Compositions and methods for stabilizing coelenterazine and analogs and derivatives thereof

Abstract

To provide compositions and methods for stabilizing coelenterazine and analogs and derivatives thereof, and for improving the solubility and reconstitution efficiency of coelenterazine and analogs and derivatives thereof.SOLUTION: A composition comprises: (a) a compound selected from among coelenterazine, coelenterazine-h, coelenterazine-h-h, furimazine, JRW-0238, JRW-1743, and JRW-1744; and (b) a surface selected from among a paper or fiber matrix, a plastic, a glass, and a metal.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Patent Application No. 62 / 740,622, filed October 3, 2018, and U.S. Provisional Patent Application No. 62 / 805,517, filed February 14, 2019, each of which is incorporated by reference in its entirety for all purposes.

[0002] Provided herein are compositions and methods for stabilizing coelenterazine and its analogs and derivatives, and for improving the solubility and reconstitution efficiency of coelenterazine and its analogs and derivatives. [Background technology]

[0003] Luminescence is used in biological assays as a measure of the activity of the reporter molecule. The reporter molecule then links the luminescence measurement to a biological process of interest, such as transcription (gene expression), translation (protein expression), protein-protein interaction, etc., thereby allowing for the quantitative measurement of changes that occur in the biological process. Reporter molecules are typically luminescent enzymes (e.g., firefly luciferase, Renilla luciferase, Oplophorus luciferase, etc.) that, when provided with their luminescent substrate, result in the production of light (i.e., luminescence). Summary of the Invention

[0004] Luminescent substrates such as coelenterazine, as well as their analogs and derivatives, can degrade during storage (e.g., storage in organic solvents, storage at higher temperatures, storage at an incorrect pH, etc.), resulting in loss of substrate before addition to or use in a biological assay. Such degradation can be the result of instability of the luminescent substrate in solution over time in a temperature-dependent manner. This degradation results in waste of the luminescent substrate, as well as reduced sensitivity and reproducibility of luminescence measurements derived from biological assays using degraded luminescent substrates. The products of this degradation also inhibit the luminescent reaction. Furthermore, some coelenterazines may have low solubility in different assay buffers or directly in test samples, or exhibit inconsistent reconstitution in different assay buffers. Although coelenterazines can be dissolved in organic solvents before dilution into an appropriate buffer, organic solutions of coelenterazine compounds may suffer from instability on storage (both thermal and photoinstability). However, although solid coelenterazine and coelenterazine analogs and derivatives (e.g., furimazine) are significantly more stable than their organic solutions, they exhibit very poor reconstitution rates and efficiencies, dissolve inconsistently, and are difficult to use directly in assays and other methods, especially when non-organic solvents are required. These shortcomings have significantly limited the number and types of applications for which coelenterazine and its analogs and derivatives have been developed.

[0005] Thus, there is a need for new compositions and / or methods for stabilizing luminescent substrates, improving their solubility, and / or increasing their reconstitution efficiency. In particular, having substrates with improved physical properties and / or solubility would be beneficial for long-term storage (e.g., 12 months or more at room temperature), assay format(s) compatibility, robustness, and user convenience.

[0006] 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 was performed using HPLC, absorbance, and mass spectrometry. Further functional characterization of the substrates under assay-relevant conditions was performed by monitoring bioluminescence via relative light units (RLU) in the presence of NanoLuc® enzyme.

[0007] Provided herein are compositions comprising a compound selected from coelenterazine and analogs or derivatives thereof, and a polymer. In some embodiments, the compound is selected from coelenterazine, coelenterazine-h, coelenterazine-hh, furimazine, JRW-0238, JRW-1743, and JRW-1744. In some embodiments, the compound is furimazine. In some embodiments, the compound is JRW-0238. In some embodiments, the compound is JRW-1743. In some embodiments, the compound is JRW-1744.

[0008] In some embodiments, the polymer is a natural biopolymer. In some embodiments, the natural biopolymer is selected from pullulan, trehalose, maltose, cellulose, dextran, and any combination thereof. In some embodiments, the natural biopolymer is pullulan. In some embodiments, the polymer is a cyclic saccharide 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(propylene oxide) block and at least one poly(ethylene oxide) block. In some embodiments, the synthetic polymer is a poloxamer.

[0009] In some embodiments, the composition further comprises a buffer, a surfactant, a reducing agent, a salt, a radical scavenger, a chelating agent, a protein, or any combination thereof. In some embodiments, the composition further comprises a buffer selected from phosphate buffer, 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 further 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 radical scavenger selected from ascorbic acid and sodium ascorbate. In some embodiments, the composition further comprises a chelating agent, the chelating agent being selected from citric acid and trans-1,2-diaminocyclohexane-tetraacetic acid. In some embodiments, the composition further comprises a protein selected from bovine serum albumin, gelatin, and a polypeptide fraction of highly purified porcine-derived skin collagen.

[0010] 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 malleable film. In some embodiments, the composition is a solution.

[0011] Provided herein is a composition comprising a compound selected from coelenterazine and analogs or derivatives thereof and a surface selected from a paper or fiber matrix, plastic, glass, or metal. In some embodiments, the compound is selected from coelenterazine, coelenterazine-h, coelenterazine-hh, furimazine, JRW-0238, JRW-1743, and JRW-1744. In some embodiments, the compound is furimazine. 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 natural biopolymer. In some embodiments, the natural biopolymer is selected from pullulan, trehalose, maltose, cellulose, dextran, and any combination thereof. In some embodiments, the natural biopolymer is pullulan. In some embodiments, the polymer is a cyclic saccharide 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(propylene oxide) block and at least one poly(ethylene oxide) block. In some embodiments, the synthetic polymer is a poloxamer.

[0012] In some embodiments, the composition further comprises a buffer, a surfactant, a reducing agent, a salt, a radical scavenger, a protein, or any combination thereof. In some embodiments, the composition further comprises a buffer selected from phosphate buffer, 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 further 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 radical scavenger selected from ascorbic acid and sodium ascorbate. In some embodiments, the composition further comprises a chelating agent, the chelating agent being selected from citric acid and trans-1,2-diaminocyclohexane-tetraacetic acid. In some embodiments, the composition further comprises a protein selected from bovine serum albumin, gelatin, and a polypeptide fraction of highly purified porcine-derived skin collagen. 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 / rayon blends, and glass microfibers.

[0013] Provided herein is a method for stabilizing a compound selected from coelenterazine and its analogs or derivatives, the method comprising contacting a 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. In some embodiments, the compound is stabilized against thermal degradation, chemical degradation, light-induced degradation, or any combination thereof.

[0014] Provided herein is a method for improving the solubility of a compound selected from coelenterazine and its analogs or derivatives, the method comprising contacting a 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. In some embodiments, the solubility of the compound is improved in aqueous solution compared to the compound not in contact with the polymer and / or the paper or fiber matrix.

[0015] Provided herein is a method for improving the reconstitution rate of a compound selected from coelenterazine and its analogs or derivatives, the method comprising contacting a 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, wherein the reconstitution rate of the compound is improved compared to the compound not in contact with the polymer or paper or fiber matrix.

[0016] In some embodiments, the compound is selected from coelenterazine, coelenterazine-h, coelenterazine-hh, furimazine, JRW-0238, JRW-1743, and JRW-1744. In some embodiments, the compound is furimazine. 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 natural biopolymer. In some embodiments, the natural biopolymer is selected from pullulan, trehalose, maltose, cellulose, dextran, and any combination thereof. In some embodiments, the natural biopolymer is pullulan. In some embodiments, the polymer is a cyclic saccharide 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(propylene oxide) block and at least one poly(ethylene oxide) block. In some embodiments, the synthetic polymer is a poloxamer.

[0017] In some embodiments, the composition further comprises a buffer, a surfactant, a reducing agent, a salt, a radical scavenger, a protein, or any combination thereof. In some embodiments, the composition further comprises a buffer selected from phosphate buffer, 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 further 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 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 selected from bovine serum albumin, gelatin, and a polypeptide fraction of highly purified porcine-derived skin collagen. In some embodiments, the paper or fiber matrix 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 / rayon blends, and glass microfibers.

[0018] In some embodiments, the contacting step includes dissolving the compound in an organic solvent to form a first solution, mixing the first solution with a polymer and / or a paper or fiber matrix to form a mixture, and drying the mixture. In some embodiments, the mixing step includes dissolving the polymer in a second solution and mixing the second solution with the first solution. In some embodiments, the mixing step includes applying the first solution to a paper or fiber matrix. In some embodiments, the drying step includes freeze drying. In some embodiments, the drying step includes air drying. In some embodiments, the drying is performed at ambient temperature in an inert atmosphere. In some embodiments, the drying includes 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 of the solutions are deoxygenated.

[0019] In some embodiments, the method includes contacting the compound with a polymer. In some embodiments, the method includes contacting the polymer with a paper or fiber matrix. In some embodiments, the method includes contacting the polymer with the polymer and the paper or fiber matrix.

[0020] Provided herein is a kit comprising any one 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]

[0021] [Figure 1A]Figure 1 shows signal kinetics when compositions according to the present disclosure were tested for luminescence output in (A) phosphate buffered saline (PBS) (pH 7.0), and (B) Nano-Glo® Luciferase Assay Buffer, as described in Example 1. Figure 1(C) shows images of furimazine substrate samples in pullulan-based lyophilized cake and pullulan film droplet formulations. [Figure 1B] Figure 1 shows signal kinetics when compositions according to the present disclosure were tested for luminescence output in (A) phosphate buffered saline (PBS) (pH 7.0), and (B) Nano-Glo® Luciferase Assay Buffer, as described in Example 1. Figure 1(C) shows images of furimazine substrate samples in pullulan-based lyophilized cake and pullulan film droplet formulations. [Figure 1C] Figure 1 shows signal kinetics when compositions according to the present disclosure were tested for luminescence output in (A) phosphate buffered saline (PBS) (pH 7.0), and (B) Nano-Glo® Luciferase Assay Buffer, as described in Example 1. Figure 1(C) shows images of furimazine substrate samples in pullulan-based lyophilized cake and pullulan film droplet formulations. [Figure 2A] 1 shows RLU values ​​at various time points after addition of purified NanoLuc® enzyme when compositions according to the present disclosure were tested for luminescence output in PBS (pH 7.0) as described in Example 1. [Figure 2B] 1 shows RLU values ​​at various time points after addition of purified NanoLuc® enzyme when compositions according to the present disclosure were tested for luminescence output in PBS (pH 7.0) as described in Example 1. [Figure 2C] 1 shows RLU values ​​at various time points after addition of purified NanoLuc® enzyme when compositions according to the present disclosure were tested for luminescence output in PBS (pH 7.0) as described in Example 1. [Figure 3A]1 shows RLU values ​​at various time points after addition of purified NanoLuc® enzyme when compositions according to the present disclosure were tested for luminescence output in Nano-Glo® Luciferase Assay Buffer as described in Example 1. [Figure 3B] 1 shows RLU values ​​at various time points after addition of purified NanoLuc® enzyme when compositions according to the present disclosure were tested for luminescence output in Nano-Glo® Luciferase Assay Buffer as described in Example 1. [Figure 3C] 1 shows RLU values ​​at various time points after addition of purified NanoLuc® enzyme when compositions according to the present disclosure were tested for luminescence output in Nano-Glo® Luciferase Assay Buffer as described in Example 1. [Figure 4A] 1 shows absorbance values ​​in aqueous solution when compositions according to the present disclosure were tested for absorbance over the range of 210-600 nm in PBS (pH 6.8) as described in Example 2. [Figure 4B] 1 shows absorbance values ​​in aqueous solution when compositions according to the present disclosure were tested for absorbance over the range of 210-600 nm in PBS (pH 6.8) as described in Example 2. [Figure 4C] 1 shows absorbance values ​​in aqueous solution when compositions according to the present disclosure were tested for absorbance over the range of 210-600 nm in PBS (pH 6.8) as described in Example 2. [Diagram 5] 1 shows images demonstrating the ability of compositions according to the present disclosure to be reconstituted in PBS (pH 7.0), as described in Example 3. [Figure 6A] Figure 3 shows absorbance values ​​over the range of 210-600 nm of pullulan in PBS (pH 6.8) as described in Example 4. [Figure 6B] Figure 3 shows absorbance values ​​over the range of 210-600 nm of pullulan in PBS (pH 6.8) as described in Example 4. [Figure 7A]1 shows representative HPLC traces for a 0% w / v pullulan-based lyophilized cake formulation containing furimazine (A) 0 hours and (B) 5 hours after reconstitution, as described in Example 5. [Figure 7B] 1 shows representative HPLC traces for a 0% w / v pullulan-based lyophilized cake formulation containing furimazine (A) 0 hours and (B) 5 hours after reconstitution, as described in Example 5. [Figure 8A] 1 shows representative HPLC traces for a 2.5% w / v pullulan-based lyophilized cake formulation containing furimazine (A) 0 hours and (B) 5 hours after reconstitution, as described in Example 5. [Figure 8B] 1 shows representative HPLC traces for a 2.5% w / v pullulan-based lyophilized cake formulation containing furimazine (A) 0 hours and (B) 5 hours after reconstitution, as described in Example 5. [Figure 9A] 1 shows representative HPLC traces for a 15% w / v pullulan-based lyophilized cake formulation containing furimazine (A) 0 hours and (B) 5 hours after reconstitution, as described in Example 5. [Figure 9B] 1 shows representative HPLC traces for a 15% w / v pullulan-based lyophilized cake formulation containing furimazine (A) 0 hours and (B) 5 hours after reconstitution, as described in Example 5. [Figure 10A] Representative HPLC traces for Nano-Glo® Luciferase Assay Substrate after (A) 0 hours and (B) 5 hours of reconstitution, as described in Example 5, are shown. [Figure 10B] Representative HPLC traces for Nano-Glo® Luciferase Assay Substrate after (A) 0 hours and (B) 5 hours of reconstitution, as described in Example 5, are shown. [Figure 11A] FIG. 1 shows an analysis of HPLC traces for formulated furimazine samples with and without pullulan, showing (A) absorbance at 254 nm over time, and (B) peak area over time, as described in Example 5. [Figure 11B] FIG. 1 shows an analysis of HPLC traces for formulated furimazine samples with and without pullulan, showing (A) absorbance at 254 nm over time, and (B) peak area over time, as described in Example 5. [Figure 12] 1 shows data from HPLC traces for formulated furimazine samples with and without pullulan, illustrating the production of aminopyrazine degradation products over time, as described in Example 5. [Figure 13A] (A) Kinetic analysis of RLU values ​​when compositions were tested for luminescence output as described in Example 6, (B) RLU values ​​at time 0 when compositions were tested for luminescence output as described in Example 6, and (C) images of paper spots made from Whatman® 903 protein saver cards for punching holes, prepared as described in Example 6. [Figure 13B] (A) Kinetic analysis of RLU values ​​when compositions were tested for luminescence output as described in Example 6, (B) RLU values ​​at time 0 when compositions were tested for luminescence output as described in Example 6, and (C) images of paper spots made from Whatman® 903 protein saver cards for punching holes, prepared as described in Example 6. [Figure 13C] (A) Kinetic analysis of RLU values ​​when compositions were tested for luminescence output as described in Example 6, (B) RLU values ​​at time 0 when compositions were tested for luminescence output as described in Example 6, and (C) images of paper spots made from Whatman® 903 protein saver cards for punching holes, prepared as described in Example 6. [Figure 14] 14A-B show images of formulated furimazine samples dried onto Whatman® 903 protein saver cards and kept at (A) 4° C. for 2 weeks or (B) 4° C. or 25° C. for 3 months, as further described in Example 7. [Figure 15]15A-D show data demonstrating the effect of additives on assay performance of formulated furimazine samples dried onto paper spots made from Whatman® 903 Protein Saver cards for hole punching, as described in Example 8. [Figure 16] 1 shows data demonstrating the RLU output of formulated furimazine samples on paper spots made from hole punched Whatman® 903 Protein Saver cards, prepared as described in Example 9. [Figure 17A] FIG. 1 shows data demonstrating the RLU output of furimazine samples on paper spots made from Whatman® 903 Protein Saver cards for hole punching as described in Example 9 and tested after 1 day of storage at (A) 4° C., (B) 25° C., and (C) 37° C. [Figure 17B] FIG. 1 shows data demonstrating the RLU output of furimazine samples on paper spots made from Whatman® 903 Protein Saver cards for hole punching as described in Example 9 and tested after 1 day of storage at (A) 4° C., (B) 25° C., and (C) 37° C. [Figure 17C] FIG. 1 shows data demonstrating the RLU output of furimazine samples on paper spots made from Whatman® 903 Protein Saver cards for hole punching as described in Example 9 and tested after 1 day of storage at (A) 4° C., (B) 25° C., and (C) 37° C. [Figure 18A] FIG. 1 shows data demonstrating the RLU output of formulated furimazine samples on paper spots made from Whatman® 903 Protein Saver cards for hole punching as described in Example 9 and tested after storage for 3 days at (A) 4° C., (B) 25° C., and (C) 37° C. [Figure 18B] FIG. 1 shows data demonstrating the RLU output of formulated furimazine samples on paper spots made from Whatman® 903 Protein Saver cards for hole punching as described in Example 9 and tested after storage for 3 days at (A) 4° C., (B) 25° C., and (C) 37° C. [Figure 18C] FIG. 1 shows data demonstrating the RLU output of formulated furimazine samples on paper spots made from Whatman® 903 Protein Saver cards for hole punching as described in Example 9 and tested after storage for 3 days at (A) 4° C., (B) 25° C., and (C) 37° C. [Figure 19A] 1 shows data for formulated furimazine samples placed on paper spots made from hole punch Whatman® 903 protein saver cards, pretreated with different protein buffers, and tested for activity with purified NanoLuc® enzyme as described in Example 10, showing RLU output after storing spots at (A) 60° C. and (B) 25° C., and % activity over time after storing spots at (C) 60° C. and (D) 25° C. [Figure 19B] 1 shows data for formulated furimazine samples placed on paper spots made from hole punch Whatman® 903 protein saver cards, pretreated with different protein buffers, and tested for activity with purified NanoLuc® enzyme as described in Example 10, showing RLU output after storing spots at (A) 60° C. and (B) 25° C., and % activity over time after storing spots at (C) 60° C. and (D) 25° C. [Figure 19C] 1 shows data for formulated furimazine samples placed on paper spots made from hole punch Whatman® 903 protein saver cards, pretreated with different protein buffers, and tested for activity with purified NanoLuc® enzyme as described in Example 10, showing RLU output after storing spots at (A) 60° C. and (B) 25° C., and % activity over time after storing spots at (C) 60° C. and (D) 25° C. [Figure 19D]1 shows data for formulated furimazine samples placed on paper spots made from hole punch Whatman® 903 protein saver cards, pretreated with different protein buffers, and tested for activity with purified NanoLuc® enzyme as described in Example 10, showing RLU output after storing spots at (A) 60° C. and (B) 25° C., and % activity over time after storing spots at (C) 60° C. and (D) 25° C. [Figure 20A] FIG. 1 shows accelerated stability data demonstrating the RLU output of formulated furimazine samples in paper spots made from Whatman® 903 Protein Saver cards for hole punching and tested for substrate activity over several days stored at 25° C. or 60° C., as described in Example 10, showing the RLU output after storing the spots at (A) 60° C. and (B) 25° C., and the % activity over time after storing the spots at (C) 60° C. and (D) 25° C. [Figure 20B] FIG. 1 shows accelerated stability data demonstrating the RLU output of formulated furimazine samples in paper spots made from Whatman® 903 Protein Saver cards for hole punching and tested for substrate activity over several days stored at 25° C. or 60° C., as described in Example 10, showing the RLU output after storing the spots at (A) 60° C. and (B) 25° C., and the % activity over time after storing the spots at (C) 60° C. and (D) 25° C. [Figure 20C] FIG. 1 shows accelerated stability data demonstrating the RLU output of formulated furimazine samples in paper spots made from Whatman® 903 Protein Saver cards for hole punching and tested for substrate activity over several days stored at 25° C. or 60° C., as described in Example 10, showing the RLU output after storing the spots at (A) 60° C. and (B) 25° C., and the % activity over time after storing the spots at (C) 60° C. and (D) 25° C. [Figure 20D]FIG. 1 shows accelerated stability data demonstrating the RLU output of formulated furimazine samples in paper spots made from Whatman® 903 Protein Saver cards for hole punching and tested for substrate activity over several days stored at 25° C. or 60° C., as described in Example 10, showing the RLU output after storing the spots at (A) 60° C. and (B) 25° C., and the % activity over time after storing the spots at (C) 60° C. and (D) 25° C. [Figure 21A] FIG. 13 shows data demonstrating the RLU output of formulated furimazine samples on paper spots made from Whatman® 903 Protein Saver cards for hole punching and prepared using different drying methods, as described in Example 11. [Figure 21B] FIG. 13 shows data demonstrating the RLU output of formulated furimazine samples on paper spots made from Whatman® 903 Protein Saver cards for hole punching and prepared using different drying methods, as described in Example 11. [Figure 21C] FIG. 13 shows data demonstrating the RLU output of formulated furimazine samples on paper spots made from Whatman® 903 Protein Saver cards for hole punching and prepared using different drying methods, as described in Example 11. [Figure 22A]

