Compositions, methods and kits for stabilizing coelenterazine and analogs and derivatives thereof

Abstract

The present application relates to compositions, methods and kits for stabilizing coelenterazine and analogs and derivatives thereof for improving the solubility and reconstitution efficiency of coelenterazine and analogs and derivatives thereof.

Description

[0001] This application is a divisional application of a patent application with application number 201980073774.8, application date October 3, 2019, and invention name “Compositions and methods for stabilizing coelenterazine and its analogs and derivatives”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to and the benefit of U.S. Provisional Patent Application No. 62 / 740,622, filed on October 3, 2018, and U.S. Provisional Patent Application No. 62 / 805,517, filed on February 14, 2019, each of which is incorporated herein by reference in its entirety and for all purposes. Field of the Invention

[0004] The present invention relates to the field of biochemistry. Specifically, provided herein are compositions, methods and kits for stabilizing coelenterazine and its analogs and derivatives and for improving the solubility and reconstitution efficiency of coelenterazine and its analogs and derivatives. Background Art

[0005] Luminescence is used as a measure of reporter activity in bioassays. Reporter molecules then link luminescence measurements with target biological processes such as transcription (gene expression), translation (protein expression), protein-protein interactions, thereby allowing quantitative measurement of the changes that occur in biological processes. Reporter molecules are typically luminescent enzymes (e.g., firefly luciferase, sea renilla luciferase, thorn shrimp luciferase, etc.), which, when provided with their luminescent substrates, cause the generation of light (i.e., luminescence). Summary of the invention

[0006] Luminescent substrates such as coelenterazine and its analogs and derivatives decompose during storage (e.g., storage in organic solvents, storage at higher temperatures, storage at inappropriate pH, etc.), resulting in loss of substrate before addition or use in bioassays. Such decomposition may be the result of the instability of the luminescent substrate in a temperature-dependent manner over time in solution. This decomposition results in waste of luminescent substrates and reduced sensitivity and reproducibility of luminescent measurements of bioassays derived from decomposed luminescent substrates. The products of this decomposition also inhibit the luminescent reaction. In addition, some coelenterazines have low solubility in different assay buffers or in directly entered test samples, or may exhibit inconsistent reconstruction in different assay buffers. Although coelenterazine can be dissolved in an organic solvent before dilution into an appropriate buffer solution, organic solutions of coelenterazine compounds may suffer from instability (thermal instability and light instability) during storage. However, although solid coelenterazine and coelenterazine analogs and derivatives (e.g., furimazine) are significantly more stable than their organic solutions, they exhibit extremely poor reconstitution speed and efficiency, dissolve inconsistently, and are difficult to use directly in assays and other methods, especially when non-organic solvents are required. These shortcomings have greatly limited the number and types of applications for which coelenterazine and its analogs and derivatives have been developed.

[0007] Therefore, new compositions and / or methods for stabilizing luminescent substrates, improving the solubility of luminescent substrates and / or increasing the reconstitution efficiency of luminescent substrates are needed. In particular, substrates with improved physical properties and / or solubility are beneficial for long-term storage (e.g., ≥12 months at room temperature), compatibility with one or more assay formats, robustness, and user-friendliness.

[0008] Provided herein are compositions and methods for stabilizing and improving the solubility and / or reconstitution efficiency of luminescent substrates such as coelenterazine or its analogs or derivatives. Characterization of the chemical integrity and / or reconstitution efficiency of the substrates in different solid compositions, formulations, and formats is performed using HPLC, absorbance, and mass spectrometry. Additional functional characterization of the substrate under assay relevant conditions was performed by monitoring bioluminescence in the presence of enzyme via relative light units (RLU).

[0009] Provided herein are compositions comprising a compound selected from coelenterazine and its analogs or derivatives and a polymer. In some embodiments, the compound is selected from coelenterazine, coelenterazine-h, coelenterazine-hh, furomazine, JRW-0238, JRW-1743, and JRW-1744. In some embodiments, the compound is furomazine. In some embodiments, the compound is JRW-0238. In some embodiments, the compound is JRW-1743. In some embodiments, the compound is JRW-1744.

[0010] In some embodiments, the polymer is a naturally occurring biopolymer. In some embodiments, the naturally occurring biopolymer is selected from pullulan, trehalose, maltose, cellulose, dextran and any combination thereof. In some embodiments, the naturally occurring biopolymer is pullulan. In some embodiments, the polymer is a cyclic sugar polymer or a derivative thereof. In some embodiments, the polymer is hydroxypropyl beta-cyclodextrin. In some embodiments, the polymer is a synthetic polymer. In some embodiments, the synthetic polymer is selected from polystyrene, poly (meth) acrylate and any combination thereof. In some embodiments, the synthetic polymer is a block copolymer comprising at least one poly (oxypropylene) block and at least one poly (oxyethylene) block. In some embodiments, the synthetic polymer is poloxamer.

[0011] In some embodiments, the composition further comprises a buffer, a surfactant, a reducing agent, a salt, a free radical scavenger, a chelating agent, a protein or any combination thereof. In some embodiments, the composition further comprises a buffer selected from phosphate buffer, tris(hydroxymethyl)methylglycine (tricine) and 2-(N-morpholino)ethanesulfonic acid. In some embodiments, the composition further comprises a surfactant selected from polysorbate 20, polysorbate 40 and polysorbate 80. In some embodiments, the composition comprises a reducing agent selected from thiourea and 6-aza-2-thiothymine. In some embodiments, the composition further comprises a salt selected from sodium chloride and sodium phosphate. In some embodiments, the composition further comprises a free radical scavenger selected from ascorbic acid and sodium ascorbate. In some embodiments, the composition further comprises a chelating agent, and the chelating agent is selected from citric acid and trans-1,2-diaminocyclohexane-tetraacetic acid. In some embodiments, the composition further comprises a protein of a polypeptide fraction of a highly purified dermal collagen selected from bovine serum albumin, gelatin and pig sources.

[0012] In some embodiments, the composition is in the form of a lyophilized powder or cake. In some embodiments, the composition is in the form of a stretchable film. In some embodiments, the composition is a solution.

[0013] Provided herein are compositions comprising: a compound selected from coelenterazine and its analogs or derivatives; and a surface selected from paper or fiber matrix, plastic, glass or metal. In some embodiments, the compound is selected from coelenterazine, coelenterazine-h, coelenterazine-hh, furomazine, JRW-0238, JRW-1743 and JRW-1744. In some embodiments, the compound is furomazine. In some embodiments, the compound is JRW-0238. In some embodiments, the compound is JRW-1743. In some embodiments, the compound is JRW-1744. In some embodiments, the composition further comprises a polymer. In some embodiments, the polymer is a naturally occurring biopolymer. In some embodiments, the naturally occurring biopolymer is selected from pullulan, trehalose, maltose, cellulose, dextran and any combination thereof. In some embodiments, the naturally occurring biopolymer is pullulan. In some embodiments, the polymer is a cyclic sugar polymer or a derivative thereof. In some embodiments, the polymer is hydroxypropyl beta-cyclodextrin. In some embodiments, the polymer is a synthetic polymer. In some embodiments, the synthetic polymer is selected from polystyrene, poly (meth) acrylate and any combination thereof. In some embodiments, the synthetic polymer is a block copolymer comprising at least one poly (oxypropylene) block and at least one poly (oxyethylene) block. In some embodiments, the synthetic polymer is a poloxamer.

[0014] In some embodiments, the composition further comprises a buffer, a surfactant, a reducing agent, a salt, a free radical scavenger, a protein or any combination thereof. In some embodiments, the composition further comprises a buffer selected from phosphate buffer, tris(hydroxymethyl)methylglycine and 2-(N-morpholino)ethanesulfonic acid. In some embodiments, the composition further comprises a surfactant selected from polysorbate 20, polysorbate 40 and polysorbate 80. In some embodiments, the composition comprises a reducing agent selected from thiourea and 6-aza-2-thiothymine. In some embodiments, the composition further comprises a salt selected from sodium chloride and sodium phosphate. In some embodiments, the composition further comprises a free radical scavenger selected from ascorbic acid and sodium ascorbate. In some embodiments, the composition further comprises a chelating agent, and the chelating agent is selected from citric acid and trans-1,2-diaminocyclohexane-tetraacetic acid. In some embodiments, the composition further comprises a protein selected from the polypeptide fraction of the highly purified dermal collagen of bovine serum albumin, gelatin and pig origin. In some embodiments, the surface is selected from cellulose paper, nitrocellulose paper, nylon paper, cotton paper, polyester paper, sodium carboxymethylcellulose, porous or polymeric membranes, high purity cotton fibers, high purity cotton of cotton / rayon blends, and glass microfibers.

[0015] Provided herein is a method of stabilizing a compound selected from coelenterazine and its analogs or derivatives, comprising contacting the coelenterazine compound or its analogs or derivatives with an effective amount of the polymer and / or the paper or fiber substrate to form a composition. In some embodiments, the compound is stabilized to prevent thermal decomposition, chemical decomposition, light-induced decomposition, or any combination thereof.

[0016] Provided herein is a method of improving the solubility of a compound selected from coelenterazine and its analogs or derivatives, comprising contacting the coelenterazine compound or its analogs or derivatives with an effective amount of the polymer and / or the paper or fibrous matrix to form a composition. In some embodiments, the solubility of the compound in an aqueous solution is improved compared to the compound that has not been contacted with the polymer and / or the paper or fibrous matrix.

[0017] Provided herein are methods of improving the reconstitution rate of a compound selected from coelenterazine and analogs or derivatives thereof, comprising contacting the coelenterazine compound or analogs or derivatives thereof with an effective amount of the polymer and / or the paper or fibrous substrate to form a composition, wherein the reconstitution rate of the compound is improved compared to the compound that has not been contacted with the polymer or the paper or fibrous substrate.

[0018] In some embodiments, the compound is selected from coelenterazine, coelenterazine-h, coelenterazine-hh, furomazine, JRW-0238, JRW-1743 and JRW-1744. In some embodiments, the compound is furomazine. In some embodiments, the compound is JRW-0238. In some embodiments, the compound is JRW-1743. In some embodiments, the compound is JRW-1744. In some embodiments, the polymer is a naturally occurring biopolymer. In some embodiments, the naturally occurring biopolymer is selected from pullulan, trehalose, maltose, cellulose, dextran and any combination thereof. In some embodiments, the naturally occurring biopolymer is pullulan. In some embodiments, the polymer is a cyclic sugar polymer or a derivative thereof. In some embodiments, the polymer is hydroxypropyl β-cyclodextrin. In some embodiments, the polymer is a synthetic polymer. In some embodiments, the synthetic polymer is selected from polystyrene, poly(meth)acrylate and any combination thereof. In some embodiments, the synthetic polymer is a block copolymer comprising at least one poly(oxypropylene) block and at least one poly(oxyethylene) block. In some embodiments, the synthetic polymer is a poloxamer.

[0019] In some embodiments, the composition further comprises a buffer, a surfactant, a reducing agent, a salt, a free radical scavenger, a protein or any combination thereof. In some embodiments, the composition further comprises a buffer selected from phosphate buffer, tris (hydroxymethyl) methylglycine and 2- (N- morpholino) ethanesulfonic acid. In some embodiments, the composition further comprises a surfactant selected from polysorbate 20, polysorbate 40 and polysorbate 80. In some embodiments, the composition comprises a reducing agent selected from thiourea and 6-aza-2-thiothymine. In some embodiments, the composition further comprises a salt selected from sodium chloride and sodium phosphate. In some embodiments, the composition further comprises a free radical scavenger selected from ascorbic acid and sodium ascorbate. In some embodiments, the composition further comprises a chelating agent, and the chelating agent is citric acid. In some embodiments, the composition further comprises a protein of a polypeptide fraction of a highly purified dermal collagen selected from bovine serum albumin, gelatin and pig sources. In some embodiments, the paper or fiber substrate is selected from cellulose paper, nitrocellulose paper, nylon paper, cotton paper, polyester paper, sodium carboxymethylcellulose, porous or polymeric membranes, high purity cotton fibers, high purity cotton of cotton / rayon blends, and glass microfibers.

[0020] In some embodiments, the contacting step comprises: dissolving the compound in an organic solvent to form a first solution; mixing the first solution with the polymer and / or the paper or fiber substrate to form a mixture; and drying the mixture. In some embodiments, the mixing step comprises dissolving the polymer in a second solution and mixing the second solution with the first solution. In some embodiments, the mixing step comprises applying the first solution to the paper or fiber substrate. In some embodiments, the drying step comprises freeze drying. In some embodiments, the drying step comprises air drying. In some embodiments, the drying is performed at ambient temperature in an inert atmosphere. In some embodiments, the drying comprises vacuum drying. In some embodiments, the drying is performed at a temperature of about 30°C to about 70°C. In some embodiments, one or all solutions are deoxygenated.

[0021] In some embodiments, the method comprises contacting the compound with the polymer. In some embodiments, the method comprises contacting the polymer with the paper or fibrous substrate. In some embodiments, the method comprises contacting the polymer with the polymer and the paper or fibrous substrate.

[0022] Provided herein are kits comprising any of the compositions disclosed herein. In some embodiments, the composition is contained in one or more containers. In some embodiments, the composition is contained in a plurality of tubes. In some embodiments, the composition is in the form of a plurality of paper spots, each spot having a diameter of about 2 mm to about 5 mm. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figures 1A to 1C Shown is the results of the experiments described in Example 1 in (A) phosphate buffered saline (PBS), pH 7.0 and (B) Signal kinetics when testing the luminescent output of compositions according to the present disclosure in luciferase assay buffer. Figure 1C Images showing furomazine substrate samples in pullulan-based lyophilized cake and pullulan film-drop formulations.

[0024] FIG. 2A to FIG. 2C It is shown that when the luminescent output of the composition according to the present disclosure is tested in PBS, pH 7.0 as described in Example 1, upon addition of purified RLU values ​​at different time points after enzyme.

[0025] FIG. 3A to FIG. 3C It is shown that when as described in Example 1 When testing the luminescent output of the composition according to the present disclosure in luciferase assay buffer, the purified RLU values ​​at different time points after enzyme.

[0026] FIG. 4A to FIG. 4C Shown are absorbance values ​​in aqueous solution when the compositions according to the present disclosure were tested for absorbance in the range of 210-600 nm in PBS, pH 6.8 as described in Example 2.

[0027] Figure 5 Shown are images demonstrating the ability of a composition according to the present disclosure to be reconstituted into PBS, pH 7.0 as described in Example 3.

[0028] FIG. 6A to FIG. 6B Shown are the absorbance values ​​in the range of 210-600 nm for pullulan in PBS, pH 6.8 as described in Example 4.

[0029] FIG. 7A to FIG. 7B Representative HPLC traces of a 0% w / v pullulan-based lyophilized cake formulation containing furazolidone as described in Example 5 are shown at (A) 0 hours and (B) 5 hours after reconstitution.

[0030] FIG. 8A to FIG. 8B Representative HPLC traces of a 2.5% w / v pullulan-based lyophilized cake formulation containing furomazine as described in Example 5 are shown at (A) 0 hours and (B) 5 hours after reconstitution.

[0031] FIG. 9A to FIG. 9B Representative HPLC traces of a 15% w / v pullulan-based lyophilized cake formulation containing furazolidone as described in Example 5 are shown at (A) 0 hours and (B) 5 hours after reconstitution.

[0032] FIG. 10A to FIG. 10B Show as described in Example 5 Representative HPLC traces of luciferase assay substrate at (A) 0 hours and (B) 5 hours after reconstitution.

[0033] FIG. 11A to FIG. 11B Shown are HPLC trace analyses of furomazine samples formulated as described in Example 5 with or without pullulan, showing: (A) absorbance at 254 nm over time and (B) peak area over time.

[0034] Fig.12 Shown are data from HPLC traces of formulated furomazine samples with or without pullulan as described in Example 5, showing the generation of aminopyrazine degradation products over time.

[0035] FIG. 13A to FIG. 13CShown are: (A) a kinetic analysis of the RLU values ​​when the luminescent output of the composition is tested as described in Example 6; (B) the RLU values ​​at time zero when the luminescent output of the composition is tested as described in Example 6; and (C) a luminescent composition prepared as described in Example 6 by punching Image of paper spots produced by 903 Protein Preservation Card.

[0036] FIG. 14A to FIG. 14B Shown is a sample of furazolidone formulated as further described in Example 7 dried to Images of samples on 903 Protein Preservation Card and stored at (A) 4°C for 2 weeks or (B) 4°C or 25°C for 3 months.

[0037] FIG. 15A to FIG. 15D It is shown that the additives described in Example 8 have an effect on the drying to the punched Data on the effect of 903 protein preservation card generated paper spots on the assay performance of formulated furazolidone samples.

[0038] Fig.16 It is shown that the punched Data for the RLU output of a formulated furazolidone sample in a paper spot generated from a 903 Protein Storage Card.

[0039] 17A to 17C It is shown that as described in Example 9, by punching Data for RLU output of furazolidone samples in paper spots generated on 903 Protein Preservation Cards and tested after one day of storage at (A) 4°C, (B) 25°C, and (C) 37°C.

[0040] 18A to 18C It is shown that as described in Example 9, by punching Data for RLU output of formulated furazolidone samples in paper spots generated on 903 Protein Preservation Cards and tested after three days of storage at (A) 4°C, (B) 25°C, and (C) 37°C.

[0041] FIG. 19A to FIG. 19D The display is placed in a punched hole as described in Example 10 Data for formulated furazolidone samples in paper spots generated with 903 Protein Preservation Cards and pretreated with different protein buffers as well as purified Activity of the enzyme assay showing RLU output after storage of the spots at (A) 60°C and (B) 25°C, and % activity over time after storage of the spots at (C) 60°C and (D) 25°C.

[0042] FIG. 20A to FIG. 20D It was shown that the pores were punched out within a few days of storage at 25°C or 60°C as described in Example 10. Accelerated stability data of RLU output of formulated furazolidone samples in paper spots generated from 903 Protein Storage Cards and tested substrate activity, showing RLU output after storage of spots at (A) 60°C and (B) 25°C, and % activity over time after storage of spots at (C) 60°C and (D) 25°C.

[0043] FIG. 21A to FIG. 21C It is shown that as described in Example 11, by punching Data for RLU output of formulated furazolidone samples in paper spots generated from 903 Protein Preservation Cards and prepared using different drying methods.

[0044] FIG. 22A to FIG. 22D It is shown that as described in Example 11, by punching Data for RLU output and % activity over days for formulated furazolidone samples in paper spots generated from 903 Protein Preservation Cards and prepared using different drying methods.

[0045] FIG. 23A to FIG. 23B Shown are HPLC traces of representative pullulan-based lyophilized furomazine samples after storage at 60°C for A - 0 hours and B - 48 hours as described in Example 12.

[0046] FIG. 24A to FIG. 24B Shown as commercially available as described in Example 12 Luciferase assay substrate samples were stored at 60°C. Fig.24A , 0 hours, and Fig. 24B , HPLC trace after 48 hours).

[0047] FIG. 25A to FIG. 25D Analysis of HPLC data is shown showing the furazolidone samples formulated as described in Example 12 as raw area ( Fig.25A , at 25°C, and Fig.25B , at 60°C) and as % area ( Fig.25C , at 25°C, and Fig.25D , thermal stability at 60°C).

[0048] FIG. 26A to FIG. 26F Shown is the use of purified RLU data for formulated furazolidone samples tested. FIG. 26A to FIG. 26C Data are shown for samples stored at 60°C for various times prior to reconstitution and tested with 50 μM substrate (A), 10 μM substrate (B), and 0.1 μM substrate (C); FIG. 26D to FIG. 26F Data are shown for samples stored at 25°C for various times prior to reconstitution and tested using 50 μM substrate (D), 10 μM substrate (E), and 0.1 μM substrate (F), as described in Example 12.

[0049] FIG. 27A to FIG. 27F It was shown that when the furazolidone samples were stored and then purified The activity of the enzyme assay formulated furazolidone samples was the % substrate activity at time zero. FIG. 27A to FIG. 27C Data are shown for samples stored at 60°C for various times prior to reconstitution and tested with 50 μM substrate (A), 10 μM substrate (B), and 0.1 μM substrate (C); FIG. 27D to FIG. 27F Data are shown for samples stored at 25°C for various times prior to reconstitution and tested with 50 μM substrate (D), 10 μM substrate (E), and 0.1 μM substrate (F). Fig.12 As described in.

[0050] FIG. 28A to FIG. 28C It was shown that when purified RLU data for a formulated pullulan film coated 96-well microtiter plate containing furazolidone substrate for enzyme assay.

[0051] Fig.29 A representative example of a furazolidone-containing pullulan-based film format coating the bottom of a standard 96-well microtiter plate within a pullulan film matrix as described in Example 13 is shown.

[0052] FIG. 30A to FIG. 30C Shown in Enzyme reaction or for simultaneous placement The wells of the enzyme and furomazine preparations were briefly reconstituted with PBS and then attached to the bottom of a standard 96-well microtiter plate containing either furomazine alone or in addition to Data for representative examples of pullulan-based membranes of enzymes; Fig. 30A Display the raw RLU; Fig. 30B Show % activity; and Fig. 30C The % activity over 10 days is shown as described in Example 13.

[0053] FIG. 31A to FIG. 31F Shown are the normalized absorbances of the degradation products of furomazine prepared as a pullulan-based lyophilized cake after storage at 25°C (first bar) or 60°C (second bar) for each condition compared to the commercial furomazine product as described in Example 14.

[0054] FIG. 32A to FIG. 32F Shown are the relative area percentages of the degradation products of furomazine prepared as a pullulan-based lyophilized cake after storage at 25°C (first bar) or 60°C (second bar) for each condition as described in Example 14 compared to the commercially available furomazine product.

[0055] FIG. 33A to FIG. 33BData are shown for a representative example of a pullulan-based form containing furazolidone stored at room temperature for 6 months as described in Example 15.

[0056] FIG. 34A to FIG. 34C Representative examples of HPLC analysis of furazolidone samples dried on different types of paper matrices as described in Example 16 are shown.

[0057] FIG. 35A to FIG. 35B Representative examples of bioluminescent signals of furazolidone samples formulated with the reporter protein LgTrip on three different solid phase materials after reconstitution are shown.

[0058] FIG. 36A to FIG. 36C A representative example of HPLC analysis of furazolidone samples stored as a 1 :1 mixture with ascorbic acid on different types of paper matrices as described in Example 17 is shown.

[0059] FIG. 37A to FIG. 37C A representative example of HPLC analysis of a furazolidone sample stored on a paper matrix pretreated with 30% citric acid as described in Example 18 is shown.

