Multiwell plate lipophilicity assay

Abstract

The present invention relates to a method for determining the lipophilicity of a test compound, comprising the steps of: a) providing a multiwell plate, the wells comprising a lipophilic membrane at the bottom and the bottom of the multiwell plate comprising a liquid-tight barrier for generating a liquid-tight bottom of the multiwell plate; b) adding a non-polar solvent to the lipophilic membrane in the wells of step a); c) adding an aqueous solution comprising the test compound to the wells of step b); and d) determining the amount of the test compound in the aqueous solution after partition equilibrium is reached. TIFF2024528879000005.tif52134

Description

Technical Field

[0001] The present invention provides a method for determining high, medium, and low lipophilicity values of compounds.

Background Art

[0002] Lipophilicity is an important molecular property in drug discovery. Accurately knowing the lipophilicity of a drug is useful for correlation with pharmaceutical processes such as membrane permeability, solubility, volume of distribution, metabolic stability, and protein binding. Lipophilicity is represented by either logP (octanol-water partition coefficient of the neutral species) or logD (octanol-water partition coefficient of the charged molecule).

[0003] Generally, lipophilicity is determined by the conventional shake-flask method (M.M. Abraham, H.S. Chadha, J.P. Dixon, and A.J. Leo. Hydrogen bonding. Part 9. The partition of solutes between water and various alcohols. Phys. Org. Chem. 7:712 - 716 (1994) (Non-Patent Document 1)). When performed manually, this method is very time-consuming (only 2 - 5 compounds per day). However, due to rapid analog synthesis and combinatorial chemistry, the number of compounds generated in drug discovery has increased dramatically. This situation has demanded a rapid and efficient method for determining the lipophilicity of compounds.

[0004] European Patent No. 1705474 (Patent Document 1) discloses an assay system for determining the lipophilicity of compounds consisting of two multi-well plates. After completion of the assay, the solvent must be removed from the plates so that the concentration of the compound in the solvent can be measured. Further, the assay system must be incubated for 12 hours until the partition equilibrium is reached.

[0005] Therefore, there is a need for a method that is rapid, easy to perform, and enables determination of the lipophilicity of low-soluble compounds.

Prior Art Documents

[0006] [Patent Document 1] European Patent No. 1705474 [Non-patent literature]

[0007] [Non-Patent Document 1] MMAbraham, HSChadha, JPDixon, and AJLeo. Hydrogen bonding. Part 9. The partition of solutes between water and various alcohols. Phys. Org. Chem. 7:712-716 (1994) [Overview of the project]

[0008] In a first aspect, the present invention relates to a method for determining the lipophilicity of a test compound, a) A step of providing a multiwell plate, wherein the wells include a lipophilic film at the bottom, and the bottom of the multiwell plate includes a liquid-tight barrier for creating a liquid-tight bottom of the multiwell plate, b) A step of adding a nonpolar solvent to the lipophilic film in the well of step a), c) the step of adding an aqueous solution containing the test compound to the well of step b), and d) A step of determining the amount of the test compound in the aqueous solution after partition equilibrium has been reached. This provides a method that includes [something].

[0009] In one embodiment of the method for determining the lipophilicity of a test compound, the nonpolar solvent is octanol.

[0010] In one embodiment of the method for determining the lipophilicity of a test compound, the lipophilic film is selected from the group consisting of polyvinylidene fluoride (PVDF), polytetrafluoroethylene (PTFE), cyclic olefin copolymer (COC), polypropylene (PP), or polycarbonate (PC), and is preferably a PVDF film.

[0011] In one embodiment of the method for determining the lipophilicity of a test compound, the multiwell plate is a 96-well plate, preferably a multiscreen hydrophobic immobilon P PVDF film plate welded at the bottom to a liquid-tight foil, preferably a heat-sealing foil.

[0012] In one embodiment of a method for determining the lipophilicity of a test compound, the method includes an additional step e) calculating the lipophilicity value logD of the test compound.