[0036] Figure 13 shows data demonstrating the RLU output and percent activity over several days of formulated furimazine samples on paper spots made from Whatman® 903 Protein Saver cards for hole punching and prepared using different drying methods, as described in Example 11. [Figure 22B]

[0036] Figure 13 shows data demonstrating the RLU output and percent activity over several days of formulated furimazine samples on paper spots made from Whatman® 903 Protein Saver cards for hole punching and prepared using different drying methods, as described in Example 11. [Figure 22C]

[0036] Figure 13 shows data demonstrating the RLU output and percent activity over several days of formulated furimazine samples on paper spots made from Whatman® 903 Protein Saver cards for hole punching and prepared using different drying methods, as described in Example 11. [Figure 22D]

[0036] Figure 13 shows data demonstrating the RLU output and percent activity over several days of formulated furimazine samples on paper spots made from Whatman® 903 Protein Saver cards for hole punching and prepared using different drying methods, as described in Example 11. [Figure 23A] FIG. 1 shows HPLC traces for a representative pullulan-based lyophilized furimazine sample after storage at 60° C. for A-0 hours and B-48 hours, as described in Example 12. [Figure 23B] FIG. 1 shows HPLC traces for a representative pullulan-based lyophilized furimazine sample after storage at 60° C. for A-0 hours and B-48 hours, as described in Example 12. [Figure 24A] 24A and 24B show HPLC traces of commercially available Nano-Glo® Luciferase Assay Substrate samples after storage (at 60° C. for 0 hours, FIG. 24A and 48 hours, FIG. 24B), as described in Example 12. [Figure 24B] 24A and 24B show HPLC traces of commercially available Nano-Glo® Luciferase Assay Substrate samples after storage (at 60° C. for 0 hours, FIG. 24A and 48 hours, FIG. 24B), as described in Example 12. [Figure 25A] 1 shows an analysis of HPLC data demonstrating the thermal stability of formulated furimazine samples as green area at 25° C., as described in Example 12. [Figure 25B] FIG. 25B shows an analysis of HPLC data demonstrating the thermal stability of formulated furimazine samples as green area at 60° C., as described in Example 12. [Figure 25C] 1 shows an analysis of HPLC data demonstrating the thermal stability of formulated furimazine samples as area percent at 25° C., as described in Example 12. [Figure 25D] 1 shows an analysis of HPLC data demonstrating the thermal stability of formulated furimazine samples as area percent at 60° C., as described in Example 12. [Figure 26A] 1 shows RLU data for formulated furimazine samples tested with purified NanoLuc® enzyme after storage of furimazine samples. Data is shown for samples stored at 60° C. for various periods of time before being reconstituted and tested with 50 μM substrate as described in Example 12. [Figure 26B] 1 shows RLU data for formulated furimazine samples tested with purified NanoLuc® enzyme after storage of the furimazine samples. Data is shown for samples stored at 60° C. for various periods of time before being reconstituted and tested with 10 μM substrate as described in Example 12. [Figure 26C] 1 shows RLU data for formulated furimazine samples tested with purified NanoLuc® enzyme after storage of furimazine samples. Data is shown for samples stored at 60° C. for various periods of time before being reconstituted and tested with 0.1 μM substrate as described in Example 12. [Figure 26D] 4 shows RLU data for formulated furimazine samples tested with purified NanoLuc® enzyme after storage of furimazine samples. Data is shown for samples stored at 25° C. for various periods of time before being reconstituted and tested with 50 μM substrate. [Figure 26E] 4 shows RLU data for formulated furimazine samples tested with purified NanoLuc® enzyme after storage of furimazine samples. Data is shown for samples stored at 25° C. for various periods of time before being reconstituted and tested with 10 μM substrate. [Figure 26F] Figure 1 shows RLU data for formulated furimazine samples tested with purified NanoLuc® enzyme after storage of furimazine samples. Data is shown for samples stored at 25°C for various periods of time before being reconstituted and tested with 0.1 μM substrate (F). [Figure 27A]1 shows percent substrate activity at time zero when formulated furimazine samples were tested for activity with purified NanoLuc® enzyme after storage of the furimazine samples. Data is shown for samples stored at 60° C. for various periods of time before being reconstituted and tested with 50 μM substrate as described in Example 12. [Figure 27B] 1 shows percent substrate activity at time zero when formulated furimazine samples were tested for activity with purified NanoLuc® enzyme after storage of the furimazine samples. Data is shown for samples stored at 60° C. for various periods of time before being reconstituted and tested with 10 μM substrate as described in Example 12. [Figure 27C] 1 shows percent substrate activity at time zero when formulated furimazine samples were tested for activity with purified NanoLuc® enzyme after storage of the furimazine samples. Data is shown for samples stored at 60° C. for various periods of time before being reconstituted and tested with 0.1 μM substrate as described in Example 12. [Figure 27D] Figure 1 shows the percent substrate activity at time zero when formulated furimazine samples were tested for activity with purified NanoLuc® enzyme after storage of the furimazine samples. Data is shown for samples stored at 25°C for various periods of time before being reconstituted and tested with 50 μM substrate. [Figure 27E] Figure 1 shows percent substrate activity at time zero when formulated furimazine samples were tested for activity with purified NanoLuc® enzyme after storage of the furimazine samples. Data is shown for samples stored at 25°C for various periods of time before being reconstituted and tested with 10 μM substrate. [Figure 27F] Figure 1 shows percent substrate activity at time zero when formulated furimazine samples were tested for activity with purified NanoLuc® enzyme after storage of the furimazine samples. Data is shown for samples stored at 25°C for various periods of time before being reconstituted and tested with 0.1 μM substrate. [Figure 28A]1 shows RLU data for formulated pullulan film-coated 96-well microtiter plates containing furimazine substrate when tested with purified NanoLuc® enzyme as described in Example 13. [Figure 28B] 1 shows RLU data for formulated pullulan film-coated 96-well microtiter plates containing furimazine substrate when tested with purified NanoLuc® enzyme as described in Example 13. [Figure 28C] 1 shows RLU data for formulated pullulan film-coated 96-well microtiter plates containing furimazine substrate when tested with purified NanoLuc® enzyme as described in Example 13. [Figure 29] As described in Example 13, a representative example of a pullulan-based film format containing furimazine coating the bottom of a standard 96-well microtiter plate in a pullulan film matrix is ​​shown. [Figure 30A] Representative example data are shown for pullulan-based films containing furimazine alone or also containing NanoLuc® enzyme attached to the bottom of a standard 96-well microtiter plate after reaction with NanoLuc® enzyme or simple reconstitution with PBS for wells in which NanoLuc® enzyme was placed simultaneously with a furimazine formulation, as described in Example 13; Figure 30A shows raw RLU, Figure 30B shows % activity, and Figure 30C shows % activity over 10 days. [Figure 30B] Representative example data are shown for pullulan-based films containing furimazine alone or also containing NanoLuc® enzyme attached to the bottom of a standard 96-well microtiter plate after reaction with NanoLuc® enzyme or simple reconstitution with PBS for wells in which NanoLuc® enzyme was placed simultaneously with a furimazine formulation, as described in Example 13; Figure 30A shows raw RLU, Figure 30B shows % activity, and Figure 30C shows % activity over 10 days. [Figure 30C] Representative example data are shown for pullulan-based films containing furimazine alone or also containing NanoLuc® enzyme attached to the bottom of a standard 96-well microtiter plate after reaction with NanoLuc® enzyme or simple reconstitution with PBS for wells in which NanoLuc® enzyme was placed simultaneously with a furimazine formulation, as described in Example 13; Figure 30A shows raw RLU, Figure 30B shows % activity, and Figure 30C shows % activity over 10 days. [Figure 31A] FIG. 1 shows the normalized absorbance of degradation products of furimazine prepared as pullulan-based lyophilized cakes after storage at 25° C. (first bar) or 60° C. (second bar) for each condition compared to the commercially available furimazine product, as described in Example 14. [Figure 31B] FIG. 1 shows the normalized absorbance of degradation products of furimazine prepared as pullulan-based lyophilized cakes after storage at 25° C. (first bar) or 60° C. (second bar) for each condition compared to the commercially available furimazine product, as described in Example 14. [Figure 31C] FIG. 1 shows the normalized absorbance of degradation products of furimazine prepared as pullulan-based lyophilized cakes after storage at 25° C. (first bar) or 60° C. (second bar) for each condition compared to the commercially available furimazine product, as described in Example 14. [Figure 31D] FIG. 1 shows the normalized absorbance of degradation products of furimazine prepared as pullulan-based lyophilized cakes after storage at 25° C. (first bar) or 60° C. (second bar) for each condition compared to the commercially available furimazine product, as described in Example 14. [Figure 31E] FIG. 1 shows the normalized absorbance of degradation products of furimazine prepared as pullulan-based lyophilized cakes after storage at 25° C. (first bar) or 60° C. (second bar) for each condition compared to the commercially available furimazine product, as described in Example 14. [Fig. 31F]FIG. 1 shows the normalized absorbance of degradation products of furimazine prepared as pullulan-based lyophilized cakes after storage at 25° C. (first bar) or 60° C. (second bar) for each condition compared to the commercially available furimazine product, as described in Example 14. [Figure 32A] Figure 1 shows the relative area percent of degradation products of furimazine 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 commercially available furimazine product, as described in Example 14. [Figure 32B] Figure 1 shows the relative area percent of degradation products of furimazine 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 commercially available furimazine product, as described in Example 14. [Figure 32C] Figure 1 shows the relative area percent of degradation products of furimazine 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 commercially available furimazine product, as described in Example 14. [Fig. 32D] Figure 1 shows the relative area percent of degradation products of furimazine 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 commercially available furimazine product, as described in Example 14. [Figure 32E] Figure 1 shows the relative area percent of degradation products of furimazine 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 commercially available furimazine product, as described in Example 14. [Fig. 32F] Figure 1 shows the relative area percent of degradation products of furimazine 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 commercially available furimazine product, as described in Example 14. [Diagram 33]33A-B show data for a representative example of a pullulan-based format containing furimazine stored at room temperature for six months, as described in Example 15. [Diagram 34] 34A-C show representative examples of HPLC analysis of furimazine samples dried onto different types of paper matrices, as described in Example 16. [Diagram 35] Figures 35A-B show representative examples of bioluminescence signals from furimazine samples formulated with the reporter protein, LgTrip, on three different solid phase materials after reconstitution. [Diagram 36] 36A-C show representative examples of HPLC analysis of furimazine samples stored as a 1:1 mixture with ascorbic acid on different types of paper matrices, as described in Example 17. [Figure 37] 37A-C show representative examples of HPLC analysis of furimazine samples stored on paper matrices pretreated with 30% citric acid, as described in Example 18. [Figure 38] 38A-C show representative examples of HPLC analysis of furimazine samples stored on paper matrices after pre-treatment of the matrices with water and drying overnight under vacuum as described in Example 19. [Figure 39A] A representative example of HPLC analysis of furimazine stored as a 1:1 mixture with citric acid on different paper matrices as described in Example 20 is shown. [Figure 39B] A representative example of HPLC analysis of furimazine stored as a 1:1 mixture with citric acid on different paper matrices as described in Example 20 is shown. [Figure 39C] A representative example of HPLC analysis of furimazine stored as a 1:1 mixture with citric acid on different paper matrices as described in Example 20 is shown. [Figure 39D] A representative example of HPLC analysis of furimazine stored as a 1:1 mixture with citric acid on different paper matrices as described in Example 20 is shown. [Figure 40A]Representative examples of maximum RLU (top) and % activity (bottom) of formulated furimazine samples on Whatman® 903 paper spots made from punched Whatman® 903 protein saver cards treated with furimazine at a 1:1 molar ratio with either citrate or ascorbate in the presence or absence of protein buffer and prepared using different drying methods as described in Example 21. [Figure 40B] Representative examples of maximum RLU (top) and % activity (bottom) of formulated furimazine samples on Whatman® 903 paper spots made from punched Whatman® 903 protein saver cards treated with furimazine at a 1:1 molar ratio with either citrate or ascorbate in the presence or absence of protein buffer and prepared using different drying methods as described in Example 21. [Figure 40C] Representative examples of maximum RLU (top) and % activity (bottom) of formulated furimazine samples on Whatman® 903 paper spots made from punched Whatman® 903 protein saver cards treated with furimazine at a 1:1 molar ratio with either citrate or ascorbate in the presence or absence of protein buffer and prepared using different drying methods as described in Example 21. [Diagram 41]

[0036] Figure 2 shows data demonstrating the RLU output and % activity over several days of formulated furimazine samples on paper spots made from hole punch Whatman® 903 Protein Saver cards stored under different conditions and sampled over several days stored at 25°C as described in Example 22. [Figure 42A]FIG. 2 shows data demonstrating the RLU output and % signal recovery of formulated furimazine samples in paper spots made from Whatman® 903 Protein Saver cards for hole punching before and after removing the spots from the original wells to determine if the substrate is released from the solid matrix support, as described in Example 23. [Figure 42B] FIG. 2 shows data demonstrating the RLU output and % signal recovery of formulated furimazine samples in paper spots made from Whatman® 903 Protein Saver cards for hole punching before and after removing the spots from the original wells to determine if the substrate is released from the solid matrix support, as described in Example 23. [Figure 42C] FIG. 2 shows data demonstrating the RLU output and % signal recovery of formulated furimazine samples in paper spots made from Whatman® 903 Protein Saver cards for hole punching before and after removing the spots from the original wells to determine if the substrate is released from the solid matrix support, as described in Example 23. [Figure 43A]

[0036] Figure 2 shows data demonstrating the RLU output and % activity at varying pH of various formulated furimazine solutions containing different sugar or polymer components, and with or without ascorbate, on paper spots made from Whatman® 903 protein saver cards for hole punching, as described in Example 24. [Figure 43B]

[0036] Figure 2 shows data demonstrating the RLU output and % activity at varying pH of various formulated furimazine solutions containing different sugar or polymer components, and with or without ascorbate, on paper spots made from Whatman® 903 protein saver cards for hole punching, as described in Example 24. [Figure 43C]

[0036] Figure 2 shows data demonstrating the RLU output and % activity at varying pH of various formulated furimazine solutions containing different sugar or polymer components, and with or without ascorbate, on paper spots made from Whatman® 903 protein saver cards for hole punching, as described in Example 24. [Figure 44A] FIG. 2 shows data demonstrating the RLU output and % activity of various formulated furimazine solution components at a fixed pH=7.0 on paper spots made from Whatman® 903 protein saver cards for hole punching, as described in Example 25. [Figure 44B] FIG. 2 shows data demonstrating the RLU output and % activity of various formulated furimazine solution components at a fixed pH=7.0 on paper spots made from Whatman® 903 protein saver cards for hole punching, as described in Example 25. [Figure 44C] FIG. 2 shows data demonstrating the RLU output and % activity of various formulated furimazine solution components at a fixed pH=7.0 on paper spots made from Whatman® 903 protein saver cards for hole punching, as described in Example 25. [Diagram 45] 45A-B show data demonstrating the RLU output of various formulated furimazine solutions in paper spots sampled over several days made from Whatman® 903 Protein Saver cards for hole punching and stored at 25° C., as described in Example 26. [Figure 46] Figures 46A-B show data demonstrating the RLU output of various formulated furimazine solutions containing Prionex, ascorbate, and / or ATT on paper spots sampled over several days made from Whatman® 903 protein saver cards for hole punching and stored at 25°C as described in Example 27. [Figure 47A]2 shows data demonstrating the RLU output of furimazine formulations that have been lyophilized directly into 96-well microtiter plates, as described in Example 28. [Figure 47B] 2 shows data demonstrating the RLU output of furimazine formulations that have been lyophilized directly into 96-well microtiter plates, as described in Example 28. [Figure 48] FIG. 13 shows a prophetic diagram of the assembly of an exemplary layered assay format in which a furimazine formulation is placed in one layer of a multi-layer device, as described in Example 29. [Figure 49] 1 shows data demonstrating the RLU output of Nano-Glo® Substrate (Promega Catalog No. N113) formulations containing sodium ascorbate at 37° C., as described in Example 30. [Figure 50] 3 shows data demonstrating the RLU output of a lyophilized Nano-Glo® Substrate (Promega Catalog No. N113) formulation containing hydroxypropyl-β-cyclodextrin, as described in Example 31. [Figure 51] 51A-B show data demonstrating the RLU output of Nano-Glo® Substrate (Promega Catalog No. N113) formulations containing certain individual and combinations of buffer additives, as described in Example 32. [Figure 52] 3 shows data demonstrating the RLU output of Nano-Glo® Substrate (Promega Catalog No. N113) formulations containing mixed polymers of pullulan and hydroxypropyl-β-cyclodextrin along with other buffer additives, as described in Example 33. [Figure 53] 1 shows images of representative examples of formulated substrates as described in Example 34. [Figure 54A] A representative example of HPLC analysis of a sample of JRW-0238 formulated with Pluronic® F-127, as described in Example 34, is shown. [Figure 54B]A representative example of HPLC analysis of a sample of JRW-0238 formulated with Pluronic® F-127, as described in Example 34, is shown. [Fig. 54C] A representative example of HPLC analysis of a sample of JRW-0238 formulated with Pluronic® F-127, as described in Example 34, is shown. [Figure 55A] 1 shows a representative example of HPLC analysis of a sample of furimazine formulated in Pluronic® F-127, as described in Example 35. [Figure 55B] 1 shows a representative example of HPLC analysis of a sample of furimazine formulated in Pluronic® F-127, as described in Example 35. [Figure 55C] 1 shows a representative example of HPLC analysis of a sample of furimazine formulated in Pluronic® F-127, as described in Example 35. [Figure 56] 1 shows a representative image of a solution sample of JRW-0238 formulated with Pluronic® F-127, as described in Example 36. [Figure 57] A representative example of HPLC analysis of a sample of JRW-0238 formulated with Pluronic® F-127, as described in Example 36, is shown. [Figure 58] 58A-B show representative images of a sample of JRW-0238 formulated with Pluronic® F-127, as described in Example 37. [Figure 59] 59A-B show representative images of samples of formulated JRW-0238, as described in Example 38. [Figure 60] 60A-B show traces and images from mice injected intraperitoneally with reconstituted formulated JRW-0238 as described in Example 38. [Figure 61] 61A-B show traces and images from mice injected subcutaneously with reconstituted formulated JRW-0238 as described in Example 38. [Figure 62] Figures 62A-B show images of formulated furimazine lyophilized cake in amber glass vials after scale-up and manufacturing, as well as the activity of this substrate at day "0" against freshly prepared NanoGlo® live cell substrate, as described in Example 39. [Figure 63] 1 shows RLU values ​​at various time points after addition of purified NanoLuc® enzyme when compositions according to the present disclosure were incubated at 25° C. or 60° C. and tested for luminescence output in PBS (pH 7.0) containing 0.01% BSA, as described in Example 39. [Figure 64] Figures 64A-C show images of JRW-1743 during various synthesis / formulation steps: (A) the vial on the left contains molten Pluronic® F-127 and the vial on the right contains JRW-1743 dissolved in EtOH; (B) JRW-1743 after removal of EtOH, the substrate / polymer mixture was reconstituted in 2.6 mL of pure water to a final concentration of 8.5 mM; (C) a representative example of formulated JRW-1743 after lyophilization: JRW-1743 in a dry Pluronic® F-127 matrix (left) and the same material after reconstitution in pure water (center and right). [Figure 65] Representative absorbance traces of JRW-1743 after formulation with Pluronic® F-127 and reconstitution in nanopure water are shown. The concentration of the substrate in solution was determined by absorbance. The average concentration of JRW-1743 was experimentally determined to be 8.5 mM in water. The calculated theoretical concentration of the dried formulated substrate was 8.7 mM.

