Solid composition of indocyanine green and sodium fluorescein

Abstract

The present invention relates to novel solid compositions of indocyanine green, a compound of formula (IA), and sodium fluorescein, a compound of formula (IB), their use in the diagnostic field, and kits containing such compositions. [Formula 1] JPEG2024510876000021.jpg61166

Description

【Technical Field】 【0001】 The present invention relates to a novel solid composition of indocyanine green which is a compound of the following formula (IA) and sodium fluorescein which is a compound of the following formula (IB), its use in the diagnostic field, and a kit containing such a composition. 【Chemical formula】 【0002】 The present invention relates to the field of contrast agents, particularly contrast agents used in ophthalmology. 【Background Art】 【0003】 Fluorescein is usually administered as a sodium salt and exists as an odorless reddish-brown solid at room temperature. When excited by ultraviolet light at 254 nm and in the blue range (465 - 490 nm), it emits strong fluorescence in the range of 520 - 530 nm (a very characteristic yellow-green color). Sodium fluorescein is a sterile aqueous solution contained in 5 ml or 2 ml vials and is commercially available in various concentrations of 0.25 g / ml, 0.2 g / ml to 0.1 g / ml in Europe or the United States. 【0004】 Such techniques provide diagnostic and useful information for subsequent treatment and enable visualization of angiogenesis in the retina and choroid. This test can highlight untreated (ischemic) areas and lesions caused by retinal angiogenesis. The yellow-green fluorescence of the drug indicates the boundary of the vascular regions of the retina and iris. Fluorescein is completely removed by the kidneys within 24 - 36 hours after intravenous administration. 【0005】 More recently, indocyanine green angiography (ICGA) has been developed. This dye was developed by Kodak Research Laboratories in 1955 for photographic purposes and was approved for clinical use in 1956, but it took 10 years before it was used in angiography, and not until the 1970s for retinal angiography. 【0006】 Indocyanine green of formula (IA) (ICG, 1H-benz[e]indole, 2-[7-[1,3-dihydro-1,1-dimethyl-3-(4-sulfobutyl)-2H-benz[e]indole-2-ylidene]-1,3,5-heptatrienyl]-1,1-dimethyl-3-(4-sulfobutyl)hydroxyl, intramolecular salt, sodium salt, CAS RN 3599-32-4) is sold in solid form as a sterile lyophilized powder containing 25 mg or 50 mg of indocyanine green in the presence of 5% or less sodium iodide. The amount that can be administered for ophthalmic angiography should not exceed 0.1 to 0.3 mg per kg of body weight as a bolus injection. Dissolve a 25 mg dose of the compound in 5 ml of water as an injection solution, and dissolve a 50 mg dose of the compound in 10 ml, so that 1 ml of the reconstituted injection solution contains 5 mg of indocyanine green of formula (IA). 【0007】 The total daily dose for adults should be kept below 5 mg per kg of body weight. The maximum absorption and emission of indocyanine green are both in the infrared range, with maximum absorption at 800 nm and maximum emission at 830 nm. When dissolved in water, indocyanine green decomposes to an undetectable level in just a few hours. 【0008】 Because the presence of sodium iodide can cause a severe anaphylactic reaction, it should only be used under the supervision of a physician. 【0009】 For injectable preparations, a maximum of 40 mg of indocyanine green dye can be used in 2 ml of sterile water. Immediately after injecting the dye, a 5 ml bolus of physiological saline must be injected. 【0010】 Because the indocyanine green molecule is the largest (molecular weight 775 Da compared to fluorescein's 332 Da), it binds more strongly to proteins in plasma compared to fluorescein and instead fluoresces in the infrared spectrum. Therefore, it is metabolized by the liver. 【0011】 Initially, ICG angiography was performed using infrared photographic film. However, due to the relatively weak fluorescence properties of the dye and the poor sensitivity of the film, this method was abandoned. 【0012】 Because indocyanine green binds strongly to plasma proteins, its metabolism is slower compared to fluorescein, resulting in a reduced amount of fluorescence available for imaging. A digital video camera was used to acquire images for ICG angiography. 