Crystalline indocyanine green, its method of manufacture, pharmaceutical compositions containing same, and methods of using them as medicines or diagnostic agents

Abstract

Crystalline forms of indocyanine green, methods for their preparation, pharmaceutical compositions containing same, and methods for their use as pharmaceuticals or diagnostic agents are provided. The present invention relates to a novel process for the preparation of indocyanine green, comprising the step of converting 4-[2-[7-[1,1-dimethyl-3-(4-sulfobutyl)benzo[e]indol-3-ium-2-yl]hepta-2,4,6-trienylidene]-1,1-dimethylbenzo[e]indol-3-yl]butane-1-sulfonic acid to indocyanine green by treatment with sodium chloride. The present invention also relates to crystalline forms of indocyanine green, pharmaceutical compositions containing same, and methods of their use as medicines or diagnostic agents.

Description

[Technical field]

[0001] The present invention relates to the fields of medical and pharmaceutical technology.The present invention relates to a crystalline form of indocyanine green, a method for producing said crystalline form and a method for using said crystalline form. [Background technology]

[0002] Indocyanine green (ICG) is a water-soluble tricarbocyanine fluorescent dye with the following chemical structure:

[0003] [ka]

[0004] ICG is a clinically approved near-infrared (NIR) fluorescent dye. ICG is commonly used in a wide range of medical imaging and diagnostic procedures, e.g., in measuring cardiac output and liver function, in visualizing hepatic blood flow and blood flow during ophthalmologic imaging, and in visualizing cancer tissue. These medical applications of ICG are based on its high absorbance in the spectral region (750-800 nm) where the penetration of human tissue is relatively high. These medical applications are made possible by ICG's extremely low toxicity.

[0005] In the above applications, ICG needs to be administered intravenously in the form of an aqueous solution. According to the U.S. Food and Drug Administration, in order to prepare a pharmaceutical composition for intravenous administration, ICG should be used in a form that is as pure as possible and has stable physical properties such as purity, solubility, and stability.

[0006] Currently, ICG is commercially available as a lyophilized powder that can be dissolved in sterile water for injection, and is sometimes supplied in kits. However, such indocyanine green is sensitive to air and light, and has poor stability not only in aqueous environments but also in the solid state. Poor stability in aqueous environments limits its usefulness in certain applications, such as time-sensitive surgical procedures. For this reason, ICG vendors give instructions that range from immediate use of the reconstituted ICG solution to use within 6 hours before patient administration (with any unused portion discarded). The poor stability of ICG in solid form presents challenges in ICG manufacture and long-term storage.

[0007] Furthermore, although currently available ICG compositions provide sufficient levels of ICG for use in the approved applications listed above, the solubility of ICG in water for injection (infusion) decreases when the concentration exceeds 5 mg / ml. The United States Pharmacopeia monograph for ICG states that the solubility of ICG in solution can be improved by the addition of up to 5 w / w% sodium iodide. However, iodine is a strong allergen. The risk of anaphylactic shock caused by intravenous injection of indocyanine green preparations containing up to 5% iodine cannot be ignored.

[0008] In other words, if the properties of ICG, particularly its long-term storage safety, solubility, and purity, can be improved, the clinical usefulness of ICG will increase, and such improved ICG properties will make treatment / diagnosis methods safer for patients in need of treatment.

[0009] In the prior art, many methods have been described with the aim of producing ICG with improved properties.

[0010] For example, WO 9507888 (Patent Document 1) discloses a method for producing high-purity substituted benzo[e]indoles, in particular ICG, in which an arylhydrazine is prepared and subjected to a Fischer indole synthesis together with a ketone. The resulting benzo[e]indole is reacted with a chemical radical and then subjected to a purification step.

[0011] US Patent No. 5,750,722 describes the preparation of highly pure indocyanine green by reacting 1,1,2-trimethyl-1H-benzo[e]indole with 1,4-butanesultone followed by treatment with N-((2E,4E)-5-(phenylamino)penta-2,4-dienylidene)aniline hydrochloride in the presence of triethylamine and sodium acetate. The resulting indocyanine green is free of iodide ions and contains less than 0.5% residual impurities. After lyophilization, the resulting product in the solid state has limited stability.

[0012] WO 2017 / 093889A1 (Patent Document 3) describes a method for producing high-purity indocyanine green, which comprises reacting 1,1,2-trimethyl-1H-benzo[e]indole with 1,4-butanesulfone to obtain 4-(1,1,2-trimethyl-1H-benzo[e]indolium-3-yl)butane-1-sulfonate, reacting the 4-(1,1,2-trimethyl-1H-benzo[e]indolium-3-yl)butane-1-sulfonate with N-phenyl-N-((1E,3E,5E)-5-(phenylimino)penta-1,3-dienyl)acetamide in the presence of sodium acetate and alcohol, and finally extracting the resulting compound with an ester solvent. The resulting ICG has a high purity of about 99.0% and is in the form of a non-crystalline (amorphous) powder. Reproduction of the process disclosed therein results in an essentially amorphous form of ICG, which is also characterized by limited stability.

[0013] US Patent Application Publication No. 2019 / 0337896 (Patent Document 4) describes a method for producing indocyanine green by reacting 1,1,2-trimethyl-1H-benzo[e]indole with 1,4-butanesultone, followed by treatment with N-((2E,4E)-5-(phenylamino)penta-2,4-dienylidene)aniline hydrochloride in the presence of triethylamine and sodium iodide. The method also includes steps for purifying intermediates and purifying the final product of ICG. The indocyanine green obtained by the method has a purity of more than 99.5% and contains less than 0.5% of impurities. Analysis of the product by X-ray diffraction reveals that it is a mostly amorphous product. The presence of two unclear peaks (2θ=3.30°, 4.86°) on the PXRD (powder X-ray diffraction) pattern cannot be concluded to be due to the crystalline form of ICG. Furthermore, reproduction of the process disclosed therein results in an essentially amorphous form of ICG, but as demonstrated in the Examples section of the present application, rapid decomposition of this solid form of ICG is observed.

[0014] Chinese Patent Application Publication No. 104130178 (Patent Document 5) and Chinese Patent Application Publication No. 106631979 (Patent Document 6) also disclose methods for producing ICG similar to the method described in U.S. Patent Application Publication No. 2019 / 0337896 (Patent Document 4). The resulting ICG has a purity of more than 98.0%.

[0015] None of these documents discloses crystalline forms of ICG or methods for their preparation.

[0016] Indeed, compared to amorphous forms, crystalline forms generally have lower impurity concentrations and more consistent product quality, with more consistent physical properties, such as color and long-term shelf life. A crystalline form of API ensures repeatable batch-to-batch quality control results in terms of physical properties, making it a more reliable API. Crystalline products are also easier to handle and manufacture on an industrial scale. Thus, the ability to provide ICG compounds in a substantially crystalline form would be an important advantage. [Prior art documents] [Patent documents]

[0017] [Patent Document 1] WO 95 / 07888 [Patent Document 2] U.S. Pat. No. 5,750,722 [Patent Document 3] International Publication No. 2017 / 093889 [Patent Document 4] US Patent Application Publication No. 2019 / 337896 [Patent Document 5] China Patent Application Publication No. 104130178 [Patent Document 6] China Patent Application Publication No. 106631979 Summary of the Invention [Problem to be solved by the invention]

[0018] The present invention provides a crystalline form of indocyanine green compound and a method for its preparation. [Means for solving the problem]

[0019] The applicants of the present application have developed a novel method for the preparation of ICG that leads directly to a crystalline form of indocyanine green not known in the art.

[0020] The method of the present invention significantly improves the ease of processing in that it reproducibly yields crystalline ICG that exhibits unique polymorphic forms, which offer many efficiency and economic advantages for industrial-scale applications.

[0021] Specifically, the discovery of such crystalline forms of ICG provides new opportunities for improving the properties of ICG not only in the solid state but also in formulations for intravenous administration. In particular, the applicants have discovered that crystalline indocyanine green, as described in detail below, exhibits significant properties, particularly in terms of purity and long-term stability, compared to the essentially amorphous forms of indocyanine green of the prior art.

[0022] In a first aspect, the present invention relates to a crystalline form of indocyanine green characterized by an X-ray powder diffraction pattern exhibiting peaks at least at 2θ=20.66°±0.2°, 14.38°±0.2°, 12.81°±0.2°, 4.22°±0.2° and 3.83°±0.2°.

[0023] Advantageously, said X-ray diffraction pattern exhibits additional peaks at 2θ=23.26°±0.2°, 20.24°±0.2°, 17.72°±0.2° and 5.30°±0.2°.

[0024] Advantageously, said X-ray diffraction pattern further exhibits additional peaks at 2θ=19.24°±0.2°, 18.22°±0.2°, 18.08°±0.2°, 7.69°±0.2°, 6.37°±0.2° and 5.01°±0.2°.

