Vitro Method and Apparatus for Analysing the Behaviour of Substances in Simulated Physiological Environment

Abstract

The invention refers to an in vitro method and apparatus for analysing the behaviour of substances in simulated physiological environment. The method comprises the steps of providing a first fluid, a gel matrix and a second fluid, separating the first fluid and the gel matrix by at least one first semi-permeable membrane and separating the gel matrix and the second fluid by at least one second semi-permeable membrane. The method further comprises the steps of injecting a substance into the first fluid, letting the substance migrate from the first fluid through the at least one first semi-permeable membrane, through the gel matrix, through the at least one second semi-permeable membrane and into the second fluid, and determining clearance of the substance from the first fluid.

Claims

1.-21. (canceled)

22. An apparatus for analyzing the behaviour of molecules in simulated physiological environment, the apparatus comprising a first compartment for receiving a first fluid, a second compartment for receiving a gel matrix, and a third compartment for receiving a second fluid; the apparatus further comprising at least one first semi-permeable membrane, and at least one second semi-permeable membrane, the at least one second semi-permeable membrane being arranged at a distance from the at least one first semi-permeable membrane, wherein the at least one first semi-permeable membrane is arranged between the first compartment and the second compartment, and wherein the at least one second semi-permeable membrane is arranged between the second compartment and the third compartment.

23. The apparatus according to claim 22, wherein the first compartment, the second compartment and the third compartment are arranged in a row, and wherein the at least one first semi-permeable membrane and the at least one second semi-permeable membrane are arranged substantially vertically between the respective compartments.

24. The apparatus according to claim 22, further comprising holders for holding the at least one first semi-permeable membrane and the at least one second semi-permeable membrane between the respective compartments.

25. The apparatus according to claim 24, wherein the holders do not extend into any of the first, second or third compartment.

26. The apparatus according to claim 22, wherein a Molecular Weight Cut Off (MWCO) of the at least one first semi-permeable membrane substantially corresponds to the Retinal Exclusion Limit (REL).

27. The apparatus according to claim 22, wherein the at least one first semi-permeable membrane has a Molecular Weight Cut Off (MWCO) being smaller than or equal to the Molecular Weight Cut Off (MWCO) of the at least one second semi-permeable membrane.

28. The apparatus according to claim 22, wherein the at least one first semi-permeable membrane has a concave shape.

29. The apparatus according to claim 22, wherein the at least one second semi-permeable membrane has a concave shape.

30. The apparatus according to claim 22, comprising or being made of glass.

31. The apparatus according to claim 22, further comprising a first support for supporting the at least one first semi-permeable membrane.

32. The apparatus according to claim 31, wherein the first support has a concave shape.

33. The apparatus according to claim 22, further comprising a second support for supporting the at least one second semi-permeable membrane.

34. The apparatus according to claim 33, wherein the second support has a concave shape.

35. The apparatus according to claim 22, wherein the first compartment comprises the first fluid, which first fluid is vitreous humor.

36. The apparatus according to claim 22, wherein the third compartment comprises the second fluid, which second fluid is a buffer solution.

37. The apparatus according to claim 36, wherein the buffer solution is a physiologically relevant buffer solution.

38. A method of using the apparatus according to claim 22 for in vitro testing a substance and for determining data on stability or bioavailability of the substance.

39. The method of claim 38, wherein the substance is a drug formulation.

40. The method of claim 38, wherein the substance comprises molecules having a size in a range between about 1 kDa and about 250 kDa.

41. The method of claim 38, wherein the substance comprises molecules having a size in a range between 4 kDa and 150 kDa.