[0022] definition Although any method and material similar or equivalent to those described herein can be used to practice or test the embodiments described herein, some preferred methods, compositions, devices, and materials are described herein. However, in 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 protocols described herein, as these may vary through routine experimentation and optimization. It should also be understood that the terms used herein are intended to describe only specific versions or embodiments, and are not intended to limit the scope of the embodiments described herein.

[0023] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. However, in case of conflict, the present specification, including definitions, shall prevail. Therefore, in the context of the embodiments described herein, the following definitions shall apply.

[0024] As used herein, the terms "Oplophorus luciferase" and "Oplophorus-derived luciferase" are used interchangeably and refer to luciferase secreted from the deep-sea shrimp Oplophorus gracilirostris (e.g., SEQ ID NO: 1), including wild-type, variants, and mutants thereof. For example, suitable Oplophorus 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 Oplophorus-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").

[0025] As used herein, the term "polymer" refers to an organic compound that contains two or more repeating units covalently linked in a chain, which may be linear or branched. Typically, a polymer is composed of one or more repeating units that are linked together by covalent chemical bonds to form a linear backbone. The repeating units may be the same or different. Thus, structures of the -AAAA- type (where A is a repeating unit) are polymers, also known as homopolymers. Structures of the -ABAB- or -AAABAAAB- type (where A and B are repeating units) are also polymers, sometimes referred to as copolymers. 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-saccharide-based 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 restricted 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 more, or in a range of, e.g., 2-10, 5-25, 10-50, greater than 100, etc.).

[0026] As used in this specification and 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 "polymer" is a reference to one or more polymers and equivalents thereof known to those skilled in the art, and so forth.

[0027] As used herein, the term "comprise" and linguistic variations thereof indicate the presence of the recited feature(s), element(s), method step(s), etc., without excluding the presence of additional feature(s), element(s), method step(s), etc. Conversely, the term "consisting of" and linguistic variations thereof indicate the presence of the recited feature(s), element(s), method step(s), etc., and excludes unrecited feature(s), element(s), method step(s), etc., except for impurities normally associated therewith. The phrase "consisting essentially of" indicates the recited feature(s), element(s), method step(s), etc., as well as any additional feature(s), element(s), method step(s), etc. that do not substantially affect the basic nature of the composition, system, or method. Many embodiments herein are described using the open-ended language of "comprising." Such embodiments include closed-ended embodiments of "consisting of" and / or "consisting essentially of" and may alternatively be claimed or described using such language. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0028] Provided herein are compositions comprising a compound selected from coelenterazine and its analogs or derivatives and a polymer and / or a paper or fiber matrix, or other surface, such as plastic or glass. In some embodiments, the composition stabilizes the compound against degradation (e.g., thermal, chemical, light-induced, etc.). In some embodiments, the composition stabilizes the compound against degradation compared to a composition that does not contain a polymer and / or a paper or fiber matrix or other surface. In some embodiments, the composition reduces or inhibits the formation of one or more degradation products from the compound (e.g., compared to a composition that does not contain a polymer or a paper or fiber matrix or other surface). In some embodiments, the composition improves the reconstitution efficiency of coelenterazine or its analogs or derivatives. In some embodiments, the composition improves kinetic solubility (e.g., compared to a composition that does not contain a polymer and / or a paper or fiber matrix or other surface).

[0029] The composition includes a compound selected from coelenterazine and analogs or derivatives thereof. When incorporated into the composition, the compound can be protected against degradation (e.g., thermal degradation, chemical degradation, light-induced degradation, etc.).

[0030] In some embodiments, the compound is coelenterazine, which has the structure: [ka] In some embodiments, the compound is a coelenterazine analog or derivative. Exemplary coelenterazine analogs include coelenterazine-h (2-deoxycoelenterazine or 2,8-dibenzyl-6-(4-hydroxyphenyl)imidazo[1,2-a]pyrazin-3(7H)-one), coelenterazine-hh (dideoxycoelenterazine or 2,8-dibenzyl-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one), furimazine (8-benzyl-2-(furan-2-ylmethyl) -6-phenylimidazo[1,2-a]pyrazin-3(7H)-one), JRW-0238 (8-benzyl-2-(furan-2-ylmethyl)-6-(3-hydroxyphenyl)imidazo[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 structure: [ka]

[0031] 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-methylcoelenterazine, and the like. In some embodiments, the compound is a compound as described in WO2003 / 040100, U.S. Patent Publication No. 2008 / 0248511 (e.g., paragraph

[0086] ), U.S. Patent No. 8,669,103, WO2012 / 061529, U.S. Patent Publication No. 2017 / 0233789, U.S. Patent No. 9,924,073, U.S. Patent Publication No. 2018 / 0030059, U.S. Patent No. 10,000,500, U.S. Patent Publication No. 2018 / 0155350, U.S. Provisional Patent Application No. 62 / 665,346, U.S. Patent Application No. 16 / 399,410, U.S. Provisional Patent Application No. 62 / 721,708, U.S. Patent Application No. 16 / 548,214, U.S. Patent Publication No. 2016 ...2016 / 0233789, U No. 014 / 0227759, U.S. Pat. No. 9,840,730, U.S. Pat. No. 7,268,229, U.S. Pat. No. 7,537,912, U.S. Pat. No. 8,809,529, U.S. Pat. No. 9,139,836, U.S. Pat. No. 10,077,244, U.S. Pat. No. 9,487,520, U.S. Pat. No. 9,924,073, U.S. Pat. No. 9,938,564, U.S. Pat. No. 9,951,373, U.S. Pat. No. 10,280,447, U.S. Pat. No. 10,308,975, U.S. Pat. No. 10,428,075, the disclosures of which are incorporated herein by reference in their entireties. In some embodiments, the coelenterazine analogs include prosubstrates such as those described in U.S. Patent Publication No. 2008 / 0248511, U.S. Patent Publication No. 2012 / 0707849, U.S. Patent Publication No. 2014 / 0099654, U.S. Patent No. 9,927,430, U.S. Patent No. 10,316,070, which are incorporated herein by reference in their entireties. In some embodiments, the compound is furimazine. In some embodiments, the compound is JRW-0238. In some embodiments, the compound is JRW-1743. In some embodiments, the compound is JRW-1744.

[0032] Coelenterazine and its analogs and derivatives may suffer from challenges associated with their reconstitution into buffer systems used in many assays, such as the bioluminescence assays and methods described herein. For example, coelenterazine or its analogs or derivatives, such as furimazine, may dissolve slowly and / or inconsistently in non-organic buffers (e.g., due to the heterogeneous microcrystalline nature of the solid material). Although dissolution in organic solvents prior to dilution with a buffer may provide faster and more consistent results, coelenterazine compounds may suffer from organic solution instability, including both thermal and photoinstability during storage. See, for example, U.S. Patent No. 9,676,997, which is incorporated herein by reference. In some embodiments, the incorporation of coelenterazine or its analogs or derivatives into the compositions described herein provides more reliable and consistent dissolution without such instability issues.

[0033] In some embodiments, the composition further comprises a polymer. As further described herein, in certain embodiments, the presence of the polymer stabilizes the compound against degradation, and the presence of the polymer improves the solubility of the compound in water or in an aqueous solution. In some embodiments, by stabilizing the coelenterazine or coelenterazine analog or derivative (e.g., compared to coelenterazine or coelenterazine analog in organic solvents), improving the water solubility of the coelenterazine or coelenterazine analog or derivative, and / or improving the reconstitution efficiency of the coelenterazine or coelenterazine analog in non-organic buffers (e.g., compared to coelenterazine or coelenterazine analog or derivative in the absence of a polymer). The compositions and systems herein allow for the use of coelenterazine or coelenterazine analog or derivative in point-of-care, prepackaged, and / or solid-phase systems, methods, and assays where unformulated and / or organic phase coelenterazine or coelenterazine analog is less suitable (e.g., not temperature-stable or photostable).

[0034] The polymer may be a natural biopolymer or a synthetic polymer. In some embodiments, the polymer is a natural biopolymer. Suitable natural 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 natural biopolymers may also be used. The polymer may be a derivative of a natural polymer, such as functionalized cellulose (e.g., hydroxypropyl cellulose, hydroxypropyl methylcellulose, etc.).

[0035] In some embodiments, the polymer is pullulan, which is a polysaccharide that contains maltotriose repeating units. Maltotriose is a trisaccharide that contains three glucose units linked via α-1,4 glycosidic bonds. The maltotriose units in a 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 is generally found in concentrations of about 4.5×10 4 ~about 6×10 5 It has a mass range of 100 Da and is commercially available from a variety of suppliers (CAS number 9057-02-7).

[0036] In some embodiments, the polymer is dextran, which is a complex branched polysaccharide containing glucose repeating units. The linear linkages are generally formed by α-1,6 glycosidic bonds, while the branches typically begin with α-1,3 linkages. Natural dextran can have a molecular weight ranging from about 9 kDa to about 2000 kDa. Dextran can be synthesized from sucrose by certain bacteria, including Leuconostoc mesenteroides and Streptococcus mutans. Commercially available dextran produced by Leuconostoc mesenteroides (CAS number 9004-54-0) can be purchased from a variety of suppliers, including Sigma Aldrich, and can have a variety of molecular weight ranges ranging from about 1 kDa to about 670 kDa.

[0037] In some embodiments, the polymer is a cyclic saccharide polymer, such as cyclodextrin. Typical cyclodextrins are α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin, which have 6, 7, and 8 glucopyranose units, respectively. The glucopyranose units can be functionalized. An exemplary cyclodextrin is hydroxypropyl β-cyclodextrin.

[0038] In some embodiments, the polymer is a non-sulfated glycosaminoglycan. Glycosaminoglycans are linear polysaccharides with repeating disaccharide units, each of which contains one amino sugar (N-acetylglucosamine or N-acetylgalactosamine) and either a uronic sugar (glucuronic acid or iduronic acid) or galactose. An exemplary non-sulfated glycosaminoglycan is hyaluronic acid, in which the repeating disaccharides contain N-acetylglucosamine and glucuronic acid linked through alternating β-(1→4) and β-(1→3) glycosidic bonds. Polymers of hyaluronic acid can range in size from 5 to 20,000 kDa.

[0039] In some embodiments, the polymer is cellulose, which is a polysaccharide of linear repeating β-1,4 linked D-glucose units. Natural fibers can have up to 10,000 glucose units and a molecular weight of more than 1000 Da.

[0040] In some embodiments, the polymer is a synthetic polymer. The synthetic polymer can be a homopolymer, copolymer, block copolymer (e.g., diblock copolymer, triblock copolymer, etc.). Non-limiting examples of suitable polymers include, but are not limited to, polyamines, polyethers, polyamides, polyesters, polycarbamates, polyureas, polycarbonates, polystyrenes, polyimides, polysulfones, polyurethanes, polyacetylenes, polyethylenes, polyethyleneimines, polyisocyanates, polyacrylates, polymethacrylates, polyacrylonitriles, 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-co-glycolic acid) (PLGA), poly(L-lactic-co-glycolic acid) (PLLGA), poly(D,L-lactide) (PDLA), poly(L-lactide) (PL LA), 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), polyalcalcianoacrylate, polyurethane, 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 their copolymers (e.g., poloxamers), polyalkylene terephthalates (e.g., poly(ethylene terephthalate) and the like), polyvinyl alcohol (PVA), polyvinyl ethers, polyvinyl esters (e.g., poly(vinyl acetate) and the like), polyvinyl halides (e.g., poly(vinyl chloride) (PVC) and the like), polyvinylpyrrolidone, polysiloxanes, polystyrene (PS), polyurethanes,derivatized celluloses (e.g., alkyl celluloses, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitrocellulose, hydroxypropyl cellulose, carboxymethyl cellulose, etc.), polymers of acrylic acid ("polyacrylic acid") (e.g., poly(methyl (meth)acrylate) (PMMA), poly(ethyl (meth)acrylate), poly(butyl (meth)acrylate), poly(isobutyl (meth)acrylate), poly(hexyl (meth)acrylate), poly( Poly(isodecyl (meth)acrylate), poly(lauryl (meth)acrylate), poly(phenyl (meth)acrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), poly(octadecyl acrylate), polydioxanone and its copolymers (e.g., polyhydroxyalkanoates, polypropylene fumarate), polyoxymethylene, poly(ortho)esters, poly(butyric acid), poly(valeric acid), poly(lactide-co-caprolactone), trimethylolpropane, trimethylolpropane, triethylamine ... methylene carbonate, polyvinylpyrrolidone (PVP), 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), hydroxymethylpropyl methylcellulose acetate succinate (HPMCAS), poly(ethylene-alt-propylene) (PEP), 2-methylacrylamido glucopyranose (MAG), dimethyl adipimidate (DMA), polyvinyl caprolactam-polyvinyl acetate, and mixtures and copolymers of any thereof.

[0041] 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(propylene oxide) block and at least one poly(ethylene oxide) block, such as a poloxamer. Poloxamers are non-ionic triblock copolymers having a central poly(propylene oxide) block adjacent to two poly(ethylene oxide) blocks. Poloxamers are also known by certain trade names, including Pluronic® and Kolliphor®. Exemplary poloxamers include poloxamer 188 (Pluronic® F-68) and poloxamer 407 (Pluronic® F-127).

[0042] In some embodiments, the compound (ie, coelenterazine or an analog or derivative thereof) and 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.

[0043] In some embodiments, the composition further comprises a paper or fiber matrix or other material, and the composition is disposed in or on the paper or fiber matrix or other material. In some embodiments, the material can allow the coelenterazine (or analogs or derivatives thereof) to be used in a wide variety of environments, such as field testing. In some embodiments, the paper or fiber matrix can be manufactured from high quality cotton linters, such as 100% pure cotton linters. In some embodiments, the paper or fiber matrix can be ash-free. In some embodiments, the paper or fiber matrix can include up to 0.06% ash by weight. In some embodiments, the paper or fiber matrix can have a thickness of about 0.1 μm to about 1 mm. In some embodiments, the paper or fiber matrix can have a pore size range of about 0.02 μm to about 12 μm. The paper or fiber matrix can have a variety of characteristics, including binding affinity, porosity, functionalization (e.g., having high acidic functional groups or high basicity), etc.

[0044] Exemplary paper or fiber matrices include Whatman® brand papers (e.g., W-903 paper, FTA paper, FTA elution paper, FTA DMPK paper, etc.), Ahlstrom papers (e.g., A-226 paper, etc.), M-TFN paper, FTA paper, FP705 paper, Bode DNA collection paper, nitrocellulose paper, nylon paper, cellulose paper and sample pads (e.g., EMD Millipore CFSP20300M), Dacron paper, cotton paper, polyester paper (e.g., Ahlstrom polyester fiber grade 6613, Ahlstrom treated polyester fiber grade 6613H), sodium carboxymethylcellulose, Noviplex™ plasma preparation cards, Ahlstrom CytoSep®, Cobas® plasma separation cards, porous and polymeric membranes, high purity cotton fibers (e.g., Ahlstrom grade 237), high purity cotton / rayon blends (e.g., Ahlstrom grade 1218), glass microfibers (e.g., Ahlstrom grade 2216), glass microfibers (e.g., Ahlstrom grade 2 ... 934-AH, EMD Millipore GFDX103000), and combinations thereof.

[0045] Other potential materials that can be used in place of the paper or fiber matrix include synthetic and / or polymeric films made from organic or inorganic materials (e.g., metals or ceramic materials), homogeneous or heterogeneous solids, liquids, or dissolvable tableting 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, and the like. Additional materials that can be used in place of the paper or fiber matrix include plastic or glass. In some embodiments, the material can be a cuvette, slide, plate, or any other suitable surface made of plastic or glass. In some embodiments, the material can be a metal surface, and the metal is a single metal or a metal alloy, for example, steel, copper, brass, bronze, or silver.

[0046] In some embodiments, the composition comprises (i) coelenterazine or a coelenterazine derivative or analog, (ii) a suitable polymer, and (iii) a paper or fiber matrix, or other surface, such as glass, plastic, or metal.

[0047] In addition to the compounds and polymers and / or the paper or fiber matrix or other surface, the compositions may include additional components such as buffers, surfactants, reducing agents, salts, radical scavengers, chelating agents, proteins, or any combination thereof.

[0048] 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 common buffers, such as bicine, tricine, 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)piperazin-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), and the like. In some embodiments, the composition comprises a phosphate buffer. In some embodiments, the composition comprises tricine. In some embodiments, the composition comprises 2-(N-morpholino)ethanesulfonic acid. The composition can also include any combination of buffers.

[0049] In some embodiments, the composition includes a detergent or surfactant. In some embodiments, the detergent or surfactant is present at 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 Triton™ surfactants such as Brij 35, Triton™ X series (octylphenyl ethoxylates such as Triton™ X-100, Triton™ X-100R, Triton™ X-114, etc.), octyl glucoside, polyoxyethylene (9) dodecyl ether, digitonin, octylphenyl polyethylene glycol (IGEPAL), and the like. CA630), n-Octyl-beta-D-glucopyranoside (betaOG), n-Dodecyl-beta-D-maltoside, Tween® 20 (Polysorbate 20 or Polyethylene Glycol (20) Sorbitan Monolaurate), Tween® 40 (Polysorbate 40 or Polyethylene Glycol (20) Sorbitan Monopalmitate), Tween® 80 (Polysorbate 80 or Polyethylene Glycol (20) Sorbitan Monooleate), Polidocanol, n-Dodecyl beta-D-maltoside (DDM), Nonidet P40-substituted groups, NP-40 nonylphenyl polyethylene glycol, C12E8 (octaethylene glycol n-dodecyl-monoether), hexaethylene glycol mono-n-tetradecyl ether (C14E06), octyl-beta-thioglucopyranoside (octylthioglucoside, OTG), Pluronic® F-68 (poloxamer 188), Pluronic® F-127 (poloxamer 407), saponin, emulgen, polyethylene glycol trimethylnonyl ether, and polyoxyethylene 10 lauryl ether (C12E10).Examples of ionic detergents (anionic or cationic) include deoxycholate, sodium cholate, sodium dodecyl sulfate (SDS), N-lauroyl sarcosine, and cetyltrimethylammonia bromide (CTAB). Examples of zwitterionic agents include Chaps, zwitterion 3-14, and 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. In some embodiments, the surfactant is polysorbate 20. The composition can also include any combination of surfactants.

[0050] 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.

[0051] 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.

[0052] In some embodiments, the composition may include a 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 radical scavengers and / or chelators.

[0053] In some embodiments, the composition can include a complete buffer composition, such as Nano-Glo® Luciferase Assay Buffer (Promega Catalog No. N112), Nano-Glo® Live Cell Substrate (LCS) Dilution Buffer (Promega Catalog No. N206), etc. The complete buffer composition can include a combination of components disclosed herein, including the buffer itself, as well as one or more of a salt, a metal chelator, a reducing agent, and a non-ionic surfactant.

[0054] 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 porcine skin collagen (e.g., Prionex). In some embodiments, the protein may be gelatin. The composition may also include any combination of proteins.

[0055] In some embodiments, the composition may include a solvent. Some compositions are completely dried so that any solvent can be removed, while other compositions may include a solvent or some 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.

[0056] As noted above, the composition can include any combination of the above components. For example, in some embodiments, the composition can include a protein, a buffer, and a reducing agent. In some embodiments, the composition can include a protein, a buffer, and a metal chelator.

[0057] The composition may be in the form of a lyophilized powder or cake. Such compositions may be prepared by lyophilizing a mixture of the components of the composition as further described below. The powder 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, in a lab-on-a-chip, etc. The powder product may be included in a plurality of snap tubes, each tube containing a predetermined amount of the composition, which may be dissolved in an appropriate amount of solution and used directly in the assay of interest.

[0058] The compositions can also be in the form of a hard but malleable material, such as a "drop" cast or film. Such compositions can 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 temperatures of about 30°C to about 70°C, or about 30°C to about 40°C, e.g., 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 drying under vacuum. Drop casts or films can be provided in containers, such as bottles, vials, snap tubes, microtiter plates, microtiter plates, on paper or fiber matrices or other solid material supports, lab-on-chips, and the like.

[0059] 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, for example, 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, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0.

[0060] The compositions may also be provided in other forms, such as tablets or capsules, including dissolvable tablets or capsules that can be dropped into a sample, such as a buffer or biological sample. The compositions may also be included as preformed films on a surface, such as the wells of a 96-well plate, so that the composition can be dissolved directly in an appropriate amount of solution and used directly in the assay of interest.