[0060] FIG. 38A to FIG. 38C A representative example of HPLC analysis of a furazolidone sample stored on a paper substrate after pre-treatment of the substrate with water and drying under reduced pressure overnight as described in Example 19 is shown.

[0061] FIG. 39A to FIG. 39D A representative example of HPLC analysis of furazolidone stored as a 1:1 mixture with citric acid on different paper matrices as described in Example 20 is shown.

[0062] FIG. 40A to FIG. 40C Display by punching 903 Protein Preservation Cards produced and prepared using different drying methods max RLU (top) and % activity of formulated furizine samples in 903 paper spots (bottom) treated with furizine at a 1:1 molar ratio with citrate or ascorbate in the presence or absence of protein buffer as described in Example 21.

[0063] FIG. 41A to FIG. 41B It is shown that the samples were stored under different conditions as described in Example 22 and stored at 25°C for several days. RLU output and % activity data over several days for formulated furazolidone samples in paper spots generated from 903 Protein Storage Cards.

[0064] FIG. 42A to FIG. 42C It is shown that as described in Example 23, the punched hole is removed from the original hole. Data on RLU output and % signal recovery of formulated furazolidone samples in paper spots before and after paper spots generated from 903 Protein Storage Cards to determine if the substrate was released from the solid matrix support.

[0065] FIG. 43A to FIG. 43C It is shown that as described in Example 24, by punching Data on RLU output and % activity of various formulated furazolidone solutions at varying pH with different sugar or polymer components and in the presence or absence of ascorbate in paper spots generated from 903 Protein Preservation Cards.

[0066] FIG. 44A to FIG. 44C It is shown that as described in Example 25, by punching Data for RLU output and % activity of various formulated furazolidone solution components at a fixed pH = 7.0 in paper spots generated from 903 Protein Preservation Cards.

[0067] FIG. 45A to FIG. 45B It is shown that as described in Example 26, by punching Data for RLU output of various formulated furazolidone solutions in paper spots generated on 903 Protein Storage Cards and sampled over several days of storage at 25°C.

[0068] FIG. 46A to FIG. 46B It is shown that as described in Example 27, by punching Data for RLU output of various formulated furazolidone solutions containing Prionex, ascorbate and / or ATT in paper spots generated on 903 Protein Preservation Cards and sampled within several days of storage at 25°C.

[0069] FIG. 47A to FIG. 47B Shown are data demonstrating the RLU output of a furazolidone formulation as described in Example 28 that has been lyophilized directly into a 96-well microtiter plate.

[0070] Fig.48 A prophetic diagram showing components of an exemplary layered assay format in which the furomazine formulation is placed in one layer of a multilayer device as described in Example 29.

[0071] Fig.49 It is shown that as described in Example 30, sodium ascorbate is contained at 37°C. Data for RLU output of substrate (Promega Cat. No. N113) preparation.

[0072] Fig.50 It is shown that the lyophilized Data for RLU output of substrate (Promega Cat. No. N113) preparation.

[0073] FIG. 51A to FIG. 51B It is shown that the buffer additives described in Example 32, either alone or in combination, Data for RLU output of substrate (Promega Cat. No. N113) preparation.

[0074] Fig.52 It is shown that the mixed polymer containing pullulan and hydroxypropyl-β-cyclodextrin as described in Example 33 Data for RLU output of substrate (Promega Cat. No. N113) preparation.

[0075] Fig.53 Images showing representative examples of substrates formulated as described in Example 34.

[0076] FIG. 54A to FIG. 54C As shown in Example 34, Representative example of HPLC analysis of a sample of F-127 formulated JRW-0238.

[0077] FIG. 55A to FIG. 55C As shown in Example 35, Representative example of HPLC analysis of a sample of F-127 formulated furomazine.

[0078] Fig.56 As shown in Example 36, Representative images of solution samples of F-127 formulated JRW-0238.

[0079] Fig.57 As shown in Example 36, Representative example of HPLC analysis of a sample of F-127 formulated JRW-0238.

[0080] FIG. 58A to FIG. 58B As shown in Example 37, Representative images of samples of F-127 formulated JRW-0238.

[0081] FIG. 59A to FIG. 59B Representative images of samples of JRW-0238 formulated as described in Example 38 are shown.

[0082] FIG. 60A to FIG. 60B Shown are traces and images from mice injected intraperitoneally with reconstituted formulated JRW-0238 as described in Example 38.

[0083] FIG. 61A to FIG. 61B Shown are traces and images from mice injected subcutaneously with reconstituted formulated JRW-0238 as described in Example 38.

[0084] FIG. 62A to FIG. 62B An image of a furazolidinone lyophilized cake formulated in an amber glass vial following scale-up and manufacture as described in Example 39 is shown, along with the substrate at time point "day 0" relative to freshly prepared Activity of living cell substrates.

[0085] Fig.63 It was shown that when the composition according to the present disclosure was incubated at 25°C and 60°C and tested for luminescent output in PBS, pH 7.0 containing 0.01% BSA as described in Example 39, upon addition of purified RLU values ​​at various time points after enzyme addition.

[0086] Figures 64A to 64C . Images depicting JRW-1743 during various synthesis / formulation steps: (A) The vial on the left contains molten F-127, while the vial on the right contains JRW-1743 dissolved in EtOH; (B) JRW-1743 after removal of EtOH and reconstitution of the substrate / polymer mixture in 2.6 mL of pure water to a final concentration of 8.5 mM; (C) Representative example of JRW-1743 formulated after lyophilization: JRW-1743 in F-127 matrix (left) and the same material after reconstitution in pure water (center and right).

[0087] Fig.65 .In use Representative absorbance trace of JRW-1743 after F-127 formulation and reconstitution in nanopure water. The concentration of the substrate in solution was determined by absorbance. The average concentration of JRW-1743 in water was experimentally determined to be 8.5 mM. The calculated theoretical concentration of the dried formulated substrate was 8.7 mM. DETAILED DESCRIPTION

[0088] definition

[0089] Although any methods and materials similar to or equivalent to those described herein can be used in the practice or testing of the embodiments described herein, some preferred methods, compositions, devices and materials are described herein. However, before describing the materials and methods of the present invention, it should be understood that the present invention is not limited to the specific molecules, compositions, methodologies or schemes described herein, as these can vary according to routine experiments and optimizations. It should also be understood that the terms used in this specification are only used to describe the purpose of specific variations or embodiments, and are not intended to limit the scope of the embodiments described herein.

[0090] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by one of ordinary skill in the art to which the invention belongs. However, in the event of a conflict, the present specification (including definitions) shall prevail. Therefore, in the context of the embodiments described herein, the following definitions apply.

[0091] As used herein, the terms "spiny shrimp luciferase" and "spiny shrimp-derived luciferase" are used interchangeably and refer to luciferase secreted from deep-sea shrimp Oplophorus gracilirostris (e.g., SEQ ID NO: 1), including wild-type, variants, and mutants thereof. For example, suitable spiny shrimp luciferase variants are described in U.S. Patent Nos. 8,557,970 and 8,669,103, each of which is incorporated herein by reference in its entirety. Exemplary spiny shrimp-derived luciferases include, for example, the luciferase of SEQ ID NO: 2 (also referred to interchangeably herein as "NanoLuc," "Nluc," "Nluc luciferase," and "Nluc enzyme").

[0092] As used herein, the term "polymer" refers to an organic compound comprising two or more repeating units covalently bonded in a chain, wherein the chain may be straight or branched. Typically, a polymer is composed of one or more repeating units, which are linked together by covalent chemical bonds to form a linear backbone. The repeating groups may be the same or different. Therefore, an -AAAA-type structure is a polymer, also referred to as a homopolymer, in which A is a repeating unit. -ABAB- or -AAABAAAB-type structures are also polymers, and are sometimes referred to as copolymers, in which A and B are repeating units. As used herein, the term "polymer" explicitly includes chains of only two repeating units, such as disaccharides, and also includes chains of more repeating units, such as oligosaccharides and polysaccharides. The term "polymer" also includes non-glycosyl polymers (and oligomers of as few as two monomeric units), such as synthetic polymers. In some embodiments, polymers (e.g., polysaccharides) and oligomers (e.g., oligosaccharides) are limited to a defined length (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 750, 1000 or longer or ranges therebetween, e.g., 2-10, 5-25, 10-50, over 100, etc.).

[0093] As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a compound" refers to one or more polymers and equivalents thereof known to those skilled in the art, and so forth.

[0094] As used herein, the term "comprising" and its language variations indicate the presence of one or more of the listed features, elements, method steps, etc., without excluding the presence of one or more additional features, elements, method steps, etc. Conversely, the term "consisting of" and its language variations indicate the presence of one or more of the listed features, elements, method steps, etc., and excludes any one or more unlisted features, elements, method steps, etc., except for impurities that are usually accompanied. The phrase "consisting essentially of" indicates one or more of the listed features, elements, method steps, etc., and any one or more additional features, elements, method steps, etc. that do not substantially affect the basic properties of the composition, system or method. Many embodiments of the present invention are described using open "comprising" language. Such embodiments cover multiple closed "consisting of" and / or "consisting essentially of" embodiments, which can alternatively use such language to claim or describe.

[0095] Provided herein are compositions comprising compounds selected from coelenterazine and its analogs or derivatives and polymers and / or paper or fiber matrices or other surfaces such as plastics or glass. In some embodiments, the composition stabilizes the compound to prevent decomposition (e.g., thermal decomposition, chemical decomposition, light-induced decomposition, etc.). In some embodiments, compared with compositions not comprising polymers and / or paper or fiber matrices or other surfaces, the composition stabilizes the compound to prevent decomposition. In some embodiments, the composition reduces or inhibits the formation of one or more decomposition products by the compound (e.g., compared with compositions not comprising polymers or paper or fiber matrices or other surfaces). In some embodiments, the composition enhances the reconstitution efficiency of coelenterazine or its analogs or derivatives. In some embodiments, the composition enhances kinetic solubility (e.g., compared with compositions not comprising polymers and / or paper or fiber matrices or other surfaces).

[0096] The composition comprises a compound selected from coelenterazine and analogs or derivatives thereof. When incorporated into the composition, the compound can be protected to prevent decomposition (e.g., thermal decomposition, chemical decomposition, light-induced decomposition, etc.).

[0097] In some embodiments, the compound is coelenterazine, which has the following structure:

[0098]

[0099] In some embodiments, the compound is a coelenterazine analog or derivative. Exemplary coelenterazine analogs include coelenterazine-h (2-deoxy coelenterazine or 2,8-dibenzyl-6-(4-hydroxyphenyl)imidazo[1,2-a]pyrazin-3(7H)-one), coelenterazine-hh (dideoxy coelenterazine or 2,8-dibenzyl-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one), furazolidinone (8-benzyl-2-(furan-2-ylmethyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one), JRW-0238 (8-benzyl-2-( -1,2-a]pyrazin-3(7H)-one), JRW-1744 (6-(3-amino-2-fluorophenyl)-8-benzyl-2-(furan-2-ylmethyl)imidazo[1,2-α]pyrazin-3(7H)-one and JRW-1743 (6-(3-amino-2-fluorophenyl)-8-(2-fluorobenzyl)-2-(furan-2-ylmethyl)imidazo[1,2-α]pyrazin-3(7H)-one), which have the following structures:

[0100]

[0101] Additional exemplary coelenterazine analogs include coelenterazine-n, coelenterazine-f, coelenterazine-hcp, coelenterazine-cp, coelenterazine-c, coelenterazine-e, coelenterazine-fcp, coelenterazine-I, coelenterazine-icp, coelenterazine-v, 2-methyl coelenterazine, etc. In some embodiments, the compound may be a coelenterazine analog described in WO 2003 / 040100; U.S. Patent Publication 2008 / 0248511 (e.g., paragraph

[0086] ); U.S. Patent No. 8,669,103; WO 2012 / 061529; U.S. Patent Publication 2017 / 0233789; U.S. Patent No. 9,924,073; U.S. Patent Publication 2018 / 0030059; U.S. Patent No. 10,000,500; U.S. Patent Publication 2018 / 0155350; U.S. Provisional Patent Application No. 62 / 665,346; U.S. Application No. 16 / 399,410; U.S. Provisional Patent Application No. 62 / 721,708; U.S. Application No. 16 / 548,214; U.S. Patent Publication 2014 / 0227759; U.S. Patent No. 9,840,73 0; U.S. Patent No. 7,268,229; U.S. Patent No. 7,537,912; U.S. Patent No. 8,809,529; U.S. Patent No. 9,139,836; U.S. Patent No. 10,077,244; U.S. Patent No. 9,487,520; U.S. Patent No. 9,924,073; U.S. Patent No. 9,938,564; U.S. Patent No. 9,951,373; U.S. Patent No. 10,280,447; U.S. Patent No. 10,308,975; U.S. Patent No. 10,428,075; the disclosures of which are incorporated herein by reference in their entirety. In some embodiments, the coelenterazine analogs include prosubstrates, such as those described in U.S. Patent Publication 2008 / 0248511; U.S. Patent Publication 2012 / 0707849; U.S. Patent Publication 2014 / 0099654; U.S. Patent No. 9,927,430; U.S. Patent No. 10,316,070, which are incorporated herein by reference in their entirety. In some embodiments, the compound is furazolidone. In some embodiments, the compound is JRW-0238. In some embodiments, the compound is JRW-1743. In some embodiments, the compound is JRW-1744.

[0102] Coelenterazine and its analogs and derivatives may suffer from challenges associated with their reconstruction in buffer systems used in many assays such as bioluminescent assays and methods as described herein. For example, coelenterazine or its analogs or derivatives such as furimazine can be slowly and / or inconsistently dissolved in non-organic buffer solutions (e.g., due to the heterogeneous microcrystalline nature of the solid material). Although dissolving in an organic solvent before dilution with a buffer can provide faster and more consistent results, coelenterazine compounds may suffer from instability in organic solutions during storage, including both thermal instability and photoinstability. See, for example, U.S. Patent No. 9,676,997, which is incorporated herein by reference. In some embodiments, coelenterazine or its analogs or derivatives are incorporated into the compositions described herein to provide more reliable and consistent dissolution without such instability issues.

[0103] In some embodiments, the composition also includes a polymer. As further described herein, in certain embodiments, the presence of a polymer stabilizes the compound to prevent decomposition, and the presence of a polymer improves the solubility of the compound in water or aqueous solution. In some embodiments, by stabilizing coelenterazine or coelenterazine analogs or derivatives (for example, compared with coelenterazine or coelenterazine analogs in organic solvents), the water solubility of coelenterazine or coelenterazine analogs or derivatives is improved, and / or the reconstitution efficiency of coelenterazine or coelenterazine analogs in non-organic buffers is improved (for example, compared with coelenterazine or coelenterazine analogs or derivatives in the absence of polymers). Compositions and systems herein allow coelenterazine or coelenterazine analogs to be used in unprepared and / or organic phases that are not suitable for (for example, not temperature or light stable) instant, prepackaged and / or solid phase systems, methods and assays to be used coelenterazine or coelenterazine analogs or derivatives.

[0104] The polymer may be a naturally occurring biopolymer or a synthetic polymer. In some embodiments, the polymer is a naturally occurring biopolymer. Suitable naturally occurring biopolymers are carbohydrates, including disaccharides (e.g., trehalose, maltose, and sucrose), polysaccharides (e.g., pullulan, dextran, and cellulose), and non-sulfated glycosaminoglycans (e.g., hyaluronic acid). Mixtures of naturally occurring biopolymers may also be used. The polymer may be a derivative of a naturally occurring polymer, such as functionalized cellulose (e.g., hydroxypropyl cellulose, hydroxypropyl methylcellulose, etc.).

[0105] In some embodiments, the polymer is pullulan, which is a polysaccharide comprising repeating units of maltotriose. Maltotriose is a trisaccharide comprising three glucose units linked via α-1,4 glycosidic bonds. The maltotriose units within the pullulan polymer are linked to each other via α-1,6 glycosidic bonds. Pullulan is naturally produced from starch by the fungus Aureobasidum pullulans and typically has a molecular weight of about 4.5 x 10 4 About 6 x 10 5 Da in the mass range and is commercially available from a number of suppliers (CAS No. 9057-02-7).

[0106] In some embodiments, the polymer is a dextran, which is a complex branched polysaccharide including glucose repeating units. Straight chain linkages are usually formed by α-1,6 glycosidic bonds, while branches usually start from α-1,3 linkages. Naturally occurring dextran may have a molecular weight ranging from about 9 kDa to about 2000 kDa. Dextran may be synthesized from sucrose by certain bacteria including Leuconostoc mesenteroides and Streptococcus mutans. Commercially available dextran (CAS No. 9004-54-0) produced by Leuconostoc mesenteroides may be purchased from multiple suppliers including Sigma Aldrich, and may have a variety of molecular weight ranges ranging from about 1 kDa to about 670 kDa.

[0107] In some embodiments, the polymer is a cyclic sugar polymer, such as cyclodextrin. Typical cyclodextrins are α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin, which have six, seven, and eight pyranose glucopyranose units, respectively. The pyranose glucopyranose units may be functionalized. An exemplary cyclodextrin is hydroxypropyl-β-cyclodextrin.

[0108] In some embodiments, the polymer is a non-sulfated glycosaminoglycan. Glycosaminoglycan is a linear polysaccharide with a repeating disaccharide unit, each repeating unit including an amino sugar (N-acetylglucosamine or N-acetylgalactosamine) and uronic acid (glucuronic acid or iduronic acid) or galactose. Exemplary non-sulfated glycosaminoglycan is hyaluronic acid, wherein the repeated disaccharide includes N-acetylglucosamine and glucuronic acid connected via alternating β-(1→4) and β-(1→3) glycosidic bonds. The size of the polymer of hyaluronic acid can be in the range of 5kDa to 20000kDa.

[0109] In some embodiments, the polymer is cellulose, which is a polysaccharide of linearly repeating β-1,4 linked D-glucose units. Natural fibers may have up to 10,000 glucose units with a molecular weight greater than 1000 Da.

[0110] In some embodiments, the polymer is a synthetic polymer. Synthetic polymers can be homopolymers, copolymers, block copolymers (e.g., diblock copolymers, triblock copolymers, etc.). Non-limiting examples of suitable polymers include, but are not limited to, polyamines, polyethers, polyamides, polyesters, polyurethanes, polyureas, polycarbonates, polystyrenes, polyimides, polysulfones, polyurethanes, polyacetylene, polyethylene, polyethyleneimine, polyisocyanates, polyacrylates, polymethacrylates, polyacrylonitrile, and polyarylates. Non-limiting examples of specific polymers include poly(caprolactone) (PCL), ethylene vinyl acetate polymer (EVA), poly(lactic acid) (PLA), poly(L-lactic acid) (PLLA), poly(glycolic acid) (PGA), poly(lactic acid-co-glycolic acid) (PLGA), poly(L-lactic acid-co-glycolic acid) (PLLGA), poly(D,L-lactide) (PDLA), poly(L-lactide) (PLLA), poly(D,L-lactide-co-caprolactone), poly(D,L-lactide-co-caprolactone-co-glycolide), poly(D,L-lactide-co-PEO-co-D,L-lactide), poly(D,L-lactide-co-PPO-co-D,L-lactide).L-lactide), polyalkyl cyanoacrylates, polyurethanes, poly-L-lysine (PLL), hydroxypropyl methacrylate (HPMA), poly(ethylene glycol), poly-L-glutamic acid, poly(hydroxy acids), polyanhydrides, polyorthoesters, poly(ester amides), polyamides, poly(ester ethers), polycarbonates, polyalkylenes (e.g., polyethylene and polypropylene), polyalkylene glycols (e.g., poly(ethylene glycol)) (PEG) and poly(propylene glycol) (PPG)) and copolymers thereof (e.g., poloxamers), polyalkylene terephthalates (e.g., poly(ethylene terephthalate)), polyvinyl alcohol (PVA), polyvinyl ethers, polyvinyl esters (e.g., poly(ethylene acrylate), poly(methyl (meth)acrylate) (PMMA), poly(ethyl (meth)acrylate), poly(butyl (meth)acrylate), poly(isobutyl (meth)acrylate), poly(hexyl (meth)acrylate), poly(isodecyl (meth)acrylate), poly( ... ester), poly(1-vinylpyrrolidone-co-vinyl acetate) (PVP-VA), poly(4-vinylpyridine), poly(4-vinylpyridine-co-butyl methacrylate), poly(4-vinylpyridine-co-styrene), Poly[4-vinylpyridinium poly(hydrogen fluoride), methyl acrylate (p(MAA-co-MMA)) copolymer, poly(1-vinylpyrrolidone-co-2-dimethylaminoethyl methacrylate), poly(1-vinylpyrrolidone-co-styrene), poly(4-vinylpyridinium p-toluenesulfonate), hydroxypropyl acetate succinate (HPMC), hydroxypropyl methylcellulose acetate succinate (HPMCAS), poly(ethylene-alt-propylene) (PEP), 2-methacrylamidopyranose (MAG), dimethyl adipate (DMA), polyvinyl caprolactam-polyvinyl acetate and any mixtures and copolymers thereof. ,

[0111] In some embodiments, the synthetic polymer is a polyalkylene glycol. In some embodiments, the synthetic polymer is a polyalkylene glycol copolymer. In some embodiments, the synthetic polymer is a block copolymer comprising at least one poly(oxypropylene) block and at least one poly(oxyethylene) block, such as a poloxamer. A poloxamer is a nonionic triblock copolymer having a central poly(oxypropylene) block flanked by two poly(oxyethylene) blocks. Poloxamers are also known as poly(oxypropylene) copolymers including and Exemplary poloxamers include poloxamer 188 ( F-68) and Poloxamer 407 ( F-127).

[0112] In some embodiments, the compound (ie, coelenterazine or an analog or derivative thereof) and the polymer may be present in the composition in a weight ratio of about 0.001:1 to about 0.50:1 or about 0.0025:1 to about 0.40:1.

[0113] In some embodiments, the composition also includes paper or fiber matrix or other materials, and the composition is placed in or on paper or fiber matrix or other materials. In some embodiments, the material can allow coelenterazine (or its analogs or derivatives) to be used in a variety of environments such as field tests. In some embodiments, paper or fiber matrix can be made of high-quality cotton wool such as 100% pure cotton wool. In some embodiments, paper or fiber matrix can be ashless. In some embodiments, paper or fiber matrix can include up to 0.06% by weight of ash. In some embodiments, paper or fiber matrix can have a thickness of about 0.1 μM to about 1 mm. In some embodiments, paper or fiber matrix can have a pore size range of about 0.02 μM to about 12 μM. Paper or fiber matrix can have a variety of characteristics, including binding affinity, porosity, functionality (e.g., with a highly acidic or alkaline functional group) etc.