【0013】 Another approach to fluorescence angiography (using fluorescein or indocyanine green) is the scanning laser ophthalmoscope. Due to recent marketing efforts, this tool has become a common part of clinical practice. Advantages of the scanning laser ophthalmoscope include the ability to use excitation light that scans the retina, thereby enabling stronger excitation (and thus a stronger emission signal) while using safe illumination levels. This is possible because the scan range illuminates each point in the retinal region for only 0.1–0.7 microseconds. Angiography using a scanning laser ophthalmoscope provides more accessible time information than still imaging systems, as it allows imaging at a rate of 20–30 photograms per second using actual video. 【0014】 While ICG angiography may be advantageous in certain cases of subretinal neovascularization and other diseases, one of its drawbacks is the long duration of the angiography (45 minutes) and the need to obtain a second angiography after the first one using fluorescein. The process of obtaining both fluorescein and ICG angiography is lengthy, requiring 2-3 injections per patient to complete a study set. Furthermore, because angiography is performed at various times, it becomes difficult to determine whether variations in fluorescence leakage are due to differences in dye properties or image quality. 【0015】 To overcome these shortcomings, in 1998, simultaneous angiography was performed using a PC-based miniature digital confocal scanning laser ophthalmoscope to administer fluorescein and indocyanine green simultaneously (WR Freeman et al., Arch. Ophysical., vol. 116, Apr. 1998, 455-463), enabling simultaneous imaging after a single intravenous injection of indocyanine green and fluorescein. Clinically, no serious side effects were observed in 169 simultaneous injections of indocyanine green and fluorescein. The time required to conduct the entire study was significantly shorter compared to conducting initial studies on fluorescein, reviewing them, and then conducting studies on indocyanine green. 【0016】 Currently, two dyes are injected simultaneously, reaching the retinal and choroidal levels within seconds of injection. Using a special device known as a fluorescence angiography system, it is possible to image the back of the eye through all steps of dye passage, thereby identifying the presence of changes such as ischemic areas, neovascularization, areas of dye diffusion (leakage) from optical discs or retinal blood vessels, and inflammatory foci. FAG and ICGA are routine but invasive tests, and therefore, since fluorescein is cleared from the kidneys within a few hours after injection, it is necessary to confirm that the patient is not allergic to the contrast agent, and thus normal renal function must be confirmed before performing the test. The test is usually performed on an empty stomach or after breakfast or a very light meal. 【0017】 With the advent of high-resolution OCT and advanced OCT angiography, this invasive test can often be avoided. However, in some situations, it remains fundamental, and a multimode approach is necessary to achieve accurate diagnosis and treatment for the patient. Ophthalmologists must determine which test is most appropriate for each individual patient on a case-by-case basis. 【0018】 The simultaneous administration of the two dyes is practically carried out by adding an aqueous solution of sodium fluorescein, the contents of a vial, to indocyanine green powder in order to obtain a sterile solution of the mixture of the two dyes without precipitate. It is clear how this preparation method implies potential problems, as it requires taking the solution containing sodium fluorescein in one syringe, adding it to the sterile indocyanine green powder, waiting for the solution to form without precipitate, and then taking the final solution again using the same or a different syringe. 【0019】 Another problem is that it is necessary to administer a different amount of dye than that which is commercially available, and therefore, mixtures that cannot be administered to other patients must be discarded. Since indocyanine green is expensive, this can be costly to discard. 【0020】 Another problem is that indocyanine green is not stable in aqueous solution over a long period of time, and thus the mixture cannot be stored. 【Summary of the Invention】 【0021】 An object of the present invention is a novel composition in solid state obtained by simultaneously lyophilizing two dyes, compounds of formula (IA) and (IB), wherein the amount of the compound of formula (IA) is 2.5 mg, 5 mg, 10 mg, 25 mg, 50 mg or 100 mg, and the amount of the compound of formula (IB) is 0.1 g, 0.2 g, 0.4 g, 2.5 g, 2.0 g, 1.25 g or 1 g. The concentration that can be obtained by dissolving the mixture in water is described in the experimental part. 