[0061] When the composition is provided on a paper or fiber matrix, the paper or fiber matrix can be in the form of a card with spots that can be punched so that the spots can be reconstituted and used directly in the assay of interest. Alternatively, the paper or fiber matrix can be provided in the form of pre-punched spots (e.g., about 1-5 mm in diameter) that can be reconstituted for use in the assay of interest. The paper or fiber matrix containing the composition can be dried, for example, by air drying, drying at ambient temperature, drying at elevated temperature (e.g., at a temperature of about 30°C to about 70°C, or about 30°C to about 40°C, e.g., 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 drying under vacuum.

[0062] The compositions of the present disclosure may be used in any method in which luciferase substrates, such as coelenterazine and its analogs and derivatives, are used. For example, they may be used in bioluminescence methods that use coelenterazine, or its analogs or derivatives, to detect one or more molecules, such as enzymes, cofactors for enzymatic reactions, enzyme substrates, enzyme inhibitors, enzyme activators, or ·OH radicals, or one or more conditions, such as redox conditions, in a sample. Samples may include animals (e.g., vertebrates), plants, fungi, physiological fluids (e.g., blood, plasma, urine, mucous secretions), cells, cell lysates, cell supernatants, or purified fractions of cells (e.g., subcellular fractions). The presence, amount, spectral distribution, emission kinetics, or specific activity of such molecules may be detected or quantified. Molecules may be detected or quantified in solution, including multiphase solutions (e.g., emulsions or suspensions), or on solid supports (e.g., particles, capillaries, or assay vessels).

[0063] In certain embodiments, the compositions can be used to quantify molecules of interest, hi some embodiments, the compositions can be used as probes for specific biochemical activities, such as apoptosis or drug metabolism.

[0064] In certain embodiments, the compositions can be used to detect luminescence in live cells or animals, e.g., in vivo. In some embodiments, luciferase can be expressed in cells (as a reporter or otherwise) and the cells are treated with the compositions. Coelenterazine, or an analog or derivative thereof, permeates cells in culture, reacts with luciferase, and generates luminescence. In some embodiments, the compositions can be used for more robust live cell luciferase-based reporter assays. In still other embodiments, samples (including cells, tissues, animals, etc.) containing luciferase and compositions of the present disclosure can be assayed using a variety of microscopy and imaging techniques, e.g., in vivo imaging. In still other embodiments, secreted luciferase is expressed in cells as part of a live cell reporter system.

[0065] Also provided herein is a method of stabilizing a compound selected from coelenterazine or an analog or derivative thereof, the method 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 against thermal degradation, chemical degradation, light-induced degradation, or any combination thereof.

[0066] In some embodiments, the compositions herein provide a method for preventing degradation of a compound (i.e., a coelenterazine or analogue or derivative) at 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 -35°C to about 80°C, about -30°C to about 80°C, about -25°C to about 80°C, about -35°C to about 80°C, about -30 ... About 80°C, about -15°C to about 80°C, about -10°C to about 80°C, about -5°C to about 80°C, about 0°C to about 80°C, about -80°C to about 75°C, about -80°C to about 70°C, about -80°C to about 65°C, about -80°C to about 60°C, about -80°C to about 55°C, about -80°C to about 50°C, about -80°C to about 45°C, about -80°C to about 40°C, The mixture is stabilized at a temperature of about -80°C to about 35°C, about -80°C to about 30°C, about -80°C to about 25°C, about -20°C to about 60°C, about -20°C to about 55°C, about -20°C to about 50°C, about -20°C to about 45°C, about -20°C to about 40°C, about -20°C to about 35°C, about -20°C to about 30°C, or about -20°C to about 25°C.

[0067] In some embodiments, the compositions herein provide a method for preventing degradation of a compound (i.e., coelenterazine or an analog or derivative thereof) at temperatures 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, -68°C, -69°C, -68 ... 6℃, -65℃, -64℃, -63℃, -62℃, -61℃, -60℃, -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℃, -32℃, -31℃, -30℃, -29℃, -28℃, -27℃, -26℃, -25℃ ,-24℃,-23℃,-22℃,-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℃, The composition stabilizes the compound at about -80°C, about -20°C, about 4°C, about 20°C, about 4°C, about 20°C, about 25°C, about 26°C, about 27°C, about 28°C, about 29°C, about 30°C, about 31°C, about 32°C, about 33°C, about 34°C, about 35°C, about 36°C, about 37°C, about 38°C, about 39°C, about 40°C, about 41°C, about 42°C, about 43°C, about 44°C, about 45°C, about 46°C, about 47°C, about 48°C, about 49°C, about 50°C, about 51°C, about 52°C, about 53°C, about 54°C, about 55°C, about 56°C, about 57°C, about 58°C, about 59°C, about 60°C, about 61°C, about 62°C, about 63°C, about 64°C, about 65°C, about 66°C, about 67°C, about 68°C, about 69°C, about 70°C, about 75°C, or about 80°C. The composition may stabilize the compound against degradation at about -80°C, about -20°C, about 4°C, about 20°C, about 25°C, or about 37°C.

[0068] In some embodiments, the compositions herein stabilize the compound (i.e., coelenterazine or an analog or derivative thereof) against degradation in the presence of light (e.g., compared to a coelenterazine compound or an analog or derivative thereof that is not in contact with a polymer and / or a paper or fiber matrix). The compositions may increase the half-life of the compound in the presence of light compared to compositions that do not contain a polymer or a paper or fiber matrix. The composition may increase the half-life of the compound in the presence of light by about 1.1x, 1.2x, 1.3x, 1.4x, 1.5x, 1.6x, 1.7x, 1.8x, 1.9x, 2.0x, 2.1x, 2.2x, 2.3x, 2.4x, 2.5x, 2.6x, 2.7x, 2.8x, 2.9x, 3.0x, 3.1x, 3.2x, 3.3x, 3.4x, 3.5x, 3.6x, 3.7x, 3.8x, 3.9x, 4.0x, 4.1x, 4.2x, 4.3x, 4.4x, 4.5x, 4.6x, 4.7x, 4.8x, 4.9x, or 5.0x or more compared to a composition that does not contain a polymer or a paper or fiber matrix.

[0069] In some embodiments, the compositions herein provide a method for protecting a compound (i.e., coelenterazine or an analog or derivative thereof) against degradation 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, 31 days, 32 days, 33 days, 34 days, 35 days, 36 days, 37 days, 38 days, 39 days, 40 days, 41 days, 42 days, 43 days, 44 days, 45 days, 46 days, 47 days, 48 ​​days, 49 days, 50 days, 51 days, 52 days, 53 days, 54 days, 55 days, 56 days, 57 days, 58 days, 59 days, 60 days, 61 days, 62 days, 63 days, 64 days, 65 days, 66 days, 67 days, 68 days, 69 days, 70 days, 71 days, 72 days, 73 days, 74 days, 75 days, 76 days, 77 days, 78 days, 79 days, 80 days, 81 days, 82 days, 83 days, 84 days, 85 days, 86 days, 87 days, 88 days, 89 days, 90 days, 91 days, 92 days, 93 days, 94 days, 95 days, 96 days, 9 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, 85 days, 90 days, 100 days, 110 days, 120 days, 130 days, 140 days, 150 days, 160 days, 170 days, 180 days, 190 days, 200 days, 210 days, 220 days, 230 days, 240 days, 250 days, 260 days, 270 days, 280 days, 290 days, 300 days, 310 days, 320 days, 330 days, 340 days, 350 days, 360 days, 1 year, 2 years, 3 years, 4 years, or 5 years.

[0070] In some embodiments, the composition increases the half-life of the compound (i.e., coelenterazine or an analog or derivative thereof) against degradation by at least about 1.25x, 1.5x, 1.75x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 11x, 12x, 13x, 14x, 15x, 16x, 17x, 18x, 19x, 20x, 21x, 22x, 23x, 24x, or 25x compared to a composition that does not include a polymer or a paper or fiber matrix (e.g., compared to a coelenterazine compound or an analog or derivative thereof that is not in contact with a polymer and / or a paper or fiber matrix).

[0071] 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, whereby the solubility of the coelenterazine compound and its analogs or derivatives is improved compared to the compound not in contact with the polymer. The solubility of the compound may be improved in aqueous solution compared to the corresponding compound not in contact with the polymer and / or a paper or fiber matrix. The solubility of the compound may be improved in the presence of a polymer after reconstitution of a lyophilized powder, a drop cast film or a "droplet", or from rehydration of a paper or fiber matrix or other solid support material on or within which the compound is disposed.

[0072] The composition may increase the solubility of the compound (i.e., coelenterazine or an analog or derivative thereof) in, for example, pure water or in an aqueous solution, such as one further comprising a buffer, a salt, a protein, a reducing agent, a radical scavenger, a surfactant, etc., or any combination of such components. The composition may increase the solubility of the compound in, for example, an aqueous buffer solution, 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 another suitable buffer, such as Nano-Glo® Luciferase Assay Buffer. The composition may increase the solubility of the compound in, for example, a biological sample from a subject, a biological or environmental fluid, such as a culture medium (e.g., tissue culture medium), etc.

[0073] For example, the composition may increase the solubility of the compound by about 1.1x, 1.2x, 1.3x, 1.4x, 1.5x, 1.6x, 1.7x, 1.8x, 1.9x, 2.0x, 2.1x, 2.2x, 2.3x, 2.4x, 2.5x, 2.6x, 2.7x, 2.8x, 2.9x, 3.0x, 3.1x, 3.2x, 3.3x, 3.4x, 3.5x, 3.6x, 3.7x, 3.8x, 3.9x, 4.0x, 4.1x, 4.2x, 4.3x, 4.4x, 4.5x, 4.6x, 4.7x, 4.8x, 4.9x, or 5.0x or more in the presence of light compared to a composition that does not contain a polymer and / or paper or fiber matrix.

[0074] Also provided herein is a method for improving the reconstitution rate of a compound selected from coelenterazine and its analogs or derivatives, the method comprising contacting the compound with an effective amount of a polymer and / or a paper or fiber matrix, wherein the reconstitution rate of the compound is improved compared to the compound not in contact with the polymer and / or a paper or fiber matrix.

[0075] The composition may increase the rate of reconstitution of the compound in, for example, pure water or in an aqueous solution, such as one further comprising a buffer, salt, protein, reducing agent, surfactant, etc., or any combination of such components. The composition may increase the rate of reconstitution of the compound in, for example, an aqueous buffer solution, 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 another suitable buffer, such as Nano-Glo® Luciferase Assay Buffer. The composition may increase the solubility of the compound in, for example, a biological sample from a subject, a biological or environmental fluid, such as a culture medium (e.g., tissue culture medium), etc.

[0076] For example, the composition may increase the rate of reconstitution of a compound (e.g., a coelenterazine compound or an analog or derivative thereof) in the presence of light by about 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2.0-fold, 2.1-fold, 2.2-fold, 2.3-fold, 2.4-fold, 2.5-fold, 2.6-fold, 2.7-fold, 2.8-fold, 2.9-fold, 3.0-fold, 3.1-fold, 3.2-fold, 3.3-fold, 3.4-fold, 3.5-fold, 3.6-fold, 3.7-fold, 3.8-fold, 3.9-fold, 4.0-fold, 4.1-fold, 4.2-fold, 4.3-fold, 4.4-fold, 4.5-fold, 4.6-fold, 4.7-fold, 4.8-fold, 4.9-fold, or 5.0-fold or more as compared to a composition not containing a polymer.

[0077] The composition can have any combination of the properties disclosed herein. For example, the composition can have increased solubility as described herein, improved reconstitution rate as described herein, increased stability as described herein, and / or increased half-life as described herein. The composition can have one of the disclosed properties or any combination of the disclosed properties, and can further have other improved properties.

[0078] In embodiments of the methods described herein, the contacting step may include 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 matrix to form a mixture, and drying the mixture. In some embodiments, the contacting step includes 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 dissolving the compound in a solvent to form a first solution, applying the first solution to a paper or fiber matrix, and drying the paper or fiber matrix. In some embodiments, the contacting step includes 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 and second solutions to form a third solution, applying the third solution to the paper or fiber matrix, and drying the paper or fiber matrix.

[0079] In some embodiments, the drying step comprises freeze-drying. In some embodiments, the drying step comprises air-drying. In some embodiments, the drying step comprises drying at ambient temperature under an inert atmosphere (e.g., under nitrogen or argon). In some embodiments, the drying step comprises drying at elevated temperature (e.g., 30° C.) under an inert atmosphere. In some embodiments, the drying step comprises vacuum drying. In some embodiments, one or all of the solutions used in the method may be deoxygenated. Deoxygenation can be achieved by degassing the solution under vacuum, or by bubbling an inert gas (e.g., nitrogen or argon) through the solution, etc.

[0080] The compositions can be tested by using them as substrates for luciferase to generate luminescence, and analyzing the luminescence from the compositions 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 analogue. Light output can be measured as an immediate or near-instantaneous measurement of light output at the start of the luminescence reaction, which can be initiated upon addition of coelenterazine substrate (sometimes referred to as "T=0" luminescence or "flash").

[0081] In various embodiments, the luminescence reaction is carried out in a solution. The solution may contain, for example, a lysate from cells in a prokaryotic or eukaryotic expression system. The solution may contain purified proteins, peptides, or small molecules tagged with luminescent enzyme components. In other embodiments, expression occurs in a cell-free system or the luciferase protein is secreted into the extracellular medium, in the latter case, no lysate needs to be generated. In some embodiments, the reaction is initiated by adding appropriate materials, such as the compositions of the present disclosure, buffers, etc., into a reaction chamber (e.g., a well of a multi-well plate such as a 96-well plate, a test tube or vial, a cuvette, etc.) containing the luminescent protein. The reaction chamber may be placed in a reading device that can measure the light output, for example, using a luminometer, a photomultiplier tube, or a camera (e.g., a smartphone camera, a CCD camera, or any other handheld device that can record an image). The light output or luminescence may also be measured over time, for example, for a period of seconds, minutes, hours, etc., in the same reaction chamber. Light output or luminescence may be reported as an average over time, a half-life of signal decay, a sum of signal over a period of time, or a peak output. Luminescence may be measured in relative light units (RLU). In certain embodiments, compositions may be tested by using them as substrates for Oplophorus luciferase.

[0082] In yet other embodiments, the luciferase and / or composition is introduced into a host and measurements of luminescence are made in the host or a part thereof, which may include a whole organism, or a cell, tissue, explant, or extract thereof.

[0083] In other embodiments, the luminescent reaction is carried out on a solid support, which can be, for example, a bead, a resin, a magnetic particle, a membrane, or a surface such as a vial, a microtiter plate, a cassette, a cuvette, or a swab. The reaction can then be placed in a reader that can measure the light output from the particular solid support format.

[0084] In other embodiments, the luminescence reaction is performed in vivo for whole animal imaging. The vehicle for injecting the substrate into the animal must be non-toxic and highly compatible with mammalian biology, significantly limiting the available options. Pullulan and many other polymers described herein are non-toxic and even approved as food additives, making them particularly suitable as components of injectable solutions. In addition, the improved solubility and reconstitution of coelenterazine analogs such as furimazine in simple buffers such as PBS makes them ideal for administration to animals by intravenous injection, intraperitoneal injection, intracranial administration, etc. The composition components can be combined immediately prior to injection, and good reconstitution allows the sample to become homogenous quickly, which is important for animal work where the presence of undissolved microcrystals can be fatal. Once the substrate formulation is introduced into the animal (e.g., by intravenous or intraperitoneal injection), the sedated animal is placed in an imaging chamber and analyzed for in vivo production of bioluminescence.

[0085] In certain embodiments, the compositions disclosed herein are provided as part of a kit. The compositions may be contained within a single container. In some embodiments, the kit may further include one or more luciferases (in the form of a polypeptide, polynucleotide, or both) along with suitable reagents and instructions to enable a user to perform an assay 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 containing the composition. Instructions included in the kits of the present disclosure may be affixed to the packaging material or included as a package insert. The instructions are typically written or printed, but are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by the present disclosure. Such media include, but are not limited to, electronic storage media (e.g., magnetic disks, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like. As used herein, the term "instructions" may include the address of an internet site providing the instructions. EXAMPLES

[0086] Experiments conducted during the development of embodiments herein demonstrate the utility of the compositions and methods described herein. Unless otherwise indicated, pullulan was obtained from Sigma-Aldrich (CAS number 9057-02-7).

[0087] Abbreviations used in the examples include: ATT is 6-aza-2-thiothymine, EtOH is ethanol, Fz is furimazine, HPLC is high performance liquid chromatography, NGB is Nano-Glo® Luciferase Assay Buffer (Promega catalog number N112), PBS is phosphate buffered saline, and TFA is trifluoroacetic acid.

[0088] Example 1 Furimazine-pullulan composition Samples were prepared as follows: For each of the following conditions, all substrates and additives were combined at the concentrations listed in solutions of various w / v percent pullulan in water. In each case, substrates were added from stock solutions in ethanol such that the total amount of ethanol (v / v) in the final solution containing the polymer did not exceed 10 v / v%.

[0089] Condition 1: Solutions were prepared with 0, 2.5, 5, or 10% (w / v) pullulan in water. A 30 mM furimazine stock solution in ethanol was prepared. 4 μL of furimazine stock was added to 46 μL of solution containing pullulan, resulting in a final concentration of furimazine of 2 mM. The total concentration of ethanol was less than 10% v / v in the final solution in all cases. Samples were frozen and then lyophilized overnight to form a powder product.

[0090] Condition 2: A solution of 15% (w / v) pullulan, 200 mM tricine, and 2 mM furimazine in <10% v / v ethanol / water was prepared as above. A series of 60 μL aliquots were pipetted onto parafilm and dried in the dark at 25° C. for at least 3 h to form a hard, malleable "drop."

[0091] Condition 3: A solution of 15% (w / v) pullulan and 2 mM furimazine in <10% v / v ethanol / water was prepared as above. A series of 60 μL aliquots were pipetted onto parafilm and dried in the dark at 25° C. for at least 3 h.

[0092] Samples were tested by dissolving formulated furimazine in NGB or PBS (pH 7.0) with vortexing as necessary. In each case, samples were diluted in 5 mL of buffer to a final working concentration of 10 μM furimazine.

[0093] The experimental results are as follows: Samples according to condition 1 containing 2.5% (w / v) pullulan dissolved easily in solutions in NGB and PBS (pH 7.0) in less than 1 minute. Samples according to condition 1 containing 5% (w / v) and 10% (w / v) pullulan dissolved completely in PBS (pH 7.0) within a few minutes. Samples according to condition 2 required additional vortexing and took about 10-15 minutes to completely dissolve in PBS (pH 7.0). Samples according to condition 1 without pullulan took about 10 minutes to completely dissolve in PBS (pH 7.0) (determined by experiment).

[0094] After storing samples at 4°C for 5 weeks, samples were diluted to 6mL in 1x NGB for 20μM stock or 6mL in 1x PBS (pH 7.0) for 20μM stock. Purified NanoLuc® (Nluc) luciferase enzyme was added at 1x final concentration (2x stock solutions were prepared in either PBS or NGB starting from a 1000x stock of NanoLuc® enzyme, Promega catalog number E499). Control samples contained Nluc in assay buffer with 10μM final Nano-Glo® substrate. Assays were performed on solid, white, non-binding surface (NBS) plates in a total assay volume of 100μL using kinetic readings on a luminometer (specifically, GloMax® Discover multimode microplate reader, Promega catalog number GM3000) collecting total luminescence. The kinetic traces for samples reconstituted in PBS are shown in Figure 1A, and for samples reconstituted in NGB are shown in Figure 1B. Figure 1C shows images of the lyophilized cake and film droplet formulations. Data from Figure 1A at specific time points are presented in the bar graphs of Figures 2A-C, and data from Figure 1B at specific time points are presented in the bar graphs of Figures 3A-C.

[0095] The results illustrated in Figures 1-3 demonstrate the increased solubility of the furimazine composition in neutral buffers without the need for organic solvents or special buffer conditions. The light emission from the furimazine composition is stronger compared to commercially available furimazine formulations.

[0096] Example 2 Absorbance of reconstituted furimazine composition Solid furimazine bulk was diluted in ethanol to a final concentration of 10 mM (solution 1). Dry pullulan was dissolved in pure water to final concentrations of 0 w / v%, 2.5 w / v%, 5 w / v%, 10 w / v%, 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 tube vials. 5 μL of solution 1 was then added to each vial and mixed by vigorously pipetting to form solutions 3a-e, each containing a final concentration of 1 mM (19.08 μg) furimazine in 50 μL of solution. After mixing, the vials containing solutions 3a-e were placed in dry ice and frozen for 1 hour. These frozen stocks were then lyophilized overnight to form dry pullulan matrices containing furimazine.