[0114] Exemplary paper or fiber substrates include, but are not limited to, Brand paper (e.g., W-903 paper, FTA paper, FTA elution paper, FTA DMPK paper, etc.), Ahlstrom paper (e.g., A-226 paper, etc.), M-TFN paper, FTA paper, FP705 paper, BodeDNA collection paper, nitrocellulose paper, nylon paper, cellulose paper and sample pad (e.g., EMD MilliporeCFSP20300M), Dacron paper, cotton paper, polyester paper (e.g., Ahlstrom polyester fiber grade 6613, Ahlstrom treated polyester fiber grade 6613H), sodium carboxymethyl cellulose, Noviplex TM Plasma preparation card, Ahlstrom Plasma separation cards, porous and polymer membranes, high purity cotton fibers (e.g., Ahlstrom grade 237), high purity cotton of cotton / rayon blends (e.g., Ahlstrom grade 1218), glass microfibers (e.g., Ahlstrom 934-AH, EMD Millipore GFDX103000), and combinations thereof.

[0115] Other potential materials that can replace paper or fiber matrix include synthetic and / or polymer films, homogeneous or heterogeneous solids, liquids or soluble tablet materials made of organic or inorganic materials (e.g., metals or ceramic materials). Exemplary additional materials include, for example, cellulose acetate, cellulose esters, cellulose ethers, polysulfones, polyethersulfones, polyacrylonitrile, polyethylene, polypropylene, polyvinylidene fluoride, polyethylene glycol, polyvinyl alcohol, starch, etc. The additional materials that can replace paper or fiber matrix include plastics or glass. In some embodiments, the material can be a cuvette, a slide, a plate or any other suitable surface made of plastics or glass. In some embodiments, the material can be a metal surface, wherein the metal is a single metal or a metal alloy, such as steel, copper, brass, bronze or silver.

[0116] In some embodiments, the composition comprises (i) coelenterazine or a coelenterazine derivative or analog, (ii) a suitable polymer, and (iii) a paper or fiber substrate or other surface such as glass, plastic, or metal.

[0117] In addition to the compound and the polymer and / or paper or fiber matrix or other surface, the composition may comprise additional components such as buffers, surfactants, reducing agents, salts, free radical scavengers, chelating agents, proteins, or any combination thereof.

[0118] In some embodiments, the composition comprises a buffer such as a phosphate buffer, a borate buffer, an acetate buffer or a citrate buffer, or other commonly used buffers such as bicine, tris(hydroxymethyl)methylglycine, tris(hydroxymethyl)aminomethane (tris), N-[tris(hydroxymethyl)methyl]-3-aminopropanesulfonic acid (TAPS), 3-[N-tris(hydroxymethyl)methylamino]-2-hydroxypropanesulfonic acid (TAPSO), 2-[4-(2-hydroxyethyl)piperazine-1-yl]ethanesulfonic acid (HEPES), N-[tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid (TES), piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES), 2-(N-morpholino)ethanesulfonic acid (MES), etc. In some embodiments, the composition comprises a phosphate buffer. In some embodiments, the composition comprises tris(hydroxymethyl)methylglycine. In some embodiments, the composition comprises 2-(N-morpholino)ethanesulfonic acid. The composition may also comprise any combination of buffers.

[0119] In some embodiments, the composition comprises a detergent or surfactant. In some embodiments, the detergent or surfactant is present in an amount of about 0.01 mol % to 5 mol % (e.g., 0.01%, 0.02%, 0.05%, 0.1%, 0.2%, 0.5%, 1%, 2%, 5%, or any range therebetween (e.g., 0.1% to 0.5%). Exemplary surfactants include nonionic surfactants, anionic surfactants, cationic surfactants, and zwitterionic surfactants. Examples of nonionic detergents include Brij 35, Triton TM Surfactants such as Triton TM X series (octylphenol ethoxylates, such as Triton TM X-100, Triton TM X-100R, Triton TM X-114, etc.), octyl glucoside, polyoxyethylene (9) lauryl ether, digitonin, octylphenyl polyethylene glycol (IGEPAL CA630), n-octyl-β-D-pyranoglucoside (betaOG), n-dodecyl-β-D-maltoside, 20 (polysorbate 20 or polyethylene glycol (20) sorbitan monolaurate), 40 (polysorbate 40 or polyethylene glycol (20) sorbitan monopalmitate), 80 (polysorbate 80 or polyethylene glycol (20) sorbitan monooleate), polydocanol, dodecyl β-D-maltoside (DDM), Nonidet P40 alternative, NP-40 nonylphenyl polyethylene glycol, C12E8 (octaethylene glycol n-dodecyl monoether), hexaethylene glycol mono-n-tetradecyl ether (C14E06), octyl-β-thiopyranoglucoside (octyl thioglucoside, OTG), F-68 (Poloxamer 188), F-127 (Poloxamer 407), saponin, Emulgen, polyethylene glycol trimethyl nonyl ether and polyoxyethylene 10 lauryl ether (C12E10). Examples of ionic detergents (anions or cations) include deoxycholate, sodium cholate, sodium dodecyl sulfate (SDS), N-lauroyl sarcosine and cetyl trimethyl ammonium bromide (CTAB). Examples of zwitterionic agents include Chaps, zwitterion 3-14 and 3-[(3-cholamidopropyl) dimethylammonium]-1-propanesulfonate. In some embodiments, the surfactant is polysorbate 20. The composition may also include any combination of surfactants.

[0120] In some embodiments, the composition may include a reducing agent such as dithiothreitol (DTT), 2-mercaptoethanol (BME), cysteamine, (2S)-2-amino-1,4-dimercaptobutane (DTBA), thiourea, 6-aza-2-thiothymine (ATT), etc. In some embodiments, the reducing agent is thiourea. In some embodiments, the reducing agent is ATT. The composition may also include any combination of reducing agents.

[0121] In some embodiments, the composition may include a salt such as sodium chloride, potassium chloride, magnesium chloride, sodium phosphate, etc. In some embodiments, the salt is sodium chloride. In some embodiments, the salt is sodium phosphate. The composition may also include any combination of salts.

[0122] In some embodiments, the composition may include a free radical scavenger, such as ascorbic acid, sodium ascorbate, etc. In some embodiments, the composition may include a metal chelator, such as citric acid, ethylenediaminetetraacetic acid, trans-1,2-diaminocyclohexane-tetraacetic acid, etc. In some embodiments, the composition includes ascorbic acid. In some embodiments, the composition includes sodium ascorbate. In some embodiments, the composition includes citric acid. In some embodiments, the composition includes trans-1,2-diaminocyclohexane-tetraacetic acid. The composition may include any combination of free radical scavengers and / or chelators.

[0123] In some embodiments, the composition may include a complete buffer composition such as Luciferase Assay Buffer (Promega Cat. No. N112), Live Cell Substrate (LCS) Dilution Buffer (Promega Catalog No. N206), etc. A complete buffer composition may comprise a combination of components disclosed herein, including the buffer itself and one or more of a salt, a metal chelator, a reducing agent, and a nonionic surfactant.

[0124] In some embodiments, the composition may include a protein. For example, the composition may include a carrier protein to prevent surface adsorption of a luminescent enzyme that may be added in a downstream assay. In some embodiments, the protein may be bovine serum albumin (BSA). In some embodiments, the protein may be a polypeptide fraction of highly purified dermal collagen of porcine origin (e.g., Prionex). In some embodiments, the protein may be gelatin. The composition may also include any combination of proteins.

[0125] In some embodiments, the composition may include a solvent. Some compositions are completely dried to remove any solvent, while other compositions may include a solvent or a certain amount of residual solvent. In some embodiments, the composition may include an organic solvent such as methanol, ethanol, isopropanol, ethylene glycol, propylene glycol, etc., or any combination thereof. For example, the composition may include a combination of ethanol and propylene glycol.

[0126] As described above, the composition may include any combination of the above components. For example, in some embodiments, the composition may include a protein, a buffer, and a reducing agent. In some embodiments, the composition may include a protein, a buffer, and a metal chelator.

[0127] The composition may be in the form of a lyophilized powder or cake. Such a composition may be prepared by freeze drying a mixture of the components of the composition, as further described below. The powdered product may be provided in a container such as a bottle, vial, snap tube, microtiter plate, on a paper or fiber matrix or other solid material support, a chip lab, etc. The powdered product may be contained in a plurality of snap tubes, each containing a predetermined amount of the composition, which is dissolved into an appropriate amount of solution and used directly for the target assay.

[0128] The composition may also be in the form of a hard but ductile material, such as a "drop" cast or film. Such a composition may be prepared by applying a solution containing the components of the composition to a surface and drying the composition, for example, by air drying, drying at ambient temperature, drying at elevated temperatures (e.g., at about 30°C to about 70°C, or about 30°C to about 40°C, such as at a temperature of about 30°C, about 35°C, about 40°C, about 45°C, about 50°C, about 55°C, about 60°C, about 65°C, or about 70°C), drying under an inert atmosphere, or vacuum drying. The drop cast or film may be provided in a container such as a bottle, vial, snap tube, microtiter plate, microtiter plate, on a paper or fiber matrix or other solid material support, in a chip lab, etc.

[0129] In some embodiments, the composition is in the form of a solution (e.g., an aqueous solution). When the composition is a solution, the composition may have a pH of about 5.5 to about 8.0, such as about 6.5 to about 7.5. In some embodiments, the composition has a pH of about 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 77.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0.

[0130] The composition may also be provided in other forms, such as a tablet or a capsule containing a dissolvable tablet or a capsule that can be dropped into a sample such as a buffer or a biological sample. The composition may also be included as a preformed film on the surface of a well such as a 96-well plate, so that the composition can be directly dissolved in an appropriate amount of solution and used directly in the target assay.

[0131] When the composition is provided on paper or fiber substrate, paper or fiber substrate can be in the form of a card with spots, and the spots can be punched so that spots can be reconstructed and directly used for target determination. Alternatively, paper or fiber substrate can be provided in the form of pre-punched spots (e.g., with a diameter of about 1-5 mm), and the spots can be reconstructed for target determination. Paper or fiber substrate with composition can be, for example, dried by air drying, dried at ambient temperature, at elevated temperatures (e.g., at about 30 ° C to about 70 ° C, or about 30 ° C to about 40 ° C, such as at a temperature of about 30 ° C, about 35 ° C, about 40 ° C, about 45 ° C, about 50 ° C, about 55 ° C, about 60 ° C, about 65 ° C or about 70 ° C), dried under an inert atmosphere or dried by vacuum drying.

[0132] The compositions of the present disclosure can be used in any manner in which luciferase substrates of coelenterazine and its analogs and derivatives have been used. For example, they can be used in a bioluminescent method using coelenterazine or its analogs or derivatives to detect one or more molecules in a sample, such as an enzyme, a cofactor of an enzyme reaction, an enzyme substrate, an enzyme inhibitor, an enzyme activator, or an OH radical, or one or more conditions, such as redox conditions. The sample may comprise an animal (e.g., a vertebrate), a plant, a fungus, a physiological fluid (e.g., blood, plasma, urine, mucous secretions), a cell, a cell lysate, a cell supernatant, or a purified fraction of a cell (e.g., a subcellular fraction). The presence, amount, spectral distribution, emission kinetics, or specific activity of such a molecule can be detected or quantified. The molecule can be detected or quantified in a solution including a multiphase solution (e.g., an emulsion or suspension) or on a solid support (e.g., a particle, a capillary, or an assay container).

[0133] In certain embodiments, the compositions can be used to quantify target molecules.In some embodiments, the compositions can be used as probes for specific biochemical activities such as apoptosis or drug metabolism.

[0134] In certain embodiments, the composition can be used for, for example, in vivo detection of luminescence in living cells or animals. In some embodiments, luciferase can be expressed in cells (as a reporter gene or other), and the cells are treated with the composition. Coelenterazine or its analog or derivative will infiltrate the cells in the culture, react with luciferase, and emit light. In some embodiments, the composition can be used for more robust, based on the reporter gene assay of living cell luciferase. In other embodiments, samples (including cells, tissues, animals, etc.) containing luciferase and the disclosed composition can use various microscopes and imaging techniques, such as in vivo imaging assays. In other embodiments, secretable luciferase can be expressed in cells as a part of a living cell reporter system.

[0135] Also provided herein is a method for stabilizing a compound selected from coelenterazine or an analog or derivative thereof, comprising contacting the compound with an effective amount of a polymer and / or a paper or fiber matrix to form a composition. The compound may be stabilized to prevent thermal decomposition, chemical decomposition, light-induced decomposition, or any combination thereof.

[0136] In some embodiments, the compositions herein stabilize the compound (i.e., coelenterazine or an analog or derivative thereof) against decomposition at temperatures (e.g., compared to a coelenterazine compound or an analog or derivative thereof that has not been contacted with a polymer and / or paper or fiber matrix) of about -80°C to about 80°C, about -75°C to about 80°C, about -70°C to about 80°C, about -65°C to about 80°C, about -60°C to about 80°C, about -55°C to about 80°C, about -50°C to about 80°C, about -45°C to about 80°C, about -40°C to about 80°C, about -35°C to about 80°C, about -30°C to about 80°C, about -25°C to about 80°C, about -20°C to about 80°C, about -15°C to about 80°C. ℃, about -10 ℃ to about 80 ℃, about -5 ℃ to about 80 ℃, about 0 ℃ to about 80 ℃, about -80 ℃ to about 75 ℃, about -80 ℃ to about 70 ℃, about -80 ℃ to about 65 ℃, about -80 ℃ to about 60 ℃, about -80 ℃ to about 55 ℃, about -80 ℃ to about 50 ℃, about -80 ℃ to about 45 ℃, about -80 ℃ to about 40 ℃, about -80 ℃ to about 35 ℃, about -80 ℃ to about 30 ℃, about -80 ℃ to about 25 ℃, about -20 ℃ to about 60 ℃, about -20 ℃ to about 55 ℃, about -20 ℃ to about 50 ℃, about -20 ℃ to about 45 ℃, about -20 ℃ to about 40 ℃, about -20 ℃ to about 35 ℃, about -20 ℃ to about 30 ℃, or about -20 ℃ to about 25 ℃.

[0137] In some embodiments, the compositions herein stabilize the compound (i.e., coelenterazine or an analog or derivative thereof) against decomposition at temperatures (e.g., compared to a coelenterazine compound or an analog or derivative thereof that has not been contacted with a polymer and / or paper or fiber) of about -80°C, -79°C, -78°C, -77°C, -76°C, -75°C, -74°C, -73°C, -72°C, -71°C, -70°C, -69°C, -68°C, -67°C, -66°C, -65°C, -64°C, -63°C, -62°C, -61°C, -6 0℃、-59℃、-58℃、-57℃、-56℃、-55℃、-54℃、-53℃、-52℃、-51℃、-50℃、-49℃、-48℃、-47℃、-46℃、-45℃、-44℃、-43℃、-42℃、-41℃、-40℃、-39℃、-38℃、-37℃、-36℃、-35℃、-34℃、-33℃、-32℃、-31℃、-30℃、-29℃、-28℃、-27℃、-26℃、-25℃、-24℃、-23℃、-2 2℃、-21℃、-20℃、-19℃、-18℃、-17℃、-16℃、-15℃、-14℃、-13℃、-12℃、-11℃、-10℃、-9℃、-8℃、-7℃、-6℃、-5℃、-4℃、-3℃、-2℃、-1℃、0℃、1℃、2℃、3℃、4℃、5℃、6℃、7℃、8℃、9℃、10℃、11℃、12℃、13℃、14℃、15℃、16℃、17℃、18℃、19℃、20℃、21℃、22℃、23℃、24℃、2 ℃, 50 ℃, 51 ℃, 52 ℃, 53 ℃, 54 ℃, 55 ℃, 56 ℃, 57 ℃, 58 ℃, 59 ℃, 60 ℃, 61 ℃, 62 ℃, 63 ℃, 64 ℃, 65 ℃, 66 ℃, 67 ℃, 68 ℃, 69 ℃, 70 ℃, 75 ℃ or 80 ℃. The composition can stabilize the compound to prevent decomposition at about -80 ℃, about -20 ℃, about 4 ℃, about 20 ℃, about 25 ℃ or about 37 ℃.

[0138] In some embodiments, the compositions herein stabilize the compound (i.e., coelenterazine or an analog or derivative thereof) against decomposition in the presence of light (e.g., compared to a coelenterazine compound or an analog or derivative thereof that has not been contacted with a polymer and / or paper or fibrous matrix). The compositions can increase the half-life of the compound in the presence of light compared to a composition that does not contain a polymer or paper or fibrous matrix. The composition can increase the half-life of the compound in the presence of light by about 1.1 times, 1.2 times, 1.3 times, 1.4 times, 1.5 times, 1.6 times, 1.7 times, 1.8 times, 1.9 times, 2.0 times, 2.1 times, 2.2 times, 2.3 times, 2.4 times, 2.5 times, 2.6 times, 2.7 times, 2.8 times, 2.9 times, 3.0 times, 3.1 times, 3.2 times, 3.3 times, 3.4 times, 3.5 times, 3.6 times, 3.7 times, 3.8 times, 3.9 times, 4.0 times, 4.1 times, 4.2 times, 4.3 times, 4.4 times, 4.5 times, 4.6 times, 4.7 times, 4.8 times, 4.9 times, or 5.0 times or more compared to a composition without a polymer or paper or fiber matrix.

[0139] In some embodiments, the compositions herein stabilize the compound (i.e., coelenterazine or an analog or derivative thereof) for at least about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 35 days, 40 days, 45 days, 50 days, 55 days, 60 days, 65 days, 70 days, 75 days, 80 days, 86 days, 87 days, 88 days, 89 days, 90 days, 91 days, 92 days, 93 days, 94 days, 95 days, 96 days, 97 days, 98 days, 99 days, 100 days, 101 days, 102 days, 103 days, 104 days, 105 days, 106 days, 107 days, 108 days, 109 days In some embodiments, the present invention provides a coelenterazine compound or an analog or derivative thereof that has been in contact with the polymer and / or paper or fiber substrate for 0, 85, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360 days, 1 year, 2 years, 3 years, 4 years, or 5 years to prevent decomposition (e.g., compared to a coelenterazine compound or an analog or derivative thereof that has not been in contact with the polymer and / or paper or fiber substrate).

[0140] In some embodiments, the composition increases the half-life of a compound (i.e., coelenterazine or an analog or derivative thereof) against degradation (e.g., compared to a coelenterazine compound or an analog or derivative thereof that has not been contacted with a polymer and / or paper or fiber matrix) by at least about 1.25-fold, 1.5-fold, 1.75-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 21-fold, 22-fold, 23-fold, 24-fold, or 25-fold compared to a composition that does not comprise a polymer or paper or fiber matrix.

[0141] Also provided herein is a method for improving the solubility of a compound selected from coelenterazine and its analogs or derivatives, comprising contacting the compound with an effective amount of a polymer and / or a paper or fiber matrix, wherein the solubility of the coelenterazine compound or its analog or derivative is improved compared to the compound not yet contacted with the polymer. The solubility of the compound in aqueous solution can be improved compared to the corresponding compound not yet contacted with the polymer and / or paper or fiber matrix. The solubility of the compound can be improved when a polymer is present after reconstitution of a lyophilized powder, a drop cast film or a "drop" or due to rehydration of a paper or fiber matrix or other solid support material on which or in which the compound has been placed.

[0142] The composition can increase the solubility of the compound (i.e., coelenterazine or its analog or derivative) in, for example, pure water or an aqueous solution, such as those that also contain a buffer, a salt, a protein, a reducing agent, a free radical scavenger, a surfactant, etc., or any combination of these components. The composition can increase the solubility of the compound in, for example, an aqueous buffer such as phosphate buffered saline (PBS) having a pH of about 6.5 to about 7.5 (e.g., a pH of about 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, or 7.5, or any range therebetween) or in a buffer such as Solubility in another suitable buffer of the luciferase assay buffer. The composition can increase the solubility of the compound in, for example, a biological or environmental fluid such as a biological sample from a subject, a culture medium (eg, tissue culture medium), and the like.

[0143] For example, the composition can increase the solubility of the compound in the presence of light by about 1.1 times, 1.2 times, 1.3 times, 1.4 times, 1.5 times, 1.6 times, 1.7 times, 1.8 times, 1.9 times, 2.0 times, 2.1 times, 2.2 times, 2.3 times, 2.4 times, 2.5 times, 2.6 times, 2.7 times, 2.8 times, 2.9 times, 3.0 times, 3.1 times, 3.2 times, 3.3 times, 3.4 times, 3.5 times, 3.6 times, 3.7 times, 3.8 times, 3.9 times, 4.0 times, 4.1 times, 4.2 times, 4.3 times, 4.4 times, 4.5 times, 4.6 times, 4.7 times, 4.8 times, 4.9 times, or 5.0 times or more compared to a composition without a polymer and / or paper or fiber matrix.

[0144] Also provided herein is a method of improving the reconstitution rate of a compound selected from coelenterazine and analogs or derivatives thereof, comprising contacting the compound with an effective amount of a polymer and / or a paper or fibrous matrix, wherein the reconstitution rate of the compound is improved compared to the compound that has not been contacted with the polymer or the paper or fibrous matrix.

[0145] The composition can increase the reconstitution rate of the compound in, for example, pure water or an aqueous solution, such as those that further comprise a buffer, a salt, a protein, a reducing agent, a surfactant, etc., or any combination of such components. The composition can increase the reconstitution rate of the compound in, for example, an aqueous buffer such as phosphate buffered saline (PBS) having a pH of about 6.5 to about 7.5 (e.g., a pH of about 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, or 7.5, or any range therebetween) or in an aqueous solution such as The reconstitution rate in another suitable buffer of the luciferase assay buffer. The composition can increase the solubility of the compound in, for example, a biological or environmental fluid such as a biological sample from a subject, a culture medium (eg, tissue culture medium), and the like.