[0025] Advantageously, the indocyanine green according to the present invention has a purity of at least 98.0%, preferably at least 99.0%, more preferably at least 99.5%, even more preferably at least 99.7% and most preferably at least 99.8%, measured as area percent by HPLC at 240 nm.

[0026] Preferably, each impurity is less than 0.50%, preferably less than 0.40%, more preferably less than 0.20%, and most preferably less than 0.15%, by area percent HPLC at 240 nm.

[0027] Advantageously, the crystalline form of ICG according to the invention has the appearance of red crystals.

[0028] The present invention further relates to a pharmaceutical composition comprising indocyanine green as described above and in more detail below, and a pharma- ceutically acceptable carrier.

[0029] Preferably, the pharma- ceutically acceptable carrier comprises at least one diluent selected from the group consisting of water for injection, in particular sterile water for injection (SWFI) and / or bacteriostatic water for injection (BWFI), ethyl alcohol, methyl alcohol, isopropyl alcohol, n-propyl alcohol, butyl alcohol, glycerin, propylene glycol, polyethylene glycol and mixtures thereof, preferably water for injection.

[0030] Preferably, the pharmaceutical composition further comprises at least one compound selected from the group consisting of histidine, ethylenediaminetetraacetic acid (EDTA) and its salts, cysteine, sodium chloride, dithiothreitol, ascorbic acid, sodium ascorbate, disodium hydrogen phosphate, sodium dihydrogen phosphate, and mixtures thereof.

[0031] In a variant, the pharmaceutical composition comprises indocyanine green according to the invention and at least one compound selected from ascorbic acid, its salts or combinations thereof.

[0032] In another variation, the pharmaceutical composition comprises indocyanine green according to the present invention and histidine.

[0033] In another variation, the pharmaceutical composition comprises indocyanine green according to the invention, histidine, ethylenediaminetetraacetic acid (EDTA) and / or salts thereof, and sodium chloride.

[0034] The present invention further relates to indocyanine green or a pharmaceutical composition containing said indocyanine green, as described above and in detail below, for use as a medicine or diagnostic agent.

[0035] Preferably, said indocyanine green or said pharmaceutical composition containing said indocyanine green is used for obtaining angiographic images of a patient's tissue, and / or for measuring cardiac output, and / or for measuring liver function and hepatic blood flow, and / or for detecting sentinel lymph nodes, and / or for assessing and / or predicting skin-flaps viability, and / or for diagnosing and treating age-related macular degeneration, and / or for diagnosing and treating choroidal neovascularization and tumors.

[0036] In a second aspect, the present invention relates to a process for the preparation of indocyanine green, preferably a crystalline indocyanine green according to the present invention, characterized in that it comprises at least a step d) of converting 4-[2-[7-[1,1-dimethyl-3-(4-sulfobutyl)benzo[e]indol-3-ium-2-yl]hepta-2,4,6-trienylidene]-1,1-dimethylbenzo[e]indol-3-yl]butane-1-sulfonic acid of formula (VI) into 3,3,3',3'-tetramethyl-1,1'-di(4-sulfobutyl)-4,5,4',5'-dibenzoindotricarbocyanine sodium salt of formula (VII) by treatment with sodium chloride.

[0037] [ka]

[0038] Advantageously, said process for the preparation of indocyanine green further comprises a step e) of purifying indocyanine green after step d), said purification step e) comprising at least the steps e1) suspending, dispersing or dissolving indocyanine green of formula (VII) in a solvent, e2) heating the composition obtained in step e1), e3) cooling the solution obtained in step e2) and e4) recovering the indocyanine green in crystalline form.

[0039] Advantageously, said solvent of step e1) is chosen from the group consisting of water, acetone, methanol, ethanol, isopropyl alcohol, n-propanol, n-butanol and mixtures thereof.

[0040] Advantageously, in step e2), the composition is heated at a temperature ranging from 40 to 80°C, preferably from 40 to 65°C.

[0041] Advantageously, in step e3), the solution is cooled to a temperature ranging from 10 to 25°C.

[0042] Advantageously, in step e3), the cooling is carried out without stirring or agitation.

[0043] The present invention further relates to indocyanine green obtained by, obtainable by, or directly obtained by the process as defined above, in particular in which in step d) the sodium salt of formula (VII) has been treated with sodium chloride. [Brief description of the drawings]

[0044] [Figure 1] FIG. 1 shows a reaction scheme for obtaining indocyanine green in acid form of formula (VI). [Diagram 2] 1 is an X-ray diffraction pattern of indocyanine green prepared by the method according to the present invention (Example 1). [Diagram 3]1 is an X-ray diffraction pattern of indocyanine green prepared according to a method of Comparative Example (Example 2) including a salt formation step using sodium iodide. [Figure 4] 1 is an X-ray diffraction pattern of indocyanine green prepared according to a method of Comparative Example (Example 3) including a salt formation step using sodium acetate. [Diagram 5] 1 is an X-ray diffraction pattern of indocyanine green prepared according to the method of Comparative Example (Example 4). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0045] The phrase "consisting essentially of," when followed by one or more features, means that the methods or materials of the invention may contain features or steps in addition to the components or steps explicitly recited that do not materially affect the properties or characteristics of the invention.

[0046] The phrase "consisting of X through Y" is inclusive unless otherwise stated. This phrase means that the range includes values ​​X and Y, as well as all values ​​from X through Y.

[0047] References to "indocyanine green" or ICG throughout this application refer to the compound having formula (VIII) below.

[0048] References herein to methods of treatment may be taken as references to the compounds, pharmaceutical compositions and medicaments of the invention for use in methods of treatment of the human and / or animal body by treatment or for diagnosis.

[0049] (Crystalline form of indocyanine green)

[0050] In a first aspect, the present invention provides indocyanine green in crystalline form.

[0051] The crystalline forms of ICG according to the present invention are characterized by an X-ray powder diffraction pattern exhibiting at least major peaks at 2θ=20.66°±0.2°, 14.38°±0.2°, 12.81°±0.2°, 4.22°±0.2° and 3.83°±0.2°.

[0052] In a preferred embodiment, the crystalline form of ICG according to the present invention is characterized by an X-ray powder diffraction pattern exhibiting at least major peaks at 2θ=20.66°±0.2°, 14.38°±0.2°, 12.81°±0.2°, 4.22°±0.2°, and 3.83°±0.2°, with additional peaks at 2θ=23.26°±0.2°, 20.24°±0.2°, 17.72°±0.2°, and 5.30°±0.2°.

[0053] In a more preferred embodiment, the crystalline form of ICG according to the present invention is characterized by an X-ray powder diffraction pattern exhibiting at least major peaks at 2θ=20.66°±0.2°, 14.38°±0.2°, 12.81°±0.2°, 4.22°±0.2°, and 3.83°±0.2°, with additional peaks at 2θ=23.26°±0.2°, 20.24°±0.2°, 19.24°±0.2°, 18.22°±0.2°, 18.08°±0.2°, 17.72°±0.2°, 7.69°±0.2°, 6.37°±0.2°, 5.30°±0.2°, and 5.01°±0.2°.

[0054] In a most preferred embodiment, the crystalline form of ICG according to the present invention is characterized by the following angular positions in the X-ray powder diffraction pattern, expressed as scattering angle 2θ, interstitial distance d, intensity, and relative intensity (percentage of the most intense peak):

[0055] [Table 1]

[0056] The intensity and relative intensity values ​​of each peak may vary by ±15%.

[0057] Advantageously, the ICG according to the invention has a crystalline form characterized by the powder X-ray diffraction pattern shown in FIG.

[0058] Preferably, the ICG of the present invention has the appearance of red crystals, whereas the amorphous ICG of the prior art has the appearance of green powder. More precisely, the color of the crystals of the ICG of the present invention can be recognized as a brilliant golden red. This characteristic can be confirmed visually or by techniques known to those skilled in the art, such as spectrophotometry.

[0059] (Method of producing crystalline indocyanine green)

[0060] In a second aspect, the present invention relates to a method for preparing crystalline indocyanine green, comprising at least the step of salifying the acid form of indocyanine green with sodium chloride to obtain the sodium salt of indocyanine green, commonly called indocyanine green or ICG.

[0061] For the purposes of the present invention, the expression "indocyanine green in acid form" corresponds to the compound of formula (VI) below defined as 4-[2-[7-[1,1-dimethyl-3-(4-sulfobutyl)benzo[e]indol-3-ium-2-yl]hepta-2,4,6-trienylidene]-1,1-dimethylbenzo[e]indol-3-yl]butane-1-sulfonic acid.

[0062] In a preferred embodiment, the method according to the invention further comprises at least a step e) of purifying the indocyanine green obtained in step d).