【0022】 The applicant has surprisingly found that a composition of the compounds of formula (IA) and (IB) is water-soluble and stable when prepared by lyophilization, without the addition of NaI as is done in the prior art to stabilize the compound of formula (IA). The composition of the present invention can contain NaI in an amount of 0% ≦ NaI ≦ 2.5%, for example 0%, 0.9%, 2.5%. The percentage refers to the amount of the compound of formula (IA) based on potentiometric titration using a silver electrode according to the United States Pharmacopeia monograph. 【0023】 The composition of the present invention appears to have not been described hitherto, can overcome the aforementioned problems, and enables the simultaneous preparation of the dosages required for diagnostic imaging. 【0024】 The compounds of formula (IB) used in the present invention are commercially available. In particular, the compounds of formula (IA) of the present invention may be commercially available, but are conveniently prepared as described in Italian Patent Application (IT102021000006794) (not published) filed by the same applicant. The aforementioned application has a total impurity content of 0.5% or less and individual impurities of 0.10% or less (purity is measured by a new analytical method HPLC at a wavelength of 254 nm), and also has a stable NaI-containing related composition that is water-soluble and has a NaI content of 2.5% or less. It relates to a new process for preparing indocyanine green of formula (IA). 【0025】 The process comprises the following steps: (a) Reacting the compound 1,1,2-trimethyl-1H-benzo[e]indole of formula (II) with 1,4-butanethione of formula (III) 【Chemical formula】 【Chemical formula】 in a suitable high-boiling solvent to provide 4-(1,1,2-trimethyl-1H-benzo[e]indol-3-yl)butane-1-sulfonate of formula (IV) according to a known method, 【Chemical formula】 (b) Reacting the compound of formula (IV) with the compound of formula (V), benzenamine, N-[(2E,4E)-5-(phenylamino)-2,4-pentadien-1-ylidene]-, hydrochloride (1:1) (known as GAD), 【Chemical formula】 in the presence of acetic anhydride and sodium acetate, using a dipolar aprotic solvent to react to provide the final compound of formula (IA) without isolating the intermediate, assuming the synthesis of the compound of formula (IA). 【0026】 Step a) is also well known, and the reaction can be carried out at a temperature depending on the temperature of the high boiling point solvent used, such as xylene and acetone, using aprotic solvents such as hexane, cyclohexane, toluene, xylene, tetrahydrofuran, acetone, acetonitrile, 1,4-dioxane, diethyl ether, dichloromethane, ethyl acetate, N,N-dimethylformamide, methyl tert-butyl ether, etc. 【0027】 The applicant used xylene as the solvent at a temperature of approximately 130°C. Anisole could also be used, yielding favorable results in terms of reaction rate; while it typically required 24 hours with xylene at 125–130°C, complete conversion occurred in 7–8 hours at 140–150°C. The intermediate compound of formula (IV) was isolated by precipitation with the addition of acetone to the reaction mixture and used directly in a wet state without recrystallization, as described in US2019 / 0337896. 【0028】 Therefore, the process is characterized by the direct implementation of step b), i.e., a "one-step" process, without the isolation of any intermediates, nor the need to purify either intermediate (VI) or intermediate (VII), as occurs in known prior art and the aforementioned synthesis. 【0029】 Step b) is carried out by condensing the compound of formula (IV) and the compound of formula (V) in the presence of a solvent (acetonitrile), acetic anhydride, and sodium acetate, as is already known. The reaction is carried out at a temperature of 40-50°C to form the crude compound of formula (IA). The compounds of formula (V) and formula (IV) are dissolved in acetonitrile in the presence of sodium acetate (4 equivalents). Then, acetic anhydride (4 equivalents) is added at a temperature lower than that published in US2019 / 0337896, and the reaction is carried out at the same temperature for 1-3 hours. By using acetonitrile in this "one-pot" step, it is possible to use acetic anhydride in the minimum amount necessary for a complete reaction, and therefore acetic anhydride is not used as the reaction solvent. This makes its removal by distillation easier, and therefore makes the use of the water / isopropanol mixture used for the isolation of the crude compound of formula (IA) safer. 【0030】 In fact, the subsequent processing was carried out using isopropanol to separate the crude solid of the compound of formula (IA). 