5 is 20 .mu.l of the prepared in Example 1 Support suspension, 115 μl PBS buffer and 15 μl a 1.0 mM solution of substance in PBS buffer / DMSO (90:10 v: v) pipetted. In the corresponding wells Another microtiter plate (without filter membranes) is 15 .mu.l of a 10 % solution of DMSO in PBS buffer and 135 μl PBS buffer pipetted.

Some wells The microtiter plate from Example 5 act here as acceptor cavities, other than Donorkavitäten.

Further wells the microtiter plate of Example 5 are as reference as their corresponding cavities in the further microtiter plate with a 1% solution of DMSO filled in PBS.

The Permeation of the substances is achieved by joining the started on both disks. After 4 hours in all filled cavities the Microtiter plate from Example 5, the fluorescence intensities in the Measuring arrangement determined "from above" intensities were the concentration-proportional sizes of the permeated substance determined.

The substances studied were desipramine, quinidine, testosterone, furosemide, terbutaline, acyclovir, ketoprofen, carbamazepine, amiloride, metoprolol, imipramine, chloramphenicol, propranolol, corticosterone, naproxen, sulfasalazine, verapamil, progesterone and warfarin. The determined logarithmic permeabilities of the substances are correlated to the measured values of the measurement from Example 6 in FIG 1 shown.

Example 8: Determination the substance permeation through a hexadecane membrane on a microtiter plate Example 5, continuously read by dye-coated silicate spheres

Also in Example 8, two permeation directions are to be considered. In the case 1 permeates the substance from below to the top "in the Microtiter plate from example 5 (acceptor), in case 2 permeated the substance from the microtiter plate from Example 5 (donor) of "from above below".

In the case 1 will be in some cavities of the filter plate from Example 5 115 .mu.l PBS buffer pH 7.4, 20 .mu.l of in Example 1 prepared carrier suspension and 15 μl a 10% solution of DMSO in PBS buffer pipetted. The corresponding cavities of a another plate with 135 ul PBS buffer and 15 μl a 1.0 mM solution of substance in PBS buffer / DMSO (90:10 v: v) filled.

In the case 2 will be in more cavities the microtiter plate from Example 5 20 .mu.l of the prepared in Example 1 Support suspension, 115 μl PBS buffer and 15 μl a 1.0 mM solution of substance Pipette. In the corresponding cavities of the further plate will be 15 μl of a 10% solution of DMSO in PBS buffer and 135 μl PBS buffer pipetted.

Further wells the microtiter plate of Example 5 are as reference as their corresponding cavities in the further microtiter plate with a 1% solution of DMSO filled in PBS.

The Permeation of the substances is achieved by joining the started on both disks. At discrete time intervals of 5 min over one Total time of 4 hours, the fluorescence intensities in the Measuring geometry "from top "in all filled cavities of Microtiter plate from Example 5 measured. From these intensities were for each measured discrete time the concentration proportional Sizes of permeated substance.

The investigated substances were warfarin, carbamazepine and propranolol. The obtained mean permeabilities of the substances were: logP warfarin = -4.7 logP carbamazepine = -3.9 logP propanolol = -3.8

The determined courses of the concentration - proportional sizes of the permeating substances are in 2 in each case, the upper measurement curve illustrates the decrease of the substance concentration in a donor cavity and the lower measurement curve illustrates the increase of the substance concentration in an acceptor cavity.

Example 9: Determination the substance permeation through a hexadecane membrane on a microtiter plate Example 5, read by dye-loaded dispersible polymer particles.

120 μl of PBS buffer, 15 μl of a 10% solution of DMSO in PBS buffer and 15 μl of a suspension of dye-loaded polystyrene particles (1 μm, 0.05%) are used in several wells of an acceptor plate prepared according to Example 5. Manufacturer Estapor). The corresponding wells of another microtiter plate serving as a donor plate are filled with 135 μl of PBS buffer and 15 μl of a 1.0 mM substance solution in PBS buffer / DMSO (90:10 v: v). Further cavities of the donor plate (and the corresponding 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 .

To determine the concentration-proportional size c ' _equilibrium , both some cavities of the donor and the corresponding wells of the acceptor plate are filled with 5E-5 molar solutions (the calculated concentration in equilibrium) of the substances to be investigated.

The Permeation of the substances is achieved by joining the started on both disks. After 4 hours in all filled cavities the Acceptor plate the fluorescence intensities of the dye-loaded Polystyrene particles in the measuring geometry were measured from the top the concentration-proportional sizes of the permeated substance determined.

Among other things, the substance warfarin was investigated. The permeability of warfarin was determined to be logP _warfarin = -4.5.

Figure Description

1 : Schematic structure of a permeation measurement with a cell monolayer as permeation barrier.

2 Schematic representation of a measuring arrangement "from above" The direction of permeation is marked by the black arrow The permeating substance is located in the left-hand permeation device while the measurement in the right-hand permeation device serves as a reference.

3 Schematic representation of a measuring arrangement for the simultaneous measurement "from above" and "from below" for the determination of the concentration-proportional quantities c ' _acceptor and c' _donor . The permeation direction is marked by the black arrow.