[0013] In a second aspect, the present invention provides a multiwell plate for use in a method for determining the lipophilicity of a test compound, wherein each well includes a lipophilic film at the bottom of the well, and the bottom of the multiwell plate includes a liquid-tight barrier.

[0014] In one embodiment of a multiwell plate for use in a method for determining the lipophilicity of a test compound, the lipophilic film is selected from the group consisting of polyvinylidene fluoride (PVDF), polytetrafluoroethylene (PTFE), cyclic olefin copolymer (COC), polypropylene (PP), or polycarbonate (PC).

[0015] In one embodiment of a multiwell plate for use in a method for determining the lipophilicity of a test compound, the liquid-tight barrier is a liquid-tight foil, preferably a heat-sealed foil.

[0016] In one embodiment of a multiwell plate for use in a method for determining the lipophilicity of a test compound, the lipophilic film is a PVDF film.

[0017] In one embodiment of the multi-well plate for use in a method for determining the lipophilicity of a test compound, the multi-well plate is a 96-well plate.

[0018] In one embodiment of the multi-well plate for use in a method for determining the lipophilicity of a test compound, the multi-well plate is a multi-screen hydrophobic Immobilon-P PVDF membrane plate, wherein the bottom of the plate is welded with a liquid-tight foil, preferably a heat-sealing foil, and is a multi-screen hydrophobic Immobilon-P PVDF membrane plate.

[0019] In one embodiment of the multi-well plate for use in a method for determining the lipophilicity of a test compound, the method for determining the lipophilicity of the compound is the method according to the method of the present invention.

[0020] In a third aspect, the present invention provides a multi-screen hydrophobic Immobilon-P PVDF membrane plate, wherein the bottom of the plate is welded with a heat-sealing foil. [Invention 1001] A method for determining the lipophilicity of a test compound, a) A step of providing a multiwell plate, wherein the wells include a lipophilic film at the bottom, and the bottom of the multiwell plate includes a liquid-tight barrier for creating a liquid-tight bottom of the multiwell plate, b) A step of adding a nonpolar solvent to the lipophilic film in the well of step a), c) the step of adding an aqueous solution containing the test compound to the well of step b), and d) A step of determining the amount of the test compound in the aqueous solution after partition equilibrium has been reached. Methods that include... [Invention 1002] The method of the present invention 1001, wherein the nonpolar solvent is octanol. [Invention 1003] The method of the present invention 1001 or 1002, wherein the lipophilic film is selected from the group consisting of polyvinylidene fluoride (PVDF), polytetrafluoroethylene (PTFE), cyclic olefin copolymer (COC), polypropylene (PP), or polycarbonate (PC), and is preferably a PVDF film. [Invention 1004] The method according to any one of the present invention 1001 to 1003, wherein the multiwell plate is a 96-well plate, preferably a multiscreen hydrophobic immobilon P PVDF membrane plate welded to a liquid-tight foil, preferably a heat-sealing foil, at the bottom. [Invention 1005] A method of any one of the present invention 1001 to 1004, comprising an additional step e) for calculating the lipophilicity value logD of the test compound. [Invention 1006] A multiwell plate for use in a method for determining the lipophilicity of a test compound, wherein each well contains a lipophilic film at the bottom of the well, and the bottom of the multiwell plate contains a liquid-tight barrier. [Invention 1007] A multiwell plate for use in a method for determining the lipophilicity of a test compound according to the present invention 1006, wherein the lipophilic film is selected from the group consisting of polyvinylidene fluoride (PVDF), polytetrafluoroethylene (PTFE), cyclic olefin copolymer (COC), polypropylene (PP), or polycarbonate (PC). [Invention 1008] A multiwell plate for use in a method for determining the lipophilicity of a test compound according to the present invention 1006 or 1007, wherein the liquid-tight barrier is a liquid-tight foil, preferably a heat-sealing foil. [Invention 1009] A multiwell plate for use in a method for determining the lipophilicity of any of the test compounds according to invention 1006 to 1008, wherein the lipophilic film is a PVDF film. [Invention 1010] A multiwell plate for use in a method for determining the lipophilicity of any of the test compounds according to invention 1006 to 1009, wherein the multiwell plate is a 96-well plate. [Invention 1011] A multiwell plate for use in a method for determining the lipophilicity of any of the test compounds of the present invention 1006 to 1010, wherein the multiwell plate is a multiscreen hydrophobic immobilon P PVDF membrane plate welded to a liquid-tight foil at the bottom. [Invention 1012] A multiwell plate for use in a method for determining the lipophilicity of a test compound according to any of the methods 1006 to 1011 of the present invention, wherein the method for determining the lipophilicity of a compound is any of the methods 1001 to 1005 of the present invention. [Invention 1013] A multi-screen hydrophobic immobilon P PVDF membrane plate, wherein the bottom of the plate is welded with a liquid-tight foil, preferably a heat-sealing foil. [Modes for carrying out the invention]