[0146] For example, the composition can increase the reconstitution rate of a compound (e.g., a coelenterazine compound or an analog or derivative thereof) in the presence of light by about 1.1 times, 1.2 times, 1.3 times, 1.4 times, 1.5 times, 1.6 times, 1.7 times, 1.8 times, 1.9 times, 2.0 times, 2.1 times, 2.2 times, 2.3 times, 2.4 times, 2.5 times, 2.6 times, 2.7 times, 2.8 times, 2.9 times, 3.0 times, 3.1 times, 3.2 times, 3.3 times, 3.4 times, 3.5 times, 3.6 times, 3.7 times, 3.8 times, 3.9 times, 4.0 times, 4.1 times, 4.2 times, 4.3 times, 4.4 times, 4.5 times, 4.6 times, 4.7 times, 4.8 times, 4.9 times, or 5.0 times or more compared to a composition without a polymer.

[0147] The composition may have any combination of properties disclosed herein. For example, the composition may have increased solubility as described herein, improved reconstitution rate as described herein, increased stability as described herein, and / or increased half-life as disclosed herein. The composition may have one of the disclosed features or any combination of the disclosed features, and may also have other improved properties.

[0148] In embodiments of the methods described herein, the contacting step may include the steps of: dissolving the compound (i.e., coelenterazine or an analog or derivative thereof) in a first solvent to form a first solution; mixing the first solution with a polymer and / or a paper or fiber substrate to form a mixture; and drying the mixture. In some embodiments, the contacting step includes the steps of: dissolving the compound in a first solvent to form a first solution; dissolving the polymer in a second solvent to form a second solution; mixing the first solution and the second solution to form a mixture; and drying the mixture. In some embodiments, the contacting step includes the steps of: dissolving the compound in a solvent to form a first solution; applying the first solution to the paper or fiber substrate; and drying the paper or fiber substrate. In some embodiments, the contacting step includes the steps of: dissolving the compound in a first solvent to form a first solution; dissolving the polymer in a second solvent to form a second solution; combining the first solution and the second solution to form a third solution; applying the third solution to a paper or fiber substrate; and drying the paper or fiber substrate.

[0149] In some embodiments, the drying step includes lyophilization. In some embodiments, the drying step includes air drying. In some embodiments, the drying step includes drying under an inert atmosphere (e.g., under nitrogen or argon) at ambient temperature. In some embodiments, the drying step includes drying at an elevated temperature (e.g., 30 ° C). In some embodiments, the drying step includes vacuum drying. In some embodiments, one or all solutions used in the method can be deoxygenated. Deoxygenation can be achieved by degassing the solution under vacuum, by bubbling an inert gas (e.g., nitrogen or argon) through the solution, etc.

[0150] Compositions can be tested by using them as substrates for luciferase to generate luminescence and analyzing the luminescence from the composition after reconstitution. "Luminescence" refers to the light output of luciferase under appropriate conditions, for example, in the presence of a suitable substrate such as a coelenterazine analog. Light output can be measured as an instantaneous or near-instantaneous measure of light output at the start of a luminescent reaction (sometimes referred to as "T=0" luminescence or "flash"), which can be started when a coelenterazine substrate is added.

[0151] In various embodiments, the luminescent reaction is carried out in a solution. The solution may contain a lysate, such as a lysate from a cell in a prokaryotic or eukaryotic expression system. The solution may contain purified proteins, peptides, or small molecules labeled with luminescent enzyme components. In other embodiments, expression occurs in a cell-free system, or the luciferase protein is secreted into an extracellular medium, so that in the latter case, it is not necessary to produce a lysate. In some embodiments, the reaction is started by adding appropriate materials, such as compositions, buffers, etc. disclosed herein to a reaction chamber containing a luminescent protein (e.g., a hole, a test tube or a vial, a cuvette, etc., of a multi-well plate such as a 96-well plate). The reaction chamber may be located in a readout device, which may, for example, measure light output using a photometer, a photomultiplier tube, or a camera (e.g., a smartphone camera, a CCD camera, or any other handheld device that can record images). Light output or luminescence may also be measured over time, such as for a period of time of several seconds, minutes, hours, etc. in the same reaction chamber. Light output or luminescence may be reported as an average value over time, a half-life of signal decay, a sum of signals over a period of time, or a peak output. Luminescence can be measured in relative light units (RLU).In certain embodiments, a composition can be tested by using it as a substrate for spiny shrimp luciferase.

[0152] In still other embodiments, the luciferase and / or composition is introduced into a host and luminescence is measured for the host or a portion thereof, which may include a whole organism or a cell, tissue, explant, or extract thereof.

[0153] In other embodiments, the luminescent reaction is carried out on a solid support. The solid support can be, for example, a bead, a resin, a magnetic particle, a membrane, or a surface such as a vial, a microtiter plate, a box, a cuvette, a swab, etc. This reaction can then be placed in a readout device that can measure the light output from the specific solid support format.

[0154] In other embodiments, luminescent reaction is carried out in vivo to facilitate whole animal imaging. The medium for substrate injection into animals must be nontoxic and highly compatible with mammalian biology, which significantly limits the available selection. Pullulan and many other polymers described herein are nontoxic and even approved as food additives, which makes them particularly suitable as components of injectable solutions. In addition, the improved solubility and reconstruction of coelenterazine analogs such as furazolidone in simple buffers such as PBS are ideal for application to animals such as by intravenous injection, intraperitoneal injection, intracranial administration, etc. The composition components can be merged before injection, and excellent reconstruction makes the sample quickly homogenized, which is important for animal work where there is insoluble microcrystals that may be fatal. Once the substrate preparation is introduced into the animal body (such as intravenous or intraperitoneal injection), the sedated animal is placed in an imaging chamber and analyzed for the in vivo generation of bioluminescence.

[0155] In certain embodiments, the compositions disclosed herein are provided as part of a kit. The compositions may be contained in a single container. In some embodiments, the kit may also include one or more luciferases (in the form of polypeptides, polynucleotides, or both) and suitable reagents and instructions for enabling the user to perform assays such as those disclosed herein. The kit may also include one or more buffers, such as those disclosed herein. The kit may include instructions for storing the composition and / or a single container for accommodating the composition. The instructions included in the kit of the present disclosure may be adhered to the packaging material or may be included as a package insert. Although the instructions are typically written or printed materials, they are not limited to such. The present disclosure contemplates any medium capable of storing such instructions and communicating them to the end user. Such media include, but are not limited to, electronic storage media (e.g., disks, tapes, magnetic cassettes, chips), optical media (e.g., CD ROMs), etc. As used herein, the term "instructions" may include the address of a website (internet site) providing the instructions.

[0156] experiment

[0157] Experiments performed during development of the embodiments herein demonstrate the utility of the compositions and methods described herein. Unless otherwise indicated, pullulan was obtained from Sigma-Aldrich (CAS No. 9057-02-7).

[0158] The abbreviations used in the examples include the following: ATT is 6-aza-2-thiothymidine; EtOH is ethanol; Fz is furazolidone; HPLC is high performance liquid chromatography; NGB is Luciferase assay buffer (Promega Cat. No. N112); PBS is phosphate buffered saline; and TFA is trifluoroacetic acid.

[0159] Example 1

[0160] Furoxazine-Pullulan Composition

[0161] Sample preparation was as follows. For each of the following conditions, all substrates and additives were combined in aqueous solutions of pullulan at different percentages w / v at the concentrations listed. In each case, the substrate was added from an ethanol stock solution so that the total amount of ethanol (v / v) in the final solution containing the polymer did not exceed 10% v / v.

[0162] Condition 1: Solutions were prepared with 0%, 2.5%, 5%, or 10% (w / v) pullulan aqueous solution. A 30 mM stock solution of furomazine in ethanol was prepared. 4 μL of the furomazine stock solution was added to 46 μL of a solution containing pullulan, where the final concentration of furomazine was 2 mM. In all cases, the total concentration of ethanol in the final solution was <10% v / v. The samples were frozen and then lyophilized overnight to form a powdered product.

[0163] Condition 2: A solution of 15% (w / v) pullulan, 200 mM tricine, and 2 mM furazolidone in <10% v / v ethanol / water was prepared as described above. A series of 60 μL aliquots were pipetted onto parafilm and allowed to dry at 25° C. in the dark for at least 3 hours to form hard, malleable “drops”.

[0164] Condition 3: A solution of 15% (w / v) pullulan and 2 mM furazolidone in <10% v / v ethanol / water was prepared as described above. A series of 60 μL aliquots were pipetted onto parafilm and allowed to dry at 25°C in the dark for at least 3 hours.

[0165] Samples were tested by dissolving formulated furomazine into NGB or PBS, pH 7.0 by vortexing as appropriate. In each case, samples were diluted in 5 mL of buffer to a final working concentration of 10 μM furomazine.

[0166] The empirical results are as follows. Samples according to condition 1 with 2.5% (w / v) pullulan easily enter NGB and PBS, pH 7.0 solutions in less than a minute. Samples according to condition 1 at 5% (w / v) and 10% (w / v) completely dissolve in PBS, pH 7.0 within a few minutes. Samples according to condition 2 require further vortexing and require about 10-15 minutes to completely dissolve in PBS, pH 7.0. Samples according to condition 1 without pullulan require about 10 minutes to completely dissolve in PBS (determined empirically), pH 7.0.

[0167] After storing samples at 4°C for five weeks, dilute samples to 6 mL with 1x NGB for a 20 μM stock or 1x PBS, pH 7.0 for a 20 μM stock. (Nluc) luciferase was added at a final concentration of 1x (where the 2x stock solution was prepared from 1000x Enzyme, Promega #E499 stock solution was prepared in PBS or NGB). Control samples contained The substrate is Nluc in the assay buffer. Total luminescence was collected using a kinetic readout on an Expedition Multimode Microplate Reader – Promega Cat. No. GM3000) and the assay was performed on solid white non-binding surface (NBS) plates in a total assay volume of 100 μL. The kinetic trace of samples reconstituted in PBS is shown in Figure 1A and the kinetic traces of the samples reconstructed in NGB are shown in Figure 1B middle. Figure 1C Images showing lyophilized cake and membrane drop preparations. Figure 1A The data at a specified time point in FIG. 2A to FIG. 2C in the bar graph and from Figure 1B The data at a specified time point in FIG. 3A to FIG. 3C in the bar chart.

[0168] The results shown in Figures 1 to 3 demonstrate the increased solubility of the furomazine composition in neutral buffers without the need for organic solvents or special buffer conditions. The luminescence from the furomazine composition was more intense relative to the commercially available furomazine formulation.

[0169] Example 2

[0170] Absorbance of reconstituted furazolidone compositions

[0171] Bulk solid furazolidinone was diluted in ethanol to a final concentration of 10 mM (solution 1). Dried pullulan was dissolved in pure water to a final concentration of 0%, 2.5%, 5%, 10%, 15% w / v (solutions 2a, 2b, 2c, 2d and 2e, respectively). 45 μL of solution 2a-e was pipetted into a separate 1.5 mL snap tube vial. Then 5 μL of solution 1 was added to each vial and pipetted vigorously to mix, thereby forming solutions 3a-e, each of which contained furazolidinone at a final concentration of 1 mM (19.08 μg) in 50 μL solution. After mixing, the vials containing solution 3a-e were placed in dry ice and frozen for 1 hour. These frozen stock solutions were then lyophilized overnight to form a dry pullulan matrix containing furazolidinone.

[0172] Powder formulations of furomazine (19.08 μg) in pullulan matrix (0%-15% w / v) were diluted in 0.5 mL PBS buffer, pH 6.8, equilibrated at room temperature for 30 minutes, and the absorbance was read at 254 nm. The absorbance spectrum of dry furomazine (50 nmol) formulated with increasing amounts of pullulan after reconstitution in PBS buffer showed Figure 4A The formulation of furomazine with pullulan resulted in an increase in the absorbance of furomazine in aqueous solution. According to Beer's law, using the extinction coefficient of furomazine in methanol (21000 M -1 cm -1 ), the concentration of furomazine was determined from the absorbance measured at 254 nm. Bulk furomazine had an absorbance of 0.0571, corresponding to a calculated concentration of 0.0082 mM. Formulations of furomazine with 2.5%-5% w / v pullulan resulted in absorbances of 0.2204 and 0.2467, giving calculated concentrations of 0.032 mM and 0.035 mM, respectively. Furomazine formulated with 10-15% w / v pullulan resulted in absorbances of 0.3964 and 0.3836, giving calculated concentrations of 0.055 mM and 0.052 mM, respectively, in PBS. Figure 4A A summary of the absorbance data presented in Figure 4C middle, Figure 4C It was shown that when furazolidone was formulated with pullulan, the concentration of furazolidone (Fz) in solution increased compared to samples without pullulan.

[0173] Separately, a dry formulation of furomazine (95.4 μg) in a pullulan matrix (0%-15% w / v) prepared similarly to the above samples was diluted in 0.5 mL PBS buffer, pH 6.8, equilibrated for 30 minutes at room temperature, and the absorbance at 254 nm was read. After reconstitution in PBS buffer, the absorbance of furomazine (95 μg) formulated with increasing amounts of pullulan was shown at Figure 4B Solid furomazine formulated with increasing concentrations of pullulan matrix resulted in increased absorbance and, therefore, increased furomazine concentrations in the PBS buffer compared to conditions containing furomazine alone without pullulan.

[0174] Example 3

[0175] Reconstitution of stored samples

[0176] Solid furomazine was dissolved in ethanol and the dissolved solution was added to an aqueous solution of pullulan (0% or 15% w / v) to give a total concentration of 1 mM furomazine in 50 μL of solution containing <10% v / v ethanol. The samples were freeze dried or dried at ambient temperature. Figure 5 Images demonstrating the ability of these compositions to reconstitute into PBS, pH 7.0 are shown. A "drop" formulation with 15% w / v pullulan dried at ambient temperature went into solution after brief pipetting. A freeze-dried sample with 15% w / v pullulan dissolved immediately upon addition of PBS. The sample without pullulan did not completely dissolve in PBS even after vortexing for 15 minutes, demonstrating lower solubility in PBS.

[0177] Example 4

[0178] Absorbance of pullulan samples

[0179] The absorbance of pure 2.5% w / v pullulan and pure 10% w / v pullulan samples in PBS, pH 6.8 was tested. The absorption spectra in the range of 210-600 nm are shown in Fig. 6A (2.5%) and Figure 6B These spectra demonstrate that pullulan does not absorb in the same wavelength range as furomazine and does not artificially enhance the absorption signal in samples containing furomazine.

[0180] Example 5

[0181] HPLC Analysis of Furomazine Samples

[0182] Bulk solid furazolidinone was diluted in ethanol to a final concentration of 10 mM (solution 1). Dry pullulan was dissolved in pure water to a final concentration of 0%, 2.5%, 5%, 10% or 15% w / v (solutions 2a, 2b, 2c, 2d and 2e, respectively). 45 μL of solution 2a-2e was pipetted into a separate 1.5 mL snap tube vial. Then 5 μL of solution 1 was added to each vial and pipetted vigorously to mix, thereby forming solutions 3a-e, each of which contained furazolidinone at a final concentration of 1 mM. After mixing, the vials containing solutions 3a-e were placed in dry ice and frozen for 1 hour. These frozen stock solutions were then lyophilized overnight to form a dry pullulan matrix containing furazolidinone.

[0183] General method for all HPLC traces: The above furazolidone sample (containing 19.08 μg furazolidone) was diluted to 38.16 μg / mL with 0.5 mL PBS, pH 6.8 in a small snap-cap tube. 15 μL of the solution was injected neat into the HPLC (vial with insert) over 5 hours to evaluate stability and solubility over time. Instrument: Synergi Max-RP50x4.6 mm, 2.54u. Solvent: 0.1% TFA / acetonitrile in water. Commercially available furazolidone (5 mM, Promega catalog number N113) was diluted to 38.16 μg / mL in PBS and also compared.

[0184] The HPLC traces of the samples were obtained immediately after dilution with 0.5 mL PBS, pH 6.8 and 5 hours after dilution, and the traces are shown in Figure 7 (0% pullulan - (A) 0 hours, (B) 5 hours), Figure 8 (2.5% pullulan - (A) 0 hours, (B) 5 hours), Figure 9 (15% pullulan - (A) 0 hours, (A) 5 hours) and Figure 10 ( Luciferase assay substrate - (A) 0 hours, (B) 5 hours). (Traces were similarly obtained for 5% pullulan and 10% pullulan formulations and commercial furomazine samples, data not shown.) Peaks at retention times 5-10-5.13 minutes (major peaks in each spectrum) represent furomazine. Peaks at retention times 5.36-5.37 minutes (marked with asterisks) represent aminopyrazine, a known degradation product of furomazine (confirmed by spectra). Specific peaks and area percentages are summarized in Table 1.

[0185] Table 1.

[0186]

[0187]

[0188] Figure 11 and Fig.12 Shown are analyses of compiled and processed data from the HPLC traces shown in Figures 7 to 10 as well as traces obtained at additional time points via the same method.

[0189] Fig.11A Purity analysis of each sample as measured by absorbance at 254 nm is shown, with each trace normalized to time 0. All conditions prepared as dry formulations with pullulan showed high purity levels in aqueous solution with no significant loss of absorbance. Conditions lacking pullulan (0% condition and commercial furazolidone solution, Promega catalog number N113) showed significant loss of absorbance in approximately 6 hours due to chemical degradation.

[0190] Fig. 11B Shown are analyses of the peak areas of furazolidone samples (50 nmol) formulated in PBS with increasing amounts of pullulan (0% - 15% w / v). (These analyses are for Fig.11A The loss of purity for commercial furazolidone and the 0% condition in the bar graph in Figure 11a also corresponds to a decrease in peak area, indicating that the signal loss is not due to a change in solubility over time, but rather to Luciferase assay substrate and 0% pullulan conditions were degraded over the course of the experiment. Therefore, the presence of pullulan not only helps improve the aqueous solubility of furomazine, but also helps prevent its degradation in solution.

[0191] Fig.12 The results show the formation of aminopyrazine byproducts of furomazine in the samples described above. The data all indicate that the presence of pullulan helps prevent the formation of aminopyrazines in solution. Within 5.5 hours after reconstitution in PBS, the furomazine formulation containing pullulan showed minimal aminopyrazine formation. In contrast, bulk furomazine lacking pullulan (0% condition) and commercially available The luciferase assay substrate preparations all showed an increase of approximately 12% in aminopyrazine over the course of the experiment. Fig. 11B The purity changes shown in Luciferase assay for degradation of furazolidinone in substrate and 0% pullulan samples.

[0192] Example 6

[0193] Furoxolotl compositions on paper substrates

[0194] By using a standard 3.2mm handheld hole punch ( Brand) from Paper spots were produced by stamping circular "spots" with a diameter of 3.2 mm on 903 Protein Preservation Card. 200 μM and 2 μM furazolidone stock solutions were prepared in ethanol. 5 μL of these solutions were applied to each paper spot and dried under vacuum for 60 minutes. The spots were then stored at 4°C in the dark until testing.

[0195] At the time of testing, each spot was placed in a single well of a standard 96-well plate and reconstituted with 100 μL of PBS buffer, pH 7.0, containing purified (Nluc) enzyme. The final working concentrations of furazolidinone were 10 μM and 0.1 μM, respectively. Freshly prepared commercially available Luciferase assay substrate was prepared at 10 μM and 0.1 μM for comparison.

[0196] The results are shown in FIG13 . Fig.13A Shown are paper spot samples and freshly prepared commercially available The RLU of the luciferase assay substrate samples changes over time. Fig. 13B The initial RLU for each sample at time 0 is shown. Fig. 13C Image showing perforated spots in tube. These results demonstrate that formulated furomazine can be dried into a solid matrix / paper and later reconstituted with non-organic aqueous buffer conditions.

[0197] Example 7

[0198] Furoxolotl compositions on paper substrates

[0199] This experiment is based on the structural complementation assay disclosed in International Patent Publication No. WO 2014 / 151736. A 4-well plate containing the assay components was prepared by first diluting 5 μL of goat anti-mouse IgG3-SmBiT (0.4 mg / mL) in 495 μL of sucrose protein buffer containing 20 mM Na3PO4, 5% w / v BSA, 0.25% v / v Tween 20, 10% w / v sucrose. 903 Protein Storage Card. Then add 5 μL of the stock solution to 903 card at positions 2 and 4 and allowed to dry at 35°C for 1 hour. 5 μL of goat anti-mouse IgG3-LgBiT (0.4 mg / mL) was diluted in 495 μL of the same sucrose protein buffer and 5 μL of the solution was added directly to Positions 2 and 4 of the 903 protein card. Then The 903 card was dried again at 35°C for 1 hour.

[0200] A 5 mM stock solution of furazolidone was prepared in ethanol and 5 μL of this stock solution was added to the card positions to conditions 1, 2, and 4. The card was then placed under high vacuum for 15 minutes.

[0201] The cards were kept at 4°C or 25°C and Enzyme-conjugated IgG was tested for activity at several time points. At position 1, 10 pg of fresh Labeled antibodies to test substrate activity. Images were recorded and shown in Fig.14A Center: Left – image taken with a standard camera; Center – image taken with the LAS300 imager; Right – image taken with an iPhone camera. Add After enzyme addition, spots 1, 2, and 4 all produced bioluminescence at this time point, indicating that the substrate remained active.

[0202] Additional sample sets were prepared similarly and stored at 4°C or 25°C for 3 months. Images were recorded and are shown in Fig. 14B Center: Left – image taken with a standard camera; Center – 10 pg added to PBS Images of cards stored at 4°C after antibody labeling to determine substrate activity for spots 1, 2 and 3, with spot 4 receiving PBS alone as a negative control; right – 10 pg added to PBS Image of a card stored at 25°C after labeling with antibody. Only spot 2 luminesces, while spot 1 does not. This example shows that all or some of the components of the sucrose protein loading buffer (20 mM Na3PO4, 5% w / v BSA, 0.25% v / v Tween 20, 10% w / v sucrose) are necessary for substrate activity at this time point and temperature, and that furazolidone can be dried together on a solid paper matrix and reconstituted after storage at 4°C or 25°C for extended periods of time.

[0203] Example 8

[0204] Furoxolotl compositions on paper substrates with buffer and additives

[0205] The purpose of this example is to demonstrate the effect of additives on overall reconstitution efficiency and assay performance. The samples were stored at different temperatures to simulate a range of thermal stressors and tested for overall performance and stability under these conditions.

[0206] 903 Protein Protect Spot Cards (3.2 mm punch), Sucrose Protein Buffer (20 mM Na3PO4, 5% w / v BSA, 0.25% v / v Tween 20, 10% w / v sucrose (prepare the evening before use)); 200 μM furazolidone solution in ethanol; 20 mM and 50 mM stocks of 6-aza-2-thiothymidine (ATT) in water; and 20 mM and 100 mM stocks of thiourea in water.