【0031】 The compound of formula (IA) obtained in this crude form already exhibits a high HPLC purity level (>90%), with the only significant impurity present being impurity A. Compound (IA) can be conveniently purified by crystallization in isopropanol / H2O rather than by known methods such as the methanol / isopropanol mixture of US2019 / 0337896, or acetone, isopropanol, or methanol used in other references. No previously mentioned references have described the use of an isopropanol / H2O mixture in appropriate ratios selected from the following, i.e., isopropanol / water: 5.9 / 3.4 or 7.4 / 3.4 or 9.9 / 3.4 in volume (liters / kg) for the crude compound of formula (IA). 【0032】 Using isopropanol / H2O has the advantage of providing the compound of formula (IA) with a purity of 99.5 or higher, despite the fact that intermediate (VI) has not been isolated. 【0033】 The solid compositions of the compounds of formulas (IA) and (IB) of the present invention, in powder form, are conveniently housed in suitable containers that can ensure the sterility of the composition and prevent contact with air, particularly oxygen. 【0034】 A further object of the present invention is a convenient "single dose" composition comprising, in particular, 5 mg of the compound of formula (IA) and 200 mg of the compound of formula (IB). Such a composition is particularly advantageous for professionals when it is necessary to prepare a mixture of two components to be injected in an appropriate dose typically used in a single injection in clinical practice. 【0035】 A further object of the present invention is a kit comprising a container containing the composition according to the present invention and a vial of sterile water suitable for reconstituting an injectable solution. 【0036】 The object of the present invention is also a kit comprising the solid compositions of the present invention of compounds of formulas (IA) and (IB), housed in a suitable container that ensures the sterility of the composition and non-contact with air, particularly oxygen, which can cause known degradation. 【0037】 [Experiment Part] [ Freeze-drying conditions ] The freeze-drying conditions used for all preparations are as follows: Freeze dryer: Edwards MINIFAST 680 Temperature: -40°C at the start of freeze-drying; +5°C at the end of freeze-drying. Pressure (vacuum): 6.6 at the start of freeze-drying. * 10 2 mbar, at the end of freeze-drying: 4.6 * 10 -2 mbar Freeze-drying time: 72 hours Nitrogen was injected into the amber-colored glass vial, and it was sealed at the end of distillation. 【0038】 [ Analytical method used to determine the purity of the compound of formula (IA) ] • Column HPLC: ODS Hypersil 4.6x250mm 5μm Column temperature: 40°C • Detector: UV254nm Step A: Ammonium formate 4.09 g / L, pH=5.0 (using formic acid) Step B: Acetonitrile ·Mixed phase: 70:30 A:B ·Flow rate: 1.5ml / min ·Injection volume: 10μL ·Analysis time: 30 minutes [Table 1] 【0039】 [ Sample preparation (Method 1) ] Dissolve 40 mg in a 50 ml flask and adjust the volume with the mixed phase to the correct level. The material is subjected to sonication for 5 minutes to confirm complete solubilization. The sample will not stabilize after 30 minutes, so inject it immediately. . 【0040】 [ Sample preparation (Method 2) ] Dissolve 40 mg in a 50 ml flask and adjust the volume with methanol. Sonicate for 5 minutes to confirm complete solubilization. Inject immediately. The sample will not stabilize after 30 minutes, so inject it immediately. . 【0041】 The use of methanol allows for better stabilization of the compound of formula (IA), [MH] + :752.5, that is, it is possible to prevent the formation of degradation impurities having "-1" with respect to the product. Inject every 1-10 minutes. 【0042】 [Example 1] [ Preparation of 4-(1,1,2-trimethyl-1H-benzo[e]indolyl-3-yl)butan-1-sulfonate of formula (IV) ] [ka] 31.1 g of compound (II) (0.15 mol, 1 equivalent, commercially available) and 40.5 g of compound (III) (0.30 mol, 2 equivalents, commercially available) were packed into a 2 L reactor under a nitrogen stream in 93 ml of xylene. The suspension was stirred and heated to approximately 130°C for 24 hours. The suspension was cooled and acetone (200 ml) was added. The resulting solid was then filtered and dried under vacuum. In this way, 48.5 g of the desired compound was obtained, which corresponds to a yield of 94.5% (HPLC purity: 97-98%). 【0043】 Alternatively, an equal amount of anisole can be used instead of xylene as the solvent. Following the same protocol, if the reaction is carried out at 140°C, the conversion of the product is completed in 6–8 hours. The yield and amount obtained are similar to those obtained in the reaction in xylene. 