[0021] Detailed description of the invention The test compound may be any chemical or biological compound. The test compound may be, for example, an organic compound, a protein, a peptide, or a nucleic acid. Organic compounds may also include organic-inorganic molecules. As used herein, the term organic-inorganic molecule refers to an organic molecule in which at least one inorganic atom is bonded to a carbon atom. The inorganic atom may be a metal atom, for example, silicon (Si) or germanium (organometallic, i.e., Si or Ge bioisoester of an organic molecule).

[0022] The test compound may be a solid or a liquid. The test compound is dissolved in an aqueous solution. The test compound may be a lipophilic or hydrophilic compound.

[0023] As used herein, the term “multiwell plate” refers to a plate having multiple wells used as small test tubes. Multiwell plates are commercially available as 24-well, 48-well, 96-well, and 384-well plates. Multiwell plates can be manufactured from a variety of materials, such as polystyrene, polypropylene, and acrylonitrile butadiene styrene (ABS).

[0024] The liquid-tight barrier is preferably a liquid-tight foil, preferably a heat-sealing foil. When heat-sealing foil, the sealant is first placed in place on the plate. Heat is applied uniformly for several seconds, resulting in the sealant bonding to the plate and achieving a complete seal. Heat-sealing foil is commercially available, for example, from Thermo Fisher Scientific.

[0025] As used herein, the term “nonpolar solvent” refers to a hydrophobic solvent. Nonpolar solvents are immiscible or only slightly immiscible with polar solvents, such as water. Lipophilic compounds tend to be more soluble in nonpolar solvents than in polar solvents. The dielectric constant of nonpolar solvents is usually lower than that of water. Examples of hydrophobic solvents are organic solvents, such as octanol or aliphatic hydrocarbons (dodecane, hexadecane, or halogenated hydrocarbons).

[0026] The aqueous solution may be a hydrophilic buffer solution consisting of, for example, a buffer salt in water having high buffering capacity within the desired pH range (i.e., an aqueous solution of phosphate or tapso salt buffered at pH 7.4). The desired pH may be in the range of pH 0 to 14, preferably about 7.4.

[0027] As used herein, the term “partition equilibrium” refers to the equilibrium of the partition between an aqueous solution containing the test compound and a nonpolar solvent used to impregnate a lipophilic film. Preferably, the partition equilibrium is achieved between 0.1 and 24 hours, and more preferably within 2 hours.

[0028] As used herein, the term “lipophilic film” refers to a film for nonpolar solvents. Such films may be formed by meshing out the lipophilic material or as a layer having pores. Preferably, the pore size or mesh size is in the range of 0.01 to 100 μm. Lipophilic film materials include, but are not limited to, polyvinylidene fluoride (PVDF), polytetrafluoroethylene (PTFE), cyclic olefin copolymer (COC), polypropylene (PP), or polycarbonate (PC).

[0029] A lipophilic film, preferably a PVDF film, can be impregnated by coating the film with a nonpolar solvent, thereby allowing the film to completely absorb the solvent. The solvent can be applied, i.e., by a dispenser, at a rate of 0.1 μl–50 μl / cm² on the film surface. 2 Further methods, such as robotic liquid handling systems, that enable the dispensing of organic modifiers are known in the art.