Description

[0045] The invention is further described with regard to examples and embodiments, which are illustrated by means of the following drawings, wherein

[0046] FIG. 1 shows a schematic illustration of a test set-up according to the invention;

[0047] FIGS. 2 and 3 show diffusion rates (FIG. 2) and permeability (FIG. 3) of dextran versus hyaluronic acid concentration in a gel matrix;

[0048] FIG. 4 shows the effect of different membranes on the diffusion of macromolecules versus time;

[0049] FIGS. 5 and 6 show diffusion (FIG. 5) and permeability (FIG. 6) of various macromolecules;

[0050] FIG. 7 shows test results performed with monoclonal antibody;

[0051] FIGS. 8a-8e show a first illustrative embodiment of an apparatus according to the invention;

[0052] FIG. 9 shows a schematic illustration of a second embodiment of the apparatus according to the invention;

[0053] FIGS. 10a, 10b show a variant of the embodiment of the apparatus of FIG. 9.

[0054] In FIG. 1 an embodiment of a set-up of the apparatus and method is schematically shown. The set-up simulates the geometry and situation of an eye and may preferably be used for in vitro tests simulating eye conditions. The first compartment 1 for receiving vitreous humor is formed by an upper wall 10 and a first semi-permeable membrane 4 covering part of said upper wall 10. The upper wall 10 is provided with an opening 101 for filling the first compartment 1 through said opening 101. The opening 101 may be closed, preferably in an air-tight manner, with a cover 6. The cover 6 may for example be a conically shaped plug, for example a glass plug. The semi-permeable membrane 4 has the form of part of a circle, for example of a circle. The form of the first compartment 1 and of the first membrane simulates the geometry of an eyeball and retina. The first compartment 1 is mounted to a third compartment 3. Thereby, the first membrane 4 is preferably completely inserted into an upper opening 303 of the third compartment 3 and inside the third compartment 3. The third compartment 3 is for receiving a buffer solution. A concave shaped second semi-permeable membrane 5 is arranged in the upper opening 303 and covers part of an upper wall 30 of the third compartment 3. The second membrane 5 also has the form of part of a circle, for example to of a circle. First and second membranes 4, 5 may be supported by respective first and second support forming compartment walls as described below with respect to FIGS. 8a-8e below. In the mounted state of the first and third compartment 1,3, the two semi-permeable membranes 4,5 are distanced from each other forming a second compartment 2 in the gap between the first and the second membrane. Gap sizes may be varied, for example adapted to a physiological system to be simulated. First and second membrane 4,5 are arranged concentrically and such as to have a predefined distance between the membranes, preferably over the entire extension of the membranes. Via inlet and outlet opening 102 provided in the upper wall 10 of the first compartment 1, a gel matrix may be filled into and removed again from the second compartment 2. The three compartments may be sealed, for example by the provision of an O-ring arranged on the upper wall 30 of the third compartment and arranged circumferentially around the gap forming the second compartment 2.

[0055] The third compartment 3 is provided with inlet and outlet openings 32, 33. Through the inlet opening the third compartment 3 may be filled with buffer solution and through the outlet opening the third compartment 3 may be emptied. Inlet and outlet are preferably designed to allow a flow through the third compartment such as to clean and replace the content of the third compartment 3. A continuous or discontinuous flow through the third compartment 3 may also be used for sampling the second fluid for subsequent analysis of the sample.

[0056] With the first compartment and first membrane 4 the geometry as well as the physical and chemical environment inside an eyeball is simulated. In combination with the gel matrix and second membrane 5, the barrier out of the tissue enveloping the eyeball, such as the anterior or posterior segment tissue, is simulated. A substance, for example macromolecules, may be injected into the vitreous humor in the first compartment 1. It migrates to the first membrane, through the first membrane 4 and the gel matrix in the second compartment 2, through the second membrane and into the buffer solution in the third compartment 3. The removable cover 6 of the first compartment 1, as well as the inlet and outlet 32,33, of the third compartment allow the extraction of samples for analysing purposes.

[0057] In the following, examples performed with the system as described in FIG. 1 are described. All experiments mentioned in the examples were performed in aseptic conditions under laminar air flow. Samples were collected from the first and the third compartment 1,3 (or VH-compartment 1 and FT-compartment 3, respectively) at different time intervals.