[0097] A powder formulation of furimazine (19.08 μg) in pullulan matrix (0 w / v%-15 w / v%) was diluted in 0.5 mL of PBS buffer (pH 6.8), equilibrated at room temperature for 30 min, and absorbance was read at 254 nm. The absorbance spectra of formulated dried furimazine (50 nmol) with increasing amounts of pullulan after reconstitution in PBS buffer are shown in Figure 4A. Formulated furimazine with pullulan resulted in an increase in furimazine absorbance in aqueous solution. The concentration of furimazine was calculated using the extinction coefficient (21,000 M) of furimazine in methanol with absorbance measured at 254 nm. -1 cm -1) was used to determine the absorbance of furimazine by Beer's law. Furimazine bulk had an absorbance of 0.0571, corresponding to a calculated concentration of 0.0082 mM. Furimazine formulations containing 2.5 w / v% to 5 w / v% pullulan yielded absorbances of 0.2204 and 0.2467, yielding calculated concentrations of 0.032 mM and 0.035 mM, respectively. Formulated furimazine with 10 to 15 w / v% pullulan yielded absorbances of 0.3964 and 0.3836, yielding calculated concentrations of 0.055 mM and 0.052 mM, respectively, in PBS. A summary of the absorbance data displayed in Figure 4A can be found in Figure 4C, showing the increase in furimazine (Fz) concentration in solution when furimazine is formulated with pullulan compared to samples that do not contain pullulan.

[0098] Separately, dry formulations of furimazine (95.4 μg) in pullulan matrix (0 w / v% - 15 w / v%) prepared similarly to the samples above were diluted into 0.5 mL of PBS buffer (pH 6.8), equilibrated at room temperature for 30 minutes, and absorbance was read at 254 nm. The absorbance of formulated furimazine (95 ug) with increasing amounts of pullulan after reconstitution in PBS buffer is shown in Figure 4B. Solid formulated furimazine with increasing concentrations of pullulan matrix resulted in increased absorbance and therefore furimazine concentration in PBS buffer compared to conditions containing only furimazine without pullulan.

[0099] Example 3 Reconstitution of preserved samples Solid furimazine was dissolved in ethanol and the dissolved solution was added to an aqueous solution of pullulan (0 or 15 w / v%) to obtain a total concentration of 1 mM furimazine in 50 μL of solution containing less than 10 v / v% ethanol. Samples were lyophilized or dried at ambient temperature. Figure 5 shows images demonstrating the ability of these compositions to be reconstituted in PBS (pH 7.0). Ambient temperature dried "droplet" formulations containing 15 w / v% pullulan dissolved in solution after a short pipetting period. Lyophilized samples containing 15 w / v% pullulan dissolved immediately after adding PBS. Samples without pullulan did not completely dissolve in PBS even after vortexing for 15 minutes, demonstrating poor solubility in PBS.

[0100] Example 4 Absorbance of pullulan sample Samples of solvent-free 2.5 w / v% pullulan and solvent-free 10 w / v% pullulan in PBS (pH 6.8) were tested for their absorbance. The absorbance spectra spanning the range of 210-600 nm are illustrated in Figures 6A (2.5%) and 6B (10%). These spectra demonstrate that pullulan does not absorb in the same wavelength range as furimazine and does not artificially enhance the absorbance signal in samples containing furimazine.

[0101] Example 5 HPLC analysis of furimazine samples Solid furimazine bulk 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 w / v%, 2.5 w / v%, 5 w / v%, 10 w / v%, or 15 w / v% (solutions 2a, 2b, 2c, 2d, and 2e, respectively). 45 μL of solutions 2a-2e were pipetted into separate 1.5 mL snap tube vials. 5 μL of solution 1 was then added to each vial and mixed by vigorously pipetting to form solutions 3a-e, each containing a final concentration of 1 mM furimazine. After mixing, the vials containing solutions 3a-e were placed in dry ice and frozen for 1 hour. These frozen stocks were then lyophilized overnight to form the dry pullulan matrix containing furimazine.

[0102] General methodology for all HPLC traces: The furimazine sample above (containing 19.08 μg furimazine) was diluted with 0.5 mL PBS (pH 6.8) to 38.16 μg / mL in a small snap-cap tube. 15 μL of this solution was injected neat onto the HPLC (vial with insert) over a 5 hour period to assess stability and solubility over time. Instrument: Synergi Max-RP 50×4.6 mm, 2.54 u. Solvent: 0.1% TFA / water, acetonitrile. Commercially available furimazine (5 mM, Promega catalog number N113) was also diluted to 38.16 μg / mL in PBS and run for comparison.

[0103] HPLC traces of samples obtained immediately after dilution with 0.5 mL of PBS (pH 6.8) and 5 hours after dilution 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, (B) 5 hours), and Figure 10 (Nano-Glo® Luciferase Assay Substrate - (A) 0 hours, (B) 5 hours), respectively. (Traces were obtained similarly for 5% pullulan and 10% pullulan formulations, as well as the commercial furimazine sample. Data not shown.) The peak at retention times 5.10-5.13 minutes (the major peak in each spectrum) represents furimazine. The peak at retention times 5.36-5.37 minutes (marked with an asterisk) represents aminopyrazine (confirmed spectroscopically), a known degradation product of furimazine. The specific peaks and area percents are summarized in Table 1. [Table 1]

[0104] Figures 11 and 12 show analyses of the compiled and processed data from the HPLC traces shown in Figures 7-10, along with traces obtained via the same methods at additional time points.

[0105] Figure 11A shows an analysis of the purity of each sample as measured by absorbance at 254 nm with each trace normalized to time 0. All conditions prepared as dry formulations containing pullulan showed high levels of purity in aqueous solution with no significant loss of absorbance. Conditions lacking pullulan (0% condition, as well as the commercially available furimazine solution, Promega catalog number N113) showed a significant loss of absorbance over approximately 6 hours due to chemical degradation.

[0106] FIG. 11B shows an analysis of the peak areas of formulated furimazine samples (50 nmol) in PBS containing increasing amounts of pullulan (0% w / v to 15% w / v). (These analyses are for the same traces analyzed in FIG. 11A). The loss of purity of commercial furimazine and the 0% condition in the graph of FIG. 11a also corresponds to a decrease in peak area, indicating that the loss of signal is not due to changes in solubility over time, but rather due to product degradation in the Nano-Glo® Luciferase Assay Substrate and 0% pullulan conditions over the course of the experiment. Thus, the presence of pullulan not only helps to improve the water solubility of furimazine, but also helps to prevent its degradation in solution.

[0107] FIG. 12 shows the formation of aminopyrazine by-products of furimazine in the samples as described above. This data suggests that the presence of pullulan helped to prevent the formation of aminopyrazine in solution. The formulation of furimazine containing pullulan showed minimal aminopyrazine formation over 5.5 hours after reconstitution in PBS. In contrast, both the furimazine bulk lacking pullulan (0% condition) as well as the commercial Nano-Glo® Luciferase Assay Substrate formulation showed an approximately 12% increase in aminopyrazine over the course of the experiment. This data is consistent with the purity changes shown in FIG. 11B due to furimazine degradation in the Nano-Glo® Luciferase Assay Substrate and 0% pullulan samples.

[0108] Example 6 Furimazine Composition on Paper Matrix Paper spots were generated by extruding 3.2 mm diameter circular "spots" from Whatman® 903 protein saver cards using a standard 3.2 mm hand-held punch (Darice® brand). Furimazine stock solutions of 200 μM and 2 μM 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 in the dark at 4° C. until tested.

[0109] At the time of testing, each spot was placed into an individual well of a standard 96-well plate and reconstituted with 100 μL of PBS buffer (pH 7.0) containing purified NanoLuc® (Nluc) enzyme at a final concentration of 2 ng / mL. The final working concentrations of furimazine were 10 μM and 0.1 μM, respectively. Freshly prepared commercial Nano-Glo® luciferase assay substrate was prepared at 10 μM and 0.1 μM for comparison.

[0110] The results are illustrated in Figure 13. Figure 13A shows the change in RLU over time for paper spot samples and freshly prepared commercial Nano-Glo® Luciferase Assay Substrate samples. Figure 13B shows the initial RLU at time 0 for each sample. Figure 13C shows an image of the punched spots in the tube. These results demonstrate that formulated furimazine can be dried onto a solid matrix / paper and later reconstituted in non-organic aqueous buffer conditions.

[0111] Example 7 Furimazine Composition on Paper Matrix This experiment is based on the structural complementation assay disclosed in International Patent Publication No. WO2014 / 151736. Whatman® 903 protein saver cards containing the assay components were loaded into a 20 mM Na 3 PO 4 A stock solution 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 5 w / v% BSA, 0.25 v / v% Tween 20, 10 w / v% sucrose. 5 μL of this stock solution was then added to positions 2 and 4 on a Whatman® 903 card and allowed to dry for 1 hour at 35° C. 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 this solution was added directly to the Whatman® 903 protein card as positions 2 and 4. The Whatman® 903 card was then allowed to dry again for 1 hour at 35° C.

[0112] A 5 mM furimazine stock was prepared in ethanol and 5 μL of this stock was added to the card positions for conditions 1, 2, and 4. The cards were then placed under high vacuum for 15 minutes.

[0113] Cards were kept at 4° C. or 25° C. and tested at several time points for activity by adding NanoLuc® enzyme conjugated IgG. 10 pg of fresh NanoLuc® labeled antibody in PBS was added to position 1 to test for substrate activity. Images were recorded and are illustrated in FIG. 14A (left - image taken with a standard camera, center - image taken using a LAS300 imager, right - image taken with an iPhone® camera). Spots 1, 2, and 4 all produce bioluminescence at this point upon addition of NanoLuc® enzyme, indicating that the substrate remains active.

[0114] An additional set of samples was similarly prepared and stored at 4° C. or 25° C. for 3 months. Images were recorded and illustrated in FIG. 14B (left - image taken with a standard camera, center - image of a card stored at 4° C. after addition of 10 pg of NanoLuc®-labeled antibody in PBS to determine substrate activity to spots 1, 2, and 3 (spot 4 received only PBS as a negative control), right - image of a card stored at 25° C. after addition of 10 pg of NanoLuc®-labeled antibody in PBS). Only spot 2 produced light, while spot 1 did not. This example was demonstrated using the components of the sucrose-protein loading buffer (20 mM Na 3 PO 4 , 5% w / v BSA, 0.25% v / v Tween 20, 10% w / v sucrose) are required for substrate activity at this time and temperature, indicating that furimazine can be co-dried onto a solid paper matrix and reconstituted after long-term storage at either 4°C or 25°C.

[0115] Example 8 Furimazine Compositions on Paper Matrix Containing Buffers and Additives The purpose of this example was to demonstrate the effect of additives on overall reconstitution efficiency and assay performance. Samples were stored at different temperatures to simulate various thermal stresses and test the overall performance and stability under these conditions.

[0116] Whatman® 903 Protein Saver Spot Cards (3.2 mm punch), sucrose protein buffer (20 mM Na 3 PO 4 , 5% w / v BSA, 0.25% v / v Tween 20, 10% w / v sucrose (prepared the night before use), 200 μM furimazine solution in ethanol, 20 mM and 50 mM stocks of 6-aza-2-thiothymine (ATT) in water, and 20 mM and 100 mM stocks of thiourea in water.

[0117] To a spot of a 3.2 mm Whatman® 903 protein saver card, 5 μL of 200 μM furimazine in ethanol was added along with various additional ingredients as follows:

[0118] Sample 1: Furimazine Sample 2: Furimazine + sucrose protein buffer Sample 3: Furimazine + ATT (20 mM) Sample 4: Furimazine + ATT (50 mM) Sample 5: Furimazine + ATT (20 mM) + sucrose protein buffer Sample 6: Furimazine + ATT (50 mM) + sucrose protein buffer Sample 7: Furimazine + thiourea (20 mM) Sample 8: Furimazine + thiourea (100 mM) Sample 9: Furimazine + thiourea (20 mM) + sucrose protein buffer Sample 10: Furimazine + thiourea (100 mM) + sucrose protein buffer

[0119] Spots containing protein buffer were dried for 1 hour at 35°C before adding other components (furimazine, ATT, and / or thiourea). When ATT was used, 5 μL of the appropriate solution was added to the spot followed by drying under vacuum for 30 minutes (if a 20 mM solution was used, a final concentration of 1 mM ATT was obtained, and if a 50 mM solution was used, a final concentration of 2.5 mM ATT was obtained). When thiourea was used, 5 μL of the appropriate solution was added to the spot followed by drying under vacuum for 30 minutes (if a 20 mM solution was used, a final concentration of 1 mM was obtained, and if a 100 mM solution was used, a final concentration of 5 mM was obtained). Spots were made and stored at 4°C for 5 days before testing.

[0120] RLU experimental conditions - Assay buffer: PBS (pH 7.0), Plates: NBS solid white plates (Corning® 3600). Various final concentrations of Nluc enzyme (20 μg / mL, 2 μg / mL, or 0.2 μg / mL) were used.

[0121] The data are presented in Figures 15A-D, where Figures 15A-C show the raw RLU from luminescence reactions at various concentrations of Nluc enzyme (20 μg / mL, 2 μg / mL, and 0.2 μg / mL, respectively), and Figure 15D shows the % activity at one concentration of Nluc enzyme (0.2 μg / mL). The data suggest that the addition of additives such as ATT or thiourea may help improve overall RLU and signal stability when reconstituted in PBS compared to other formulations.

[0122] Example 9 Furimazine compositions on paper matrices with different polymers Materials and Methods: Whatman® 903 Protein Saver Spot Cards (3.2 mm punch), Furimazine. Protein buffer was prepared the day before testing with the components listed below.

[0123] Protein Buffer 1: Purified water Protein Buffer 2: 20 mM Na 3 PO 4, 5w / v% BSA, 0.25v / v% Tween 20, 10w / v% sucrose Protein Buffer 3: 20 mM Na 3 PO 4 , 5w / v% BSA, 0.25v / v% Tween 20, 2.5w / v% Pullulan Protein Buffer 4: 20 mM Na 3 PO 4 , 5w / v% BSA, 0.25v / v% Tween 20, 2.5w / v% Trehalose

[0124] 5 μL of one of protein buffers 1-4 was applied to each spot, and the spots were dried for 1 hour at 35° C. Then, 5 μL of a freshly prepared 200 μM furimazine solution in ethanol was applied to each spot, and the spots were dried under vacuum for 30 minutes. The spots were stored in the dark at 4° C., 25° C., and 35° C.

[0125] For luminescence measurements, at the time of testing, each spot was placed into an individual well of a standard 96-well plate and reconstituted with 100 μL of PBS buffer (pH 7.0) containing purified NanoLuc® (Nluc) enzyme at a final concentration of 8 ng / mL. Kinetic readings were started immediately.

[0126] Results from spots tested immediately after preparation are shown in FIG. 16, with traces of freshly prepared Nano-Glo® substrate shown for comparison. Results from spots tested after 1 day storage at 4° C., 25° C., and 37° C. are shown in FIG. 17A, FIG. 17B, and FIG. 17C, respectively. Results from spots tested after 3 days storage at 4° C., 25° C., and 37° C. are shown in FIG. 18A, FIG. 18B, and FIG. 18C, respectively. These data show that the signal is more stable for the furimazine composition on the paper matrix, but the overall signal is lower. Adding protein buffer with additives before adding furimazine to the spots may have prevented a sufficient amount of furimazine from completely penetrating the paper, but the samples still produced a useful and stable signal.

[0127] Example 10 Accelerated stability studies for formulated furimazine substrates in paper matrices Paper furimazine samples were tested to determine the effect of the formulation on furimazine thermal stability and functional integrity as measured by RLU in comparison to known furimazine formulations (Nano-Glo® Substrate, Promega catalog number N113, and Nano-Glo® Live Cell Substrate, Promega catalog number N205).

[0128] Whatman® 903 Protein Saver Spot Cards (3.2 mm punches) were processed as follows.

[0129] For conditions 1 and 2, the paper spots were filled with 5 μL of water (condition 1) or protein buffer (20 mM Na 3 PO 4 Condition 1 was pretreated with either 5 μL of protein buffer (20 mM NaCl, 5 w / v % BSA, 0.25 v / v % Tween 20, 10 w / v % sucrose - condition 2), or 5 μL of protein buffer (20 mM NaCl, 5 w / v % BSA, 0.25 v / v % Tween 20, 10 w / v % sucrose - condition 3) that lacked the sucrose component. 3 PO 4 , 5 w / v% BSA, 0.25 v / v% Tween 20). All conditions were then dried for 60 min at 35°C. A 200 μM furimazine stock solution was prepared in ethanol as above, and 5 μL of this stock was added to conditions 1 and 2. For condition 3, a 200 μM furimazine stock was prepared in a mixture of 2.5% pullulan in water and less than 10 v / v% ethanol. 5 μL of this solution was then added to condition 3, and all spots were then dried for another 30 min under vacuum. The spots were then stored in the dark at either 25°C or 60°C. At the time of measurement, one spot from each condition was placed in an individual well and diluted in PBS containing Nluc. The final theoretical concentration of furimazine is 10 μM, and the final concentration of Nluc is 1 ng / mL.

[0130] The compiled RLU data is shown in Figure 19 along with data for samples stored at (A) 60°C and (B) 25°C for various periods of time before reconstitution and testing. Figure 19 also shows percent enzyme activity data at time 0 for samples stored at (C) 60°C and (D) 25°C for various periods of time before reconstitution and testing.

[0131] The above experiment was extended to include high (1 mM and 100 μM final concentrations) and low (10 μM final concentration) concentrations of furimazine (FIG. 20). Each condition was prepared as above. The spots were pretreated with water, protein buffer, or protein buffer lacking sucrose. In the first two conditions, 5 μL of either a 2 mM or 200 μM solution of furimazine in ethanol was added to each spot and then dried for an additional 30 minutes at 35° C. In the third condition, either a 20 mM or 200 μM stock of furimazine was prepared in a mixture of 2.5% pullulan in water and less than 10 v / v% ethanol. 5 μL of this 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 either 25° C. or 60° C.

[0132] Figure 20 shows compiled RLU data for samples stored for various periods at (A) 60° C. and (B) 25° C. before reconstitution and testing. Figure 20 also shows percent enzyme activity data at time 0 for samples stored for various periods at (C) 60° C. and (D) 25° C. before reconstitution and testing.

[0133] By increasing the loading concentration of furimazine, there is an overall improvement in both maximum RLU as well as percent activity. However, spots that received no pretreatment (water) still performed better overall compared to both pretreatments containing protein buffer or protein buffer with pullulan at equivalent concentrations.

[0134] Example 11 Effect of drying methods on furimazine formulations in paper samples 3.2 mm punched Whatman® 903 protein saver card spots were washed with water or protein buffer (20 mM Na 3 PO 4 , 5% w / v BSA, 0.25% v / v Tween 20, 10% w / v sucrose) and dried at 35°C for 1 h. A stock solution of 10 mM furimazine in ethanol was prepared and 20 μL of this solution was added to 980 μL of either 2.5% (w / v) pullulan or 5% (w / v) pullulan in water. After thorough mixing, 5 μL of this solution was added to each spot. The spots were dried in the dark under vacuum or at ambient temperature for 2 h. After drying, the spots were stored in the dark at 4°C overnight.

[0135] 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. Triplicates were performed for each spot.

[0136] The results are shown in Figure 21, where Figure 21A shows data for samples dried under vacuum and Figure 21B shows data for samples dried under ambient air. Substrate performance does not appear to be significantly affected by whether the spots were dried under vacuum or at ambient temperature.

[0137] Figure 21C shows the summary data of Figure 21A, which indicates that the presence of pullulan reduces the overall RLU output. Experimental observations showed that the presence of pullulan made the surface of the paper matrix hard and waxy, which may have hindered the accessibility of the substrate to proteins, resulting in lower light output. This observation also indicates that the order of adding different components to the paper matrix may play a role in the overall function.