[0063] [Salt production process d)]

[0064] Specifically, the process according to the present invention comprises at least a step d) of converting 4-[2-[7-[1,1-dimethyl-3-(4-sulfobutyl)benzo[e]indol-3-ium-2-yl]hepta-2,4,6-trienylidene]-1,1-dimethylbenzo[e]indol-3-yl]butane-1-sulfonic acid of formula (VI) into 3,3,3',3'-tetramethyl-1,1'-di(4-sulfobutyl)-4,5,4',5'-dibenzoindotricarbocyanine sodium salt of formula (VII) by treatment with or use of sodium chloride, according to the following scheme:

[0065] [ka]

[0066] Advantageously, in step d), sodium chloride is used in an amount such that the molar ratio of indocyanine green in acid form of formula (VI) to said sodium chloride is less than or equal to 1:1, preferably ranging from 1:5 to 1:1, more preferably from 1:3 to 1:1.

[0067] Advantageously, the reaction of step d) is carried out in a solvent selected from the group consisting of water, methanol, ethanol, isopropyl alcohol, n-propanol, n-butanol and mixtures thereof.

[0068] In a preferred embodiment, step d) is carried out in a mixture of water and methanol, preferably in a volume ratio ranging from 5:1 to 1:5, preferably from 2:1 to 1:5, more preferably from 2:1 to 1:2.

[0069] Advantageously, step d) is carried out at a temperature ranging from 25 to 65°C, preferably from 40 to 55°C.

[0070] Advantageously, after completion of the reaction of step d), the reaction mixture is cooled to room temperature and the product of formula (VII) is isolated from the reaction solvent by precipitation, preferably by adding a solvent selected from the group consisting of ethers, water, acetone and mixtures thereof.

[0071] In a preferred embodiment, the indocyanine green of formula (VII) is isolated from the reaction solvent by precipitation with acetone.

[0072] Alternatively, it may be envisaged to isolate the product of formula (VII) from the reaction solvent by another purification technique such as solvent extraction or column chromatography.

[0073] [Preparation steps a), b) and c) of indocyanine green in acid form] The indocyanine green in acid form of formula (VI) may be obtained by any process known to a person skilled in the art, such as, for example, the processes described in WO 2017 / 093889 A1 and U.S. Pat. No. 5,750,722 A1.

[0074] In one preferred embodiment of the present invention, indocyanine green in acid form of formula (VI) is prepared according to the reaction scheme depicted in FIG. 1, comprising steps a), b) and c).

[0075] In step a), 4-(1,1,2-trimethyl-1H-benzo[e]indolium-3-yl)butane-1-sulfonate of formula (IIII) is obtained by reacting 1,2,2-trimethylbenzo[e]indole of formula (I) with 1,4-butanesultone of formula (II) in an equimolar ratio according to the following manner:

[0076] [ka]

[0077] This reaction is known to those skilled in the art and is described, for example, in U.S. Pat. No. 5,750,722 (Patent Document 2), WO 2017 / 093889 (Patent Document 3), Chem. Eur. J. 2021, 27, 14535-14542, etc.

[0078] The reaction of step a) may be carried out in a solvent selected from the group consisting of pentane, hexane, heptane, cyclohexane, cyclopentane, petroleum ether, xylene, toluene and mixtures thereof. In a preferred embodiment, step a) is carried out in toluene.

[0079] Advantageously, the reaction of step a) is carried out at a temperature ranging from 100°C to 180°C, preferably from 120°C to 160°C, more preferably from 120°C to 150°C.

[0080] Advantageously, after completion of the reaction, the reaction mixture is cooled to room temperature and the product of formula (III) is isolated from the reaction solvent by precipitation, preferably by adding a solvent selected from the group consisting of ethers, water, acetone and mixtures thereof.

[0081] Step b) consists in condensing the intermediate of formula (III) obtained in step a) with glutaconaldehyde dianil hydrochloride of formula (IV) in an equimolar ratio in the presence of acetic anhydride to obtain 4-(1,1-dimethyl-2-((1E,3E,5E)-6-(N-phenylacetamido)hexa-1,3,5-trienyl)-1H-benzo[e]indolium-3-yl)butane-sulfonate of formula (V) according to the following manner:

[0082] [ka]

[0083] This reaction is described, for example, in EP2764861, U.S. Pat. No. 5,750,722 (Patent Document 2), U.S. Patent Application Publication No. 2019 / 337896 (Patent Document 4), and the like.

[0084] Alternatively, the reaction of step b) may be carried out in the presence of maleic anhydride instead of acetic anhydride.

[0085] Preferably, the reaction in step b) is carried out at a temperature in the range of 50°C to 170°C, preferably 60°C to 150°C, more preferably 70°C to 130°C.

[0086] Advantageously, after completion of the reaction, the reaction mixture is cooled to room temperature and the product of formula (V) is isolated from the reaction solvent by precipitation, preferably by adding a solvent selected from the group consisting of ethers, water, acetone and mixtures thereof.

[0087] Step c) consists in reacting the intermediate of formula (V) obtained in step b) with 4-(1,1,2-trimethyl-1H-benzo[e]indolium-3-yl)butane-1-sulfonate of formula (III) in an equimolar ratio in the presence of a base to obtain 4-[2-[7-[1,1-dimethyl-3-(4-sulfobutyl)benzo[e]indol-3-ium-2-yl]hepta-2,4,6-trienylidene]-1,1-dimethylbenzo[e]indol-3-yl]butane-1-sulfonic acid of formula (VI), according to the following manner:

[0088] [ka]

[0089] This reaction is described, for example, in EP2764861, U.S. Pat. No. 5,750,722 (Patent Document 2), U.S. Patent Application Publication No. 2019 / 337896 (Patent Document 4), and the like.

[0090] The reaction in step c) may be carried out in a solvent selected from the group consisting of water, methanol, ethanol, isopropyl alcohol, propanol, butanol, and mixtures thereof. In a preferred embodiment, the reaction in step c) is carried out in a mixture of water and methanol. Preferably, water and methanol are mixed in a volume ratio ranging from 1:1 to 1:3.

[0091] Preferably, the base in step c) is selected from the group consisting of triethylamine, pyridine, ammonia, sodium carbonate, sodium bicarbonate and sodium hydroxide. More preferably, the base is triethylamine.

[0092] Preferably, the reaction of step c) is carried out at a temperature in the range of 20°C to 40°C, preferably at 25°C.

[0093] Advantageously, each intermediate of formula (III), (V) and (VI) can be purified after each step of step a), step b) and step c). The purification of these intermediates can be carried out by at least one common purification step such as precipitation, crystallization, solvent extraction, etc. For example, the purification of these intermediates can be carried out according to the procedure disclosed in the examples of US Patent Application Publication No. 2019 / 337896 (Patent Document 4).

[0094] [Purification step e)] In a preferred embodiment, the method for producing indocyanine green according to the present invention further comprises a step e) of purifying indocyanine green after step d).

[0095] In a preferred embodiment, purification step e) is carried out by recrystallization of indocyanine green of formula (VII).

[0096] Preferably, the purification step e) comprises e1) suspending, dispersing or dissolving indocyanine green of formula (VII) in a solvent; e2) heating the composition obtained in step e1); e3) cooling the solution obtained in step e2), and e4) recovering the indocyanine green in crystalline form; At least

[0097] Advantageously, the solvent of step e1) is selected from the group consisting of water, acetone, methanol, ethanol, isopropyl alcohol, n-propanol, n-butanol and mixtures thereof.

[0098] Preferably, the solvent in step e1) is a mixture of water and acetone.

[0099] More preferably, in step e1), water and acetone are used in a ratio ranging from 10:1 to 1:10 (v / v), preferably from 4:1 to 1:6 (v / v), more preferably from 1:1 to 1:5 (v / v).

[0100] Advantageously, when the composition obtained in step e1) is a suspension or dispersion, in step e2), said composition is heated to a temperature allowing the solubilization of the indocyanine green.

[0101] Advantageously, in step e2) the reaction medium is heated to a temperature greater than or equal to 40°C, preferably to a temperature ranging from 40 to 80°C, preferably from 40°C to 65°C.

[0102] Advantageously, in step e3), the solution is cooled or decreased in temperature to a temperature between 10°C and 25°C, preferably between 15°C and 25°C.

[0103] "Cooling" means allowing the reaction solvent to cool to room temperature, or actively lowering the temperature, for example by placing the reactor in an environment that is lower than room temperature to accelerate the temperature drop.

[0104] In a preferred embodiment, the solution is then maintained at that temperature for a time sufficient to form ICG crystals. Preferably, the solution is maintained at a temperature of 10-25° C., preferably 15-25° C., for a time in the range of 10-120 hours, preferably 24-96 hours, more preferably 48-80 hours.

[0105] Optionally, the solution is then further cooled to a temperature below 10°C, preferably to a temperature in the range of -5 to 10°C, more preferably 0 to 10°C.

[0106] In a preferred embodiment, step e3) is carried out without stirring or agitating the solution.

[0107] "Step e3) is carried out without stirring or agitating the solution" means that each of the substeps, i.e., cooling to a temperature between 10 and 25°C, maintaining the solution at that temperature for a certain period of time, and further, the optional cooling to a temperature below 10°C, is carried out without stirring or agitating the reaction medium.

[0108] Advantageously, in step e4), the crystalline indocyanine green is isolated from the reaction solvent by filtration.