EXAMPLE 1

[0058] Example 1 was performed in order to optimize a hyaluronic acid gel matrix concentration. For this, impact of different concentrations of hyaluronic acid (HA) (molecular weight 1.410.sup.6 Daltons) gel-matrix (GM) on the diffusion rate of FITC-Dextran (40 kDa) from vitreous humor (VH)-compartment 1 to flow through (FT)-compartment 3 was studied. VH-compartment and FT-compartment were separated by GM-compartment 2, which acts as a diffusion controlling barrier. Two dialysis membranes 4,5 were utilized to separate these three compartments 1, 2, 3. The first dialysis membrane 4 separating VH-compartment 1 from GM-compartment 2 was denoted DM-1 (dialysis controlling membrane 4), and the second dialysis membrane separating GM-compartment from FT-compartment was named DM-2 (dialysis controlling membrane 5). Molecular weight cut-off (MWCO) for DM-1 and DM-2 were 50 kDa and 100 kDa, respectively. FT-compartment 3 was filled with sterile phosphate buffer saline (PBS) (pH 7.4) and GM-compartment 2 was filled with different concentrations of about 3 mL sterile HA gel prepared in PBS (ranging from 0-0.9% w/v). About 3.5 mL of sterile porcine VH was added in VH-compartment. The device was sealed and VH was conditioned overnight at 37 C. At the end of incubation, 50 L of 80 mg/mL of FITC-Dextran (40 kDa) was injected into the VH-compartment 1. The apparatus was sealed and incubated at 37 C. throughout the study. Samples were evaluated for the concentration of FITC-Dextran by fluorescence spectrophotometer at excitation wavelength of 490 nm and emission wavelength of 520 nm. FIG. 2 illustrates the rate of diffusion versus concentration of HA and FIG. 3 illustrates apparent permeability (P.sub.app) versus concentration of HA. Permeability is the property of a diffusion controlling barrier (single barrier) to allow transfer of components from one side to the other side of the barrier. When more than one diffusion-controlling barriers are involved, permeability is calculated as apparent permeability (Papp (apparent) or Peff (effective)). In the present experiments, Papp (apparent) has been implemented to facilitate correlation between the in vitro/ex vivo diffusion data with reported in vivo diffusion data in literature. Apparent permeability, for example, can be calculated from the relationship Papp=Q/[At(CoCi)], where Q is the quantity of permeant transported through the membrane with the area (A) in time t. Co and Ci are the donor concentration (concentration in the VH chamber) and receiver concentration (concentration in the FT compartment), respectively. Papp can be represented in cm/sec, cm/min or cm/hr.

[0059] Results described in FIG. 2 suggest that increase in the concentration of HA gel matrix significantly reduces the rate of diffusion as well as apparent permeability of macromolecule. The plausible explanation could be, increase in the HA concentration may result in reduced porosity of the matrix and/or may increase the interaction of dextran with matrix components resulting in slower diffusion of FITC-Dextran. These results indicate that by changing the GM concentration, it is possible to tune the rate of diffusion of macromolecules across the system.

EXAMPLE 2

[0060] Example 2 was performed in order to analyse the effect of dialysis membranes on the diffusion of macromolecules.

[0061] Diffusion of bovine serum albumin (BSA) and Immunoglobulin G (IgG) in the flow direction from VH-compartment to FT-compartment was investigated by varying MWCO of the dialysis membrane. The experiment was performed similarly as described in example 1 with minor modifications. Two different MWCO dialysis membranes were used as DM-1, with MWCO 50 kDa and 100 kDa, whereas the MWCO of DM-2 was kept constant at 100 kDa. VH-compartment was filled with 3.5 mL of sterile porcine VH and GM-compartment was filled with about 3 mL of sterile HA gel (0.6% w/v). FT-compartment was filled with sterile PBS. The device was sealed and VH was conditioned overnight at 37 C. At the end of incubation, 50 L of FITC-BSA (80 mg/mL) or 200 L of FITC-IgG (20 mg/mL) was injected into the VH-compartment. The apparatus was sealed and incubated at 37 C. throughout the study. Samples were evaluated for the concentration of FITC-BSA and FITC-IgG by fluorescence spectrophotometer at excitation wavelength of 490 nm and emission wavelength of 520 nm.