【0044】 [Example 2] [ Preparation of the compound of formula (IA) (one-pot synthesis) ] [ka] 20.0 g of compound (V) (0.07 mol, 1 equivalent, commercially available), 48.5 g of compound (IV) (prepared as described in Example 1, 0.14 mol, 2 equivalents), 23 g of sodium acetate (0.28 mol, 4 equivalents), and 180 ml of acetonitrile are packed into a 1 L reactor under a nitrogen stream. The suspension is stirred at 20-25°C, and 28.8 g of acetic anhydride (0.28 mol, 4 equivalents) is added dropwise over 5-10 minutes. The suspension is heated to 45-50°C, and stirring is continued for approximately 2 hours. The reaction mixture is concentrated in vacuum while maintaining the temperature at 40-50°C. Then, the mixture is returned to atmospheric pressure, 100 ml of isopropanol is added, and the reaction mixture is concentrated again in vacuum while maintaining the temperature at 40-50°C. 【0045】 Subsequently, water (180 ml) and isopropanol (320 ml) are added, the product is dissolved at 50-55°C, then isopropanol (100 ml) is added, and the mixture is gradually cooled to 20-25°C. The mixture is filtered and washed with isopropanol. In this way, the desired wet compound is obtained (yield after weight loss: 46.3 g, 85.1% relative to compound (V), HPLC purity 80-85%). 【0046】 [Example 3] [ Purification of the compound of formula (IA) (without NaI) ] Fill a 1-liter flask with 49 g of wet indocyanine green (prepared as described in Example 2, equivalent to 24.4 g (dry)), 130 ml of isopropanol, and 77 ml of water. Heat to 50-55°C and stir until completely dissolved. Calibrate the pH of the solution to 7.5-8.5 with 5% NaOH and cool to 40-45°C. 【0047】 While maintaining a temperature of 40-45°C, add isopropanol (48 ml), then gradually cool to 20-25°C, continue stirring for 1.5 hours, then filter and wash with isopropanol (2 x 48 ml). The powder is dried in a vacuum at 60°C for 40 hours. Yield: 20g (82.9%). HPLC purity: 99.5%; Impurity A: 0.40%. Sodium iodide content: 0%. 【0048】 [Example 4] [ Preparation of the compound of formula (IA) using NaI (crystallization of I). ] 93.6 g of the crude compound of formula (IA) (46.3 g of the theoretically dry compound, prepared as described in Example 2) and sodium iodide (1.39 g; 3% w / w) are suspended in 250 ml of isopropanol and 148 ml of water. The suspension is heated to a temperature of 55-60°C and stirred until completely dissolved. The pH is calibrated to 7.5-8.5 using a 2.5% w / w sodium hydroxide solution. The solution is cooled to a temperature of 45-50°C, and 93 ml of isopropanol is added after 15-30 minutes. The solution is slowly cooled to a temperature of 20-25°C, and stirring is continued for 30 minutes. The suspension is then brought to 35-40°C and stirred for about 1 hour, then cooled again to 20-25°C for about 2 hours, and finally filtered at 20-30°C and washed with isopropanol. 【0049】 The above materials were dried in a vacuum at 50-80°C for 8-48 hours to obtain 35.89 g of the desired product in a yield of 77.5% (for each packed crude dried product). HPLC purity: 99.6%; Impurity A: 0.28% Iodide (potentiometric titration with silver electrode): 1%. 【0050】 When known impurities exceed 0.15% and unknown impurities exceed 0.1%, a second crystallization can be carried out using less sodium iodide, and reducing the amount of sodium iodide is essential to keep the amount of sodium iodide in the final product below 2.5% (Example 6). 【0051】 [Example 5] [ Preparation of the compound of formula (IA) using NaI (II crystallization) ] The wet compound of formula (IA) obtained from the first crystallization (prepared as described in Example 4) (63.7%, equivalent to the corresponding dry product of 35.9 g on a weight loss basis), sodium iodide (0.54 g; 1.5% w / w), isopropanol (194 ml), and water (115 ml) are packed into a 1 liter reactor. The mixture is heated to 55-60°C until completely dissolved, then the solution is filtered on cardboard, the filter is washed with water (7 ml), then with isopropanol (18 ml). The filtrate is brought to 55-60°C, the pH is calibrated to the range of 7.5-8.5 with diluted NaOH as needed, then cooled to 45-50°C, and isopropanol (54 ml) is added after about 30 minutes. 