[0109] Advantageously, after step e4), the crystalline indocyanine green is further purified by at least one step e5) of washing the crystalline indocyanine green with a solvent.

[0110] Advantageously, washing is carried out with a solvent chosen from aqueous acidic solutions, acetone, acetonitrile and mixtures thereof.

[0111] Preferably, at least one wash with acetone and at least one wash with acetonitrile are performed.

[0112] More preferably, washing with acetone is performed 1 to 3 times, and washing with acetonitrile is performed 1 to 3 times.

[0113] (Properties of the obtained indocyanine green)

[0114] In a third aspect, the present invention relates to an ICG compound obtained or obtainable by a process comprising at least a step of converting 4-[2-[7-[1,1-dimethyl-3-(4-sulfobutyl)benzo[e]indol-3-ium-2-yl]hepta-2,4,6-trienylidene]-1,1-dimethylbenzo[e]indol-3-yl]butane-1-sulfonic acid of formula (VI) into 3,3,3',3'-tetramethyl-1,1'-di(4-sulfobutyl)-4,5,4',5'-dibenzoindotricarbocyanine sodium salt of formula (VII) by treatment with sodium chloride.

[0115] The applicant of the present application has surprisingly found that the ICG obtained or obtainable by this method is crystalline. Specifically, the ICG obtained or obtainable by this method has a specific crystalline morphology as described above.

[0116] The applicant has further surprisingly found that the ICG obtained or obtainable by this method exhibits a higher purity and / or physical stability and / or solubility than the amorphous form of ICG obtained by the methods of the prior art, and the results of this method are highly reproducible, so that ICG of consistent quality is available.

[0117] These improved properties, as well as the novel crystalline form of ICG, are a direct result of the unique method of producing ICG according to the present invention.

[0118] [ICG Purity]

[0119] Specifically, the method of the present invention makes it possible to produce an ICG compound with a higher purity than that obtained by the conventional methods described in the prior art that use sodium iodide or an organic sodium salt as a salt-forming agent.

[0120] In particular, the indocyanine green of formula (VII) obtained by the process according to the present invention is substantially pure, having a purity of more than 99.0%, preferably more than 99.5%, more preferably more than 99.7% and most preferably more than 99.8% as measured by HPLC.

[0121] As used herein, the term "purity" refers to the degree to which indocyanine green is free of undesirable or extraneous chemicals.

[0122] In the present invention, the purity and impurities of the obtained indocyanine green are evaluated by area percent at 240 nm by HPLC.

[0123] Preferably, the indocyanine green obtained by carrying out the process of the present invention contains less than 1.0%, preferably less than 0.5% impurities as measured by HPLC.

[0124] Preferably, the indocyanine green obtained by carrying out the process of the present invention contains less than 0.50%, preferably less than 0.40%, more preferably less than 0.20%, and most preferably less than 0.15% of each impurity as measured by HPLC.

[0125] The impurities contained in the indocyanine green may be any reaction by-product or intermediates occurring in the process detailed above. For example, the impurities may include, but are not limited to, N-phenylacetamide, 4-(1,1,2-trimethyl-1H-benzo[e]indolium-3-yl)butane-1-sulfonate, and 4-(1,1,2-dimethyl-2-((1E,3E,5E)-6-(N-phenylacetamide)hexa-1,3,5-trienyl)-1H-benzo[e]indolium-3-yl)butane-1-sulfonate.

[0126] The indocyanine green obtained by carrying out the method according to the present invention is substantially free, preferably completely free, of iodine and / or iodide, in particular sodium iodide. The absence of iodine / iodide in the indocyanine green can be confirmed, for example, by ion exchange chromatography.

[0127] Preferably, the amount of foreign metals in the resulting indocyanine green is less than 200 ppm by weight, preferably less than 100 ppm by weight, more preferably less than 50 ppm by weight, and even more preferably less than 20 ppm by weight, based on the weight of the indocyanine green. The amount of foreign metals in the indocyanine green can be measured by inductively coupled plasma mass spectrometry (ICP-MS).

[0128] "Metallic foreign bodies" are to be understood as meaning the so-called "heavy" metals, in particular Al, As, Cd, Cr, Cu, Fe, Sn, Mn, Hg, Mo, Ni, Pb, Zn and their organic and inorganic derivatives.

[0129] More preferably, the indocyanine green contains no or less than 2 ppm lead and arsenic.

[0130] [Stability of ICG]

[0131] The applicant has found that the crystalline indocyanine green obtained by the method of the present invention has significantly superior stability compared to the indocyanine green of the prior art.

[0132] The terms "stable" and "stability" mean that the amount of indocyanine green in the solid state and / or in a liquid composition, particularly in an aqueous composition, remains stable after storage for a certain period of time.

[0133] An "aqueous composition" is to be understood as meaning an aqueous solution, an aqueous suspension, an aqueous colloidal suspension, an aqueous dispersion, preferably an aqueous solution.

[0134] The stability of the ICG according to the present invention is evaluated by measuring the change in the amount of indocyanine green before and after long-term storage when indocyanine green is stored in the solid state or after dissolution in an aqueous composition.

[0135] It should be understood that storage of the indocyanine green in a solid state refers to storage in the aforementioned crystalline form (not in a freeze-dried form). The crystalline indocyanine green of the present invention is advantageous in that it shows almost no evidence of aggregation or decomposition as a result of chemical modification of the indocyanine green even after long-term storage, as well as virtually no or minimal generation of impurities. The crystalline ICG of the present invention is further advantageous in that it does not show significant discoloration or loss of fluorescence intensity or peak absorbance even after long-term storage.

[0136] Advantageously, the indocyanine green of the present invention has much greater stability in the solid state than prior art indocyanine green compounds, particularly prior art freeze-dried ICG.

[0137] Advantageously, the crystalline indocyanine green of the present invention remains stable when stored in solid state at room temperature for a period of up to 1 month, preferably up to 2 months, preferably up to 3 months, more preferably up to 6 months, even more preferably up to 12 months and most preferably up to 2 years.

[0138] Advantageously, the crystalline indocyanine green according to the present invention remains stable when stored at room temperature after dissolution in an aqueous solvent for up to 24 hours, preferably up to 48 hours, more preferably up to 3 days, most preferably up to 6 days. The term "room temperature" means regulated room temperature between 20-25°C, between 15-30°C as specified by the United States Pharmacopoeia, or between 15-25°C as specified by the European Pharmacopoeia.

[0139] Advantageously, such stability of indocyanine green is observed in a container that protects said indocyanine green from natural light and / or UV light and / or fluorescence, preferably said container is tightly closed or sealed.

[0140] The stability of the indocyanine green of the present invention can be evaluated by visual inspection of color and / or transparency and / or other analytical techniques. Those skilled in the art are familiar with analytical techniques for evaluating the stability of indocyanine green. Such analytical techniques may include nuclear magnetic resonance spectroscopy (NMR), high performance liquid chromatography (HPLC), size exclusion chromatography (SEC), liquid chromatography mass spectrometry (LC-MS), dynamic light scattering (DLS), differential scanning calorimetry (DSC), ultraviolet spectroscopy, Fourier transform infrared spectroscopy (FTIR), or a combination of these techniques.

[0141] Advantageously, the stability of indocyanine green is assessed by measuring the change in the amount of indocyanine green before and after long-term storage by HPLC. More advantageously, the amount of indocyanine green and the change in said amount are assessed by HPLC area percentage at 240 nm.

[0142] Preferably, the indocyanine green has a stability corresponding to a decrease in the amount of indocyanine green of not more than 10%, preferably not more than 5%, more preferably not more than 2%, as measured by HPLC area percent at 240 nm, when the indocyanine green is stored in a solid state at room temperature for a period of not more than 1 month, preferably not more than 3 months, more preferably not more than 6 months, even more preferably not more than 12 months, and most preferably not more than 2 years.

[0143] Preferably, the indocyanine green has a stability corresponding to a decrease in the amount of indocyanine green of no more than 10%, preferably no more than 5%, more preferably no more than 2%, as measured by HPLC area percent at 240 nm, when the indocyanine green is stored at room temperature in an aqueous solvent for up to 24 hours, preferably no more than 48 hours, more preferably no more than 3 days, and most preferably no more than 6 days.

[0144] The stability of indocyanine green according to the present invention in aqueous media can be greatly improved by adding at least one stabilizer compound selected from the group consisting of histidine, ethylenediaminetetraacetic acid (EDTA) and its salts, cysteine, sodium chloride, dithiothreitol, ascorbic acid, sodium ascorbate, disodium hydrogen phosphate, sodium dihydrogen phosphate and mixtures thereof.

[0145] The presence of such additives makes the aqueous ICG formulation more stable, exhibiting a decrease in the amount of indocyanine green of no more than 10%, preferably no more than 5%, more preferably no more than 2% when stored at room temperature for up to 1 month, preferably no more than 3 months, more preferably no more than 6 months, even more preferably no more than 12 months, and most preferably no more than 2 years, as measured by HPLC area percent at 240 nm. In the sense of this application, "decrease in amount" is understood to mean the % difference between the measured amount of indocyanine green before storage and the measured amount of indocyanine green after a given storage period, as measured by HPLC area percent at 240 nm.