[0138] An additional set of spots that were dried a second time after substrate addition, either at ambient temperature or at 35° C., were compared. Each spot was dried in water, sucrose protein buffer (20 mM Na3 PO 4 , 5 w / v% BSA, 0.25 v / v% Tween 20, 10 w / v% sucrose), or pullulan protein buffer (20 mM Na 3 PO 4 , 5 w / v% BSA, 0.25 v / v% Tween 20, 2.5% pullulan) and dried at 35°C for 1 hour. A 200 μM furimazine stock was prepared in ethanol and 5 μL of this furimazine stock was added to each spot. The spots were then left in the dark at ambient temperature or at 35°C for 30 minutes. The spots were then stored in the dark at 25°C or 60°C for up to 5 days. At the time of testing, the spots corresponding to each condition were placed in wells of a standard 96-well plate and rehydrated with 100 μL of PBS solution (pH 7.0) and 2 ng / mL Nluc in each well to a final concentration of 10 μM furimazine in solution.

[0139] The results are shown in Figure 22. Spots dried a second time at 35°C showed higher RLU output, followed by spots dried a second time at ambient temperature. These results were consistent between conditions (protein buffer pre-treatment or water control) or whether the spots were stored at 25°C or 60°C for up to 5 days (maximum RLU values ​​are shown in Figures 22A and 22B, and % activity is shown in Figures 22C and 22D). These results suggest that the difference in drying method has an effect on overall substrate performance, with a second drying of the spots at 35°C being favorable for substrate performance.

[0140] Example 12 Accelerated substrate testing for powdered pullulan formulations. Powdered furimazine samples were tested to determine the effect of pullulan formulation on the thermal stability and functional integrity of furimazine as measured by both RLU and HPLC in comparison to known furimazine formulations (Nano-Glo® Substrate, Promega catalog number N113, and Nano-Glo® Live Cell Substrate, Promega catalog number N205).

[0141] Materials and Methods: Solid furimazine bulk was diluted in ethanol to a final concentration of 10 mM (solution 1), and dried pullulan was dissolved in pure water to final concentrations of 0 w / v%, 2.5 w / v%, 5 w / v%, 10 w / v%, 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 tube vials. 5 μL of solution 1 was then added to each vial and mixed by vigorously pipetting to form solutions 3a-e, each containing a final concentration of 1 mM furimazine.

[0142] After mixing, the vials containing Solutions 3a-e were placed in dry ice and frozen for 1 hour. These frozen stocks were then lyophilized overnight to form a dry pullulan matrix containing furimazine.

[0143] A particular powdered furimazine sample for testing was prepared as follows. 1) 1 mM (50 nmol total) furimazine stock prepared as a powder formulation containing 0% pullulan 2) 1 mM (50 nmol total) furimazine stock prepared as a powder formulation containing 2.5% pullulan 3) 1 mM (50 nmol total) furimazine stock prepared as a powder formulation containing 5% pullulan 4) 1 mM (50 nmol total) furimazine stock prepared as N113 solution (Promega catalog number N113) 5) 1 mM (50 nmol total) furimazine stock prepared as N205 solution (Promega catalog N205) (Note: N205 solution was made approximately 15 hours after N113 solution) 6) Furimazine bulk (50 nmol, taken from a stock solution in ethanol)

[0144] Half of the samples were stored at 25°C prior to HPLC testing, and half were stored at 60°C for extended periods prior to testing. For HPLC testing, formulated furimazine (19.08 μg) was diluted in 0.5 mL of PBS buffer (pH 6.8) in a small snap-cap tube. The tubes were vortexed for approximately 15 seconds and then equilibrated at room temperature for 30 minutes in the dark. 15 μL of sample was injected neat onto a HPLC (vial without insert), 0.1% TFA / water, acetonitrile, Synergi Max-RP 50×4.6 mm, 2.54 u. The HPLC traces of samples containing 5% w / v pullulan after storage at 60°C for 0 hours (FIG. 23A) or 48 hours (FIG. 23B) show only minimal degradation. The HPLC traces of N113 samples after storage at 60°C for 0 hours (FIG. 24A) or 48 hours (FIG. 24B) show significantly more degradation.

[0145] HPLC data was obtained for other samples (not shown) and the data was processed to provide thermal stability traces shown in FIG. 25. The area under the curve was measured and plotted over a 35-day period. "Bulk" refers to the solid furimazine as produced. "0% pullulan" refers to furimazine dissolved in a stock solution of ethanol, added to water (no pullulan), and lyophilized. FIGS. 25A and 25B show the thermal stability at 25° C. and 60° C. as raw peak area, while FIGS. 25C and 25D show the thermal stability at 25° C. and 60° C. as peak area percent. Formulations consisting of solid furimazine showed high levels of consistent chemical integrity when stored at room temperature or 60° C. In contrast, Nano-Glo® Luciferase Assay Substrate (Promega Cat. No. N113) and Nano-Glo® Live Cell Substrate (Promega Cat. No. N205) solutions showed significant loss of peak height and area over the measured time when stored at elevated temperatures.

[0146] For luminescence measurements, powdered furimazine samples no. 1-5 were reconstituted in PBS and sample no. 6 was reconstituted in ethanol (all 500 μL). Samples were equilibrated at room temperature for 30 minutes. Samples were then further diluted 1:5 (100 μM to 20 μM) and then 1:100 (20 μM to 0.2 μM). 50 μL of this solution was added to wells of a 96-well plate, a background reading was taken, and then NanoLuc® (Nluc) enzyme was added. (Before addition, the commercial Nluc sample stock was diluted to a concentration of 2 ng / mL in PBS and 50 μL was added to each well.) After dilution, the final concentrations were 0.1 μM furimazine and 1 ng / mL Nluc. RLU was then determined. (Background is the reading of 2x substrate solution without added Nluc.)

[0147] Compiled RLU data is shown in Figure 26. The numbers in the legend for each graph correspond to the following formulations: 1-0% pullulan (Note - this sample 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-Glo® Luciferase Assay Substrate (Promega Catalog No. N113), 5- Nano-Glo® Live Cell Substrate (N205), 6- Furimazine bulk (reconstituted in ethanol). Shown in Figures 26A-C are data after samples were stored at 60°C for various periods of time before being reconstituted and tested with 50 μM substrate (Figure 26A), 10 μM substrate (Figure 26B), or 0.1 μM substrate (Figure 26C), as described above. Shown in Figures 26D-F are data after samples were stored at 25°C for various periods of time before being reconstituted and tested with 50 μM substrate (Figure 26D), 10 μM substrate (Figure 26E), or 0.1 μM substrate (Figure 26F), as described above.

[0148] Figure 27 shows data for percent enzyme activity. The numbers in the legend for each graph correspond to the following formulations: 1-0% pullulan (Note - this sample 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-Glo® Luciferase Assay Substrate (Promega Catalog No. N113), 5-Nano-Glo® Live Cell Substrate (N205), 6-Furimazine Bulk (reconstituted in ethanol). 27A-C show enzyme activity at time 0 after samples were stored at 60° C. for various periods of time before being reconstituted and tested with 50 μM substrate (FIG. 27A), 10 μM substrate (FIG. 27B), or 0.1 μM substrate (FIG. 27C) as described above, and FIGS. 27D-F show enzyme activity at time 0 after samples were stored at 25° C. for various periods of time before being reconstituted and tested with 50 μM substrate (FIG. 27D), 10 μM substrate (FIG. 27E), or 0.1 μM substrate (FIG. 27F) as described above.

[0149] The solid furimazine samples demonstrated consistent chemical integrity as indicated by the RLU output in the luciferase assay after exposure to elevated temperatures. In contrast, furimazine formulated in commercially available N113 and N205 solutions demonstrated a loss of luminescence signal over time after storage at elevated temperatures. (Note: Sample 6 was dissolved in ethanol, which inhibited Nluc enzyme activity at higher concentrations.)

[0150] Example 13 Formulated furimazine film-coated microtiter plates Formulated furimazine films were formed directly on microtiter plates. Films containing 200 mM furimazine in either 2.5% (w / v) pullulan or 5% (w / v) pullulan were prepared directly in the wells of a microtiter plate. A representative image of this format is seen in FIG. 29 (artificially colored for clarity and presentation purposes). Well coatings were prepared as follows: A 2 mM furimazine stock in ethanol was prepared (Solution 1). Separately, solutions of 2.5% w / v and 5% w / v pullulan were prepared in water (Solution 2 and Solution 3, respectively). 45 μL of either 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 of the wells containing either Solution 2 or Solution 3 and mixed thoroughly by pipetting. The ethanol concentration in the final solution should be less than 5 v / v%. Higher ethanol concentrations will cause the pullulan to precipitate out of solution.

[0151] The plates were then dried in the dark under ambient conditions for 3 hours. The films in the wells were rehydrated to a final concentration of 10 μM furimazine in solution for all conditions by adding 100 μL of PBS (pH 7.0) and 2 ng / mL Nluc to each well either immediately or after pre-equilibration in 50 μL / well of PBS on a shaker for 30 minutes. RLUs were read and compared to freshly prepared commercial furimazine substrate (Nano-Glo® Live Cell Substrate, Promega Cat. No. N205). Data are shown in FIG. 28, where (a) shows data as raw RLU without pre-equilibration, (b) shows data as activity without pre-equilibration, and (c) shows data as raw RLU with pre-equilibration. This example highlights that furimazine can be dried onto pullulan-based films on a hard surface and reconstituted at the specified times. Based on visual observation, the films also reconstituted more quickly and completely than solid furimazine bulk. The data also show that pre-equilibration of furimazine-pullulan based films in PBS microtiter plates resulted in a significant decrease in light output.

[0152] FIG. 29 shows an image of a furimazine filmed plate made using the same method as above, but with the addition of food coloring so that the film coating could be visualized.

[0153] Figure 30A shows kinetic readouts of preparations in the same format as described for the data presented in Figure 28, except that the loading concentration of furimazine was higher (20 μM final concentration in 100 μL) and in some cases the furimazine formulation contained NanoLuc® enzyme co-filmed as a complete solution. Figure 30B shows the percent activity of the same experiment described in Figure 30A. Figure 30C shows the results of a stability study of the filmed microtiter plate after a storage period, with approximately 35% activity remaining at 10 days.

[0154] Example 14 HPLC and Mass Spectrometric Analysis of Purity, Stability, and By-Product Formation of Formulated Furimazine Formulated furimazine in lyophilized pullulan matrix was prepared as described in Example 12 at 19.07 μg furimazine in 0 w / v%, 2.5 w / v%, and 5 w / v% pullulan. Samples containing furimazine bulk, Nano-Glo® Luciferase Assay Substrate (Promega Catalog No. N113), and Nano-Glo® Live Cell Substrate (Promega Catalog No. N205) were stored for 35 days at either 25° C. or 60° C. Samples were reconstituted in PBS buffer or in ethanol in the case of furimazine bulk and 0% pullulan samples, equilibrated at room temperature for 30 minutes, and then analyzed on HPLC for known by-products of the furimazine degradation pathway. The absorbance data is shown in Figure 31 (A-Furimazine Bulk, B-0% Pullulan, C-2.5% Pullulan, D-5% Pullulan, E-Nano-Glo® Luciferase Assay Substrate, and F-Nano-Glo® Live Cell Substrate). In each case, the solid form of the formulated furimazine showed significantly less degradation products compared to the commercially available solution-based storage formulations (Promega Cat. No. N113 and Cat. No. N205).

[0155] FIG. 32 shows the area percent of the individual by-products relative to the furimazine peak (A-furimazine bulk, B-0% pullulan, C-2.5% pullulan, D-5% pullulan, E-Nano-Glo® Luciferase Assay Substrate, and F-Nano-Glo® Live Cell Substrate). In the solid pullulan formulations, furimazine is the major peak with minimal by-product formation, especially when stored at room temperature (left bar for each condition). In contrast, after 35 days when stored at either 25° C. (left bar) or 60° C. (right bar), there was a near complete loss of furimazine when stored in the commercial formulation.

[0156] Example 15 Substrate activity of formulated furimazine compositions stored at room temperature for 6 months 19 μg of furimazine was formulated as a lyophilized cake or film droplets were prepared in 15 w / v% pullulan as described in Example 1, conditions 1 and 3. Samples were stored at 25° C. for 6 months in the presence of ambient light. Both formulations were reconstituted with 100 μL of PBS (pH 7.0) and 1 ng / mL NanoLuc (Nluc) to a final concentration of 10 μM furimazine in solution. RLUs were read and compared to freshly prepared commercial furimazine substrate (Nano-Glo® Live Cell Substrate, Promega Cat. No. N205). The results of this experiment are shown in FIG. 33 (A-Raw RLU, B-Percent activity from time 0). After 6 months of storage at ambient temperature and ambient light, the formulated solid furimazine in pullulan matrix was still viable when exposed to luciferase.

[0157] Example 16 Activity and stability of furimazine formulated on different solid support matrices Four different types of paper were tested for different properties including substrate retention and effect on substrate integrity. The paper types included: 1. Thick glass fiber: Glass Microfiber 934-AH (Ahlstrom, particle retention: 1.5 μM, thickness: 435 μm), 2. Glass fiber thin: Glass fiber diagnostic pad (EMD Millipore), GFDX103000, Lot number 495362, 3. Cellulose: Cellulose sample pad (EMD Millipore), CFSP20300M Lot No. 11065, and 4. Whatman® 903 Protein Saver Cards

[0158] Each paper sample was cut into 7x7mm squares. A 10mM furimazine stock was prepared and 10μL of this stock was added to each paper matrix. Samples were dried at 35°C for 30 minutes. Cards were stored in the dark at either 25°C or 60°C for 72 hours. Samples were then placed in glass vials and 1mL of ethanol was added. Vials were sonicated for 10 seconds, the solvent was extracted, filtered and analyzed by analytical HPLC. The results of these experiments are shown in Figure 34. Figure 34A shows the raw area of ​​the furimazine peak after extraction from the paper or fiber matrix. The amount of furimazine extracted from the paper and analyzed from solution is also affected by the type of paper (Figure 34B). For substrates extracted and put back into solution, there is a slightly faster rate of decomposition of furimazine when dried onto paper or fiber matrix compared to furimazine bulk. In addition, furimazine substrates can be effectively dried and reconstituted from a variety of solid surfaces (Figure 34C).

[0159] Additional experiments were performed to determine substrate stability on paper in conjunction with the reporter protein LgTrip. To prepare the paper surface, a vial was prepared containing 200 μL of 5 uM LgTrip(3546) (SEQ ID NO: 3, see, e.g., U.S. Patent Application No. 62 / 684,014, the entire contents of which are incorporated herein by reference), 5 mM ATT, and 5 mM ascorbic acid. Approximately 5 μL of this solution was added to each spot, and the spots were then dried at 35° C. for 1 hour. After drying, a 1 mM furimazine stock in ethanol was prepared. Approximately 5 μL of this solution was added to each spot, and the spots were dried at 35° C. for an additional 30 minutes.

[0160] Different materials were tested with substrate and LgTrip input. At the time of testing, fresh Nluc was added and substrate was isolated. Figure 35A shows the bioluminescence signal on three different solid phase materials (Whatman 903, Ahlstrom 237, and Ahlstrom 6613H) resulting from surface reconstitution when fresh NanoLuc was added to the dry LgTrip and substrate. Ahlstrom 6613H appears to be detrimental to the signal output over time. Overall, the stability of the assay components can be affected by the composition of the solid matrix material in which they are immersed.

[0161] Figure 35B shows the bioluminescence signal from Whatman 903 paper containing both LgTrip as well as substrate and stored under ambient conditions for 25 days. The spots were exposed to 1 nM dipeptide in PBS at the time of testing. Overall, this experiment shows that there is no significant loss of signal from the material after extended storage time at ambient temperature.

[0162] Example 17 Effect of additives on the activity and stability of furimazine formulated on different solid support matrices Different additives were combined with furimazine in solution and allowed to dry on the paper surface. These experiments aimed to improve the overall substrate integrity while drying within the paper matrix. A 10 mM ascorbic acid solution was prepared in ethanol. This solution was then added to the furimazine bulk to create a solution containing 1:1 ascorbic acid and furimazine in ethanol. 10 μL of this solution was then added to the same paper matrix described in the previous example. The samples were allowed to dry at 35° C. for 30 minutes. The cards were stored in the dark at either 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, the solvent was extracted, filtered, and analyzed by analytical HPLC.

[0163] The results of these experiments are set forth in Figure 36. The raw area of ​​the furimazine peak is plotted in Figure 36A, showing better absorbance of the furimazine than the sample that did not contain the ascorbic acid additive. This effect was also observed in the overall percent recovery of furimazine back into solution (Figure 36B), as well as in furimazine purity (Figure 36C). Purity increased by approximately 15-20%, suggesting that the presence of ascorbic acid helps to limit thermal or chemical degradation of the furimazine substrate when stored on paper.

[0164] Example 18 Effect of chemical pretreatment of different solid support matrices on formulated furimazine activity and stability Paper matrices (Ahlstrom Glass Microfiber 934-AH and Whatman® 903 Protein Saver cards) were soaked in a solution of 30 w / v% citric acid for 30 minutes and then dried overnight at 35° C. A 10 mM furimazine stock in EtOH was prepared and 10 μL of the stock was added to a 7×7 mm paper card and dried at 35° C. for 30 minutes. The cards were then stored in the dark at room temperature or at 60° C. for 72 hours. At the time of reading, the cards were extracted with 1 mL of ethanol and sonicated for 15 seconds. The extracted solvent was then filtered and injected onto an analytical HPLC.

[0165] The results of these experiments are shown in Figure 37. The raw area of ​​the furimazine peak is shown in Figure 37A. Pretreatment of the paper matrix with 30% citric acid solution prior to application of the furimazine substrate had minimal impact on overall substrate purity after extraction into ethanol compared to paper matrix that was not pretreated with citric acid (Figure 37C). There was also limited improvement in the amount of substrate recovered in solution after extraction with ethanol (Figure 37B).

[0166] Example 19 Effect of mechanical pretreatment of different solid support matrices on formulated furimazine activity and stability Paper matrices (Ahlstrom Glass Microfiber 934-AH and Whatman® 903 Protein Saver cards) were soaked in water for 30 minutes and then dried overnight under vacuum to collapse or shrink the pores present in the paper matrix. A 10 mM furimazine stock in ethanol was prepared and 10 μL of the stock solution was added to a 7×7 mm paper card and dried at 35° C. for 30 minutes. The card was then stored in the dark at room temperature or at 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 solution was filtered and injected onto an analytical HPLC for analysis.

[0167] The results of this experiment are shown in Figure 38, with the raw area of ​​the furimazine peak shown in Figure 38A, the percent recovery shown in Figure 38B, and the purity shown in Figure 38C. There is no significant improvement in the purity of the substrate, or recovery into solution, between substrate added and dried on paper previously dried under pressure versus paper that was not pretreated.

[0168] Example 20 Effect of additives on the activity and stability of furimazine formulated on different solid support matrices Different additives were combined with furimazine in solution and allowed to dry on the paper surface. This series of experiments was aimed at helping to improve the overall substrate integrity while drying within the solid matrix. A 10 mM citric acid solution was prepared in ethanol. This solution was added to the furimazine bulk to create a solution containing 1:1 citric acid and furimazine in ethanol. 10 μL of this solution was then added to the same paper matrix described in the previous example. The samples were allowed to dry at 35° C. for 30 minutes. The cards were stored in the dark at either 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, the solvent was filtered, and analyzed by analytical HPLC.

[0169] The results of these experiments are set forth in FIG. 39. The raw area of ​​the furimazine peak is plotted in FIGS. 39A and 39B for furimazine dried on paper in the presence (A) and absence (B) of citric acid. The purity at 254 nm is plotted in FIGS. 39C and 39D for furimazine dried on paper in the presence (C) and absence (D) of citric acid. The plots show better absorbance of furimazine when dried in a mixture with citric acid. This increase in absorbance corresponds to approximately a 10-20% increase in purity, suggesting that the presence of citric acid helps limit the thermochemical decomposition of the furimazine substrate over the course of this experiment.

[0170] Example 21 Effect of citric acid and ascorbic acid on formulated furimazine activity and stability in the presence of sucrose protein loading buffer The effect of citric acid and ascorbic acid on furimazine activity and stability was examined in sucrose protein buffer (20 mM Na 3 PO 4 , 5% w / v BSA, 0.25% v / v Tween 20, 10% w / v sucrose). Spots were prepared from Whatman® 903 protein saver cards as described above. Each spot was pretreated with either sucrose protein buffer or water and dried at 35° C. for 1 hour. 200 μM furimazine stock was prepared in ethanol or in an ethanol solution containing either 200 μM citric acid or 200 μM ascorbate. 5 μL of furimazine or a furimazine 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 in the dark at 25° C. for up to 12 days.