[0207] To 3.2mm To the 903 Protein Preservation Card spot, 5 μL of 200 μM furazolidone in ethanol was added, along with various additional components, as follows:

[0208] Sample 1: Furoxetine

[0209] Sample 2: Furoxil + Sucrose Protein Buffer

[0210] Sample 3: Furoxolotl + ATT (20 mM)

[0211] Sample 4: Furoxolotl + ATT (50 mM)

[0212] Sample 5: furazolidinone + ATT (20 mM) + sucrose protein buffer

[0213] Sample 6: Furoxil + ATT (50 mM) + Sucrose Protein Buffer

[0214] Sample 7: Furoxil + Thiourea (20 mM)

[0215] Sample 8: Furoxil + Thiourea (100 mM)

[0216] Sample 9: furazolidinone + thiourea (20 mM) + sucrose protein buffer

[0217] Sample 10: furazolidinone + thiourea (100 mM) + sucrose protein buffer

[0218] Spots containing protein buffer were dried at 35°C for 1 hour prior to adding other components (furoxil, ATT, and / or thiourea). When ATT was used, 5 μL of the appropriate solution was added to the spot and then dried under vacuum for 30 minutes (a final concentration of 1 mM ATT was provided when a 20 mM solution was used, and a final concentration of 2.5 mM ATT was provided when a 50 mM solution was used). When thiourea was used, 5 μL of the appropriate solution was added to the spot and then dried under vacuum for 30 minutes (a final concentration of 1 mM was provided when a 20 mM solution was used, and a final concentration of 5 mM was provided when a 100 mM solution was used). Spots were made and stored at 4°C for 5 days prior to testing.

[0219] RLU experimental conditions – Assay buffer: PBS, pH 7.0; Plates: NBS solid white plates ( 3600). Different final concentrations of Nluc enzyme were used (20 μg / mL, 2 μg / mL or 0.2 μg / mL).

[0220] The data is presented in FIG. 15A to FIG. 15D Among them FIG. 15A to FIG. 15C The raw RLU from the luminescent reaction at different concentrations of Nluc enzyme (20 μg / mL, 2 μg / mL, and 0.2 μg / mL, respectively) are shown, and Fig.15D The % activity of the Nluc enzyme at one concentration (0.2 μg / mL) is shown. This data suggests that once reconstituted in PBS, the addition of additives such as ATT or thiourea can help improve the overall RLU and signal stability compared to other formulations.

[0221] Example 9

[0222] Furoxolotl compositions on paper substrates with different polymers

[0223] Materials and methods: 903 Protein Protect Spot Cards (3.2 mm punch); Furoxil. One day prior to testing, prepare a protein buffer with the following components.

[0224] Protein buffer 1: purified water

[0225] Protein buffer 2: 20 mM Na3PO4, 5% w / v BSA, 0.25% v / v Tween 20, 10% w / v sucrose

[0226] Protein buffer 3: 20 mM Na3PO4, 5% w / v BSA, 0.25% v / v Tween 20, 2.5% w / v pullulan

[0227] Protein buffer 4: 20 mM Na3PO4, 5% w / v BSA, 0.25% v / v Tween 20, 2.5% w / v trehalose

[0228] 5 μL of one of the protein buffers 1-4 was applied to each spot and the spot was allowed to dry at 35°C for 1 hour. Then, 5 μL of a freshly prepared 200 μM solution of furazolidone in ethanol was applied to each spot and the spot was dried under vacuum for 30 minutes. The spots were stored in the dark at 4°C, 25°C and 35°C, respectively.

[0229] For luminescence measurements, at the time of testing, each spot was placed in a single well of a standard 96-well plate and reconstituted with 100 μL of PBS buffer, pH 7.0, containing purified (Nluc) enzyme. Start kinetic reading immediately.

[0230] Results for spots tested immediately after preparation are shown in Fig.16 In which freshly prepared The trace of the substrate is used for comparison. The results of the spots tested after one day of storage at 4°C, 25°C and 37°C are shown in Fig.17A , Fig. 17B and Fig. 17C The results of the spots tested after three days of storage at 4°C, 25°C and 37°C are shown in Fig.18A , Fig.18B and Fig. 18C These data indicate that the signal is more stable for the furomazine composition on the paper matrix, but the overall signal is lower. Although the sample still produces a usable and stable signal, the addition of the protein buffer with the additive before adding furomazine to the spot may prevent enough furomazine from fully entering the paper.

[0231] Example 10

[0232] Accelerated stability study of formulated furazolidone substrate in paper matrix

[0233] Test paper furomazine samples to determine the compatibility with known furomazine formulations ( Substrate, Promega Cat. No. N113, and Effects of formulation on the thermal stability and functional integrity of furazolidone as measured by RLU were compared to those of furazolidone (Live Cell Substrate, Promega Cat. No. N205).

[0234] 903 Protein Protect Spot Cards (3.2 mm punch) were processed as follows.

[0235] For conditions 1 and 2, paper spots were pretreated with 5 μL of water (condition 1) or protein buffer (20 mM Na3PO4, 5% w / v BSA, 0.25% v / v Tween 20, 10% w / v sucrose - condition 2). Condition 3 was prepared by pretreatment with 5 μL of protein buffer (20 mM Na3PO4, 5% w / v BSA, 0.25% v / v Tween 20) lacking a sucrose component. All conditions were then dried at 35°C for 60 minutes. A stock solution of 200 μM furazolidone was prepared in ethanol, and 5 μL of the stock solution was added to conditions 1 and 2 as described above. For condition 3, a stock solution of 200 μM furazolidone was prepared in a mixture of 2.5% pullulan in water with <10% v / v ethanol. 5 μL of the solution was then added to condition 3, and all spots were then dried additionally under reduced pressure for 30 minutes. The spots were then stored in the dark at 25°C or 60°C. At the time of measurement, one spot from each condition was placed in a single well and diluted with PBS containing Nluc. The final theoretical concentration of furazolidinone was 10 μM and the final concentration of Nluc was 1 ng / mL.

[0236] Compiled RLU data for samples stored at (A) 60°C and (B) 25°C for various time periods prior to reconstitution and testing are shown in Figure 19. Figure 19 also shows data for percent enzyme activity at time 0 after storing samples at (C) 60°C and (D) 25°C for various time periods prior to reconstitution and testing.

[0237] The above experiment was expanded to include high concentrations (final concentrations of 1 mM and 100 μM) of furazolidone and low concentrations (final concentration of 10 μM) of furazolidone (Figure 20). Each condition was prepared as described above: the spots were pretreated with water, protein buffer, or protein buffer lacking sucrose. Under the first two conditions, 5 μL of a 2 mM or 200 μM solution of furazolidone in ethanol was added to each spot and then dried for an additional 30 minutes at 35°C. Under the third condition, a 20 mM furazolidone stock solution or a 200 μM furazolidone stock solution was prepared in a mixture of 2.5% pullulan in water with <10% v / v ethanol. 5 μL of the solution was then added to condition 3, and all spots were dried for an additional 30 minutes at 35°C. The spots were then stored in the dark at 25°C or 60°C.

[0238] Figure 20 shows compiled RLU data for samples stored at (A) 60°C and (B) 25°C for various time periods prior to reconstitution and testing. Figure 20 also shows data for percent enzyme activity at time 0 after samples were stored at (C) 60°C and (D) 25°C for various time periods prior to reconstitution and testing.

[0239] There was an overall improvement in both max RLU and percent activity by increasing the furazolidone loading concentration. However, spots that received no pretreatment (water) still performed better overall compared to both pretreatments containing protein buffer or protein buffer and pullulan at comparable concentrations.

[0240] Embodiment 11

[0241] Effect of drying method of furazolidone formulations in paper samples

[0242] 3.2 mm punched cells were treated with water or protein buffer (20 mM Na3PO4, 5% w / v BSA, 0.25% v / v Tween 20, 10% w / v sucrose). 903 protein preservation card spots were placed and dried at 35°C for 1 hour. A 10 mM stock solution of furazolidone in ethanol was prepared and 20 μL of the solution was added to 980 μL of a 2.5% (w / v) pullulan or 5% (w / v) pullulan solution in water. After thorough mixing, 5 μL of the solution was added to each spot. The spots were dried in a vacuum or in the dark at ambient temperature for 2 hours. After drying, the spots were stored overnight at 4°C in the dark.

[0243] For testing, spots were added to individual wells of a 96-well NBS plate. 100 μL of a 1.068 nM Nluc solution in PBS buffer, pH 7.4 was added to each well. The plate was placed in a luminometer and read for up to 60 minutes. Each spot was run in triplicate.

[0244] The results are shown in Figure 21, where Fig.21A Data for samples dried under vacuum are shown, and Fig. 21B Data are shown for samples dried under ambient air. The substrate properties did not appear to be significantly affected whether the spots were dried in vacuum or at ambient temperature.

[0245] Fig. 21C show Fig.21A The data summarized above indicate that the presence of pullulan reduces the overall RLU output. Empirical observations indicate that the presence of pullulan makes the surface of the paper substrate hard and waxy. This may have prevented the substrate from accessing the protein, resulting in lower light output. The observations also indicate that the order in which the different components are added to the paper substrate may play a role in the overall function.

[0246] Compare another set of spots, after adding substrate, at ambient temperature or at 35 ℃, carry out second drying.Each spot is pretreated with water, sucrose protein buffer (20mM Na3PO4, 5%w / v BSA, 0.25%v / v Tween 20, 10%w / v sucrose) or pullulan protein buffer (20mM Na3PO4, 5%w / v BSA, 0.25%v / v Tween 20, 2.5%w / v pullulan) and allowed to dry at 35 ℃ for 1 hour.Prepare 200μM furazolidinone stock solution in ethanol, and 5μL of the furazolidinone stock solution is added to each spot.Then allow the spot to stand for 30 minutes at ambient temperature or at 35 ℃ in the dark.Then store the spot at 25 ℃ or at 60 ℃ in the dark for up to 5 days. At the time of testing, spots corresponding to each condition were placed in wells of a standard 96-well plate and rehydrated in each well with 100 μL of PBS solution, pH 7.0, and 2 ng / mL Nluc, with a final furazolidone concentration of 10 μM.

[0247] The results are shown in Figure 22. Spots dried a second time at 35°C showed higher RLU output compared to spots dried a second time at ambient temperature. These results were consistent across conditions (protein buffer pretreatment or water control) or whether spots were stored at 25°C or 60°C for up to 5 days (max RLU values ​​are shown in Fig.22A and Fig. 22B and the % activity is shown in Fig. 22C and Fig.22D These results indicate that differences in drying methods have an impact on overall substrate performance and that the second drying spot at 35 °C is beneficial for substrate performance.

[0248] Example 12

[0249] Accelerated substrate testing of powdered pullulan preparations

[0250] Powdered furomazine samples were tested to determine the compatibility with known furomazine formulations ( Substrate, Promega Cat. No. N113 and Effects of pullulan formulations on the thermal stability and functional integrity of furomazine as measured by RLU and HPLC compared to live cell substrate (Promega Cat. No. N205).

[0251] Materials and Methods: Bulk solid furazolidinone was diluted in ethanol to a final concentration of 10 mM (solution 1); dry pullulan was dissolved in purified water to a final concentration of 0%, 2.5%, 5%, 10% and 15% w / v (solutions 2a, 2b, 2c, 2d and 2e, respectively). 45 μL of solutions 2a-e were pipetted into separate 1.5 mL snap-fit ​​vials. 5 μL of solution 1 was then added to each vial and mixed by vigorous pipetting to form solutions 3a-e, each containing a final concentration of 1 mM furazolidinone.

[0252] After mixing, the vials containing solutions 3a-e were frozen in dry ice for 1 hour. These frozen stock solutions were then lyophilized overnight to form dry pullulan matrix containing furazolidone.

[0253] The specific powdered furomazine sample used for testing was prepared as follows:

[0254] 1) Prepare 1 mM (50 nmol total) furazolidone stock solution as a powder formulation with 0% pullulan

[0255] 2) Prepare a 1 mM (50 nmol total) furazolidone stock solution as a powder formulation with 2.5% pullulan

[0256] 3) Prepare 1 mM (50 nmol total) furazolidone stock solution as a powder formulation with 5% pullulan

[0257] 4) Prepare 1 mM (50 nmol total) furazolidone stock solution as N113 solution (Promega catalog number N113)

[0258] 5) Prepare 1 mM (50 nmol total) furazolidone stock solution as N205 solution (Promega catalog number N205) (Note: prepare N205 solution approximately 15 hours after N113 solution)

[0259] 6) Bulk furazolidinone (50 nmol, aliquoted from a stock solution in ethanol)

[0260] Prior to HPLC testing, half of the sample was stored at 25°C and the other half was stored at 60°C for a longer period of time before testing. For HPLC testing, formulated furazolidone (19.08 μg) was diluted with 0.5 mL PBS buffer, pH 6.8 in a small snap-top tube. The tube was vortexed for approximately 15 seconds and then allowed to equilibrate in the dark at room temperature for 30 minutes. 15 μL of sample was injected neat into the HPLC (vial without insert), 0.1% TFA / acetonitrile in water, Synergi Max-RP50x4.6 mm, 2.54u. At 0 hours ( Fig.23A) or after storage at 60°C for 48 hours ( Fig. 23B ), the HPLC trace of the sample containing 5% w / v pullulan showed only minimal degradation. Fig.24A ) or after storage at 60°C for 48 hours ( Fig. 24B ), the HPLC trace of the N113 sample showed significantly more degradation.

[0261] HPLC data were obtained for other samples (not shown) and processed to show the thermal stability trace in Figure 25. The area under the curve was measured and plotted for 35 days. "Bulk" refers to solid furazolidone as manufactured. "0% pullulan" refers to furazolidone dissolved in a stock solution in ethanol, added to water (without pullulan) and lyophilized. Fig.25A and Fig.25B The thermal stability is shown as the raw peak area at 25 °C and 60 °C, while Fig.25C and Fig.25D Thermal stability is shown as peak area percentage at 25°C and 60°C. Formulations consisting of solid furazolidinone showed high and consistent chemical integrity when stored at room temperature or 60°C. Luciferase Assay Substrate (Promega Cat. No. N113) and Furoxolotl formulated in live cell substrate (Promega Cat. No. N205) solution showed a significant loss of peak height and peak area over the time measured when stored at elevated temperature.

[0262] For luminescence measurements, powdered furimazine samples #1-5 were reconstituted in PBS, while sample #6 was reconstituted in ethanol (500 μL total). The samples were equilibrated at room temperature for 30 minutes. The samples were then further diluted 1:5 (from 100 μM to 20 μM) and then 1:100 (from 20 μM to 0.2 μM). 50 μL of the solutions were added to the wells of a 96-well plate, the background was read, and then added (Nluc) enzyme. (Before addition, the commercial Nluc stock sample was diluted to a concentration of 2 ng / mL in PBS and 50 μL was added to each well.) After dilution, the final concentration was 0.1 μM furazolidinone and 1 ng / mL Nluc. The RLU was then determined. (The background is the reading of the 2X substrate solution without the addition of Nluc.)

[0263] The compiled RLU data are shown in Figure 26. The numbers in each figure legend correspond to the following formulations: 1 - 0% Pullulan (Note - the sample encountered solubility issues and may not have been fully reconstituted); 2 - 2.5% Pullulan lyophilized cake formulation; 3 - 5% Pullulan lyophilized cake formulation; 4 - Nano- Luciferase Assay Substrate (Promega Catalog No. N113); 5-Nano- Living cell substrate (N205); 6 - Bulk furazolidinone (reconstituted in ethanol). FIG. 26A to FIG. 26C As shown above, 50 μM substrate ( Fig.26A ), 10 μM substrate ( Fig.26B ) or 0.1 μM substrate ( Fig.26C ) Data after storing samples at 60°C for different periods of time before reconstitution and testing. FIG. 26D to FIG. 26F As shown above, 50 μM substrate ( Fig.26D ), 10 μM substrate ( Fig.26E ) or 0.1 μM substrate ( Fig.26F ) Data after storing samples at 25°C for different periods of time before reconstitution and testing.

[0264] Figure 27 shows the data for percent enzyme activity. The numbers in each figure legend correspond to the following formulations: 1 - 0% Pullulan (Note - the samples described all experienced solubility issues and may not have been fully reconstituted); 2 - 2.5% Pullulan lyophilized cake formulation; 3 - 5% Pullulan lyophilized cake formulation; 4 - Nano- Luciferase Assay Substrate (Promega Catalog No. N113); 5-Nano- Living cell substrate (N205); 6 - Bulk furazolidinone (reconstituted in ethanol). FIG. 27A to FIG. 27C As shown above, 50 μM substrate ( Fig.27A ), 10 μM substrate ( Fig.27B ) or 0.1 μM substrate ( Fig.27C ) enzyme activity at time 0 after storage of samples at 60°C for various periods of time prior to reconstitution and testing, and FIG. 27D to FIG. 27F As shown above, 50 μM substrate ( Fig.27D ), 10 μM substrate ( Fig.27E ) or 0.1 μM substrate ( Fig.27F ) Enzyme activity at time 0 after storage of samples at 25°C for various periods of time prior to reconstitution and testing.

[0265] Solid furazolidinone samples showed consistent chemical integrity after exposure to high temperatures, as indicated by the RLU output in the luciferase assay. In contrast, furazolidinone formulated in the commercially available N113 solution and N205 solution showed a loss of luminescent signal over time after storage at high temperatures. (Note: Sample 6 was dissolved in ethanol, which inhibited Nluc enzyme activity at higher concentrations.)

[0266] Embodiment 13

[0267] Formulated furazolidinone film-coated microtiter plates

[0268] Formulated furomazine films were formed directly onto microtiter plates. Films containing 200 mM furomazine in 2.5% (w / v) pullulan or 5% (w / v) pullulan were prepared directly in the wells of microtiter plates. Fig.29 Representative images of this format can be seen in (artificially colored for clarity and presentation purposes). Well coatings were prepared as follows: A stock solution of 2 mM furazolidone in ethanol was prepared (Solution 1). Separately, solutions of 2.5% and 5% w / v pullulan were prepared in water (Solution 2 and Solution 3, respectively). 45 μL of Solution 2 or Solution 3 were added to individual wells of a standard 96-well plate. 5 μL of Solution 1 was then added to each well containing Solution 2 or Solution 3 and pipetted thoroughly to mix. The concentration of ethanol in the final solution must be less than 5% v / v. Higher concentrations of ethanol will cause pullulan to precipitate out of solution.

[0269] The plates were then allowed to dry for 3 hours in the dark under ambient conditions. The membranes in the wells were rehydrated with 100 μL PBS, pH 7.0, and 2 ng / mL Nluc was added to each well immediately or after a 30-minute pre-equilibration period in 50 μL / well PBS on a shaker, with a final concentration of 10 μM furimazole in solution for all conditions. The RLU was read and compared to freshly prepared commercial furimazole substrate ( Live Cell Substrate, Promega Catalog No. N205) for comparison. The data are shown in Figure 28, where (a) the data are shown as raw RLU without pre-equilibrium, (b) the data are shown as activity without pre-equilibrium, and (c) the data are shown as raw RLU after pre-equilibrium. The examples emphasize that furazolidone can be dried in a pullulan-based film on a hard surface and reconstituted at a specified time. Based on visual observation, the film also reconstitutes faster and more completely than bulk solid furazolidone. The data also show that pre-equilibrium of a furazolidone-pullulan-based film in a PBS microtiter plate results in a significant reduction in light output.

[0270] Fig.29 Shown is an image of a furazolidone film-coated plaque made using the same method as above, but with the addition of food coloring to enable visualization of the film coating.

[0271] Fig. 30A The kinetic readouts for the same format formulations as described for the data presented in FIG28 were shown, but with a higher loading concentration of furimazine (finally 20 μM in 100 μL) and, in some cases, with The furazolidone formulations for the films were filmed together as a complete solution. Fig. 30B Display and Fig. 30A Percent activity from the same experiments described in . Fig. 30C Results of stability studies showing coated microtiter plates after storage for a period of time showed that approximately 35% of activity was retained at day 10.

[0272] Embodiment 14

[0273] HPLC and Mass Spectrometry Analysis of Purity, Stability and By-Product Formation of Formulated Furezine Furezine formulated in a lyophilized pullulan matrix was prepared as described in Example 12 using 19.07 μg of furazine in 0%, 2.5% and 5% w / v pullulan. Samples, including bulk furazine, Luciferase Assay Substrate (Promega Cat. No. N113) and Live cell substrate (Promega catalog number N205) and stored at 25°C or 60°C for 35 days. Samples were reconstituted in PBS buffer or, in the case of bulk furomazine and 0% pullulan samples, in ethanol, allowed to equilibrate at room temperature for 30 minutes and then analyzed on HPLC for known byproducts of the furomazine degradation pathway. Absorbance data are shown in Figure 31: A - bulk furomazine; B - 0% pullulan; C - 2.5% pullulan; D - 5% pullulan; E - Nano- luciferase assay substrate; and F-Nano- Living Cell Substrates. In each case, the solid form of formulated furazolidone showed significantly reduced degradation products relative to the commercially available solution-based depot formulations (Promega Cat. No. N113 and Cat. No. N205).

[0274] Figure 32 shows the area percentage of individual byproducts relative to the furomazine peak: A - bulk furomazine; B - 0% pullulan; C - 2.5% pullulan; D - 5% pullulan; E - Nano- luciferase assay substrate; and F-Nano- Living cell substrate. In the solid pullulan formulation, furimazine was the main peak, with minimal formation of by-products, especially when stored at room temperature (left bar in each case). In contrast, after 35 days, furimazine was almost completely lost when stored at 25°C (left bar) or 60°C (right bar) in the commercial formulation.

[0275] Embodiment 15

[0276] Substrate activity of formulated furazolidone compositions when stored at room temperature for 6 months

[0277] 19 μg of furimazine was formulated as a lyophilized cake or film drop prepared in 15% w / v pullulan as described in Example 1, Condition 1 and Condition 3. The samples were stored at 25°C in the presence of ambient light for six months. Both formulations were reconstituted with 100 μL PBS, pH 7.0 and 1 ng / mL NanoLuc (Nluc) to a final concentration of 10 μM furimazine in solution. The RLU was read and compared to freshly prepared commercial furimazine substrate ( The results of the experiment are shown in Figure 33 (A - original RLU, B - percent activity from time 0). After 6 months of storage at ambient temperature and light, solid furazolidinone formulated in a pullulan matrix was still viable when exposed to luciferase.