[0030] The amount of the test compound in an aqueous solution can be determined by methods consisting of, but not limited to, UV and / or mass spectrometry, capillary electrophoresis (CE), and high-pressure liquid chromatography (HPLC).

[0031] Lipophilicity test compounds may include, for example, polycyclic aromatic or aliphatic hydrocarbons, fat-soluble vitamins, hydrophobic drugs such as antifungal agents, halogen-containing aromatic or aliphatic hydrocarbons, and nitrogen and oxygen-containing aromatic or aliphatic hydrocarbons.

[0032] Nonpolar solvents are immiscible or only slightly immiscible with aqueous solutions containing the test compound. A preferred nonpolar solvent is octanol (octan-1-ol). The preferred aqueous solution may be water or a buffer solution.

[0033] The test compound may be a solid or a liquid. The test compound may be soluble in a suitable solvent, such as DMSO (dimethyl sulfoxide). A suitable solvent for hydrophilic compounds is preferably a polar solvent. A suitable solvent for lipophilic compounds is preferably a nonpolar solvent.

[0034] The method of the present invention offers the following advantages compared to the method disclosed in European Patent No. 1705474: Separation of solvents A and B is not required to enable measurement of the concentration of the test compound in solvent B. The concentration of the test compound can be measured after partition equilibrium is reached by directly inserting a multiwell plate containing an aqueous solution of the test compound into a suitable apparatus. Furthermore, the incubation time to reach partition equilibrium is about 1.5 hours, compared to 12 hours in the method of European Patent No. 1705474. [Brief explanation of the drawing]

[0035] [Figure 1] Figure 1: Schemes of various types of wells used in the previous CAMDIS (see European Patent No. 1705474) and the novel CAMDIS filter bottom plate (FBP). In the previous CAMDIS, octanol (red) is poured into the DIFI tube that comes into contact with the aqueous solution. In the CAMDIS FBP, the filter is directly coated with octanol. [Figure 2] Figure 2 shows an exemplary 96-multiwell plate having a PVDF membrane (red) sealing the bottom wells. The bottom of such a plate is welded to foil to have liquid-tight wells. A preferred foil is a heat-sealing foil. The resulting liquid-tight 96-multiwell plate can be used in the lipophilic method of the present invention. [Figure 3] Figure 3 shows a detailed view of the wells of a multiwell plate with a sealed bottom. The PVDF film at the bottom of each well is used as a carrier for octanol (red). The aqueous sample solution covers the PVDF film (blue). The bottom of the plate is welded with heat-sealing foil. Same plate configuration as CAMDIS. [Figure 4]Figure 4: CAMDIS FBP assay plate format. LogD values ​​are measured in triple replicates under hydrophilic and lipophilic conditions. [Examples]

[0036] Carrier-mediated distribution system (CAMDIS) filter bottom plate The method of the present invention retains the principle used in the classical shaking flask method, such as the conventional CAMDIS (see Wagner et al.: Eur J Pharm Sci. 2015 Feb 20;68:68-77), namely the principle of analyzing the concentration ratio of a drug partitioned into two phases (an aqueous buffer and an organic solvent, typically octanol). The main difference between the CAMDIS method of the present invention and the CAMDIS method disclosed in European Patent No. 1705474 is the use of a filter bottom plate (e.g., Millipore multiscreen filter plate, MSIPN45) instead of the two plates previously used. The filter of this novel plate is made of the same material as DIFI tubes, namely hydrophobic PVDF. This hydrophobic material was used to prevent the octanol phase from mixing with the aqueous phase. A membrane can be coated with octanol and then covered with an aqueous solution without mixing the two phases.

[0037] Incubation time The main advantage of the present invention is that the use of the new plate results in a larger exchange surface between the two phases than in previous configurations. In fact, the DIFI tube used in the last CAMDIS version has a diameter of 2.3 mm, while the new filter bottom plate has a diameter of 6.6 mm. Therefore, because there is a larger surface area available for exchange between the two phases, the incubation time required to reach equilibrium of distribution is expected to be shorter with the CAMDIS filter bottom plate.