【0052】 The above material is slowly cooled to 20-25°C, then heated again to 35-40°C for about 1 hour, then returned to 20-25°C for about 1 hour, and stirred for 30 minutes. The suspension is filtered, washed with isopropanol to obtain a wet product, which is then dried in a vacuum at 50-80°C for 24-48 hours. Yield: 26.9g (75%). The purity of the product obtained in this way is 99.5% or higher when measured using the HPLC method of the present invention. HPLC purity: 99.92% Impurities A, B, C, D, E, and F: Not quantifiable (HPLC). Maximum unknown impurity: 0.084% (HPLC; rrt: 0.45). Sodium iodide (potentiometric titration using USP monograph): 0.9%. Residual isopropanol: 1597 ppm. 【0053】 [Example 5] [ Drying freeze test of compounds of formulas (IA) and (IB) ] First, 5 mg / ml containing 0.9% sodium iodide. 水 A 10 ml aqueous solution of compound (IA) prepared as described in Example 5 at the specified concentration, and a 5 mg / ml solution prepared without using sodium iodide. 水 A 10 ml aqueous solution of the compound of formula (IA) prepared as described in Example 3 at the specified concentration, and 200 mg / ml水 A dry-freeze test was performed on 10 ml aqueous solutions of the commercially available compound (IB) at the specified concentration. 【0054】 Dry freezing was completed in 72 hours, and all three dry frozen products were water-soluble. 【0055】 In particular, the compound of formula (IA) obtained without using sodium iodide (sodium iodide = 0% by potentiometric titration using a silver electrode) does not dissolve in water at 20-25°C at clinically usable concentrations of 5 mg / ml or 2.5 mg / ml when used as an isolated dried powder after crystallization. 【0056】 Surprisingly, the same dried, frozen powder dissolves at concentrations of 2.5 mg / ml, 5 mg / ml, and even 10 mg / ml. 【0057】 Solubility was evaluated by filtering all obtained solutions through a syringe filter with a 0.45 μm pore. For the 5 mg / ml concentration of lyophilized powder, a filter with a 0.2 μm pore was used for evaluation, regardless of the presence or absence of NaI. It was observed that the filtration was performed fluidly and that no residue remained on the filter or in the vial from which the solution was extracted. 【0058】 This molecule is likely soluble in itself, but for kinetic reasons, it does not dissolve on a reasonable timescale. On the other hand, the dried frozen powder tends to dissolve completely and instantly without even requiring sonication, due to its large relative surface area in contact with water. These results are summarized in Table 1. [Table 2] 【0059】 Surprisingly, it was also discovered that the two components could be freeze-dried simultaneously and administered in appropriate amounts in the same vial. The freeze-dried product obtained in this way is soluble in water even at low concentrations of sodium iodide, making it suitable for simultaneous administration of the two dyes by injection after reconstitution of the aqueous solution. 【0060】 The solubilization of the lyophilized formulations 13-18 (Table 2) is also immediate, regardless of the presence or absence of NaI. 【0061】 In particular, it is possible to prepare lyophilized products containing the compound of formula (IA), the compound of formula (IB), and sodium iodide (expressed as a weight percent relative to the compound of formula (IA)), as reported in Table 2 below: [Table 3] 【0062】 [Example 6] [ Preparation of the dried freeze product of the compound of formula (IB) ] Dissolve fluorescein sodium (commercially available, 5.0 g), the compound of formula (IB), in water (25 ml) and sonicate for 1 minute. Fill five different amber-colored glass vials with this solution (approximately 5 ml of solution per vial). Freeze-dry the vials using the freeze-drying conditions already described. 【0063】 [Example 7] [ Preparation of the dried freeze product of the compound of formula (IA) ] Dissolve the compound of formula (IA) (prepared according to Example 5 or Example 3, 125 mg) in water (25 ml) and sonicate for 1 minute. 【0064】 This solution is used to fill five different amber-colored glass vials (approximately 5 ml of solution per vial). The vials are then freeze-dried using the freeze-drying conditions previously described. 【0065】 [Example 8] [ Preparation of solid compositions of compounds of formulas (IA) and (IB) ] Preparation of NaI solution Weigh 30 mg of sodium iodide into a 100 ml volumetric flask and dilute to the desired volume with water for HPLC. Sonicate for 5 minutes. 【0066】 A general method for preparing a mixture of compounds of formulas (IA) and (IB). The exact amounts of each component in the mixture are shown in Table 2. Accurately weigh the compound of formula (IA) or (IB) into a 20 ml amber glass vial. Add the correct amount of sodium iodide solution that was prepared earlier. Add the appropriate amount of water. The vial is sonicated under nitrogen at 20-25°C for approximately 1 minute. Freeze-drying is performed using the freeze-drying conditions already described. Five duplicates were prepared for each solution. 