[0146] In addition, indocyanine green may have a stability corresponding to the total amount of impurities contained in the indocyanine green not increasing during storage, particularly not increasing to an amount that reduces the usability of the indocyanine green for medical and / or diagnostic purposes. For example, when the indocyanine green is stored in a solid state at room temperature for one month or more and then the amount of such impurities is measured by HPLC, the amount of impurities observed in the composition is not only very small but also does not increase significantly with time.

[0147] In the context of the present invention, the term "impurities" is to be understood to refer to chemical substances that are not indocyanine green or excipients or other additives of indocyanine green according to the present invention. The impurities may be process-related impurities such as by-products, intermediates, etc. that may be formed during the preparation of indocyanine green, or degradation-related impurities resulting from chemical transformations of indocyanine green during storage.

[0148] In one embodiment, the indocyanine green of the present invention exhibits an increase in impurities of not more than 10%, preferably not more than 5%, more preferably not more than 2%, when the indocyanine green is stored in solid state at room temperature for up to 1 month, preferably not more than 2 months, more preferably not more than 6 months, and most preferably not more than 2 years, as measured as change in HPLC area percent at 240 nm.

[0149] (Composition containing crystalline indocyanine green)

[0150] In a fourth aspect, the present invention relates to a pharmaceutical composition containing the crystalline indocyanine green disclosed above and / or the indocyanine green obtained or obtainable by the above process.

[0151] The pharmaceutical compositions of the present invention may be provided in any suitable form, such as a liquid solution or a solid powder to be dissolved in a solution and administered intravenously.

[0152] The pharmaceutical composition may further comprise at least one pharma- ceutically acceptable carrier and / or at least one pharma- ceutically acceptable excipient.

[0153] As used herein, the term "pharmacologically acceptable" means that a compound, substance or composition is suitable for use in contact with the tissues of human or animals without excessive toxicity, irritation or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0154] As used herein, the term "carrier" means a pharma- ceutically acceptable substance or composition, or a liquid vehicle, such as a liquid or solid filler, diluent, excipient, solvent, encapsulating material, or the like.

[0155] Advantageously, the pharma- ceutically acceptable carrier consists of at least one diluent.

[0156] The term "diluent" includes pharma- ceutically acceptable solvents that are safe and non-toxic when administered to humans and animals and are useful in preparing pharmaceutical formulations.

[0157] A suitable diluent may be any biologically acceptable liquid capable of completely dissolving the composition of the present invention. Water, particularly sterile water for injection (SWFI) and / or bacteriostatic water for injection (BWFI), is suitable as a diluent since it does not contain salts or other compounds that may affect the stability of indocyanine green, but other diluents such as sterile saline solution, Ringer's solution, dextrose solution, glucose solution, ethyl alcohol, methyl alcohol, isopropyl alcohol, n-propyl alcohol, butyl alcohol, glycerin, propylene glycol, polyethylene glycol and mixtures thereof may also be used. Those skilled in the art can select a diluent according to the desired application and in relation to the other compounds of the composition.

[0158] In a preferred embodiment, the diluent is selected from the group consisting of water for injection, bacteriostatic water for injection, sterile saline, Ringer's solution, dextrose solution, and glucose solution.

[0159] In a preferred embodiment, the diluent is water for injection, in particular sterile water for injection (SWFI) and / or bacteriostatic water for injection (BWFI).

[0160] The term "additive" includes compounds contained in the pharmaceutical composition other than indocyanine green. Examples of such additives include buffers, pH adjusters, isotonicity agents, surfactants, preservatives, tonicification agents, antibacterial agents, wetting agents, emulsifiers, etc. These additives are widely available to those skilled in the art and can be in any form, such as solid, liquid, or semi-solid.

[0161] In one embodiment, the pharmaceutical composition containing indocyanine green does not contain sodium iodide.

[0162] In a preferred embodiment, the pharmaceutical composition containing indocyanine green according to the present invention further comprises at least one stabilizer compound selected from the group consisting of histidine, ethylenediaminetetraacetic acid (EDTA) and its salts, cysteine, sodium chloride, dithiothreitol, ascorbic acid, sodium ascorbate, disodium hydrogen phosphate, sodium dihydrogen phosphate, and mixtures thereof.

[0163] Such stabilizer compounds are described in, for example, PCT / EP2022 / 056443, PCT / EP2022 / 056445, PCT / EP2022 / 056446, etc. By adding such compounds, it has become possible to further improve the long-term stability of the aqueous pharmaceutical composition containing indocyanine green according to the present invention.

[0164] Preferably, in one embodiment, the pharmaceutical composition contains, and preferably consists of, indocyanine green according to the present invention and at least one compound selected from ascorbic acid, a salt thereof, or a combination thereof.

[0165] Preferably, in the second instance, the pharmaceutical composition comprises, preferably consists of, indocyanine green according to the invention and histidine.

[0166] Preferably, in the third embodiment, the pharmaceutical composition contains, preferably consists of, indocyanine green according to the invention, histidine, ethylenediaminetetraacetic acid (EDTA) and / or salts thereof, and sodium chloride.

[0167] Preferably, the amount of additives in the composition of the present invention is within a range that does not substantially adversely affect the activity of indocyanine green. Preferably, the composition of the present invention contains the minimum number and amount of additives necessary to form an effective and stable composition. Those skilled in the art can adjust the amount of additives depending on the solubility of the additive in the carrier. The pharmaceutical composition of the present invention may be prepared by any known method.

[0168] The concentration of indocyanine green in the composition may be any concentration suitable for medical or diagnostic applications, particularly suitable for producing angiographic images of satisfactory quality.

[0169] In a preferred embodiment, the pharmaceutical composition according to the invention is in the form of a kit comprising the indocyanine green according to the invention and at least one optional additive in a first part, and the carrier as described above in a second part.

[0170] In another preferred embodiment, the pharmaceutical composition according to the invention is in the form of a kit comprising the indocyanine green according to the invention in a first part, and a carrier and at least one optional additive as described above in a second part.

[0171] All of the preferred embodiments described above for indocyanine green and pharmaceutical compositions apply to each of these kit embodiments.

[0172] (Use of indocyanine green according to the present invention)

[0173] The crystalline indocyanine green according to the present invention, in particular the pharmaceutical composition containing said crystalline indocyanine green, may be used in a method for diagnosing and / or treating (as a therapeutic agent) a patient. The method comprises administering an effective amount of an aqueous composition of indocyanine green. The administration may be by enteral route, intravenous route (in particular by intravenous injection) or topical application. Preferably, the administration is by intravenous injection.

[0174] The term "effective amount" refers to that amount of indocyanine green that produces the biological or pharmacological response in a tissue, system, animal or human that is desired, for example, by a researcher or clinician.

[0175] For example, the crystalline indocyanine green according to the present invention, particularly a pharmaceutical composition containing the crystalline indocyanine green, can be used in hyperthermia, indocyanine green-enhanced selective photocoagulation, photodynamic therapy (PDT), photohyperthermia therapy (PHT), etc. More specifically, the indocyanine green according to the present invention can be used in the treatment of infectious diseases, acne, macular surgery, cancer treatment, etc. as applications of PDT. The indocyanine green according to the present invention can be used in conjunction with other therapies such as immunotherapy, radiation therapy, ultrasound therapy, chemotherapy, etc.

[0176] The crystalline indocyanine green of the present invention may also be used to obtain angiographic images of tissues of patients, to measure cardiac output, to measure liver function and hepatic blood flow, to detect sentinel lymph nodes, to evaluate and / or predict survival of skin flaps, to diagnose and treat age-related macular degeneration, and to diagnose and treat associated choroidal neovascularization and tumors.Preferably, the crystalline indocyanine green of the present invention is used to obtain angiographic images of tissues, and / or to measure cardiac output, and / or to measure liver function and hepatic blood flow, and / or to detect sentinel lymph nodes, and / or to evaluate and predict survival of skin flaps.

[0177] For diagnostic purposes, the amount of indocyanine green composition administered to a patient should be sufficient to cause the dye to fluoresce when irradiated with a suitable wavelength, taking into account that the peak absorbance and peak emission of indocyanine green are within the range of 800-850 nm. The same criteria apply to treatments using ICG solutions, and it is desirable to administer a sufficient amount of dye to perform the treatment efficiently. The dosage of indocyanine green can be easily determined by a person skilled in the art, and is preferably at least a concentration currently approved for use in ophthalmic imaging examinations, for example, a 20 mg / mL indocyanine green solution (2 ml) for diagnostic purposes. As a person skilled in the art will appreciate, the higher the concentration of dye used in each of the above diagnostic and treatment techniques, the more advantageous it may be.