[0062] In FIG. 4 results for diffusion measurements in the FT compartment 3 (named as PBS compartment in the figure) versus time for BSA with DM-1 and DM-2 MWCO 100 kDa 81, of IgG (about 144 kDa) with DM-1 and DM-2 MWCO 100 kDa 82, for BSA with DM-1 50 kDa and DM-2 MWCO 100 kDa 83 and for IgG with DM-1 50 kDa and DM-2 MWCO 100 kDa 84 is depicted. Results shown in FIG. 4 suggest that DM-1 with 50 kDa MWCO (83,84) restrained the diffusion of both IgG and BSA significantly when compared with the diffusion observed with DM-1 with 100 kDa MWCO (81,82). Hence, by changing the dialysis membrane MWCO, it will be further possible to tune the diffusion of macromolecules.

EXAMPLE 3

[0063] Example 3 was performed in order to observe the diffusion of different macromolecules (linear and globular).

[0064] This experiment was performed similarly as described in example 1 with minor modifications. 50 kDa MWCO dialysis membrane was used as DM-1 and 100 kDa MWCO dialysis membrane was used as DM-2. VH-compartment was filled with 3.5 mL of sterile porcine VH and GM-compartment was filled with about 3 mL of sterile HA gel (0.6% w/v). FT-compartment was filled with about 35 mL of sterile PBS. The device was sealed and VH was conditioned overnight at 37 C. At the end of incubation, 50 L of 80 mg/mL of FITC-dextran 4 kDa 91, FITC-dextran 40 kDa 92, FITC-dextran 70 kDa 94, FITC-BSA 93, or 200 L of FITC-IgG (20 mg/mL) 95 was injected into the VH-compartment. The apparatus was sealed and incubated at 37 C. throughout the study. Samples were evaluated for the concentration of FITC-Dextran by fluorescence spectrophotometer at excitation wavelength of 490 nm and emission wavelength of 520 nm.

[0065] As shown in FIG. 5, where the amount of the different diffused macromolecules versus time is depicted, macromolecules exhibited different rate of diffusion. Smaller molecules diffused at faster rate compared to larger molecules as expected. The same phenomenon was observed for linear and globular kinds of macromolecules. Similarly, smaller molecules exhibited higher P.sub.app relative to large molecules, which is depicted in FIG. 6. This may be due to the fact that large molecules face more resistance by the GM due to their higher molecular radius compared to the smaller molecules resulting in low P.sub.app. Interestingly, FITC-Dextran 40 kDa and FITC-BSA (66 kDa) exhibited similar values of P.sub.app. Similarity of molecular radius (FITC-Dextran 40 kDa: 4.5 nm, and FITC BSA: 3.62 nm) may attribute to their similar P.sub.app.

EXAMPLE 4

[0066] In this experiment, diffusion of a monoclonal antibody (mAb1) in the presence or absence of GM was investigated in the direction from VH-compartment to FT-compartment. The experiment was performed similarly as described in example 1 with minor modifications. Again, 50 kDa MWCO dialysis membrane was used as DM-1 and 100 kDa MWCO dialysis membrane was used as DM-2. VH-compartment was filled with 3.5 mL of sterile porcine VH and GM-compartment was filled with about 3 mL of sterile HA gel (0.6% w/v) or sterile PBS (without matrix). FT-compartment was filled with sterile PBS. The device was sealed and VH was conditioned overnight at 37 C. At the end of incubation, 33 L of mAb1 (120 mg/mL) was injected into the VH-compartment. The apparatus was sealed and incubated at 37 C. throughout the study. Samples were evaluated for the concentration of mAb1 by size exclusion chromatography (SEC).