[0171] At the time of testing, the spots corresponding to each condition were placed into wells of a standard 96-well plate and rehydrated with 100 μL of PBS solution (pH 7.0) and 2 ng / mL of Nluc in each well to a final concentration of 10 μM furimazine in solution. RLUs were read and compared to freshly prepared commercial furimazine substrate (Nano-Glo® Live Cell Substrate, Promega Cat. No. N205). The results of these experiments are shown in FIG. 40.

[0172] Paper spots pretreated with sucrose protein buffer showed a significant loss of signal over 12 days (Figure 40A). These results correspond to a near complete loss of percent activity of the substrate compared to conditions pretreated with water or with water in the presence of ascorbic acid or citric acid, which showed significant stability and signal output over 12 days (Figure 40B). A summary of these results is shown in Figure 40C. Ascorbic acid and citric acid may help maintain the integrity of the substrate when dried and stored on the paper surface, especially compared to water-only pretreatment. However, one or more components in the sucrose protein buffer may adversely affect the viability of the substrate for long-term storage and reconstitution.

[0173] Example 22 Preservation of isolated or bulk spots for furimazine activity and stability The effect of a particular storage procedure was tested on paper spots prepared from Whatman® 903 protein saver cards as described above. Each spot was pretreated with water and then dried at 35° C. for 1 hour. A 200 μM furimazine stock was prepared in ethanol and 5 μL of this solution was added to each spot. The spots were then dried for an additional 30-60 minutes at 35° C. The spots were then separated and stored in capped tubes individually or together in one vial in the dark at 25° C. for up to 12 days (bulk storage). At the time of testing, the spots corresponding to each condition were placed in wells of a standard 96-well plate and rehydrated with 100 μL of PBS solution (pH 7.0) and 2 ng / mL of Nluc in each well to a final concentration of 10 μM furimazine in solution. The results of these experiments are depicted in FIG. 40.

[0174] Individually stored spots exhibited higher maximum RLU than bulk stored spots (Figure 41A). These results are consistent with the observed percent activity (Figure 41B). These results suggest that storage method may also have an effect on overall substrate performance. Storage in individual containers may help limit environmental exposure to harmful factors such as light, air, and moisture compared to bulk stored spots. Bulk stored spots are exposed to these environmental factors every time the spots are removed for testing.

[0175] Example 23 Signal generation after removal of spots from reaction wells Spots were prepared from Whatman® 903 protein saver cards as described in Example 6. Each spot was diluted with sucrose protein buffer (20 mM Na 3 PO 4The spots were pretreated with either 5% BSA, 0.25% Tween 20, 10% sucrose) or water and dried at 35° C. for 1 hour. A 200 μM furimazine stock was prepared in ethanol or in an ethanol solution containing either 200 μM citrate or 200 μM ascorbate. 5 μL of furimazine or a furimazine solution containing an equal molar ratio of citrate or ascorbate was added to the spots. The spots were then dried at 35° C. for an additional hour. The spots were then stored in the dark at 25° C. for up to 5 days.

[0176] At the time of testing, the spots were reconstituted in PBS (pH 7.0) containing 2 ng / mL Nluc enzyme and RLUs were read kinetically. After 45 min, the spots were physically removed from the wells and placed into new wells containing fresh PBS solution (pH 7.0) and 2 ng / mL Nluc, and the kinetic RLU signals continued to be read on the wells that previously contained the spots and on the new wells containing the transferred paper spots. Figure 42A shows the kinetic RLU values ​​of the wells that previously contained the spots and the new wells to which the spots were transferred (indicated by + after the substrate formulation). A summary of the RLU results is shown in Figure 42B, comparing the RLU results from the original RLU read at 45 min, the now empty wells immediately after removal, and the RLU values ​​taken immediately after transferring the spots to the new wells containing new enzyme. There was no change in the RLU values ​​from the original spot read to the wells from which the spots were removed, indicating that the substrate was released from the paper matrix and equilibrated into the surrounding solution. Lower signals were recovered in the wells containing the transferred spots, indicating that some of the substrate formulation was retained within the paper matrix itself. Percent signal recovery was calculated for each condition by comparing the RLU signal present before the spots were transferred from the wells to the signal remaining after the spots were removed or placed into new wells (Figure 42C). After the spots were transferred to new wells containing fresh PBS solution (pH 7.0) and 2ng / mL Nluc, approximately half of the signal percentage was observed in the new wells. This indicates that most of the substrate was released into the solution in the original wells, while residual substrate remained on the paper itself.

[0177] Example 24 Effect of BSA and sugars on furimazine activity and stability. To determine whether the BSA and sugar components had an effect on furimazine activity and stability after drying on a solid surface and reconstitution, ten different versions of the protein loading buffer were prepared. The buffers prepared and tested were as follows: 1. Protein Buffer 1: 20 mM Na 3 PO4 , 5w / v% BSA, 0.25v / v% Tween 20, 10w / v% sucrose 2. Protein Buffer 2: 20 mM Na 3 PO 4 , 5w / v% BSA, 0.25v / v% Tween 20, 10w / v% sucrose, 5mM ascorbate 3. Protein Buffer 3: 20 mM Na 3 PO 4 , 0.25v / v% Tween 20, 10w / v% sucrose 4. Protein Buffer 4: 20 mM Na 3 PO 4 , 0.25 v / v% Tween 20, 10 w / v% sucrose, 5 mM ascorbate 5. Protein Buffer 5: 20 mM Na 3 PO 4 , 5w / v% BSA, 0.25v / v% Tween 20 6. Protein Buffer 6: 20 mM Na 3 PO 4 , 5w / v% BSA, 0.25v / v% Tween 20, 5mM ascorbate 7. Protein Buffer 7: 20 mM Na 3 PO 4 , 5w / v% BSA, 0.25v / v% Tween 20, 2.5% pullulan 8. Protein Buffer 8: 20 mM Na 3 PO 4 , 5w / v% BSA, 0.25v / v% Tween 20, 2.5% pullulan, 5mM ascorbate 9. Protein Buffer 9: 20 mM Na 3 PO 4 , 0.25 v / v% Tween 20, 2.5% pullulan 10. Protein Buffer 10: 20 mM Na 3 PO 4 , 0.25 v / v% Tween 20, 2.5% pullulan, 5 mM ascorbate The pH of each buffer was determined and is listed in Table 2. [Table 2]

[0178] Spots were prepared from Whatman® 903 protein saver cards as described in Example 6. Each spot was treated with one of buffers 1-10 and then dried at 35°C for 1 hour. A 200 μM furimazine stock was prepared in ethanol and 5 μL of this solution was added to each spot. The spots were then dried for an additional hour at 35°C. At the time of testing, spots corresponding to each condition were placed into wells of a standard 96-well plate and rehydrated with 100 μL of PBS solution (pH 7.0) and 2 ng / mL Nluc in each well to a final concentration of 10 μM furimazine in solution. RLUs were read and compared to freshly prepared commercial furimazine substrate (Nano-Glo® Live Cell Substrate, Promega Cat. No. N205).

[0179] These results are depicted in Figure 43. By removing BSA, there was a slight decrease in signal, which was restored in the presence of ascorbate (Buffer 3 and Buffer 4, Figure 43A). However, a significant decrease in signal was observed when the sucrose component was removed (Buffer 5 and Buffer 6). This signal was not restored in the presence of ascorbate or when the sucrose component was replaced with 2.5 w / v% pullulan (Buffer 7 and Buffer 8). The lowest signal was observed when both BSA and sucrose were absent from the loading buffer.

[0180] The kinetic results are shown in Figure 43B. Conditions lacking sucrose, BSA, or both showed a rapid decrease in signal over the course of the experiment, which 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 correspond to clear changes in percent activity (Figure 43C). Buffers 3, 5, and 9 performed worse in the RLU readings of the solution kinetics. These buffers also had the highest pH values, indicating that pH may also play a role in substrate performance on Whatman® 903 paper (Table 2).

[0181] Example 25 Effect of individual buffer components on furimazine activity and stability To determine whether specific buffer components had an effect on furimazine activity and stability during storage on the solid paper surface, eight different protein loading buffers were prepared. The buffers prepared and tested were as follows: 1.Buffer 1: Water 2. Buffer 2: Water + 5 mM ascorbate 3.Buffer 3:BSA 4. Buffer 4: BSA + 5mM ascorbate 5.Buffer 5:Na 3 PO 4 +Tween 20 6.Buffer 6:Na 3 PO 4 + Tween 20 + 5mM ascorbate 7.Buffer 7:BSA+Na 3 PO 4 +Tween 20 8.Buffer 8:BSA+Na 3 PO 4 + Tween 20 + 5mM ascorbate The pH of all buffers was fixed at 7 before addition to the paper spots.

[0182] Spots were prepared from Whatman® 903 protein saver cards as described in Example 6. Each spot was treated with one of buffers 1-8 and then dried at 35° C. for 1 hour. A 200 μM furimazine stock was prepared in ethanol and 5 μL of this solution was added to each spot. The spots were then dried at 35° C. for an additional hour. At the time of testing, spots corresponding to each condition were placed in separate wells of a standard 96-well plate and rehydrated with 100 μL of PBS solution (pH 7.0) and 2 ng / mL Nluc in each well to a final concentration of 10 μM furimazine in solution. RLUs were read and compared to freshly prepared commercial furimazine substrate (Nano-Glo® Live Cell Substrate, Promega Cat. No. N205).

[0183] These results are shown in FIG. 44. Spots pretreated with buffers containing either ascorbate, BSA, or a combination of the two showed good stability over 8 days while stored at 25° C. in the dark (FIG. 44A). Conditions lacking either BSA or ascorbate and containing Tween 20 with high levels of salt showed a significant loss of raw signal. There was also a significant loss in percent activity over the first few days (FIG. 44B). These results suggest that the presence of Tween 20 and / or high salt may adversely affect the integrity of the substrate while it is dried and stored on a solid surface, as seen by the decrease in overall RLU output. The presence of ascorbic acid helps to counter this effect. Representative kinetic traces from day 0 are shown in FIG. 44C. The rate of signal loss is faster in conditions lacking components of the protein buffer (Buffer 1 or Buffer 2).

[0184] Example 26 Effect of Prionex on furimazine activity and stability. To determine whether replacing BSA with Prionex had an effect on furimazine activity and stability while stored on a solid surface, six different protein loading buffers were prepared. The buffers tested were: 1.Buffer 1: Water 2. Buffer 2: Water + 5 mM ascorbate 3. Buffer 3: 1% v / v Prionex 4. Buffer 4: 1% v / v Prionex + 5 mM ascorbate 5. Buffer 5: 0.5% v / v Prionex 6. Buffer 6: 0.5 v / v% Prionex + 5 mM ascorbate The pH of each buffer was maintained at pH 7.

[0185] Spots were prepared from Whatman® 903 protein saver cards as described in Example 6. Each buffer was treated with one of buffers 1-6 and then dried at 35° C. for 1 hour. A 200 μM furimazine stock was prepared in ethanol and 5 μL of this solution was added to each spot. The spots were then dried at 35° C. for an additional 30 minutes. The spots were then stored in the dark at 25° C. for up to 20 days. At the time of testing, spots corresponding to each condition were placed in separate wells of a standard 96-well plate and rehydrated with 100 μL of PBS solution (pH 7.0) and 2 ng / mL of Nluc in each well to a final concentration of 10 μM furimazine in solution. RLUs were read and compared to freshly prepared commercial furimazine substrate (Nano-Glo® Live Cell Substrate, Promega Cat. No. N205). The results are depicted in FIG. 44.

[0186] In all cases, only the water pretreated condition, which observed high levels of RLU output over the course of the experiment, showed some signal loss over the three week period (Figure 45A). Kinetic data from day 1 of the study is shown in Figure 45B. The presence of Prionex stabilizes the signal once reconstituted, helping to limit signal decay compared to conditions lacking Prionex.

[0187] Example 27 Effect of ATT on formulated furimazine activity and stability. To determine whether the presence of ATT had an effect on furimazine activity and stability during storage on the solid paper surface, six different protein loading buffers were prepared. The loading buffers prepared and tested were as follows: 1. Water + 5mM ascorbate 2. Water + 5 mM ascorbate + 5 mM ATT 3.1% Prionex + 5mM Ascorbate 4.1% Prionex + 5mM ascorbate + 5mM ATT 5. 0.5% Prionex + 5mM Ascorbate 6. 0.5% Prionex + 5mM Ascorbate + 5mM ATT The pH of each buffer solution was controlled at pH 7.

[0188] Spots were prepared from Whatman® 903 protein saver cards as described in Example 6. Each buffer was treated with one of buffers 1-6 and then dried at 35° C. for 1 hour. A 200 μM furimazine stock was prepared in ethanol and 5 μL of this solution was added to each spot. The spots were then dried at 35° C. for an additional hour. The spots were then stored in the dark at 25° C. for up to 23 days. At the time of testing, spots corresponding to each condition were placed in separate wells of a standard 96-well plate and rehydrated with 100 μL of PBS solution (pH 7.0) and 2 ng / mL of Nluc in each well to a final concentration of 10 μM furimazine in solution. RLUs were read and compared to freshly prepared commercial furimazine substrate (Nano-Glo® Live Cell Substrate, Promega Cat. No. N205). The results are depicted in FIG. 45.

[0189] In all cases, high RLU output was observed (Figure 46A). Kinetic traces from spots after 22 days of storage also show high and stable signals over the course of the experiment (Figure 46B). All of the conditions of the present invention are favorable for substrate activity and storage stability on solid surfaces at ambient temperature.

[0190] Example 28 Formulated furimazine lyophilized for microtiter plates Furimazine was prepared and lyophilized directly into microtiter plate wells. The preparation resulted in wells containing 200 μM or 2 mM lyophilized powder formulations of furimazine in 5% (w / v) pullulan, prepared directly into the wells of a standard 96-well microtiter plate (Costar catalogue no. 3912). A representative image of this format is shown in FIG. 47A. The plates were prepared as follows: 2 mM and 200 μM furimazine stocks in ethanol were prepared (Solution 1). Separately, a solution of 5% w / v pullulan was prepared in water (Solution 2). 45 μL of Solution 2 was added to each well. 5 μL of Solution 1 was then added to each of the wells containing Solution 2 and mixed thoroughly by pipetting. 5 μL of pure ethanol was added to Solution 2 as a negative control. The plates were placed on dry ice to freeze for 1 hour, then lyophilized overnight.

[0191] The plates containing the furimazine cakes were rehydrated with 100 μL of PBS (pH 7.0) and 2 ng / mL of Nluc in each well to a final concentration of 10 μM or 100 μM furimazine, respectively. RLUs were read and compared to freshly prepared commercial furimazine substrate (Nano-Glo® Live Cell Substrate, Promega Cat. No. N205) or fresh Nano-Glo® Live Cell Substrate in the presence of 5 w / v% pullulan. Kinetic data is shown in FIG. 47B. This example shows that a lyophilized powder formulation format can be prepared directly on a solid surface such as a microtiter plate and reconstituted using an aqueous buffer such as PBS.

[0192] Example 29 Layered format for substrate addition Figure 48 shows a prophetic example of a two-component layered system, including separate surface components on individual paper cards, each containing either a substrate or a detection component, or separately treated components on the same surface. In use, both sides of the surface are folded together, holding each surface in intimate contact with one another. A sample solution containing the analyte of interest is then added to the folded surface material. The presence of the solution causes the different components to rehydrate and mix within the solid matrix, resulting in the complementary induced formation of bioluminescent complexes. This process, in combination with the substrate, produces light, which can then be detected and analyzed.

[0193] Example 30 Effect of sodium ascorbate on matrix preparations One volume of Nano-Glo® Luciferase Assay Substrate (Promega Catalog No. N113) was combined with 50 volumes of Nano-Glo® Luciferase Assay Buffer (Promega Catalog No. N112), containing sodium ascorbate at concentrations ranging from 0 to 300 mM. The solution was incubated at 37°C and then assayed at several time points. Nano-Glo® Luciferase Assay Substrate used according to the manufacturer's instructions (stored at -20°C during the course of the experiment and reconstituted for each time point) was used as a positive control. Cell cultures expressing the NanoLuc® enzyme were used as samples for each time point. One volume of reconstituted Nano-Glo® Luciferase Assay Buffer was mixed with one volume of sample. After 3 minutes, luminescence intensity was measured on a Bio-Tek Synergy® H1 96-well plate reader. For each sample, the luminescence intensity was background subtracted and normalized to the −20° C. control signal.

[0194] The addition of sodium ascorbate to the Nano-Glo® Luciferase Assay Buffer reduces the loss of reagent activity after reconstitution, as shown in Figure 49. When the substrate is reconstituted in Nano-Glo® Luciferase Assay Buffer containing 300 mM sodium ascorbate and held at 37°C for 23 hours, the luminescence intensity is 66% of the control compared to 38% in the absence of sodium ascorbate. After 41 hours at 37°C, the luminescence intensity is 31% compared to 9%. The stabilizing effect decreases with decreasing amounts of sodium ascorbate. Note that the results from the buffer condition containing 3 mM sodium ascorbate are most likely due to experimental error.

[0195] Example 31 Effect of hydroxypropyl β-cyclodextrin on substrate formulations A 4x furimazine solution was prepared by diluting the stock 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 for 48 hours using a Virtis Advantage Pro® freeze dryer. These lyophilized preparations were then stored at elevated temperature (37° C.) for the duration of the experiment. After 24 and 48 hours, the pellets were reconstituted in Nano-Glo® Luciferase Assay Buffer, so that the final concentrations of the components in the solution were 2x furimazine (diluted 1:50 from stock), 100 mM MES (pH 6.0), 100 mM HP-β-CD, and 300 mM sodium ascorbate. For comparison, Nano-Glo® substrate was prepared in Nano-Glo® Luciferase Assay Buffer and incubated at 37° C. for the duration of the experiment. Nano-Glo® substrate used according to the manufacturer's instructions (stored at −20° C. during the course of the experiment and reconstituted for each time point) was used as a positive control. In each case, one volume of reconstituted Nano-Glo® Luciferase Assay Substrate was mixed with one volume of sample. After 3 minutes, the luminescence intensity was measured in 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.

[0196] As shown in Figure 50, the pellets could be dissolved directly in Nano-Glo® buffer (without adding solvent) and maintained stability for 48 hours at 37°C by adding HP-β-CD and sodium ascorbate to the buffer prior to lyophilization. A non-lyophilized solution of working concentration Nano-Glo® Substrate in Nano-Glo® Luciferase Assay Buffer incubated at 37°C showed a 90% decrease in activity after the same time period. In addition, it is important to note that some signal enhancement was observed when comparing the pellets to the standard kit preparation.

[0197] Example 32 Effect of individual and combined buffer additives on matrix formulations Furimazine was diluted 1:50 in buffer with the following final composition: -Nano-Glo® Buffer - Nano-Glo® + 300 mM Sodium Ascorbate - Nano-Glo® Buffer + 100 mM Hydroxypropyl β-Cyclodextrin (HP-β-CD) - Nano-Glo® + 300 mM sodium ascorbate + 100 mM HP-β-CD

[0198] One volume of cell culture expressing NanoLuc was added to one volume of each buffer and mixed. After 3 minutes, the luminescence intensity was measured in a standard plate reader. Following the same procedure, background intensity was measured by mixing one volume of cell culture medium with each buffer.

[0199] These experimental results suggest that HP-β-CD is the primary contributor to the signal enhancement. As shown in Figure 51A, HP-β-CD enhances the signal by 15-20% compared to a solution containing Nano-Glo® buffer alone. The background signal in the absence of reporter enzyme (Figure 51B) indicated that the increase in signal was not due to an increase in background signal.

[0200] Example 33 Effect of mixed polymer matrix formulation on matrix stability A preparation of Nano-Glo® Substrate (1:50 dilution) was lyophilized in a MES (pH 6.0) solution containing 200 mM HP-β-CD, 600 mM sodium ascorbate, and 10 w / v% pullulan. After lyophilization, the vials were manually capped (not under vacuum). Some vials were stored in a 37° C. incubator, while others were left on the lab bench at room temperature. Before each measurement, the vials were rehydrated with twice the original volume so that the final concentrations of each component were 100 mM HP-β-CD, 300 mM sodium ascorbate, and 5% pullulan. Nano-Glo® Substrate (stored at −20° C. during the course of the experiment and reconstituted for each time point) used according to the manufacturer's instructions was used as a positive control. In each case, one volume of reconstituted Nano-Glo® Luciferase Assay Substrate was mixed with one volume of the 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 control signal at -20°C.