[0178] The superior stability of the indocyanine green of the present invention allows for improved diagnosis / treatment of patients compared to prior art indocyanine green formulations for the same amount of injected compound, because the increased stability of indocyanine green results in a stronger response to fluorescent irradiation. EXAMPLES

[0179] (I-Ingredients and Methods)

[0180] [I-1. Reagents and Chemicals]

[0181] 1,2,2-Trimethylbenz[e]indole (CAS No. 41732-24-7) is available from Matrix Fine Chemical (purity: >99.0% by HPLC). 1,4-Butanesultone (CAS No. 1633-83-6) is available from Matrix Fine Chemical (purity: >99.9% by HPLC). Glutaconaldehyde dianil hydrochloride (CAS No. 1497-49-0) is available from Matrix Fine Chemical.

[0182] Other reagents and solvents (acetone, methanol, triethylamine, sodium chloride, etc.) were purchased from commercial suppliers such as Sigma-Aldrich and TCI.

[0183] [I-2. Chromatography conditions]

[0184] As a test for purity and stability, the amount of indocyanine green in the above examples was measured by high performance liquid chromatography (HPLC). The purity of indocyanine green is expressed as HPLC area percent at 240 nm. The analytical conditions are as given in Table 1 below.

[0185] [Table 2]

[0186] The elution was performed with a gradient. The mobile phase consisted of ammonium formate buffer (100 mM, pH = 4.5) (eluent A) and acetonitrile (eluent B). During the elution process, the composition of the mobile phase was changed continuously according to Table II below.

[0187] [Table 3]

[0188] Impurities that may be detected by this HPLC method in the context of the present invention are summarized below.

[0189] [ka]

[0190] [ka]

[0191] [ka]

[0192] [ka]

[0193] [ka]

[0194] [I-3. Powder X-ray Diffraction]

[0195] Powder X-ray diffraction data were obtained using a Panalytical Empyrean S3 (DY2626) powder X-ray diffractometer equipped with a PIXcel 1D-Medipix3 detector, using techniques known in the art. Each sample was mounted between two sheets of Kapton and polypropylene. Kapton can produce artifacts (2θ=5.5°). Scanning parameters were: source: copper (1.54 A); X-ray tube power: 40 kV / 40 mA; range: 2.0-50.0° (2θ); scanning mode: continuous scan; step size: 0.026°; number of scans: 5; collection time: 20.40 s. Results are expressed as scattering angle (2θ°), lattice spacing d (Angstroms), peak intensity, and relative intensity (percentage of the most intense peak). Each peak position is within 0.2° (2θ).

[0196] [I-4.NMR spectroscopy]

[0197] NMR spectra were recorded on a Bruker Advanced NanoBay 300 MHZ equipped with manual shims. 1H chemical shifts (δ) relative to TMS are reported in ppm. Proton NMR information is given in the format: multiplicity (s=singlet, d=doublet, t=triplet, q=quartet, m=multiplet); coupling constant J; integral;

[0198] [I-5. Sodium content by atomic absorption spectrometry]

[0199] The sodium content of the above examples was measured by an atomic absorption spectrometer Agilent AA 240FS connected to the software SpectrAA. The flame composition was air / acetylene, and each sample was dissolved in acidic water.

[0200] II-Example 1 (Invention)

[0201] [II-1. Synthesis of 4-(1,1,2-trimethyl-1H-benzo[e]indolium-3-yl)butane-1-sulfonate (step a)]

[0202] A mixture of 20.0 g of 1,1,2-trimethylbenz[e]indole (95.6 mmol; 1 equivalent (eq)) and 19.52 g of 1,4-butanesultone (142.4 mmol; 1.5 eq) was vigorously stirred in toluene (40 mL) at 140° C. for 12 h. After cooling to room temperature, 400 mL of diethyl ether was charged. The solid was collected by filtration and dried. A blue / grey powder was obtained (yield=97%): 1 H NMR(DMSO d6,300MHz):δ(ppm):8.35(d,J=8.5Hz,1H),8.27(d,J=8.5Hz,1H),8.21 - 8.19(m,2H),7.77(dd,J=8.5,8.5Hz 1H),7.71(dd,J=8.5,8.5Hz 1H),4.59(t,J=7.8Hz,2H),2.93(s,3H),2.51(t,J=7.8Hz,2H),2.01(tt,J=7.8,7.8Hz,2H),1.77(m,1H),1.74(s,6H) 13 C NMR(MeOD,300MHz):δ(ppm):21.05,21.95,26.35, 48.5, 49.75, 55.80, 112.6, 123.0, 127.30, 127.75,129.62,131.15,133.60,137.31,138.5,196.40 HPLC retention time = 7.6 min (relative retention time for ICG = 0.26)

[0203] [II-2. Synthesis of 4-(1,1-dimethyl-2-((1E,3E,5E)-6-(N-phenylacetamido)hexa-1,3,5-trienyl)-1H-benzo[e]indolium-3-yl)butane-sulfonate (step b)]

[0204] In a 50 mL four-neck flask, 2.08 g of 4-(1,1,2-trimethyl-1H-benzo[e]indolium-3-yl)butane-1-sulfonate (6.0 mmol) was mixed with 1.88 g of glutaconaldehyde dianil hydrochloride (6.6 mmol) and then stirred in 20 mL of acetic anhydride at 120° C. for 1 h. The mixture was cooled to room temperature and stirred for 1 h, after which the crystals were isolated by filtration. The crystals were suspended and washed with acetone, isolated by filtration, and air-dried. A light purple powder was isolated (yield=56%): 1 H NMR(DMSO d6,300MHz):δ(ppm):1.97(two m,4H),2.00(s, 6H),2.12(s, 3H), 2.89(t,J=6.9Hz,2H),4.56(Br t,J=7.4Hz,2H),5.40(dd,J=13.7,11.3Hz,1H),6.60(dd,J=14.4,11.2Hz,1H),6.97(d,J=15.2Hz,1H),7.25(m,2H),7.20-8.40(m,12H) HPLC retention time = 21.8 min (relative retention time for ICG = 0.76)

[0205] [II-3. Synthesis of Indocyanine Green (Steps c and d)]

[0206] Step c): A 2 L double-jacketed borosilicate glass reactor equipped with a mechanical stirrer was charged with 28.646 g of 4-(1,1,2-trimethyl-1H-benzo[e]indolium-3-yl)butane-1-sulfonate (82.92 mmol; 0.9 equiv.) and 50.000 g of 4-(1,1-dimethyl-2-((1E,3E,5E)-6-(N-phenylacetamido)hexa-1,3,5-trienyl)-1H-benzo[e]indolium-3-yl)butane-sulfonate (92.13 mmol; 1.0 equiv.) and diluted with 75.0 mL of distilled water and 125.0 mL of MeOH. The mixture was stirred (150 rpm) at room temperature for 5 min. 12.8 mL of triethylamine (92.13 mmol; 1.00 equiv.) was added in one go, causing the mixture to turn green. The mixture was stirred at 25°C for 3 hours and then warmed to 50°C.

[0207] Step d): A solution of 8.615 g sodium chloride (147.41 mmol; 1.70 equiv.) in 50.0 mL distilled water was charged in one go. When the mixture thickened in appearance, 1.5 L acetone was slowly charged while maintaining the reaction mixture above 40° C. The mixture was stirred (200 rpm) for an additional hour. The reaction was cooled to 25° C. and stirred (200 rpm) overnight before filtering and washing with 3×250 mL acetone. The solids were dried under vacuum at 40° C. to give 64.3 g of crude indocyanine green as a green powder: The purity of the crude product was controlled by HPLC (HPLC profile: ICG 97.25%; impurity A 2.41%, impurity E 0.10%; impurity K 0.11%; impurity M 0.13%). Sodium content = 3.90%

[0208] [II-4. Purification of indocyanine green (step e)]

[0209] A 2 L double jacketed borosilicate glass reactor equipped with a mechanical stirrer was charged with 50.0 g of indocyanine green crude product and 350.0 mL of distilled water. The mixture was heated to 50°C and condensation was observed at 35-37°C. 1.5 L of acetone was added in portions while maintaining the temperature above 40°C (green homogeneous mixture). After stirring at 50°C for 1 h, the stirring system was stopped and the mixture was allowed to cool to room temperature without stirring. Golden red crystals appeared on the liquid surface. The mixture was kept at room temperature for 60 h, cooled to 5°C for 16 h without stirring, filtered and the filtrate washed three times with acetone (5 vol) and then with acetonitrile (5 vol). The filter cake was dried at 70°C under vacuum (50 mbar) for 72 h. 34.3 g of indocyanine green was obtained as a brilliant golden red solid (total isolated yield=64%, isolated purified yield=68%): Purity = 99.95% (HPLC profile: ICG 99.95%; each impurity less than 0.05%) Sodium content = 2.82% (water content corrected by thermogravimetry analysis) 1 H NMR (300MHz, methanol-d4) δ(ppm):8.22(d,J=7.23Hz,2H),8.03(m,2H),7.98(m,4H),7.61(m,5H),7.46(t,J=6.85Hz,2H),6.60(t,J=11 .03Hz,2H),6.35(d,J=11.7Hz,2H),4.24(t,J=5.3Hz,4H),2.96(t,J=7.23Hz,4H),2.03(m,8H),1.92(m,12H).