[0278] Example 16

[0279] Activity and stability of formulated furazolidinone on different solid support matrices

[0280] Four different types of paper were tested for different properties including substrate retention and effect on substrate integrity. The paper types included:

[0281] 1. Thick glass fiber: Glass Microfiber 934-AH (Ahlstrom, particle retention: 1.5 μM, thickness: 435 μm);

[0282] 2. Fine glass fiber: glass fiber diagnostic pad (EMD Millipore), GFDX103000, batch number 495362;

[0283] 3. Cellulose: Cellulose sample pad (EMD Millipore), CFSP20300M, batch number 11065; and

[0284] 4. 903 Protein Preservation Card

[0285] Each paper sample was cut into a square of 7 x 7 mm. A 10 mM stock solution of furazolidone was prepared and 10 μL of the stock solution was added to each paper matrix. The samples were dried at 35 ° C for 30 minutes. The cards were stored in the dark at 25 ° C or 60 ° C for 72 hours. The samples were then placed in glass vials and 1 mL of ethanol was added. The vials were ultrasonically treated for 10 seconds, and the solvent was extracted, filtered and analyzed by analytical HPLC. The results of these experiments are shown in Figure 34. Fig.34A The original area of ​​the furazolidinone peak after extraction from paper or fiber matrix is ​​shown. The amount of furazolidinone extracted from paper and analyzed from solution is also affected by the type of paper ( Fig.34B). For substrates extracted back into solution, the degradation rate of furazolidone was slightly faster when dried onto paper or fiber substrates compared to bulk furazolidone. Additionally, furazolidone substrates can be efficiently dried and reconstituted by a variety of solid surfaces ( Fig.34C ).

[0286] Additional experiments were performed in conjunction with the reporter protein LgTrip to determine substrate stability on paper. To prepare the paper surface, a vial containing 200 μL of 5uM LgTrip (3546) (SEQ ID NO: 3; see, e.g., U.S. Patent Application No. 62 / 684,014, incorporated herein by reference in its entirety), 5mM ATT, and 5mM ascorbic acid was prepared. Approximately 5 μL of the solution was added to each spot, and the spot was then allowed to dry at 35°C for 1 hour. After drying, a stock solution of 1 mM furazolidone in ethanol was prepared. Approximately 5 μL of the solution was added to each spot and allowed to dry for an additional 30 minutes at 35°C.

[0287] Different materials were tested with substrate and LgTrip input. When testing, fresh Nluc was added to separate the substrate. Fig.35A Bioluminescent signals in three different solid phase materials (Whatman 903, Ahlstrom 237, and Ahlstrom 6613H) resulting from reconstitution of the surface are shown when fresh NanoLuc is added to dried LgTrip and substrate. Ahlstrom 6613H appears to be detrimental to signal output over time. In general, the stability of assay components can be affected by the composition of the solid matrix material in which they are embedded.

[0288] Fig.35B Bioluminescent signal from Whatman 903 paper containing both LgTrip and substrate and stored at ambient conditions for more than 25 days is shown. When tested, the spots were exposed to 1 nM dipeptide in PBS. Overall, the experiments showed that there was no significant loss of signal from the material after extended storage time at ambient temperature.

[0289] Embodiment 17

[0290] Effects of additives on the activity and stability of furazolidinone formulated on different solid supports

[0291] Different additives were mixed with solutions with furazolidone and dried onto paper surfaces. These experiments were designed to improve overall substrate integrity when dried within a paper matrix. A 10 mM ascorbic acid solution was prepared in ethanol. The solution was then added to bulk furazolidone to make a solution containing 1:1 ascorbic acid and furazolidone in ethanol. 10 μL of the solution was then added to the same paper matrix described in the previous example. The samples were dried at 35°C for 30 minutes. The spots were then stored at 25°C or at 60°C in the dark for up to 72 days. The samples were placed in glass vials and 1 mL of ethanol was added. The vials were sonicated for 10 seconds, and the solvent was extracted, filtered and analyzed by analytical HPLC.

[0292] The results of these experiments are depicted in Figure 36. The raw area of ​​the furazolidone peak is plotted on Fig.36A In the samples without ascorbic acid additive, the absorbance of furazolidone was better. The overall recovery percentage of furazolidone into solution ( Fig.36B ) and furazolidone purity ( Fig.36C This effect was also observed for the furazolidinone substrate. The purity increased by about 15-20%, suggesting that the presence of ascorbic acid helped to limit thermal or chemical degradation of the furazolidinone substrate when stored on paper.

[0293] Embodiment 18

[0294] Effects of chemical pretreatment of different solid support matrices on the activity and stability of formulated furazolidinone

[0295] The paper substrate (Ahlstrom Glass Microfiber 934-AH and 903 protein preservation card) was soaked in 30% w / v citric acid solution for 30 minutes and then allowed to dry overnight at 35°C. A stock solution of 10 mM furazolidinone in EtOH was prepared and 10 μL of the stock solution was added to a 7x7 mm paper card and dried at 35°C for 30 minutes. The cards were then stored in the dark at room temperature or 60°C for 72 hours. At the time of reading, the card was extracted with 1 mL of ethanol and sonicated for 15 seconds. The extracted solvent was then filtered and injected into analytical HPLC.

[0296] The results of these experiments are shown in Figure 37. The raw area of ​​the furimazine peak is shown in Fig.37A Pretreatment of the paper substrate with 30% citric acid solution prior to application of the furazolidone substrate had minimal effect on the overall purity of the substrate after extraction into ethanol compared to paper substrates without citric acid pretreatment ( Fig.37C There was also limited improvement in the amount of substrate recovered into solution after extraction with ethanol ( Fig.37B ).

[0297] Embodiment 19

[0298] Effects of mechanical pretreatment of different solid support matrices on the activity and stability of furazolidone

[0299] The paper substrate (Ahlstrom Glass Microfiber 934-AH and 903 protein preservation card) was soaked in water for 30 minutes, then dried overnight under reduced pressure to collapse or shrink the holes present in the paper matrix. A stock solution of 10mM furazolidone in ethanol was prepared, and 10 μL of the stock solution was added to a 7x7 mm paper card and dried at 35°C for 30 minutes. The cards were then stored in the dark at room temperature or 60°C for 72 hours. When read out, the card was extracted with 1 mL of ethanol and ultrasonically treated for 15 seconds. The extracted solution was filtered and injected into analytical HPLC for analysis.

[0300] The results of the experiment are shown in Figure 38, where the raw area of ​​the furazolidinone peak is shown in Fig.38A The recovery percentage is shown in Fig.38B and the purity is shown in Fig.38C There was no significant improvement in substrate purity or recovery into solution when the substrate had been added and dried onto paper previously dried under pressure compared to non-pretreated paper.

[0301] Embodiment 20

[0302] Effects of additives on the activity and stability of furazolidinone formulated on different solid supports

[0303] Different additives were mixed with a solution with furazolidone and dried onto a paper surface. This series of experiments was designed to help improve the overall substrate integrity when dried within a paper matrix. A 10 mM citric acid solution was prepared in ethanol. The solution was added to bulk furazolidone to make a solution containing 1:1 citric acid and furazolidone in ethanol. 10 μL of the solution was then added to the same paper matrix described in the previous example. The samples were dried at 35°C for 30 minutes. The cards were stored in the dark at 25°C or 60°C for 72 hours. The samples were placed in glass vials and 1 mL of ethanol was added. The vials were sonicated for 10 seconds, and the solvent was filtered and analyzed by analytical HPLC.

[0304] The results of these experiments are depicted in Figure 39. The raw area of ​​the furazolidone peak for furazolidone dried on paper in the presence (A) and absence (B) of citric acid is plotted on Fig.39A and Fig.39BThe purity at 254 nm of furazolidone dried in paper in the presence (C) and absence (D) of citric acid is plotted in Fig.39C and Fig.39D These plots show that the absorbance of furazolidone is better when dried in a mixture with citric acid. This increase in absorbance corresponds to an increase in purity of about 10-20%, indicating that the presence of citric acid helps limit the thermochemical degradation of the furazolidone substrate during the course of the experiment.

[0305] Embodiment 21

[0306] Effects of citric acid and ascorbic acid on the activity and stability of formulated furazolidone in the presence of sucrose protein loading buffer

[0307] The effects of citric acid and ascorbic acid on the activity and stability of furazolidone were tested in the presence or absence of sucrose buffer (20 mM Na3PO4, 5% w / v BSA, 0.25% v / v Tween 20, 10% w / v sucrose). 903 Protein Preservation Card Preparation. Each spot was pretreated with sucrose protein buffer or water and allowed to dry at 35°C for 1 hour. A stock solution of 200 μM furazolidone was prepared in ethanol or in an ethanol solution with 200 μM citric acid or 200 μM ascorbic acid. 5 μL of furazolidone or furazolidone solution containing citric acid or ascorbic acid at equal molar concentrations was added to each spot. The spots were then dried again at 35°C for 1 hour. The spots were then stored at 25°C in the dark for up to 12 days.

[0308] At the time of testing, spots corresponding to each condition were placed in wells of a standard 96-well plate and rehydrated in each well with 100 μL of PBS solution, pH 7.0, and 2 ng / mL Nluc, with a final furazolidone concentration of 10 μM. The RLU was read and compared to freshly prepared commercial furazolidone substrate ( The results of these experiments are shown in Figure 40.

[0309] On paper, spots pretreated with sucrose protein buffer showed significant signal loss over a 12-day period ( Fig.40A These results correspond to an almost complete loss of percent substrate activity compared to conditions pretreated with water or water in the presence of ascorbic acid or citric acid, which showed significant stability and signal output over 12 days ( Fig.40B ). A summary of these results is shown in Fig.40CAscorbic acid and citric acid may help maintain substrate integrity when dried and stored on a paper surface, especially compared to water pretreatment alone. However, for long-term storage and reconstitution, one or more components within the sucrose protein buffer may have a negative impact on substrate viability.

[0310] Embodiment 22

[0311] Effects of spot storage, either individually or in batches, on the activity and stability of furazolidone

[0312] As mentioned above, the specific storage process of the test The influence of paper spots prepared by 903 protein preservation cards. Each spot was pretreated with water and then dried at 35°C for 1 hour. A stock solution of 200 μM furimox was prepared in ethanol, and 5 μL of the solution was added to each spot. The spot was then dried additionally for 30-60 minutes at 35°C. The spots were then separated and stored individually in capped tubes, or stored together (batch storage) in a vial in the dark at 25°C for up to 12 days. When tested, the spots corresponding to each condition were placed in the wells of a standard 96-well plate, and rehydrated with 100 μL PBS solution, pH 7.0 and 2ng / mL Nluc in each well, and the final concentration of furimox in the solution was 10 μM. The results of these experiments are described in Figure 40.

[0313] Spots stored individually showed higher max RLU than spots stored in batches ( Fig.41A These results are consistent with the observed percentage activity ( Fig.41B ). These results suggest that storage methods can also have an impact on overall substrate performance. Storage in individual containers can help limit environmental exposure to adverse factors such as light, air, and moisture, compared to spots stored in batches, to which the spots are exposed each time they are removed for testing.

[0314] Embodiment 23

[0315] Signal generation after removal of spots from the wells

[0316] As described in Example 6, the spots are 903 protein preservation card preparation. Each spot was pretreated with sucrose protein buffer (20mM Na3PO4, 5% w / v BSA, 0.25% v / v Tween 20, 10% w / v sucrose) or water and allowed to dry for 1 hour at 35°C. A stock solution of 200 μM furazolidone was prepared in ethanol or an ethanol solution with 200 μM citrate or 200 μM ascorbate. 5 μL of furazolidone or a furazolidone solution containing an equal molar ratio of citrate or ascorbate was added to the spot. The spot was then dried for an additional hour at 35°C. The spot was then stored in the dark at 25°C for up to 5 days.

[0317] At the time of testing, the spots were reconstituted with PBS, pH 7.0 containing 2 ng / mL Nluc enzyme and the RLU was read kinetically. After 45 minutes, the spots were physically removed from the wells and placed in new wells containing fresh PBS solution, pH 7.0 and 2 ng / mL Nluc, and the kinetic RLU signal was continued to be read in the wells containing the previous spots and the new wells containing the transferred paper spots. Fig.42A The kinetic RLU values ​​for the wells that previously contained the spots and the new wells to which the spots were transferred are shown (indicated by a + after substrate preparation). The aggregated RLU results are shown in Fig.42B In the RLU results, the original readout RLU at 45 minutes, the now empty well just after removal, and the RLU values ​​taken just after the spot was transferred to a new well containing fresh enzyme were compared. There was no change in the RLU value from the original spot reading to the well with the spot removed, indicating that the substrate was released from the paper matrix and equilibrated into the surrounding solution. A lower signal was recovered in the well containing the transferred spot, indicating that some of the substrate preparation was retained in the paper matrix itself. The percentage signal recovery for each condition ( ) was calculated by comparing the RLU signal present before the spot was transferred from the well to the signal retained after the spot was removed or placed in a new well. Fig.42C ). After transferring the spots to new wells containing fresh PBS solution, pH 7.0 and 2 ng / mL Nluc, approximately half of the signal percentage was observed in the new wells. This indicates that residual substrate is retained in the paper itself, while most of the substrate is released into the solution of the original well.

[0318] Embodiment 24

[0319] Effects of BSA and sugar on the activity and stability of furazolidone

[0320] Ten different versions of protein loading buffer were prepared to determine if BSA and sugar components had an effect on furazolidone activity and stability after drying and reconstitution on a solid surface. The following buffers were prepared and tested:

[0321] 1. Protein buffer 1: 20 mM Na3PO4, 5% w / v BSA, 0.25% v / v Tween 20, 10% w / v sucrose

[0322] 2. Protein buffer 2: 20 mM Na3PO4, 5% w / v BSA, 0.25% v / v Tween 20, 10% w / v sucrose, 5 mM ascorbate

[0323] 3. Protein buffer 3: 20 mM Na3PO4, 0.25% v / v Tween 20, 10% w / v sucrose

[0324] 4. Protein buffer 4: 20 mM Na3PO4, 0.25% v / v Tween 20, 10% w / v sucrose, 5 mM ascorbate

[0325] 5. Protein buffer 5: 20 mM Na3PO4, 5% w / v BSA, 0.25% v / v Tween 20

[0326] 6. Protein buffer 6: 20 mM Na3PO4, 5% w / v BSA, 0.25% v / v Tween 20, 5 mM ascorbate

[0327] 7. Protein buffer 7: 20 mM Na3PO4, 5% w / v BSA, 0.25% v / v Tween 20, 2.5% pullulan

[0328] 8. Protein buffer 8: 20 mM Na3PO4, 5% w / v BSA, 0.25% v / v Tween 20, 2.5% pullulan, 5 mM ascorbate

[0329] 9. Protein buffer 9: 20 mM Na3PO4, 0.25% v / v Tween 20, 2.5% pullulan

[0330] 10. Protein buffer 10: 20 mM Na3PO4, 0.25% v / v Tween 20, 2.5% pullulan, 5 mM ascorbate

[0331] The pH of each buffer was determined and listed in Table 2.

[0332] Table 2.

[0333] Buffer pH 1 9.93 2 9.04 3 11.11 4 10.53 5 11.69 6 9.00 7 9.89 8 9.00 9 11.45 10 10.45

[0334] As described in Example 6, 903 protein preservation cards were used to prepare spots. Each spot was treated with buffer 1-10 and then dried at 35°C for 1 hour. A 200 μM furimox stock solution was prepared in ethanol, and 5 μL of the solution was added to each spot. The spots were then dried for an additional hour at 35°C. When tested, the spots corresponding to each condition were placed in wells of a standard 96-well plate and rehydrated in each well with 100 μL PBS solution, pH 7.0 and 2 ng / mL Nluc, with a final concentration of 10 μM furimox in the solution. The RLU was read and compared with freshly prepared commercially available furimox substrate ( Live Cell Substrate, Promega Cat. No. N205).

[0335] These results are depicted in Figure 43. By removing BSA, there was a small decrease in signal, which was restored in the presence of ascorbate (Buffer 3 and Buffer 4, Fig.43A ). However, when the sucrose component was removed, a significant decrease in the signal was observed (Buffer 5 and Buffer 6). In the presence of ascorbate, or if the sucrose component was replaced by 2.5% w / v pullulan, the signal was not restored (Buffer 7 and Buffer 8). The lowest signal was observed when neither BSA nor sucrose was present in the loading buffer.

[0336] The kinetic results are shown in Fig.43B In conditions lacking sucrose, BSA, or both, the signal dropped dramatically over the course of the experiment, whereas this was not observed in the other conditions. The presence of ascorbate limited the rate of signal decay in these conditions (buffer 3 vs. buffer 4, buffer 5 vs. buffer 6, or buffer 9 vs. buffer 10). These differences corresponded to significant changes in percent activity ( Fig.43C ). Buffers 3, 5, and 9 had poor solution kinetic RLU readings. These buffers also had the highest pH values ​​(Table 2), indicating that pH The substrate properties on the 903 paper may also play a role.

[0337] Embodiment 25

[0338] Effects of individual buffer components on the activity and stability of furazolidone

[0339] Eight different protein loading buffers were prepared to determine if specific buffer components had an effect on the activity and stability of furazolidone while stored on a solid paper surface. The buffers prepared and tested were as follows:

[0340] 1. Buffer 1: Water

[0341] 2. Buffer 2: water + 5 mM ascorbate

[0342] 3. Buffer 3: BSA

[0343] 4. Buffer 4: BSA + 5mM ascorbate

[0344] 5. Buffer 5: Na3PO4 + Tween 20

[0345] 6. Buffer 6: Na3PO4 + Tween 20 + 5mM ascorbate

[0346] 7. Buffer 7: BSA + Na3PO4 + Tween 20

[0347] 8. Buffer 8: BSA + Na3PO4 + Tween 20 + 5mM ascorbate

[0348] All buffers were fixed at pH 7 before adding them to the paper spots.

[0349] The spots were generated as described in Example 6. 903 protein preservation card. Each spot was treated with buffer 1-8 and then dried at 35°C for 1 hour. A 200 μM furimox stock solution was prepared in ethanol, and 5 μL of the solution was added to each spot. The spots were then dried for an additional hour at 35°C. When tested, the spots corresponding to each condition were placed in separate wells of a standard 96-well plate and rehydrated in each well with 100 μL PBS solution, pH 7.0 and 2 ng / mL Nluc, with a final concentration of 10 μM furimox in the solution. Read the RLU and compare it with freshly prepared commercially available furimox substrate ( Live Cell Substrate, Promega Cat. No. N205).

[0350] These results are shown in Figure 44. Spots pretreated with buffer containing ascorbate, BSA, or a combination of both showed good stability over 8 days while stored in the dark at 25°C ( Fig.44A Conditions lacking BSA or ascorbate and containing Tween 20 and high levels of salt showed a significant loss of the original signal. There was also a significant loss in percent activity during the first few days ( Fig.44B ). These results suggest that the presence of Tween 20 and / or high salt can have a negative impact on substrate integrity while drying and storing on a solid surface, as seen by the decrease in overall RLU yield. The presence of ascorbic acid helps to offset this effect. A representative kinetic trace from day 0 is shown in Fig.44C In conditions lacking components of the protein buffer (buffer 1 or buffer 2), the rate of signal loss was higher.

[0351] Embodiment 26

[0352] Effects of Prionex on the Activity and Stability of Furoxolotl

[0353] Six different protein loading buffers were prepared to determine if replacing BSA with Prionex had an effect on the activity and stability of furazolidone while stored on a solid surface. The buffers tested were as follows:

[0354] 1. Buffer 1: Water

[0355] 2. Buffer 2: water + 5 mM ascorbate

[0356] 3. Buffer 3: 1% v / v Prionex

[0357] 4. Buffer 4: 1% v / v Prionex + 5 mM ascorbate

[0358] 5. Buffer 5: 0.5% v / v Prionex

[0359] 6. Buffer 6: 0.5% v / v Prionex + 5 mM ascorbate

[0360] The pH of each buffer was maintained at pH 7.

[0361] The spots were generated as described in Example 6. 903 protein preservation card. Each buffer was treated with buffer 1-6 and then dried at 35°C for 1 hour. A 200 μM furimox stock solution was prepared in ethanol, and 5 μL of the solution was added to each spot. The spots were then dried for an additional 30 minutes at 35°C. The spots were then stored in the dark at 25°C for up to 20 days. When tested, the spots corresponding to each condition were placed in separate wells of a standard 96-well plate and rehydrated in each well with 100 μL PBS solution, pH 7.0 and 2 ng / mL Nluc, with a final concentration of 10 μM furimox in the solution. Read the RLU and compare it with freshly prepared commercially available furimox substrate ( The results are described in Figure 44.

[0362] In all cases, high levels of RLU output were observed over the course of the experiment. The water-only pretreatment condition showed some signal loss over the three weeks ( Fig.45A ). The kinetic data for the first day of testing are shown in Fig.45B Compared with the condition without Prionex, the presence of Prionex helps stabilize the reconstructed signal and limits signal attenuation.

[0363] Embodiment 27

[0364] Effects of ATT on the activity and stability of formulated furazolidone

[0365] Six different protein loading buffers were prepared to determine if the presence of ATT had an effect on furazolidone activity and stability while stored on a solid paper surface. The loading buffers prepared and tested were as follows:

[0366] 1. Water + 5mM ascorbate

[0367] 2. Water + 5mM ascorbate + 5mM ATT

[0368] 3.1% Prionex + 5mM ascorbate

[0369] 4.1% Prionex + 5mM ascorbate + 5mM ATT

[0370] 5.0.5% Prionex + 5mM ascorbate

[0371] 6.0.5% Prionex + 5mM ascorbate + 5mM ATT

[0372] The pH of each buffer was controlled to pH 7.

[0373] The spots were generated as described in Example 6. 903 protein preservation cards. Each buffer was treated with buffer 1-6 and then dried at 35°C for 1 hour. A 200 μM furimox stock solution was prepared in ethanol, and 5 μL of the solution was added to each spot. The spots were then dried for an additional hour at 35°C. The spots were then stored in the dark at 25°C for up to 23 days. When tested, the spots corresponding to each condition were placed in separate wells of a standard 96-well plate and rehydrated in each well with 100 μL PBS solution, pH 7.0 and 2 ng / mL Nluc, with a final concentration of 10 μM furimox in the solution. Read the RLU and compare it with freshly prepared commercially available furimox substrate ( The results are described in Figure 45.

[0374] In all cases, high RLU output was observed ( Fig.46A After 22 days of storage, the kinetic trajectory of the spots also showed a high and stable signal over the course of the experiment ( Fig.46B ). All of the conditions of the present invention are favorable for the activity and storage stability of the substrate on a solid surface at ambient temperature.