[0038] The UV absorption of three standard compounds covering a wide range of logD values ​​was measured using LC-MS / UV. All compounds were ordered directly from the Roche internal library as storage solutions (10 mM) and successfully passed quality control before use. Analysis was performed at various time points (various incubation times) and compared with logD values ​​determined by the CAMDIS method previously. The experiment is described in detail in attached section 5.1.3. Based on the results shown in Figure 3, an incubation time of 90 minutes with shaking at 1000 rpm was recommended to obtain the most accurate logD values ​​for both phase volume ratios. Further experiments should be conducted to measure the evaporation effect on compound concentration, particularly under hydrophilic conditions.

[0039] CAMDIS Filter Bottom Plate Protocol (CAMDIS FBP) Before the experiment, the bottom of the filter bottom plate was welded with heat-sealing foil to prevent leakage or evaporation of the octanol phase. As with the previous CAMDIS version, the octanol and phosphate buffer (25 mM, pH 7.4) were mutually saturated at room temperature. 14 μL of the drug was introduced into 1200 μL of aqueous buffer as DMSO stock (10 mM). The solution was filtered in the same manner as in the previous CAMDIS and a second dilution was performed to reach a final volume of 1400 μL. The filter bottom plate was coated with 4 μL of octanol using an automated liquid dispenser according to the plate layout shown in Figure 4. Aliquots of 50 or 200 μL of filtrate were transferred to the pre-coated plate. The reference Rb consisted of 150 μL of aqueous buffer in the uncoated wells. A 1:5 dilution called Ra was also performed as a reference for Rb. The plate was sealed and shaken at 1000 rpm, 21°C for 90 minutes. Equilibrium aqueous drug concentrations were analyzed by LC-MS / UV. Several injections were performed into Rb with four injection volumes (1, 2, 3, and 4 μL). These were used to create a calibration curve of peak area as a function of injection volume required for MS analysis. In fact, when using UV data, the Lambert-Beer law can be directly applied by substituting the UV peak area for concentration, as Equation 2 shows that absorption is directly proportional to the compound concentration, which does not apply to MS. Also, UV detection exhibits a wide range of linearity, which is independent of compound properties. In contrast, the MS method used had a limited range of linearity with respect to its readout, strongly dependent on compound properties. Therefore, when using MS data, calibration of peak area versus concentration was necessary for each compound. Calibration was performed using four injections of Rb and the Ra criterion to fit a quadratic polynomial function. This equation allows the injection volume to be determined as a function of MS peak area (proportional to the amount of drug), and then the injection volume is used instead of the amount of substance to calculate logD as follows. TIFF0007901960000001.tif13128

[0040] Assay validation To validate the novel experimental setup of the CAMDIS filter bottom plate, the same drug set used with the latest version of CAMDIS (Wagner et al., Eur J Pharm Sci, 68:68-77, 2015) was used (see Table 1). The set consisted of 52 drugs with known logD values ​​determined by the shaking flask method or miniature shaking flask method at pH 7.4. Literature mean values ​​were obtained and compared to the mean logD value obtained from the new CAMDIS setup for each compound. The standard deviation of the entire literature was also calculated as an additional quality parameter, and was significant for some compounds.

[0041] Table 1 shows the measured compounds along with their literature, logD values, and standard deviations from the CAMDIS filter bottom plate. Dexamethasone was used as a standard, and the volume of octanol was adjusted to compensate for errors in the automated liquid dispenser. The same corrected volume was then applied to all compounds. Due to insufficient data quality, it was not possible to calculate the LogD values ​​for three compounds (cimetidine, disopyramide, and erythromycin). Previous CAMDIS methods could not measure their logD, and the literature standard deviations for these compounds were very high.