[0210] The resulting PXRD pattern is shown in Figure 2 and represents a crystalline form of indocyanine green characterized by the following peak values:

[0211] [Table 4]

[0212] The intensity and relative intensity values ​​of each peak above may vary by ±15%.

[0213] Solid state stability: 5.0 g of crystalline indocyanine green powder was stored in a 20 mL amber type 2 glass in the dark, and the results are summarized in Table IV.

[0214] [Table 5]

[0215] (III-Example 2 (Comparative Example))

[0216] The preparation procedure for indocyanine green disclosed in the examples of the prior art US2019 / 0337896A1 (Patent Document 4) was reproduced.

[0217] [III-1. Synthesis of Indocyanine Green]

[0218] A 2 L double-jacketed borosilicate glass reactor equipped with a mechanical stirrer was charged with 20.0 g of 4-(1,1-dimethyl-2-((1E,3E,5E)-6-(N-phenylacetamido)hexa-1,3,5-trienyl)-1H-benzo[e]indolium-3-yl)butane-sulfonate (0.037 mol; 1.0 equiv.) and diluted with 20.0 mL of methanol. To the reaction mixture was successively charged 12.73 g of 4-(1,1,2-trimethyl-1H-benzo[e]indolium-3-yl)butane-1-sulfonate (0.037 mol; 1.0 equiv.) and 1.0 mL of triethylamine (0.007 mol; 0.20 equiv.) (liquefaction was confirmed). After stirring at 60-65°C for 1.5 hours, no change to green was observed, suggesting that the reaction had not started. When 4.0 mL of triethylamine (0.028 mol; 0.8 equiv.) was added to the mixture, the color immediately changed to green.

[0219] The mixture was cooled to 10-15°C and charged with a solution of 5.52 g of sodium iodide (0.037 moles; 1.0 equiv.) in 100.0 mL of methanol. The reactor was heated at 60-65°C for 1.5 hours and then cooled to room temperature. Methanol was distilled under reduced pressure to produce a viscous green residue. 400 mL of acetone was poured into the reactor to obtain a homogenous mixture, which was heated at 50-60°C for 1 hour, filtered at 50°C and washed with 40 mL of acetone to obtain 23.5 g of crude indocyanine green as a green powder.

[0220] [III-2. Purification of Indocyanine Green]

[0221] 23.5 g of crude indocyanine green was diluted with 94 mL of methanol (4 volumes) and 141 mL of isopropyl alcohol (6 volumes). The mixture was heated at 60-70 °C for 1 h and filtered at 50 °C. The filter cake was washed with 45 mL of isopropyl alcohol to obtain 21.2 g of pure indocyanine green as a green powder (isolation and purification yield = 90%). Purity = 99.21% (HPLC profile ICG 99.21%; impurity A < 0.30%; impurity C < 0.07%; impurity M < 0.07%; impurity L < 0.06%; total unknown impurities 0.29%) Sodium content = 3.27%

[0222] The resulting PXRD pattern is shown in Figure 3 and shows a mostly amorphous product with two indistinct peaks (2θ=3.30°, 4.86°).

[0223] Solid state stability: 5.0 g of indocyanine green powder was stored in a 20 mL amber type 2 glass in the dark, and the results are summarized in Table V.

[0224] [Table 6]

[0225] (IV-Example 3 (Comparative))

[0226] The preparation procedure for indocyanine green disclosed in the examples of the prior art WO 2017 / 093889 (Patent Document 3) was reproduced.

[0227] [IV-1. Synthesis of Indocyanine Green]

[0228] A 2 L double jacketed borosilicate glass reactor equipped with a mechanical stirrer was charged with 56.17 g of 4-(1,1,2-trimethyl-1H-benzo[e]indolium-3-yl)butane-1-sulfonate (0.163 mol; 1.89 equiv.) and diluted with 140.0 mL of methanol. 35.10 g of sodium acetate (0.428 mol; 4.97 equiv.) was charged and the reaction was stirred at room temperature for 5 min. The mixture was heated at 65° C. and charged with a solution of 25.0 g of N-phenyl-N-((1E,3E,5E)-5-(phenylimino)penta-1,3-dienyl)acetamide (0.086 mol; 1.0 equiv.) in 25.0 mL of methanol. A green color change occurred. The reaction mixture was stirred at 65° C. for 2 h, cooled to room temperature, diluted with 1.0 L of ethyl acetate, and stirred at room temperature overnight. The mixture was filtered and dried at 40° C. to give 92 g of crude indocyanine green as a sticky green solid (yield=100%).

[0229] [IV-2. Purification of Indocyanine Green]

[0230] 60.0 g of crude indocyanine green was diluted with 420 mL of distilled water and treated with 1.2 g of sodium iodide (2 w / w%). The mixture was heated to 55-60°C and 1.8 L of acetone was charged while maintaining the reaction mixture above 45°C. The reaction was heated at 65°C for 1 hour, cooled to room temperature, and stirred at room temperature over the weekend. The green solids were filtered and washed with acetone to give 30.9 g of indocyanine green as a green / red powder (isolated yield=52%). Purity = 98.60% (HPLC profile: ICG 98.60%; impurity E less than 0.16%; impurity L less than 0.11%, impurity M less than 0.17%) Sodium content = 7.04%

[0231] The PXRD pattern obtained is shown in Figure 4. The pattern indicates a mostly amorphous product. The pattern also shows some very weak and unclear peaks that may be due to traces of crystalline forms. However, the peaks are different from those observed in the product obtained according to Example 1. The observed peaks are summarized in Table VI.

[0232] [Table 7]

[0233] Solid state stability: 5.0 g of indocyanine green powder was stored in a 20 mL amber type 2 glass in the dark.

[0234] [Table 8]

[0235] (V-Example 4 (Comparative Example))

[0236] The preparation procedure for indocyanine green disclosed in the examples of the prior art WO 95 / 07888 (Patent Document 1) was reproduced.

[0237] [V-1. Synthesis of Indocyanine Green]

[0238] A 0.5 L double-jacketed borosilicate glass reactor equipped with a mechanical stirrer was charged with 10.0 g of 4-(1,1-dimethyl-2-((1E,3E,5E)-6-(N-phenylacetamido)hexa-1,3,5-trienyl)-1H-benzo[e]indolium-3-yl)butane-sulfonate (0.184 mol; 1.00 equiv.) and 58 mL of EtOH. The mixture was stirred for 5 min and then charged with 6.36 g of 4-(1,1,2-trimethyl-1H-benzo[e]indolium-3-yl)butane-1-sulfonate (0.184 mol; 1.00 equiv.) and 2.6 mL of triethylamine (0.184 mol; 1.00 equiv.). The reaction mixture turned green and was heated at 78° C. for 15 min and cooled to 20° C. with stirring. A solution of 1.52 g AcONa (0.184 mol; 1.0 equiv.) in 105 mL EtOH was charged and the mixture was stirred at 20° C. for 30 min. The mixture was filtered, washed twice with 50 mL acetone and dried under reduced pressure (50 mbar) at 40° C. to give 11 g of red-green solid. The purity of the crude product was controlled by HPLC analysis (typical HPLC profile: ICG 92.62%; impurity A 4.82%; impurity C 1.07%).

[0239] [V-2. Purification of indocyanine green]

[0240] 11 g of the red-green ICG crude product was diluted with 110 mL of acetone, heated to reflux for 30 min, cooled at room temperature, filtered, and washed with 20 mL of acetone. This process was repeated twice to obtain 10.3 g of purified ICG, a red-green solid (isolation yield = 72%). Purity = 95.98% (HPLC profile: ICG 95.98%; impurity A 1.36%, impurity C 1.08%, impurity E 0.37%, impurity B 0.07%, impurity H 0.25%, impurity K 0.06%, impurity J 0.05%, impurity M less than 0.17%) Sodium content = 2.43%

[0241] The resulting PXRD pattern is shown in Figure 5 and indicates an amorphous product.

[0242] Solid state stability: 5.0 g of indocyanine green powder was stored in a 20 mL amber type 2 glass in the dark, and the results are shown in Table VIII.

[0243] [Table 9]

[0244] (VI-Example 5: Comparison of salt-forming reagents)

[0245] The procedure of Example 1 was reproduced by replacing NaCl with several other salt-forming reagents: sodium bromide (NaBr), sodium acetate (NaOAc), sodium ascorbate (NaOAsc), and sodium ethanolate (NaOEt).