[0375] Embodiment 28

[0376] Lyophilization of formulated furazolidinone directed into microtiter plates

[0377] Furoxolotl was prepared and lyophilized directly in microtiter plate wells. Preparations produced wells containing lyophilized powdered formulations of 200 μM or 2 mM furoxolotl in 5% (w / v) pullulan prepared directly in the wells of a standard 96-well microtiter plate (Costar Cat. No. 3912). Representative images of this format are shown in Fig.47A Prepare the plate as follows: Prepare 2 mM and 200 μM furazolidone stock solutions in ethanol (Solution 1). Separately, prepare a 5% w / v solution of pullulan in water (Solution 2). Add 45 μL of Solution 2 to a single well. Then add 5 μL of Solution 1 to each well containing Solution 2 and pipette thoroughly to mix. Add 5 μL of pure ethanol to Solution 2 as a negative control. Freeze the plate on dry ice for 1 hour and then lyophilize overnight.

[0378] The plates containing furimazine cakes were rehydrated with 100 μL PBS, pH 7.0 and 2 ng / mL Nluc in each well, and the final concentration of furimazine was 10 μM or 100 μM, respectively. RLU was read and compared with freshly prepared commercial furimazine substrate ( Live Cell Substrate, Promega Cat. No. N205) or fresh in the presence of 5% w / v pullulan The kinetic data are shown in Fig.47B The examples show that a lyophilized powder formulation can be prepared directly on a solid surface such as a microtiter plate and reconstituted using an aqueous buffer such as PBS.

[0379] Embodiment 29

[0380] Layered format for substrate addition

[0381] Fig.48 A prophetic embodiment of a two-part layered system is shown, which includes separate surface components on a single paper card, or components that are separately processed on the same surface, which contain substrates or detection components respectively. When used, the two sides of the surface are folded together so that each surface is in close contact with each other. A sample solution containing the target analyte is then added to the folded surface material. The presence of the solution will cause the different components to rehydrate and mix within the solid matrix, resulting in the complementary induced formation of a bioluminescent complex. The process combined with the substrate will produce light that can be detected and analyzed.

[0382] Embodiment 30

[0383] Effect of Sodium Ascorbate on Substrate Preparations

[0384] A volume of Luciferase assay substrate (Promega catalog number N113) was mixed with 50 volumes of 5% paraformaldehyde containing sodium ascorbate at concentrations ranging from 0 mM to 300 mM. luciferase assay buffer (Promega catalog # N112). The solution was incubated at 37°C and then assayed at several time points. Luciferase assay substrate (stored at -20°C during the course of the experiment and reconstituted at each time point) was used as a positive control. A cell culture of the enzyme was used as a sample. A volume of reconstituted Luciferase assay buffer was mixed with one volume of sample. After 3 minutes, the luciferase assay buffer was added to the sample using a Bio-Tek Luminescence intensity was measured by H1 96-well plate reader. For each sample, the luminescence intensity was background subtracted and normalized to the -20°C control signal.

[0385] like Fig.49 As shown in luciferase assay buffer to reduce the loss of reagent activity after reconstitution. When reconstituted in luciferase assay buffer and maintained at 37°C for 23 hours, the luminescence intensity was 66% of the control, compared to 38% in the absence of sodium ascorbate. After 41 hours at 37°C, the luminescence intensity was 31% compared to 9%. The stabilization effect diminished as the amount of sodium ascorbate was reduced. Please note that the results from the buffer condition containing 3 mM sodium ascorbate are most likely due to experimental error.

[0386] Embodiment 31

[0387] Effect of Hydroxypropyl-β-Cyclodextrin on Substrate Preparation

[0388] Prepare a 4x furazolidinone solution by diluting the stock solution 1:25 in a buffer containing 200 mM MES pH 6.0, 200 mM hydroxypropyl-β-cyclodextrin (HP-β-CD), and 600 mM sodium ascorbate. The solution was lyophilized in a freeze dryer for 48 hours. These lyophilized preparations were then stored at elevated temperature (37°C) for the duration of the experiment. After 24 and 48 hours, the precipitate was The luciferase assay buffer was reconstituted so that the final concentrations of the components in the solution were 2x furazolidone (diluted 1:50 from the stock solution), 100 mM MES pH 6.0, 100 mM HP-β-CD, and 300 mM sodium ascorbate. Preparation of Luciferase Assay Buffer The substrate was added and incubated at 37°C for the duration of the experiment. Substrate (stored at -20°C during the course of the experiment and reconstituted at each time point) was used as a positive control. In each case, one volume of the reconstituted Luciferase assay substrate was mixed with one volume of sample. After 3 minutes, the luminescence intensity was measured with a Bio-Tek Synergy H1 96-well plate reader. Cell cultures expressing NanoLuc were used as samples. For each sample, the luminescence intensity was background-subtracted and normalized to the -20°C control signal.

[0389] Prior to lyophilization, the addition of HP-β-CD and sodium ascorbate to the buffer allowed the precipitate to be dissolved directly in buffer (without adding solvent) and kept stable at 37°C for 48 hours. Fig.50 Incubate at 37°C at the working concentration indicated in Substrate in The non-lyophilized solution in luciferase assay buffer showed a 90% reduction in activity after the same period of time. Additionally, it is important to note that some signal enhancement was observed when comparing the precipitate to the standard kit preparation.

[0390] Embodiment 32

[0391] Effects of buffer additives alone and in combination on substrate formulations

[0392] Dilute furazolidone 1:50 into a buffer with the following final composition:

[0393] - Buffer

[0394] - Buffer + 300mM sodium ascorbate

[0395] - Buffer + 100 mM hydroxypropyl-β-cyclodextrin (HP-β-CD)

[0396] - Buffer + 300 mM sodium ascorbate + 100 mM HP-β-CD.

[0397] One volume of NanoLuc expressing cell culture was added to one volume of each buffer and mixed. After 3 minutes, the luminescence intensity was measured using a standard plate reader. Following the same procedure, background intensity was measured by mixing one volume of cell culture medium with each buffer.

[0398] These experimental results indicate that HP-β-CD is the main cause of signal enhancement. Fig.51A As shown in HP-β-CD enhanced the signal by 15% to 20% compared to the solution in buffer. Fig.51B ) indicates that the increase in signal is not due to an increase in background signal.

[0399] Embodiment 33

[0400] Effect of mixed polymer substrate formulation on substrate stability

[0401] Lyophilized in MES, pH 6.0 solution containing 200 mM HP-β-CD, 600 mM sodium ascorbate and 10% w / v pullulan Preparation of substrates (1:50 dilution). After freeze drying, the vials were capped by hand (no vacuum was applied). Some vials were stored in an incubator at 37 °C, while others were left on the laboratory bench at room temperature. Prior to each measurement, the vials were rehydrated to twice their original volume, resulting in a final concentration of each component of 100 mM HP-β-CD, 300 mM sodium ascorbate, and 5% pullulan. The assay was used according to the manufacturer's instructions. Substrate (stored at -20°C during the course of the experiment and reconstituted at each time point) was used as a positive control. In each case, one volume of the reconstituted Luciferase assay substrate was mixed with one volume of sample. After 3 minutes, the luminescence intensity was measured with a Bio-Tek Synergy H1 96-well plate reader. Cell cultures expressing NanoLuc were used as samples. For each sample, the luminescence intensity was background-subtracted and normalized to the -20°C control signal.

[0402] like Fig.52 As shown in , the presence of pullulan in the lyophilized formulation allows the substrate to retain its activity over a period of 15 days when stored at room temperature and at 37°C. It is believed that the addition of pullulan provides a barrier to oxygen and moisture and, when combined with the previously described additives, provides a stable matrix that has the potential to retain the activity of furazolidone for weeks to months. When combined with an inert gas, this storage method shows promise for achieving particularly long term stability of the substrate.

[0403] Embodiment 34

[0404] F-127 formulated JRW-0238

[0405] 2.5 mg F-127 (Sigma Aldrich) was loaded in batches into 5 mL snap-top Eppendorf tubes. The polymer was heated to 70°C in a water bath until melted and a clear solution was obtained. A 174 mM stock solution of the coelenterazine analog JRW-0238 was prepared in EtOH. 5 μL of the stock solution was added to the molten polymer and pipetted to mix. Two separate conditions were prepared: Condition 1 - After adding the substrate, the substrate / polymer solution was dried under high vacuum for 30 minutes. Condition 2 - After adding the substrate, the substrate / polymer solution was further diluted with 45 μL of water, frozen and lyophilized overnight. A representative example of the final dry formulated substrate is shown in Fig.53 middle.

[0406] Samples from both Condition 1 and Condition 2 were reconstituted in water, diluted to 100 μM, and analyzed for chemical integrity via analytical HPLC ( FIG. 54 ). Fig.54A ) compared to the formulated substrate conditions, none of them showed any significant chemical degradation ( Fig.54B and Fig.54C ). The peak information is summarized in Table 3.

[0407] Table 3.

[0408]

[0409] The reconstituted samples from both conditions were placed in the dark at ambient temperature. After 24 hours, a small amount of precipitation was observed in the sample prepared by condition 1. During the experiment, the solution prepared by condition 2 remained clear. This series of experiments shows that the solid formulation of coelenterazine analogs can be prepared with synthetic polymers and improves the overall kinetic solubility in aqueous media without the need for organic solvents or stabilizers. However, the preparation method may have an impact on thermodynamic solubility. After 24 hours at ambient temperature, the lyophilized condition (condition 2) was still in solution. This is in contrast to the sample from condition 1, which began to precipitate out of solution within 24 hours after reconstitution in water.

[0410] Embodiment 35

[0411] use F-127 formulated furazolidone

[0412] For the coelenterazine analog furomazine, the formulation was also prepared. 2.5 mg of F-127 (Sigma Aldrich) was loaded in bulk into 1.5 mL pop-top Eppendorf tubes. The polymer was heated to 70°C in a water bath until melted. A 10 mM stock solution of furazolidone was prepared in EtOH. 5 μL of the stock solution was added to the molten polymer and pipetted to mix. Two separate conditions were prepared: Condition 1 - After adding the substrate, the solution was vacuum dried for 30 minutes. Condition 2 - After adding the substrate, the substrate / polymer solution was further diluted with 45 μL of water, frozen, and then lyophilized overnight.

[0413] Samples from both condition 1 and condition 2 were diluted with water to 100 μM and analyzed for substrate integrity by analytical HPLC ( FIG. 55 ). Fig.55A ), condition 1 showed considerable degradation ( Fig.55B This may be due to the extensive sonication required to reconstitute the sample. In contrast, the reconstituted sample from condition 2 showed no significant degradation ( Fig.55C ). The peak information is summarized in Table 4.

[0414] Table 4.

[0415]

[0416] This series of experiments demonstrated that solid formulations of coelenterazine analogs, including furazolidone, can be prepared using synthetic polymers and have improved overall kinetic solubility in aqueous media without the need for organic solvents or stabilizers.

[0417] Embodiment 36

[0418] Maximum concentration of prepared JRW-0238 in water

[0419] 25 mg F-127 was loaded in bulk into 1.5 mL snap-top Eppendorf tubes. The polymer was heated to 70 °C in a water bath until melted. 3.4 mg of JRW-0238 was dissolved in 50 μL of EtOH and then added to the hot polymer and mixed by pipetting. An additional 50 μL of EtOH was used to wash and help transfer the substrate into the polymer solution. The solvent was removed under reduced pressure without heating.

[0420] Four vials were prepared in a similar manner and all contained 7.3:1 w / w polymer to substrate. The initial aqueous stock solutions were prepared using different volumes of water as follows:

[0421] Vial 1: After injecting the polymer and substrate together, the solution was dissolved in 500 μL of water. After some sonication, everything was dissolved. The sample was frozen and lyophilized overnight. Using 5% w / v polymer, the calculated concentration of substrate was determined to be 17.4 mM.

[0422] Vial 2: After injecting the polymer and substrate together, the solution was dissolved in 400 μL of water. After some sonication, everything was dissolved. The sample was frozen and lyophilized overnight. Using 6.25% w / v polymer, the calculated concentration of substrate was determined to be 21.4 mM.

[0423] Vial 3: After injecting the polymer and substrate together, the solution was dissolved in 250 μL of water. After some sonication, everything was dissolved. The sample was frozen and lyophilized overnight. Using 10% w / v polymer, the calculated concentration of substrate was determined to be 34.4 mM.

[0424] Vial 4: After injecting the polymer and substrate together, the solution was dissolved in 100 μL of water. After some sonication, everything was dissolved. The sample was frozen and lyophilized overnight. Using 25% w / v polymer, the calculated concentration of substrate was determined to be 85.4 mM.

[0425] After lyophilization, each sample was reconstituted with 500 μL, 400 μL, 250 μL, or 100 μL of water. All material was dissolved in each condition. Representative images of these solutions are shown in Fig.56 After 24 hours at ambient temperature, the reconstituted stock solution was centrifuged and no precipitation was observed. A representative HPLC trace showing the chemical integrity of the reconstituted substrate after standing in solution for 24 hours is shown in Fig.57 No significant chemical degradation was observed. The peak information is summarized in Table 5.

[0426] Table 5.

[0427]

[0428]

[0429] These experiments indicate that by formulating with a solid polymer, high concentrations of the coelenterazine analog JRW-0238 can be achieved in water without any loss of chemical integrity under ambient conditions.

[0430] Embodiment 37

[0431] Lower polymer / substrate ratios without observable loss of substrate solubility

[0432] 23.8mg, 20.4mg, 17mg, 13.6mg, 10.2mg and 6.8mg F-127 was batched into a single 1.5 mL pop-top Eppendorf tube. The polymer was heated to 70 ° C in a water bath until melted. 23.7 mg JRW-0238 was dissolved in 350 μL EtOH, and 50 μL of the stock solution was added to each vial containing the hot polymer; mixed thoroughly by pipetting. The vials were then placed under high vacuum for 30 minutes to remove all organic solvents. Each vial was diluted with 500 μL water to a final concentration of 17.4 mM JRW-0238, with 7x, 6x, 5x, 4x, 3x or 2x w / w polymer / substrate, respectively. Each tube was frozen and lyophilized overnight.

[0433] At the time of testing, 500 μL of water was added to each vial and vortexed until all material was dissolved. After initial reconstitution, all samples were clear except for the sample containing 2x w / w polymer relative to substrate ( Fig.58A After 1 hour in solution at room temperature, the reconstituted substrate was observed to remain in solution except for the sample containing 2x w / w polymer relative to the substrate ( Fig.58B ).

[0434] Embodiment 38

[0435] Solid Formulations for Whole Animal Imaging

[0436] For whole animal imaging in the mouse model, a stock sample of solid formulated JRW-0238 was prepared as follows: 90 mg of F-127 was batched into glass screw-cap vials. The polymer was then heated to 70°C in a water bath until melted (became a clear solution). 12.5 mg of JRW-0238 was dissolved in 250 μL of EtOH and added to the hot polymer, mixing thoroughly with a thin spatula. The solvent was then removed under reduced pressure. The concentrated sample was diluted with 3.646 ml of water to prepare a mother solution of 8.7 mM substrate in water. 480 μL of the aqueous stock solution was then aliquoted into 1.5 mL screw-cap vials, frozen and lyophilized overnight. Representative images of the formulations are shown in Fig.59A At the time of testing, 480 μL of water was added to the vial and vortexed for approximately 15 seconds until all material was dissolved ( Fig.59B ).

[0437] Transgenic mouse subjects (average age: 6 months) engineered to express the Antares protein construct (see U.S. Pat. No. 9,908,918), a fusion of NanoLuc and cyan-excitable orange-red fluorescent protein (CyOFP), were anesthetized with isoflurane and injected with 480 μL of the reconstituted substrate solution via intraperitoneal (IP) or subcutaneous (SC) injection. Each mouse was then imaged every minute after injection using the Ami imaging system. Fig.60A Shown are traces of the average RLU from five animals injected IP with reconstituted JRW-0238. Fig.60B are representative images of each mouse when maximum light output was measured. Fig.61A Shown are traces of the mean RLU from five animals that were injected SC with reconstituted JRW-0238. Fig.61B Representative images of each mouse subject are shown when maximum light output was measured. Taken together, these results indicate that in vivo imaging can be achieved with coelenterazine analogs that are prepared as dry formulations, reconstituted in water at the time of use, and injected into live animal subjects via IP or SC injection routes.

[0438] It should be understood that the foregoing detailed description and accompanying examples are illustrative only and should not be taken as limiting the scope of the present disclosure, which is defined only by the appended claims and their equivalents.

[0439] Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Such changes and modifications may be made without departing from the spirit and scope of the present disclosure, including but not limited to those related to the chemical structures, substituents, derivatives, intermediates, syntheses, compositions, preparations or methods of use of the present invention.

[0440] Embodiment 39

[0441] Large-scale preparation of polymer furazolidinone formulations

[0442] The dried furomazine formulation was scaled up to a larger volume and demonstrated that these compositions can be prepared under manufacturing conditions. The concentration of furomazine in the cake is 200uM. The cake can be reconstituted into a 10mL stock solution, providing a 2x furomazine stock solution (20uM). This can then be diluted 1:1 with the sample to a final concentration of 10uM.

[0443] To prepare the bulk solution, 50 mL of milli-Q purified water was added to 1.25 g pullulan, 35.7 mg ATT and 44 mg ascorbate and mixed until all solids were dissolved. The final solution contained 2.5% w / v pullulan and 5 mM ATT and 5 mM ascorbate, respectively.

[0444] 29.4 mL of pullulan solution was measured into a 50 mL plastic vial. 600 μL of furazolidone prepared as a 10 mM EtOH stock solution was added and mixed thoroughly. A small amount of thin needle-like precipitates were observed in the solution. The precipitation was most likely due to the interaction of the pullulan polymer with the EtOH in the solution. This will not affect the success of the preparation or the properties of the final material.

[0445] Use 10 mL amber glass vials. Aliquot 1 mL of the furazolidone-pullulan stock solution into 10 mL amber glass vials and partially insert the rubber stopper into the vial.

[0446] The freeze dryer used (Virtis Genesis 12EL freeze dryer) had a 4 ft 2 The shelf surface and a total of three shelves. The refrigeration system consists of 2 two-stage compressors. Vacuum / pressure control is achieved by a single vacuum pump and a regulating control valve, which discharges nitrogen into the freeze drying chamber to balance the suction of the vacuum pump and maintain the pressure at a specified set point. The shelf can be compressed via a hydraulic piston. A small tray of manually dispensed products containing 178x 10 vials (combinations of fourteen different formulations) is loaded onto a single shelf in a freeze dryer at a temperature of +4.7°C. The product is then subjected to a freezing step for 2 hours at a shelf temperature of -50°C, and then the condenser step is started. During the run, the condenser temperature is between -5°C and -87°C. Next, evacuate and run at pressure set points of 75 mTorr and 200 mTorr. Good control on these two pressure set points is shown throughout the run. All steps of the freeze-drying formula / cycle are performed as programmed. Based on the average temperature of the product probe, sublimation lasts for about 7.5 hours, and desorption lasts for about 16.1 hours. At the end of the run, the vial was backfilled with nitrogen and sealed with the stopper fully inserted at a pressure of about 600 Torr (about 740 Torr is atmospheric pressure).

[0447] After lyophilization, nitrogen was applied to each glass vial containing 20x furimazine lyophilized cake to fill the headspace of the vial, completely sealed with a cap, and the vials were stored at 25°C or 60°C, respectively. At various time points after lyophilization, the formulated furimazine was reconstituted with 10 ml of PBS, pH 7.0 containing 0.01% BSA, and the vials were manually shaken and allowed to equilibrate at room temperature for 5 minutes. 50 ul of the formulated furimazine stock solution was added to 50 ul of 1 ng / ml purified in PBS, pH 7 containing 0.01% BSA. Enzyme (Nluc) (Promega catalog number E499) (final [Nluc] = 0.5 ng / ml). For each time point data collection, the control used was freshly sampled from -20°C 10uM final solution of Live Cell Substrate (Promega Cat. No. N205). The assay was performed and analyzed on solid white non-binding surface (NBS) plates using a kinetic readout to collect total luminescence on an Expedition Multimode Microplate Reader – Promega Cat. No. GM3000).

[0448] Fig.62A The lyophilized formulated furazolidone at time point "Day 0" is shown, showing that the vial contained a uniform cake evenly distributed on the bottom of the vial without any obvious defects in appearance, indicating that the formulation and lyophilization protocol were appropriate. Fig.62B The results are shown as raw RLU after reconstitution with buffer using furazolidone formulated as described above. Activity results. Formulated furazolidone and control substrate ( This was the baseline reading to start the accelerated stability study. A portion of the vials or control substrate was then placed at either 60°C or 25°C. A new vial was reconstituted at different time points and purified Enzyme activity analysis. Fig.63 Shown are the raw RLU from formulated samples stored at 25°C (blue closed circles) or 60°C (red squares) as monitored for 34 days, the RLU from formulated samples stored at 25°C (green triangles) or 60°C (orange inverted triangles) as monitored for 34 days. Raw RLU of live cell substrate and freshly prepared control kept at -20°C (black diamonds) Raw RLU of live cell substrate. The vials reconstituted on day 0 were kept in solution, kept at room temperature, and sampled over 18 days to determine activity (light blue open rings). The data show that the formulated furimoxazine remained active at both test temperatures over the time tested and showed improvement over furimoxazine dissolved in an organic solvent. After adding the buffer, all formulated furimoxazine reconstituted within 5 minutes, which is in stark contrast to the behavior of solid furimoxazine.

[0449] Figure 62 and Fig.63The results in demonstrate that the furazolidone composition can be prepared on a larger scale and under more stringent quality control conditions, including in glass vials and under an inert atmosphere. The composition can be stored for extended periods of time at ambient or elevated temperatures and reconstituted in a neutral buffer without the need for organic solvents or special buffer conditions. Even after reconstitution in an aqueous buffer, the composition can be stored in solution and under ambient conditions for up to 24-48 hours without any significant loss of performance and maintains some activity for up to 18 days.