4 Schematic representation of a luminescence measurement exclusively "from above" to the simul Determination of concentration-proportional quantities c ' _acceptor and c' _donor The permeation directions are marked by the black arrows.

Claims (34)

Method for measuring permeation at least one in a solvent contained substance through a permeation barrier and / or to Determination of permeation-influencing parameters, in particular for Determination of permeability the barrier for the at least one substance, using at least one luminescent probe comprising - the provision of at least a substance on one side of the permeation barrier, - Incubation the permeation barrier with the at least one, in the solvent contained substance and - Measurement of luminescence signals of the probe on at least one side of the Permeation barrier, which depends on the respective Concentration of the substance are modulated, where the excitation and / or the emission spectrum of the at least one probe with the Absorption spectrum of at least one substance overlaps. Method according to claim 1, characterized in that that the Permeationsbarriere comprises an artificial membrane. Method according to claim 2, characterized in that that the artificial membrane a porous Support material in particular a filter membrane. Method according to one of claims 2 or 3, characterized that the artificial membrane comprises organic material, in particular as a layer on the surface of the porous one Carrier material and / or in the pores of the porous Support material. Method according to claim 4, characterized in that that it Substantially, the organic material is at least at least a hydrocarbon having a preferred chain length of 8 or more carbons and / or at least one alcohol with a preferred chain length of 4 or more carbons. Method according to claim 4, characterized in that that it the organic material is essentially a lipid or a lipid mixture is. Method according to Claim 6, characterized that this Lipid or the lipid mixture membranous, membrane integral and / or having transmembrane proteins. Method according to claim 4, characterized in that that it the organic material is essentially cells, in particular in the form of a cell mono- or a cell Cell multilayer are present. Method according to claim 8, characterized in that that it the cell monolayer is a CACO-2 or an MDCK monolayer. Method according to claim 4, characterized in that that it The organic material is essentially cell fragments is. Method according to claim 1, characterized in that that the Permeationsbarriere comprises a non-artificial membrane, in particular a membrane of organ tissue. Method according to one of claims 1 or 11, characterized that the Permeation barrier is prepared from a gut tissue. Method according to one of claims 1 or 11, characterized that the Permeation barrier is prepared from skin tissue. Method according to one of the preceding claims, characterized characterized in that at least one probe is part of the permeation barrier. A method according to claim 14, characterized in that the porous support material of a kindifiziel len membrane is marked. Method according to one of claims 14 or 15, characterized that this organic material of an artificial membrane is probe-marked. Method according to one of the preceding claims, characterized characterized in that at least one probe is applied to the permeation barrier, especially in the form of sedimented on the permeation barrier, probe-labeled solid. Method according to one of the preceding claims, characterized characterized in that at least one probe in the solvent is included. Method according to one of the preceding claims, characterized characterized in that at least one probe is contained in a gel. Method according to one of the preceding claims, characterized characterized in that it is performed in at least one permeation device, the at least one donor compartment in which the at least one Substance is provided, the permeation barrier and at least an acceptor compartment into which the substance is optionally at least partially permeated. Method according to one of the preceding claims, characterized characterized in that at least one probe in the form of a probe-labeled insert in the at least one donor and / or in the at least one acceptor compartment is introduced. Method according to one of the preceding claims, characterized characterized in that it carried out in parallel becomes. Method according to one of the preceding claims, characterized characterized in that Lumineszenzsignale at one or more discrete wavelengths and / or be measured as a spectrum. Method according to one of the preceding claims, characterized characterized in that at least one luminescent probe fluorescent and / or phosphorescent Features. Method according to one of the preceding claims, characterized characterized in that a single probe or a combination of probes is used. Use of at least one microtiter plate in a method according to any one of the preceding claims, in particular as a component of at least one permeation device. Use of at least one dye in one Method according to one of the claims 1 to 25, in particular as at least one luminescent probe. Use of at least one porous support material, in particular at least one filter membrane, in a method according to one of claims 1 to 25, in particular as part of at least one permeation barrier. Use of at least one lipid or a lipid mixture in a method according to any one of claims 1 to 25, in particular as part of at least one permeation barrier. Use of at least one hydrocarbon with a preferred chain length of 8 or more carbons and / or at least one alcohol with a preferred chain length of 4 or more carbons in a process according to one of claims 1 to 25, in particular as part of at least one permeation barrier. Use of cells, in particular in the form of a Cell mono- or one Cell multilayer, in a method according to one of claims 1 to 25, in particular as part of at least one permeation barrier. Use of cell fragments in a procedure according to one of the claims 1 to 25, in particular as part of at least one permeation barrier. Use of at least one membrane of organ tissue in a method according to any one of claims 1 to 25, in particular as a permeation barrier. Kit to carry out a method according to a the claims 1 to 25, comprising at least 2 members from the group with - at least a microtiter plate, - at least a luminescent probe, At least one porous carrier material, - at least a probe-marked insert, At least one solvent, - at least a suitable buffer system, - At least one hydrocarbon with a preferred chain length of 8 or more carbons and / or at least one alcohol with a preferred chain length of 4 or more carbons, At least one lipid or a lipid mixture, - cells, in particular in the form of a cell monolayer or a cell multilayer, - cell fragments and - at least a non-artificial membrane, in particular at least one Membrane of organ tissue.