[0246] [VI-1. Synthesis of Indocyanine Green]

[0247] A 500 mL three-neck round bottom flask was charged with 5.73 g of 4-(1,1-dimethyl-2-((1E,3E,5E)-6-(N-phenylacetamido)hexa-1,3,5-trienyl)-1H-benzo[e]indolium-3-yl)butane-sulfonate (16.59 mmol; 0.90 equiv.) and 10.0 g of 4-(1,1,2-trimethyl-1H-benzo[e]indolium-3-yl)butane-1-sulfonate (18.43 mmol; 1.00 equiv.). The reaction mixture was charged with 15.0 mL of HO (1.5 vol.) and 25.0 mL of MeOH (2.5 vol.). The mixture was charged with 2.6 mL of Et3N (18.43 mmol; 1.00 equiv.) and the reaction was stirred at 25° C. for 2 h and then heated to 50° C. A solution of the target sodium salt (31.33 mmol; 1.70 equiv.) in 10 mL of H2O was charged while maintaining the reaction mixture above 45° C. The reaction mixture was maintained at 50° C. until the mixture was observed to concentrate. 300 mL of acetone (30 vol.) was charged in portions while maintaining the temperature above 40° C. and the mixture was maintained at 50° C. for 1 h 30 min and then cooled to 25° C. The solid was filtered through sintered glass (POR 3), washed three times with 50 mL of acetone (5 vol.), and dried under vacuum at 40° C. for 16 h to give crude indocyanine green as a green powder.

[0248] [VI-2. Purification of Indocyanine Green]

[0249] A 500 mL three-neck round bottom flask was charged with a solution of 12 g of crude indocyanine green in 84 mL of HO (7 vol) and the mixture was warmed to 50° C. 360 mL of acetone (30 vol) was added in portions while maintaining the temperature above 40° C. The mixture was stirred at 50° C. for 30 min. Stirring was stopped and the mixture was allowed to cool to room temperature overnight. The mixture was then cooled (ice bath) to 0-5° C. for 2 h. The mixture was filtered through sintered glass (POR 3), washed three times with 60 mL of acetone (5 vol), and dried under vacuum at 50° C. The results are shown in Table IX.

[0250] [Table 10]

[0251] These results show that replacement of NaCl in step d) of the method according to the invention with other organic or inorganic salts, as are widely used in the art, results in ICG with poor storage stability.

[0252] VII - Example 6: Solubility Testing

[0253] A solubility test was carried out on the ICG obtained by the method of the present invention (Example 1) and the ICG obtained by changing the salt forming agent (Example 5) of the same method. 250 mg of the obtained ICG, 100 mg of sodium ascorbate, and water for injection were charged into a 100 mL round-bottom flask. The mixture was stirred at room temperature under N2 atmosphere for 5 hours and 20 minutes. The solution was filtered through a PA filter (0.22 μm). The results are shown in Table X.

[0254] [Table 11]

[0255] These results show that replacement of NaCl in step d) of the method according to the invention with other organic salts, as are widely used in the art, results in ICG that is not fully soluble in aqueous solution.

Claims

1. Indocyanine green characterized by a powder X-ray diffraction pattern showing peaks at least at 2θ = 20.66°±0.2°, 14.38°±0.2°, 12.81°±0.2°, 4.22°±0.2°, and 3.83°±0.2° in crystalline indocyanine green.

2. Indocyanine green according to claim 1, wherein the X-ray diffraction pattern shows additional peaks at 2θ = 23.26°±0.2°, 20.24°±0.2°, 17.72°±0.2°, and 5.30°±0.2°.

3. Indocyanine green according to claim 1 or 2, wherein the X-ray diffraction pattern shows additional peaks at 2θ = 19.24°±0.2°, 18.22°±0.2°, 18.08°±0.2°, 7.69°±0.2°, 6.37°±0.2°, and 5.01°±0.2°.

4. Indocyanine green according to claim 1, wherein the purity of the indocyanine green, as measured as an area percentage at 240 nm by HPLC, is 98.0% or higher.

5. Indocyanine green according to claim 1, wherein each impurity is less than 0.50% in area percentage at 240 nm on an HPLC.

6. The indocyanine green according to claim 1, wherein the crystalline form has the appearance of a red crystal.

7. A pharmaceutical composition comprising the indocyanine green described in claim 1 and a pharmaceutically acceptable carrier.

8. A pharmaceutical composition according to claim 7, wherein the pharmaceutically acceptable carrier comprises water for injection, particularly sterile water for injection (SWFI) and / or antimicrobial water for injection (BWFI), ethyl alcohol, methyl alcohol, isopropyl alcohol, n-propyl alcohol, butyl alcohol, glycerin, propylene glycol, polyethylene glycol, and mixtures thereof, wherein the pharmaceutical composition comprises at least one diluent selected from the group consisting of these.

9. A pharmaceutical composition according to claim 7 or 8, further comprising histidine, ethylenediaminetetraacetic acid (EDTA) and its salts, cysteine, sodium chloride, dithiothreitol, ascorbic acid, sodium ascorbate, disodium hydrogen phosphate, sodium dihydrogen phosphate, and mixtures thereof, wherein the pharmaceutical composition further contains at least one compound selected from the group consisting of these.

10. A pharmaceutical composition according to claim 9, comprising at least one compound selected from ascorbic acid, a salt thereof, or a combination thereof.

11. A pharmaceutical composition according to claim 9, wherein the pharmaceutical composition contains histidine.

12. A pharmaceutical composition according to claim 9, comprising histidine, ethylenediaminetetraacetic acid (EDTA) and / or a salt thereof, and sodium chloride.

13. The indocyanine green according to claim 1 or the pharmaceutical composition according to claim 7, for use as a pharmaceutical or diagnostic agent.

14. Indocyanine green or pharmaceutical composition for the use described in claim 13, for obtaining angiographic images of a patient's tissue and / or for measuring cardiac output and / or for measuring liver function and hepatic blood flow and / or for detecting sentinel lymph nodes and / or for evaluating and / or predicting skin flap viability and / or for diagnosing and treating age-related macular degeneration and / or for diagnosing and treating choroidal neovascularization and tumors.

15. In the method for producing indocyanine green according to claim 1, A method for producing indocyanine green, comprising at least step d) converting 4-[2-[7-[1,1-dimethyl-3-(4-sulfobutyl)benzo[e]indole-3-ium-2-yl]hepta-2,4,6-trienylidene]-1,1-dimethylbenzo[e]indole-3-yl]butan-1-sulfonic acid of formula (VI) to 3,3,3',3'-tetramethyl-1,1'-di(4-sulfobutyl)-4,5,4',5'-dibenzoindotricarbocyanine sodium salt of formula (VII) by treatment with sodium chloride. 【Chemistry 1】

16. In the method for producing indocyanine green according to claim 15, Step d) Purification step of indocyanine green after step e), The purification step e) further comprises, e1) A step of suspending, dispersing, or dissolving indocyanine green of formula (VII) in a solvent, e2) A step of heating the composition obtained in step e1), e3) A step of cooling the solution obtained in step e2), and e4) A step to recover indocyanine green in crystalline form, A method for producing indocyanine green, comprising at least [a certain element].

17. A method for producing indocyanine green according to claim 16, wherein the solvent in step e1) is selected from the group consisting of water, acetone, methanol, ethanol, isopropyl alcohol, n-propanol, n-butanol, and mixtures thereof.

18. A method according to claim 16 or 17, wherein in step e2) the composition is heated to a temperature in the range of 40 to 80°C, and in step e3) the solution is cooled to a temperature in the range of 10 to 25°C.

19. A method for producing indocyanine green according to claim 16, wherein cooling is performed in step e3) without stirring or mixing.

20. A method for producing indocyanine green, comprising at least step d) converting 4-[2-[7-[1,1-dimethyl-3-(4-sulfobutyl)benzo[e]indole-3-ium-2-yl]hepta-2,4,6-trienylidene]-1,1-dimethylbenzo[e]indole-3-yl]butan-1-sulfonic acid of formula (VI) to 3,3,3',3'-tetramethyl-1,1'-di(4-sulfobutyl)-4,5,4',5'-dibenzoindotricarbocyanine sodium salt of formula (VII) by treatment with sodium chloride. 【Chemistry 2】

21. In the method for producing indocyanine green according to claim 20, Step d) Purification step of indocyanine green after step e), The purification step e) further comprises, e1) A step of suspending, dispersing, or dissolving indocyanine green of formula (VII) in a solvent, e2) A step of heating the composition obtained in step e1), e3) A step of cooling the solution obtained in step e2), and e4) A step to recover indocyanine green in crystalline form, A method for producing indocyanine green, comprising at least [a certain element].

22. A method for producing indocyanine green according to claim 21, wherein the solvent in step e1) is selected from the group consisting of water, acetone, methanol, ethanol, isopropyl alcohol, n-propanol, n-butanol, and mixtures thereof.

23. The method according to claim 21, wherein in step e2) the composition is heated to a temperature in the range of 40 to 80°C, and in step e3) the solution is cooled to a temperature in the range of 10 to 25°C.

24. A method for producing indocyanine green according to claim 21, wherein cooling is performed in step e3) without stirring or mixing.

25. Indocyanine green obtained by the method of claim 20.

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