[0450] Embodiment 40

[0451] Preparation of formulated JRW-1744

[0452]

[0453] 6-(3-amino-2-fluorophenyl)-8-benzyl-2-(furan-2-ylmethyl)imidazo[1,2-α]pyrazin-3(7H)-one

[0454] A formulated example of JRW-1744 was prepared in a similar manner to JRW-0238 in Examples 34 and 38. A stock sample of solid formulated JRW-1744 was prepared as follows: 77 mg of F-127 (about 7.2xw / w) was loaded into glass screw cap vials in batches. The polymer was then heated to 80°C in a water bath until it melted (became a clear solution). 10.8mg of JRW-1744 was dissolved in a small amount of EtOH and added to the hot polymer, mixed thoroughly with a thin spatula. Additional EtOH (total amount up to 2mL) was used to completely transfer and dissolve all substrates into the polymer solution. The solvent was then removed under reduced pressure. The concentrated sample was then placed under high vacuum for 1 hour to remove residual EtOH, producing an orange solid. The solid was diluted and sonicated in 3.0mL water to prepare a mother solution of 8.7mM JRW-1744 in water. Then an aliquot of 480μL of the aqueous stock solution was transferred to a 1.5mL screw cap vial, frozen and lyophilized overnight.

[0455] Embodiment 41

[0456] Preparation of formulated JRW-1743

[0457]

[0458] 6-(3-amino-2-fluorophenyl)-8-(2-fluorobenzyl)-2-(furan-2-ylmethyl)imidazo[1,2-α]pyrazin-3(7H)-one

[0459] A formulated example of JRW-1743 was prepared in a similar manner as JRW-0238 in Examples 34 and 38.

[0460] A stock sample of formulated JRW-1743 was prepared as follows: 72 mg (7.2 x w / w) of F-127 was batched and placed in glass screw-top vials. The polymer was then heated in a water bath at 80°C until it was completely melted ( Fig.64A ).

[0461] The solid JRW-1743 of 10.0mg is dissolved in a small amount of EtOH, and is transferred to the hot polymer while stirring with a thin scraper. Extra EtOH (total amount up to 2mL) is used to help substrate is transferred to the polymer solution. Then under reduced pressure, the solvent is removed, and the polymer / substrate mixture is condensed into an orange-red gel. Then the concentrated sample is placed in a high vacuum for 1 hour to remove any residual EtOH.

[0462] In order to A masterbatch of formulated JRW-1743 was prepared in F-127, 2.6 mL of water was added to the gel, and the resulting solution was sonicated until it was completely homogeneous ( Fig.64B ). The final concentration of JRW-1743 at this volume was calculated to be 8.7 mM. A 480 μL aliquot of the aqueous stock solution was then transferred to a 1.5 mL screw cap vial, frozen and lyophilized overnight to produce a lyophilized cake containing JRW-1743 ( Fig.64C ).

[0463] Reconstitute one vial containing formulated JRW-1743 in 480 uL of water ( Fig.64C , middle and right). Absorbance measurements of substrate concentration in water were performed and indicated that the working concentration of JRW-1743 in the solution was found to be 8.5 mM, compared to a theoretical concentration of approximately 8.7 mM ( Fig.65 ).

[0464] This application can be implemented by:

[0465] 1. A composition comprising:

[0466] A compound selected from coelenterazine and its analogs or derivatives; and

[0467] polymer.

[0468] 2. The composition of item 1, wherein the compound is selected from coelenterazine, coelenterazine-h, coelenterazine-hh, furomazine, JRW-0238, JRW-1743 and JRW-1744.

[0469] 3. The composition according to claim 1, wherein the compound is furazolidone.

[0470] 4. The composition of any one of items 1-3, wherein the polymer is a naturally occurring biopolymer.

[0471] 5. The composition of item 4, wherein the naturally occurring biopolymer is selected from the group consisting of pullulan, trehalose, maltose, cellulose, dextran, and any combination thereof.

[0472] 6. The composition of item 5, wherein the naturally occurring biopolymer is pullulan.

[0473] 7. The composition according to any one of items 1 to 3, wherein the polymer is a cyclic sugar polymer or a derivative thereof.

[0474] 8. The composition of item 7, wherein the polymer is hydroxypropyl β-cyclodextrin.

[0475] 9. The composition of any one of items 1-3, wherein the polymer is a synthetic polymer.

[0476] 10. The composition of item 9, wherein the synthetic polymer is selected from the group consisting of polystyrene, poly(meth)acrylate, and any combination thereof.

[0477] 11. The composition of item 9, wherein the synthetic polymer is a block copolymer comprising at least one poly(oxypropylene) block and at least one poly(oxyethylene) block.

[0478] 12. The composition of item 11, wherein the synthetic polymer is poloxamer.

[0479] 13. A composition as described in any one of items 1-12, wherein the composition further comprises a buffer, a surfactant, a reducing agent, a salt, a free radical scavenger, a chelating agent, a protein or any combination thereof.

[0480] 14. A composition as described in item 13, wherein the composition further comprises a buffer selected from phosphate buffer, tris(hydroxymethyl)methylglycine and 2-(N-morpholino)ethanesulfonic acid.

[0481] 15. The composition of item 13 or item 14, wherein the composition further comprises a surfactant selected from polysorbate 20, polysorbate 40 and polysorbate 80.

[0482] 16. The composition of any one of items 13-15, wherein the composition comprises a reducing agent selected from thiourea and 6-aza-2-thiothymidine.

[0483] 17. A composition as described in any of items 13-16, wherein the composition further comprises a salt selected from sodium chloride and sodium phosphate.

[0484] 18. The composition of any one of items 13-17, wherein the composition further comprises a free radical scavenger selected from ascorbic acid and sodium ascorbate.

[0485] 19. The composition of any one of items 13-18, wherein the composition further comprises a chelating agent, and the chelating agent is selected from citric acid and trans-1,2-diaminocyclohexane-tetraacetic acid.

[0486] 20. The composition of any one of items 13-19, wherein the composition further comprises a protein selected from the group consisting of bovine serum albumin, gelatin, and a polypeptide fraction of highly purified dermal collagen of porcine origin.

[0487] 21. The composition of any one of items 1-20, wherein the composition is in the form of a lyophilized powder or a cake.

[0488] 22. The composition of any one of items 1-20, wherein the composition is in the form of an extensible film.

[0489] 23. The composition of any one of items 1-20, wherein the composition is a solution.

[0490] 24. A composition comprising:

[0491] A compound selected from coelenterazine and its analogs or derivatives; and

[0492] Surfaces selected from paper or fiber substrates, plastics, glass or metal.

[0493] 25. The composition of claim 24, wherein the compound is selected from coelenterazine, coelenterazine-h, coelenterazine-hh, furomazine, JRW-0238, JRW-1743, and JRW-1744.

[0494] 26. The composition of claim 24, wherein the compound is furazolidone.

[0495] 27. A composition as described in any of items 24-26, wherein the composition further comprises a polymer.

[0496] 28. The composition of item 27, wherein the polymer is a naturally occurring biopolymer.

[0497] 29. The composition of item 28, wherein the naturally occurring biopolymer is selected from the group consisting of pullulan, trehalose, maltose, cellulose, dextran, and any combination thereof.

[0498] 30. The composition of item 29, wherein the naturally occurring biopolymer is pullulan.

[0499] 31. The composition of item 27, wherein the polymer is a cyclic sugar polymer or a derivative thereof.

[0500] 32. A composition as described in item 31, wherein the polymer is hydroxypropyl β-cyclodextrin.

[0501] 33. The composition of claim 27, wherein the polymer is a synthetic polymer.

[0502] 34. The composition of item 33, wherein the synthetic polymer is selected from the group consisting of polystyrene, poly(meth)acrylate, and any combination thereof.

[0503] 35. A composition as described in item 33, wherein the synthetic polymer is a block copolymer comprising at least one poly(oxypropylene) block and at least one poly(oxyethylene) block.

[0504] 36. The composition of item 35, wherein the synthetic polymer is a poloxamer.

[0505] 37. A composition as described in any of items 24-36, wherein the composition further comprises a buffer, a surfactant, a reducing agent, a salt, a free radical scavenger, a protein or any combination thereof.

[0506] 38. A composition as described in item 37, wherein the composition further comprises a buffer selected from phosphate buffer, tris(hydroxymethyl)methylglycine and 2-(N-morpholino)ethanesulfonic acid.

[0507] 39. The composition of item 37 or item 38, wherein the composition further comprises a surfactant selected from polysorbate 20, polysorbate 40 and polysorbate 80.

[0508] 40. A composition as described in any of items 37-39, wherein the composition comprises a reducing agent selected from thiourea and 6-aza-2-thiothymidine.

[0509] 41. A composition as described in any of items 37-40, wherein the composition further comprises a salt selected from sodium chloride and sodium phosphate.

[0510] 42. A composition as described in any of items 37-41, wherein the composition further comprises a free radical scavenger selected from ascorbic acid and sodium ascorbate.

[0511] 43. A composition as described in any of items 37-42, wherein the composition further comprises a chelating agent, and the chelating agent is selected from citric acid and trans-1,2-diaminocyclohexane-tetraacetic acid.

[0512] 44. A composition as described in any of items 37-43, wherein the composition further comprises a protein selected from bovine serum albumin, gelatin and a polypeptide fraction of highly purified dermal collagen of porcine origin.

[0513] 45. A composition as described in any of items 24-44, wherein the surface is selected from cellulose paper, nitrocellulose paper, nylon paper, cotton paper, polyester paper, sodium carboxymethylcellulose, porous or polymer membranes, high-purity cotton fibers, high-purity cotton of cotton / rayon blends, and glass microfibers.

[0514] 46. ​​A method of stabilizing a compound selected from coelenterazine and an analog or derivative thereof, comprising contacting the coelenterazine compound or an analog or derivative thereof with an effective amount of a polymer and / or a paper or fibrous substrate to form a composition.

[0515] 47. The method of item 46, wherein the compound is stabilized to prevent thermal decomposition, chemical decomposition, light-induced decomposition, or any combination thereof.

[0516] 48. A method of improving the solubility of a compound selected from coelenterazine and an analog or derivative thereof, comprising contacting the coelenterazine compound or an analog or derivative thereof with an effective amount of a polymer and / or a paper or fibrous substrate to form a composition.

[0517] 49. The method of item 48, wherein the solubility of the compound in aqueous solution is improved compared to the compound that has not been contacted with the polymer and / or the paper or fiber substrate.

[0518] 50. A method of improving the reconstitution rate of a compound selected from coelenterazine and its analogs or derivatives, comprising contacting the coelenterazine compound or its analogs or derivatives with an effective amount of a polymer and / or a paper or fiber matrix to form a composition,

[0519] wherein the reconstitution rate of the compound is improved compared to a compound which has not been contacted with the polymer and / or the paper or fiber matrix.

[0520] 51. The method of any one of items 46-50, wherein the compound is selected from coelenterazine, coelenterazine-h, coelenterazine-hh, furazolidone, JRW-0238, JRW-1743, and JRW-1744.

[0521] 52. The method of claim 51, wherein the compound is furazolidone.

[0522] 53. A method as described in any of items 46-52, wherein the polymer is a naturally occurring biopolymer.

[0523] 54. The method of claim 53, wherein the naturally occurring biopolymer is selected from the group consisting of pullulan, trehalose, maltose, cellulose, dextran, and any combination thereof.

[0524] 55. The method of claim 54, wherein the naturally occurring biopolymer is pullulan.

[0525] 56. The method of any one of items 46-52, wherein the polymer is a cyclic sugar polymer or a derivative thereof.

[0526] 57. The method of claim 56, wherein the polymer is hydroxypropyl β-cyclodextrin.

[0527] 58. A method as described in any of items 46-52, wherein the polymer is a synthetic polymer.

[0528] 59. The method of item 58, wherein the synthetic polymer is selected from the group consisting of polystyrene, poly(meth)acrylate, and any combination thereof.

[0529] 60. The method of any one of items 46-52, wherein the synthetic polymer is a block copolymer comprising at least one poly(oxypropylene) block and at least one poly(oxyethylene) block.

[0530] 61. The method of item 60, wherein the synthetic polymer is a poloxamer.

[0531] 62. A method as described in any of items 46-61, wherein the composition further comprises a buffer, a surfactant, a reducing agent, a salt, a free radical scavenger, a protein or any combination thereof.

[0532] 63. A method as described in item 62, wherein the composition further comprises a buffer selected from phosphate buffer, tris(hydroxymethyl)methylglycine and 2-(N-morpholino)ethanesulfonic acid.

[0533] 64. A method as described in item 62 or item 63, wherein the composition further comprises a surfactant selected from polysorbate 20, polysorbate 40 and polysorbate 80.

[0534] 65. A method as described in any of items 62-64, wherein the composition comprises a reducing agent selected from thiourea and 6-aza-2-thiothymidine.

[0535] 66. A method as described in any of items 62-65, wherein the composition further comprises a salt selected from sodium chloride and sodium phosphate.

[0536] 67. A method as described in any of items 62-66, wherein the composition further comprises a free radical scavenger selected from ascorbic acid and sodium ascorbate.

[0537] 68. A method as described in any of items 62-67, wherein the composition further comprises a chelating agent, and the chelating agent is selected from citric acid and trans-1,2-diaminocyclohexane-tetraacetic acid.

[0538] 69. A method as described in any of items 62-68, wherein the composition further comprises a protein selected from bovine serum albumin, gelatin and a polypeptide fraction of highly purified dermal collagen of porcine origin.

[0539] 70. A method as described in any of items 46-69, wherein the paper or fiber substrate is selected from cellulose paper, nitrocellulose paper, nylon paper, cotton paper, polyester paper, sodium carboxymethyl cellulose, porous or polymer membranes, high purity cotton fibers, high purity cotton of cotton / rayon blends, and glass microfibers.

[0540] 71. The method of any one of items 46-70, wherein the contacting step comprises:

[0541] dissolving the compound in an organic solvent to form a first solution;

[0542] mixing the first solution with the polymer and / or the paper or fiber substrate to form a mixture; and

[0543] The mixture was dried.

[0544] 72. The method of item 71, wherein the mixing step comprises dissolving the polymer in a second solution, and mixing the second solution with the first solution.

[0545] 73. The method of clause 71, wherein the mixing step comprises applying the first solution to the paper or fiber substrate.

[0546] 74. A method as described in any of items 71-73, wherein the drying step comprises freeze-drying.

[0547] 75. A method as described in any of items 71-74, wherein the drying step comprises air drying.

[0548] 76. A method as described in any of items 71-74, wherein the drying is carried out at ambient temperature in an inert atmosphere.

[0549] 77. A method as described in any of items 71-74, wherein the drying comprises vacuum drying.

[0550] 78. The method of any one of items 71-74, wherein the drying is carried out at a temperature of about 30°C to about 70°C.

[0551] 79. A method as described in any of items 71-78, wherein one or all of the solutions are deoxygenated.

[0552] 80. The method of any one of items 46-79, wherein the method comprises contacting the compound with the polymer.

[0553] 81. The method of any one of items 46-79, wherein the method comprises contacting the polymer with the paper or fiber substrate.

[0554] 82. The method of any one of items 46-79, wherein the method comprises contacting the polymer with the polymer and the paper or fiber substrate.

[0555] 83. A kit comprising the composition of any one of items 1-45.

[0556] 84. A kit as described in item 83, wherein the composition is included in one or more containers.

[0557] 85. A kit as described in item 84, wherein the composition is included in multiple tubes.

[0558] 86. A kit as described in any of items 83-85, wherein the composition is in the form of a plurality of paper spots, each spot having a diameter of about 2 mm to about 5 mm.

[0559] sequence

[0560] SEQ ID NO: 1 - Amino acid sequence of natural mature spiny shrimp luciferase

[0561] FTLADFVGDWQQTAGYNQDQVLEQGGLSSLFQALGVSVTPIQKVVLSGENGLKADIHVIIPYEGLSGFQMGLIEMIFKVVYPVDDHHFKIILHYGTLVIDGVTPNMIDYFGRPYPGIAVFDGKQITVTGTLWNGNKIYDERLINPDGSLLFRVTINGVTRL

[0562] SEQ ID NO:2—Nluc amino acid sequence

[0563] MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIPIIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFIFVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILA

[0564] SEQ ID NO:3-LgTrip(3546)

[0565] MKHHHHHHVFTLDDFVGDWEQTAAYNLDQVLEQGGVSSLLQNLAVSVTPIMRIVRSGENALKIDIHVIIPYEGLSADQMAQIEEVFK VVYPVDDHHFKVILPYGTLVIDGVTPNKLNYFGRPYEGIAVFDGKKITTTGTLWNGNKIIDERLITPDD

Claims

1. A composition comprising: a compound selected from the group consisting of coelenterazine, coelenterazine-h, coelenterazine-hh, furomazine, JRW-0238, JRW-1743, and JRW-1744; and Surfaces selected from paper or fiber substrates, plastics, glass or metal.

2. The composition of claim 1, wherein the compound is furazolidone.

3. The composition of claim 1 or 2, wherein the composition further comprises a polymer.

4. The composition of claim 3, wherein the polymer is a naturally occurring biopolymer selected from the group consisting of pullulan, trehalose, maltose, cellulose, dextran, and any combination thereof.

5. The composition of claim 4, wherein the naturally occurring biopolymer is pullulan.

6. The composition of claim 3, wherein the polymer is a cyclic sugar polymer selected from the group consisting of α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin and hydroxypropyl-β-cyclodextrin, or a derivative thereof.

7. The composition of claim 6, wherein the polymer is hydroxypropyl β-cyclodextrin.

8. The composition of any one of claims 1-7, wherein the composition further comprises a buffer, a surfactant, a reducing agent, a salt, a free radical scavenger, a protein, or any combination thereof.

9. The composition of claim 8, wherein the composition further comprises a buffer selected from the group consisting of phosphate buffer, tris(hydroxymethyl)methylglycine and 2-(N-morpholino)ethanesulfonic acid.

10. The composition of claim 8 or claim 9, wherein the composition further comprises a surfactant selected from polysorbate 20, polysorbate 40 and polysorbate 80.

11. The composition of any one of claims 8-10, wherein the composition comprises a reducing agent selected from thiourea and 6-aza-2-thiothymidine.

12. The composition of any one of claims 8-11, wherein the composition further comprises a salt selected from sodium chloride and sodium phosphate.

13. The composition of any one of claims 8-12, wherein the composition further comprises a free radical scavenger selected from ascorbic acid and sodium ascorbate.

14. The composition of any one of claims 8-13, wherein the composition further comprises a chelating agent, and the chelating agent is selected from citric acid and trans-1,2-diaminocyclohexane-tetraacetic acid.

15. The composition of any one of claims 8 to 14, wherein the composition further comprises a protein selected from the group consisting of bovine serum albumin, gelatin, and a polypeptide fraction of highly purified dermal collagen of porcine origin.

16. The composition of any one of claims 1-15, wherein the surface is selected from cellulose paper, nitrocellulose paper, nylon paper, cotton paper, polyester paper, sodium carboxymethylcellulose, porous or polymer membranes, high purity cotton fibers, high purity cotton of cotton / rayon blends, and glass microfibers.

17. A method of stabilizing a compound selected from the group consisting of coelenterazine, coelenterazine-h, coelenterazine-hh, furomazine, JRW-0238, JRW-1743, and JRW-1744, comprising contacting the compound with an effective amount of a paper or fiber substrate to form a composition.

18. The method of claim 17, wherein the compound is stabilized against thermal decomposition, chemical decomposition, light-induced decomposition, or any combination thereof.

19. A method of improving the solubility of a compound selected from the group consisting of coelenterazine, coelenterazine-h, coelenterazine-hh, furomazine, JRW-0238, JRW-1743, and JRW-1744, comprising contacting the compound with an effective amount of a paper or fiber substrate to form a composition.

20. The method of claim 19, wherein the solubility of the compound in aqueous solution is improved compared to the compound which has not been contacted with the paper or fiber substrate.

21. A method of increasing the reconstitution rate of a compound selected from the group consisting of coelenterazine, coelenterazine-h, coelenterazine-hh, furomazine, JRW-0238, JRW-1743, and JRW-1744, comprising contacting the compound with an effective amount of a paper or fiber substrate to form a composition, Therein the reconstitution rate of the compound is increased compared to the compound which has not been contacted with the paper or fiber substrate.

22. The method of any one of claims 17-21, wherein the compound is furazolidone.

23. The method of any one of claims 17-22, wherein the composition further comprises a buffer, a surfactant, a reducing agent, a salt, a free radical scavenger, a protein, or any combination thereof.

24. The method of claim 23, wherein the composition further comprises a buffer selected from the group consisting of phosphate buffer, tris(hydroxymethyl)methylglycine, and 2-(N-morpholino)ethanesulfonic acid.

25. The method of claim 23 or 24, wherein the composition further comprises a surfactant selected from polysorbate 20, polysorbate 40 and polysorbate 80.

26. The method of any one of claims 23-25, wherein the composition comprises a reducing agent selected from thiourea and 6-aza-2-thiothymidine.

27. The method of any one of claims 23-26, wherein the composition further comprises a salt selected from sodium chloride and sodium phosphate.

28. The method of any one of claims 23-27, wherein the composition further comprises a free radical scavenger selected from ascorbic acid and sodium ascorbate.

29. The method of any one of claims 23-28, wherein the composition further comprises a chelating agent, and the chelating agent is selected from citric acid and trans-1,2-diaminocyclohexane-tetraacetic acid.

30. The method of any one of claims 23-29, wherein the composition further comprises a protein selected from the group consisting of bovine serum albumin, gelatin, and a polypeptide fraction of highly purified dermal collagen of porcine origin.

31. The method of any one of claims 17-30, wherein the paper or fiber substrate is selected from cellulose paper, nitrocellulose paper, nylon paper, cotton paper, polyester paper, sodium carboxymethylcellulose, porous or polymeric membranes, high purity cotton fibers, high purity cotton of cotton / rayon blends, and glass microfibers.

32. The method of any one of claims 17 to 31, wherein the contacting step comprises: dissolving the compound in an organic solvent to form a first solution; mixing the first solution with the paper or fiber substrate to form a mixture; as well as The mixture was dried.

33. The method of claim 32, wherein the mixing step comprises applying the first solution to the paper or fiber substrate.

34. The method of claim 32 or 33, wherein the drying step comprises air drying.

35. The method of claim 32 or 33, wherein the drying is performed at ambient temperature in an inert atmosphere.

36. The method of claim 32 or 33, wherein the drying comprises vacuum drying.

37. The method of claim 32 or 33, wherein the drying is carried out at a temperature of about 30°C to about 70°C.

38. The method of any one of claims 32-37, wherein the solution is deoxygenated.

39. A kit comprising the composition of any one of claims 1-16.

40. The kit of claim 39, wherein the composition is included in one or more containers.

41. The kit of claim 40, wherein the composition is included in a plurality of tubes.

42. The kit of any one of claims 39-41, wherein the composition is in the form of a plurality of paper spots, each spot having a diameter of about 2 mm to about 5 mm.

Images