【0067】 [Example 9] [ General method for preparing mixtures of compounds of formulas (IA) and (IB): Formulations 11, 12, 13, 14, 15, 16, 17, 18 ] The exact amounts of each component in the mixture are shown in Table 2. Accurately weigh the compound of formula (IA) or (IB) into a 10 ml flask. Add the appropriate amount of water. The vial is sonicated under nitrogen at 20-30°C for approximately 1 minute. If necessary, filter the entire solution obtained above through a 0.2 μm syringe filter in a new vial. Freeze-drying is performed using the freeze-drying conditions already described. 【0068】 [Example 10] [ Stability of solid compositions of compounds of formulas (IA) and (IB) ] Compounds of formula (IA) (with or without iodide) and compound (IB) were analyzed by HPLC both as is and after lyophilization according to the following method. The analysis demonstrated that such compounds are stable. 【0069】 Analysis method (HPLC) • Column HPLC: ODS Hypersil 4.6x250mm 5μm Column temperature: 40°C • Detector: UV254nm Step A: Ammonium formate 4.09 g / L, pH=5.0 (using formic acid) Step B: Acetonitrile ·Mixed phase: 70:30 A:B ·Flow rate: 1.5ml / min ·Injection volume: 10μL ·Analysis time: 30 minutes [Table 4] 【0070】 Formulations 13-18 and the compounds used in their preparation were analyzed using the following analytical methods. Based on the fact that no peaks other than Ia and Ib were present in the chromatogram, the purity of the injected IA was >99.5%, and the formulations after lyophilization were confirmed to be stable. 【0071】 Analysis method (HPLC) • Column HPLC: Polaris3 C18-A 150x4.6mm Column temperature: 20°C • Detector: UV254nm Step A: Adjust the pH of 2.3g of ammonium acetate in 1000ml to 6.8±0.05 with dilute acetic acid or ammonia. Step B: Acetonitrile • Diluent: methanol ·Flow rate: 1.5ml / min ·Injection volume: 10μL ·Analysis time: 34 minutes • Autosampler temperature: 5℃ [Table 5] 【0072】 Mixtures of formulas (IA) and (IB) listed in Table 2 were prepared, then appropriately diluted and injected into an HPLC system to evaluate the profile at T0. 【0073】 The same mixture was precisely prepared, dried, and frozen as described in the experimental section, and controlled by HPLC after lyophilization (7 days later). During these 7 days, the samples were stored in amber glass vials at room temperature under a protective atmosphere. The lyophilized product was dissolved in water at the same dilution ratio as the non-lyophilized sample. 【0074】 This analysis clearly shows that lyophilization does not degrade the compound of formula (IA), either in its original form or as a mixture with the compound of formula (IB), regardless of the presence or absence of sodium iodide. The stability of the lyophilized powder stored in vials was evaluated using HPLC after 6 months. The lyophilized product is stable even in the absence of sodium iodide (formulation 9). 【0075】 The reconstituted solutions were stable when stored at 4–10°C for at least 24 hours (formulations 1, 3, 5, and 7). [Table 6] [Table 7] [Table 8] [Table 9] [Table 10] [Table 11]

Claims

[Claim 1] A solid composition obtained by freeze-drying a compound of indocyanine green of formula (IA) and sodium fluorescein of formula (IB). 【Chemistry 1】 [Claim 2] The composition according to claim 1, wherein the amount of indocyanine green is 2.5 mg, 5 mg, 10 mg, 25 mg, 50 mg, or 100 mg, and the amount of fluorescein sodium is 0.1 g, 0.2 g, 0.4 g, 2.5 g, 2.0 g, 1.25 g, or 1 g. [Claim 3] The composition according to claim 1 or 2, comprising an amount of NaI equal to 0% ≤ NaI ≤ 2.5% relative to the amount of compound (IA), based on a potentiometric measurement method using a silver electrode in accordance with the USP monograph. [Claim 4] The composition according to each of claims 1 to 3, wherein the amount of indocyanine green is 5 mg and the amount of fluorescein sodium is 0.2 g. [Claim 5] A process for preparing the compositions according to claims 1 to 3 by freeze-drying aqueous solutions formed by compounds of formulas (IA) and (IB). [Claim 6] The process according to claim 5, wherein the aqueous solution is microfiltered before freeze-drying. [Claim 7] The compositions according to claims 1 to 3 for use in ophthalmic imaging diagnosis. [Claim 8] A kit comprising the solid composition according to claims 1 to 4 and a vial of sterile water suitable for producing an intravenous injection solution for use in ophthalmic imaging.

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