[0067] In FIG. 7 the amount of mAB1 in VH compartment versus time is depicted. As shown, diffusion of mAb1 was significantly reduced in the presence of GM (about 51% mAb1 diffused out from VH-compartment at 120 h), indicated by curve 85, when compared to the diffusion observed in absence of GM (about 87% mAb1 diffused out from VH-compartment at 120 h), which is indicated by curve 86. Reduction in the diffusion may be attributed to the resistance provided by the gel-matrix. This result clearly indicates that it is possible to tailor the residence time of protein therapeutics into the VH compartment 1 by altering the concentration of gel-matrix.

[0068] In FIG. 8a to FIG. 8e an illustrative embodiment of an apparatus according to the invention and its parts are shown. The same reference numbers as in FIG. 1 are used for the same or similar features. FIG. 8a shows the cover 6 having a cylindrical form and having a circumference corresponding to the size of the opening 101 in the upper wall 10 of the first compartment 1. When in a mounted state as shown in FIG. 8c, the cover preferably forms an air-tight seal with the upper wall 101. Macromolecules 7, representing a substance, in a first fluid such as vitreous humor 11 are indicated with circles. In FIG. 8c, the macromolecules have already migrated through the first fluid 11, for example vitreous humor, in the first compartment 1 into the direction of the first membrane 4. The first membrane is arranged on a first support forming part of the upper wall 101 of the first compartment 1. In FIG. 8d the second membrane 5 is arranged on a second support 15. The second support 15 forms part of the upper wall 30 of the third compartment 3. The third compartment is cylindrically shaped but may basically have any other form. The third compartment is filled with a second fluid, preferably a physiologically relevant buffer solution 31. Inlet and outlet 33,32 are formed by tube sections attached to or preferably integrated into the side wall 34 of the third compartment 3. First and second support 14, 15 are arranged equidistantly in the mounted state of first and third compartment as shown in FIG. 8e. There, some macromolecules are indicated as having migrated to the first membrane 4, some are shown as having partly passed the gel matrix 21 in the second compartment 2 formed by the first and second membrane 4,5 (supported by the first and second support 14,15) and some are about to diffuse through the second membrane 5.

[0069] The three compartments 1, 2, 3, are arranged above or on top of each other. The semi-permeable membranes 4,5 are arranged substantially horizontally between the respective compartments forming a horizontal arrangement of the apparatus.

[0070] All parts of the apparatus, next to the membranes may be made of glass. Preferably, the apparatus is made of three separate parts only: cover, first compartment and third compartment, wherein the separate parts may be mounted to each other preferably in an air-tight manner. Also the apparatus as shown in FIG. 9 is preferably made of glass.

[0071] The apparatus of FIG. 9 comprises a receptacle 100, for example of cuboid or tubular form. A first semi-permeable membrane 4 and a second semi-permeable membrane 5 are arranged in the receptacle such as to divide the interior of the receptacle 100 in a first compartment 1, a second compartment 2 and a third compartment 3. The first 4 and the second 4 semi-permeable membrane 5 are arranged vertically in the receptacle 100 and perpendicular to a longitudinal axis 300 of the receptacle 100.

[0072] The first compartment 1 comprises a first inlet 17 arranged on top of the receptacle 100 for supplying a first fluid, for example, vitreous humor into the first compartment 1. One side wall of the first compartment 1 is formed by the first semi-permeable membrane 4.

[0073] The two semi-permeable membranes 4,5 are arranged distanced to each other, forming the second compartment 2 between the two membranes. A distance between the membranes 4,5 may be varied, for example, adapted to a physiological system to be simulated. Preferably, first and second membrane 4,5 have a predefined distance between the membranes, preferably over the entire extension of the membranes. The second compartment 2 comprises a second inlet 18 arranged on top of the receptacle 100 for supplying a gel matrix into and removing same from the second compartment 2.