[0042] conclusion The results in this section demonstrate that the novel CAMDIS filter-bottom plate method is a promising new method for measuring logD values. Compared to the previous CAMDIS method, handling is simplified by using only one filter-bottom plate instead of a sandwich plate. The novel CAMDIS filter-bottom plate method enables the determination of logD values ​​eight times faster than the CAMDIS pooling method and yields values ​​that closely match literature-based shaking flask values ​​and previous CAMDIS values. This method makes it possible to measure a wide range of logD values ​​from -0.2 to 4, depending on the readout of each compound, which is a typical range for molecules within the drug discovery scope. Compared to the shaking flask method, as with the previous CAMDIS version, this method reduces the time required to obtain logD values ​​by eliminating the phase separation process. In fact, with CAMDIS, in contrast to the shaking flask method, there is no need to separate the two phases, octanol and aqueous buffer, to determine the drug concentration. Compared to the previous CAMDIS pooling version, this method is even faster due to the increased surface area available for compound exchange between the two phases, which reduces the incubation time from 12 hours to 90 minutes.

[0043] (Table 1) Comparison of CAMDIS FBP logD (pH 7.4) with the logD value of a shaking flask (pH 7.4) in the literature. TIFF0007901960000002.tif228170TIFF0007901960000003.tif156170 * If only two logD values ​​are available, the standard deviation is not calculated. [1] Wagner, B. et al.: Carrier Mediated Distribution System (CAMDIS): A new approach for the measurement of octanol / water distribution coefficients, European Journal of Pharmaceutical Sciences 68 (2015) 68-77”

Claims

1. A method for determining the lipophilicity of a test compound, a) A step of providing a multiwell plate, wherein the wells include a lipophilic film at the bottom, and the bottom of the multiwell plate includes a liquid-tight barrier for creating a liquid-tight bottom of the multiwell plate, b) A step of adding a nonpolar solvent to the lipophilic film in the well of step a), c) the step of adding an aqueous solution containing the test compound to the well in step b), and d) A step of determining the amount of the test compound in the aqueous solution after partition equilibrium has been reached. Methods that include...

2. The method according to claim 1, wherein the nonpolar solvent is octanol.

3. The method according to claim 1 or 2, wherein the lipophilic film is selected from the group consisting of polyvinylidene fluoride (PVDF), polytetrafluoroethylene (PTFE), cyclic olefin copolymer (COC), polypropylene (PP), or polycarbonate (PC), and is preferably a PVDF film.

4. The method according to any one of claims 1 to 3, wherein the multiwell plate is a 96-well plate, preferably a multiscreen hydrophobic Imobilon® P PVDF film plate welded to a liquid-tight foil and preferably a heat-sealing foil at the bottom.

5. The method according to any one of claims 1 to 4, further comprising the additional step e) of calculating the lipophilicity value logD of the test compound.

6. A multiwell plate for use in a method for determining the lipophilicity of a test compound, wherein each well contains a lipophilic film at the bottom of the well, and the bottom of the multiwell plate contains a liquid-tight barrier.

7. A multiwell plate for use in a method for determining the lipophilicity of a test compound according to claim 6, wherein the lipophilic film is selected from the group consisting of polyvinylidene fluoride (PVDF), polytetrafluoroethylene (PTFE), cyclic olefin copolymer (COC), polypropylene (PP), or polycarbonate (PC).

8. A multiwell plate for use in a method for determining the lipophilicity of a test compound according to claim 6 or 7, wherein the liquid-tight barrier is a liquid-tight foil, preferably a heat-sealing foil.

9. A multiwell plate for use in a method for determining the lipophilicity of a test compound according to any one of claims 6 to 8, wherein the lipophilic film is a PVDF film.

10. A multiwell plate for use in a method for determining the lipophilicity of a test compound according to any one of claims 6 to 9, wherein the multiwell plate is a 96-well plate.

11. A multiwell plate for use in a method for determining the lipophilicity of a test compound according to any one of claims 6 to 10, wherein the multiwell plate is a multiscreen hydrophobic Imobilon® P PVDF film plate welded to a liquid-tight foil at the bottom.

12. A multiwell plate for use in a method for determining the lipophilicity of a test compound according to any one of claims 6 to 11, wherein the method for determining the lipophilicity of a compound is the method according to any one of claims 1 to 5.

13. A multiscreen hydrophobic Immobilon® P PVDF membrane plate, wherein the bottom of the plate is welded with a liquid-tight foil, preferably a heat-sealing foil.

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