[0201] As shown in Figure 52, the presence of pullulan in the lyophilized preparation allows the substrate to retain its activity for over 15 days when stored at room temperature and at 37°C. The addition of pullulan appears to provide a barrier to oxygen and moisture, which when combined with the previously mentioned additives provides a stabilizing matrix that has the potential to retain the activity of furimazine for weeks to months. When combined with an inert gas, this storage method shows promise for achieving very long term stability of this substrate.

[0202] Example 34 JRW-0238 formulated with Pluronic® F-127 2.5 mg of Pluronic® F-127 (Sigma Aldrich) was bulked and placed into a 5 mL snap-top Eppendorf tube. The polymer was heated to 70° C. in a water bath until dissolved, resulting in a clear solution. A 174 mM stock of JRW-0238, a coelenterazine analog, was prepared in EtOH. 5 μL of this stock was added to the molten polymer and mixed by pipetting. Two separate conditions were prepared. Condition 1 - After the substrate was added, the substrate / polymer solution was dried under high vacuum for 30 minutes. Condition 2 - After the substrate was added, the substrate / polymer solution was further diluted with 45 μL of water and lyophilized overnight. A representative example of the final dry formulated substrate is shown in FIG. 53.

[0203] Samples from both conditions 1 and 2 were reconstituted in water, diluted to 100 μM, and analyzed for chemical integrity via analytical HPLC (Figure 54). Compared to freshly prepared substrate (100 μM JRW-0238 in EtOH, Figure 54A), none of the formulated substrate conditions showed significant chemical degradation (Figures 54B and 54C). Peak information is summarized in Table 3. [Table 3]

[0204] Reconstituted samples from both conditions were left in the dark at ambient temperature. After 24 hours, a small amount of precipitation was observed in the sample prepared from condition 1. The solution prepared from condition 2 remained clear over the course of the experiment. This series of experiments shows that solid formulations of coelenterazine analogs can be prepared with synthetic polymers without the need for organic solvents or stabilizers to improve overall kinetic solubility in aqueous media. However, the preparation method may have an effect on thermodynamic solubility. The lyophilized condition (condition 2) was still in solution after 24 hours at ambient temperature. This is in contrast to the sample from condition 1, which was reconstituted in water and began to precipitate out of solution within 24 hours.

[0205] Example 35 Furimazine formulated with Pluronic® F-127 This formulation was also prepared for the coelenterazine analog, furimazine. 2.5 mg of Pluronic® F-127 (Sigma Aldrich) was combined and placed in a 1.5 mL snap-top Eppendorf tube. The polymer was heated to 70° C. in a water bath until melted. A 10 mM furimazine stock solution was prepared in EtOH. 5 μL of this stock was then added to the molten polymer and mixed by pipetting. Two separate conditions were prepared. Condition 1 - After the substrate was added, the solution was dried under vacuum for 30 minutes. Condition 2 - After the substrate was added, the substrate / polymer solution was further diluted with 45 μL of water, frozen, and then lyophilized overnight.

[0206] Samples from both conditions 1 and 2 were reconstituted in water, diluted to 100 μM, and analyzed for substrate integrity via analytical HPLC (Figure 55). Compared to freshly prepared furimazine (Figure 55A), condition 1 showed significant degradation (Figure 55B). This may be due to the extensive sonication required to reconstitute this sample. In contrast, the reconstituted sample from condition 2 showed no significant degradation (Figure 55C). Peak information is summarized in Table 4. [Table 4]

[0207] This series of experiments shows that solid formulations of coelenterazine analogues, including furimazine, can be prepared with synthetic polymers without the need for organic solvents or stabilizers, improving overall kinetic solubility in aqueous media.

[0208] Example 36 Maximum concentration of formulated JRW-0238 in water 25 mg of Pluronic® F-127 was packaged together and placed in a 1.5 mL snap-top Eppendorf tube. 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 the substrate and aid in transfer to the polymer solution. The solvent was removed under reduced pressure without heating.

[0209] Four vials were prepared in a similar manner, all containing a polymer to substrate ratio of 7.3:1 w / w. Different volumes of water were used to make the initial aqueous stocks, as described below.

[0210] Vial 1: After injecting the substrate into the polymer, the solution was taken up in 500 μL of water. All materials were dissolved in the solution after sonication. The sample was frozen and lyophilized overnight. The calculated concentration of substrate was determined to be 17.4 mM with 5 w / v % polymer.

[0211] Vial 2: After injecting the substrate into the polymer, the solution was taken up in 400 μL of water. All materials were dissolved in the solution after sonication. The sample was frozen and lyophilized overnight. The calculated concentration of substrate in water was determined to be 21.4 mM with 6.25 w / v % polymer.

[0212] Vial 3: After injecting the substrate into the polymer, the solution was taken up in 250 μL of water. All materials were dissolved in the solution after sonication. The sample was frozen and lyophilized overnight. The calculated concentration of substrate in water was determined to be 34.4 mM with 10 w / v % polymer.

[0213] Vial 4: After injecting the substrate into the polymer, the solution was taken up in 100 μL of water. All materials were dissolved in the solution after sonication. The sample was frozen and lyophilized overnight. The calculated concentration of substrate in water was determined to be 85.4 mM with 25 w / v % polymer.

[0214] After lyophilization, each sample was reconstituted in either 500 μL, 400 μL, 250 μL, or 100 μL of water, respectively. All material for each condition was dissolved in solution. Representative images of these solutions are shown in FIG. 56. After 24 hours at ambient temperature, the reconstituted stock 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. Peak information is summarized in Table 5. [Table 5]

[0215] These experiments demonstrate that high concentrations of the coelenterazine analog JRW-0238 can be achieved in water without any loss of chemical integrity under ambient conditions by formulating it with a solid-state polymer.

[0216] Example 37 Lower polymer / substrate ratios without observable loss of substrate solubility 23.8, 20.4, 17, 13.6, 10.2, and 6.8 mg of Pluronic® F-127 were combined and placed into individual 1.5 mL snap-top Eppendorf tubes. The polymer was heated to 70° C. in a water bath until melted. 23.7 mg of JRW-0238 was dissolved in 350 μL of EtOH, and 50 μL of this stock was added to each vial containing hot polymer and 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 of water to a final concentration of 17.4 mM JRW-0238 with either 7x, 6x, 5x, 4x, 3x, or 2x w / w polymer / substrate, respectively. Each tube was frozen and lyophilized overnight.

[0217] 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). Only this sample was observed to be slightly cloudy. After 1 hour in solution at room temperature, the reconstituted substrate was observed to still be in solution except for the sample containing 2x w / w polymer relative to substrate (FIG. 58B).

[0218] Example 38 Solid Formulations Used for Whole Animal Imaging For whole animal imaging in mouse models, a stock sample of solid-formulated JRW-0238 was prepared as follows: 90 mg of Pluronic® F-127 was bulked and placed in a glass screw-cap vial. The polymer was then heated to 70° C. in a water bath until dissolved (a clear solution). 12.5 mg of JRW-0238 was dissolved in 250 μL of EtOH and added to the hot polymer and mixed thoroughly with a thin spatula. The solvent was then removed under reduced pressure. This concentrated sample was diluted with 3.646 mL of water to create a master stock of 8.7 mM substrate in water. 480 μL of this aqueous stock was then taken into a 1.5 mL screw-cap vial, frozen, and lyophilized overnight. A representative image of this formulation is 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).

[0219] Transgenic mouse subjects (mean 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 using isoflurane and injected with 480 μL of the reconstituted substrate solution via either intraperitoneal (IP) or subcutaneous (SC) injection. Each mouse was then imaged every minute after injection using an Ami Imaging System. FIG. 60A shows the average RLU traces from five animals injected intraperitoneally with reconstituted JRW-0238. FIG. 60B is a representative image of each mouse when the light output was measured at its maximum value. FIG. 61A shows the average RLU traces from five animals injected subcutaneously with reconstituted JRW-0238. FIG. 61B shows a representative image of each mouse subject when the light output was measured at its maximum value. Together, these results indicate that in vivo imaging can be achieved with coelenterazine analogs prepared as dry formulations, reconstituted in water at the time of use, and injected into live animal subjects via intraperitoneal or subcutaneous injection routes.

[0220] It should be understood that the foregoing detailed description and accompanying examples are merely illustrative and should not be construed as limitations on the scope of the present disclosure, which is defined solely by the appended claims and their equivalents.

[0221] Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Such changes and modifications, including but not limited to those with respect to the chemical structures, substituents, derivatives, intermediates, syntheses, compositions, formulations, or methods of use of the present disclosure, may be made without departing from the spirit and scope thereof.

[0222] Example 39 Preparation of larger scale polymeric furimazine formulations The dried furimazine formulations were scaled up to larger volumes to demonstrate that these compositions can be prepared under manufacturing conditions. The furimazine concentration in the cake is 200uM. The cake can be reconstituted to a 10mL volume stock to obtain a 2x (20uM) furimazine stock. This can then be diluted 1:1 with the sample to a final concentration of 10μM.

[0223] 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 with 5 mM ATT and 5 mM ascorbate, respectively.

[0224] 29.4 mL of pullulan solution was weighed into a 50 mL plastic vial. 600 μL of furimazine, prepared as a 10 mM stock in EtOH, was added and mixed thoroughly. A small amount of thin needle-like precipitate was observed in the solution. This precipitate was most likely due to pullulan polymers interacting with the EtOH in the solution. This did not affect the success of the preparation or the properties of the final material.

[0225] 10 mL amber glass vials were used. 1 mL of the furimazine-pullulan stock solution was taken into a 10 mL amber glass vial and a rubber stopper was partially inserted into the vial.

[0226] The freeze dryer used (Virtis Genesis 12EL freeze dryer) has a shelf surface of 4 square feet and a total of three shelves. The refrigeration system consists of two two-stage compressors. Vacuum / pressure control is achieved with a single vacuum pump and an adjustable control valve that bleeds nitrogen into the freeze dryer chamber to balance the suction of the vacuum pump and hold the pressure at a specified set point. The shelves are compressible via hydraulic pistons. One small tray containing 178 x 10 vials of manually dispensed product (containing 14 different formulation combinations) was loaded onto a single shelf in the freeze dryer, which was at a temperature of +4.7°C. The product then underwent a freezing step at a shelf temperature of -50°C for 2 hours, after which the condenser step was then started. The condenser temperature was run from -5°C to 87°C during the run. The vacuum was then pulled down and run at pressure set points of 75 and 200 mTorr. Good control at both these pressure set points was demonstrated throughout the run. All steps of the lyophilization recipe / cycle were run as programmed. Based on the average temperature of the product probe, sublimation lasted approximately 7.5 hours and desorption lasted approximately 16.1 hours. At the end of the run, the vials were backfilled with nitrogen and sealed with fully inserted stoppers at approximately 600 Torr pressure (approximately 740 Torr is atmospheric pressure).

[0227] After lyophilization, nitrogen gas was administered to each glass vial containing 20x furimazine-containing lyophilized cake to fill the head space of the vial, the cap was completely sealed, and the vial was stored at either 25°C or 60°C. At various time points after lyophilization, the formulated furimazine was reconstituted with 10ml of PBS (pH 7.0) containing 0.01% BSA, and the vial was manually shaken and equilibrated at room temperature for 5 minutes. 50ul of the formulated furimazine stock solution was added to 50ul of 1ng / ml purified NanoLuc® enzyme (Nluc) (Promega catalog number E499) in PBS (pH 7.0) containing 0.01% BSA (final [Nluc] = 0.5ng / ml). The control used was a 10uM final solution of Nano-Glo® Live Cell Substrate (Promega catalog number N205) freshly sampled from -20°C for each time point of data collection. Assays were performed in solid, white, non-binding surface (NBS) plates and analyzed using a kinetic readout on a luminometer (GloMax® Discover Multimode Microplate Reader, Promega Cat# GM3000) collecting total luminescence.

[0228] FIG. 62A shows the freeze-dried formulated furimazine cake at time point "Day 0", which indicates that the vials contain an evenly distributed uniform cake at the bottom of the vial, and vials without any obvious defects in appearance indicate that the formulation and freeze-drying protocol were appropriate. FIG. 62B shows the results of NanoLuc® activity expressed as raw RLU using formulated furimazine after reconstitution with buffer as described above. Formulated furimazine was run similarly to the control substrate (NanoGlo® Live Cell Substrate, Promega Cat#N205). This is the baseline reading to start the accelerated stability study. The vials or portions of the control substrate were then placed at 60° C. or 25° C. New vials were reconstituted at various time points and analyzed for activity using purified NanoLuc® enzyme. FIG. 63 shows the raw RLU from formulated samples stored at either 25° C. (blue, filled circles) or 60° C. (red squares), from NanoGlo® Live Cell Substrates stored at 25° C. (green triangles) or 60° C. (inverted orange triangles), and from freshly prepared control NanoGlo® Live Cell Substrates kept at −20° C. (black diamonds) when monitored for 34 days. Vials reconstituted on day 0 were kept in solution and at room temperature and similarly sampled for activity over 18 days (light blue, open circles). The data show that formulated furimazine maintained activity over the period tested at both temperatures tested, showing an improvement over furimazine dissolved in organic solvent. All formulated furimazine reconstituted within 5 minutes after addition of buffer, in stark contrast to the behavior of solid furimazine.

[0229] The results in Figures 62 and 63 demonstrate that the furimazine compositions can be prepared on a larger scale as well as under more stringent quality control conditions, e.g., in glass vials and under an inert atmosphere. The compositions can be stored at ambient or elevated temperatures for extended periods and reconstituted in neutral buffers without the need for organic solvents or special buffer conditions. Even after reconstitution in aqueous buffers, the compositions do not lose any significant performance while stored in solution and under ambient conditions for up to 24-48 hours, and maintain some activity for up to 18 days.

[0230] Example 40 Preparation of Formulated JRW-1744 [ka] 6-(3-amino-2-fluorophenyl)-8-benzyl-2-(furan-2-ylmethyl)imidazo[1,2-α]pyrazin-3(7H)-one Formulation examples of JRW-1744 were 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 Pluronic® F-127 (approximately 7.2 times w / w) was combined and placed in a glass screw-cap vial. The polymer was then heated to 80° C. in a water bath until dissolved (a clear solution results). 10.8 mg of JRW-1744 was dissolved in a small amount of EtOH and added to the hot polymer and mixed thoroughly with a thin spatula. Additional EtOH (up to 2 mL total) was used to completely transfer and dissolve all the substrate into the polymer solution. The solvent was then removed under reduced pressure. This concentrated sample was then placed under high vacuum for 1 hour to remove residual EtOH and yield an orange solid. The solid was diluted in 3.0 mL of water and sonicated to make a master stock of 8.7 mM JRW-1744 in water. 480 μL aliquots of this aqueous stock were then transferred to 1.5 mL screw-cap vials, frozen, and lyophilized overnight.

[0231] Example 41 Preparation of Formulated JRW-1743 [ka] 6-(3-amino-2-fluorophenyl)-8-(2-fluorobenzyl)-2-(furan-2-ylmethyl)imidazo[1,2-α]pyrazin-3(7H)-one Formulation examples of JRW-1743 were prepared in a similar manner to JRW-0238 in Examples 34 and 38.

[0232] A stock sample of formulated JRW-1743 was prepared as follows: 72 mg (7.2x w / w) of Pluronic® F-127 was bulked into a glass screw-cap vial. The polymer was then heated at 80° C. in a water bath until completely dissolved (FIG. 64A).

[0233] 10.0 mg of solid JRW-1743 was dissolved in a small amount of EtOH and transferred to the hot polymer while stirring with a thin spatula. Additional EtOH (up to 2 mL total) was used to aid in transferring the substrate to the polymer solution. The solvent was then removed under reduced pressure and the polymer / substrate mixture was concentrated to a reddish orange gel. This concentrated sample was then placed under high vacuum for 1 hour to remove any residual EtOH.

[0234] To prepare a master stock of formulated JRW-1743 in Pluronic® F-127, 2.6 mL of water was added to the gel and the resulting solution was sonicated until completely homogenous (Figure 64B). The final concentration of JRW-1743 in this volume was calculated to be 8.7 mM. A 480 μL aliquot of this aqueous stock was then transferred to a 1.5 mL screw-cap vial, frozen, and lyophilized overnight to obtain a lyophilized cake containing JRW-1743 (Figure 64C).

[0235] One vial containing formulated JRW-1743 was reconstituted in 480 μL of water (FIG. 64C center and right). Absorbance measurements of the substrate concentration in water were performed, showing that the working concentration of JRW-1743 in this solution was measured at 8.5 mM, compared to a theoretical concentration of approximately 8.7 mM (FIG. 65). array SEQ ID NO:1 - Native mature Oplophorus luciferase amino acid sequence FTLADFVGDWQQTAGYNQDQVLEQGGLSSLFQALGVSVTPIQKVVLSGENGLKADIHVIIPYEGLSGFQMGLIEMIFKVVYPVDDHHFKIILHYGTLVIDGVTPNMIDYFGRPYPGIAVFDGKQITVTGTLWNGNKIYDERLINPDGSLLFRVTINGVTGWRLCENILA SEQ ID NO:2 - Nluc amino acid sequence MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLFRVTINGVTGWRLCERILA SEQ ID NO:3 - LgTrip(3546) MKHHHHHHVFTLDDFVGDWEQTAAYNLDQVLEQGGVSSLLQNLAVSVTPIMRIVRSGENALKIDIHVIIPYEGLSADQMAQIEEVFKVVYPVDDHHFKVILPYGTLVIDGVTPNKLNYFGRPYEGIAVFDGKKITTTGTLWNGNKIIDERLITPD

Claims

1. (a)a compound selected from selenotellurazine, selenotellurazine-h, selenotellurazine-h-h, furimazine, JRW-0238, JRW-1743, and JRW-1744, and (b)one or more additional components selected from the following: (i)a buffer selected from phosphate buffer, tris, and 2-(N-morpholino)ethanesulfonic acid; (ii)a surfactant selected from polysorbate 20, polysorbate 40, and polysorbate 80; (iii)a reducing agent selected from thiourea and 6-aza-2-thiothymine; (iv)a salt selected from sodium chloride and sodium phosphate; (v)a radical scavenger selected from ascorbic acid and sodium ascorbate; and (vi)a chelating agent selected from citric acid and trans-1,2-diaminocyclohexane-tetraacetic acid; and (c)paper, and A composition comprising the same, for use in a point-of-care luminescence assay, wherein the compound and the one or more additional components are dried on the paper.

2. Use according to Claim 1, wherein the compound is furimazine.

3. Use according to Claim 1 or 2, wherein the composition further comprises a polymer.

4. Use according to Claim 3, wherein the polymer is a natural biopolymer selected from pullulan, trehalose, maltose, cellulose, dextran, and any combination thereof.

5. Use according to Claim 4, wherein the natural biopolymer is pullulan.

6. Use according to Claim 3, wherein the polymer is a cyclic saccharide polymer or a derivative thereof selected from α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, and hydroxypropyl β-cyclodextrin.

7. Use according to Claim 6, wherein the polymer is hydroxypropyl β-cyclodextrin.

8. Use according to any one of Claims 1 to 7, wherein the paper is selected from cellulose paper, nitrocellulose paper, nylon paper, cotton paper, and polyester paper.

9. A kit for use in a point-of-care luminescence assay, comprising the composition, wherein the composition comprises (a)a compound selected from selenotellurazine, selenotellurazine-h, selenotellurazine-h-h, furimazine, JRW-0238, JRW-1743, and JRW-1744, and (b) One or more additional components selected from the following: (i) A buffer selected from phosphate buffer, tris, and 2-(N-morpholino)ethanesulfonic acid; (ii) A surfactant selected from polysorbate 20, polysorbate 40, and polysorbate 80; (iii) A reducing agent selected from thiourea and 6-aza-2-thiothymine; (iv) A salt selected from sodium chloride and sodium phosphate; (v) A radical scavenger selected from ascorbic acid and sodium ascorbate; and (vi) A chelating agent selected from citric acid and trans-1,2-diaminocyclohexane-tetraacetic acid; and (c) Paper, and A kit, wherein the compound and the one or more additional components are dried on the paper.

10. The kit according to claim 9, wherein the composition is contained in one or more containers.

11. The kit according to claim 10, wherein the composition is contained in a plurality of tubes.

12. The kit according to any one of claims 9 to 11, wherein the composition is in the form of a plurality of paper spots, and each spot has a diameter of 2 mm to 5 mm.

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