[0074] The third compartment 3 is formed as a flow-through chamber comprising inlet and outlet 32,33, which are arranged in FIG. 9 on top of the receptacle 100 for supplying and removing a third fluid, for example, a buffer solution. However, inlet and outlet may also be arranged in preferably different (solid) side walls of the third compartment. One side wall of the third compartment 3 (not formed by a receptacle wall) is formed by the second semi-permeable membrane 5.

[0075] The three compartments 1, 2, 3, are arranged next to each other in a side-by-side manner. The semi-permeable membranes 4,5 are arranged vertically in the receptacle forming a vertical arrangement of the apparatus.

[0076] The semi-permeable membranes 4,5 are held by holders 24,25. The holders 24,25 are arranged on the top and below the bottom of the receptacle 100 outside of the receptacle 100. The holders 24,25 may also be arranged circumferentially around the receptacle 100. The holders may, for example, be clamps.

[0077] A clamping of the membranes 4,5 may also be achieved by the receptacle 100 itself. For example, the receptacle 100 may be made of several, for example, three parts. A semi-permeable membrane may then be clamped between two parts upon mounting and fixing the parts to each other. Each part then basically forms a compartment. Such an embodiment of the apparatus is illustrated in FIG. 10a and FIG. 10b. FIG. 10a shows the individual three parts 101, 102, 103 of the receptacle 100, which may be mounted to the apparatus according to the invention with a first semi-permeable membrane 4 clamped between first and second part 101,102 and with a second semi-permeable membrane 5 clamped between second and third part 102,103. Each two parts of the receptacle may be provided with clamping means (not shown) for holding the two parts to each other and for achieving a liquid tight connection between the two parts. To support clamping, each part 101, 102, 103 is provided with circumferentially running rims 1010, 1020, 1021, 1030. In the mounted state of the receptacle, shown in FIG. 10b, each two rims 1010,1020; 1021,1030 come to lie against each other, clamping the semi-permeable membrane along a ring portion. Preferably, clamping means are provided to clamp each two rim portions.

[0078] Preferably, the semi-permeable membranes 4,5 are plane membranes. However, if the material of the membranes allows, the membranes may also be pre-shaped, for example, concave or convex to simulate the geometry of portions of an eyeball. Preferably, the forms of the semi-permeable membranes 4,5 correspond to each other. While vertically arranged membranes are preferably held in the receptacle by clamping or other holding means, the first and second membranes 4, 5 may also be supported by respective first and second supports forming compartment walls as described above with respect to FIGS. 8a-8e. Holders 24,25 may then be omitted.

[0079] A substance, for example macromolecules, may be injected through the first opening 17 into the first fluid in the first compartment 1. It migrates to all sides. Due to gravitational force, migration to the bottom of the first compartment is preferred. However, the bottom being formed by a closed receptacle wall does not allow diffusion of the substance out of the first fluid through the bottom. Only upon migration of the substance to the first semi-permeable membrane 4, a diffusion out of the first compartment, through the first membrane 4, into and through the gel matrix in the second compartment 2, through the second membrane and into the buffer solution in the third compartment 3 occurs. Inlet 17, as well as the inlet and outlet 32,33 of the third compartment also allow the extraction of samples for analysing purposes.

[0080] The invention has been described relating to specific embodiments. However, further embodiments may be realized without departing from the scope of the invention. For example, form and size of the apparatus may be adapted to a specific application a substance shall be tested for. Especially, a geometry of the apparatus may be varied. For example, compartments and membranes may be arranged in an essentially flat manner, such that the apparatus is formed by a stack of compartment separated by the membranes. The geometry of an arrangement may also have influence on the materials used in the method and apparatus according to the invention. For example, in a flat configuration, a substance may provide sufficient support for a membrane such that for example a first support may possibly be omitted or limited to small edge portions. In addition, the fluid or semi-fluid material of the gel matrix may be replaced by a porous solid material forming the barrier. For example, open-pored ceramic or open plastic materials may be favorable when combined with cell growth in the barrier material. Yet further one membrane may be replaced by two or more membranes having the same or different MWCOs.