Crystalline indocyanine green and preparation method thereof

Abstract

The present invention relates to a novel method for preparing indocyanine green, the method comprising a step of converting 4-[2-[7-[1, 1-dimethyl-3-(4-sulfobutyl) benzo [e] indol-3-onium-2-yl] hept-2, 4, 6-trienylene]-1, 1-dimethylbenzo [e] indol-3-yl] butane-1-sulfonic acid into indocyanine green by treatment with sodium chloride. The invention also relates to indocyanine green in crystalline forms, pharmaceutical compositions comprising the same and methods of using the same as a medicament or diagnostic agent.

Description

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

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

[0003]

[0004] Chemical structure of indocyanine green, ICG

[0005] ICG is a clinically approved near-infrared (NIR) fluorescent dye. It is commonly used in various medical imaging and diagnostic procedures, for example, to determine cardiac output or liver function, to visualize liver blood flow and blood flow during ophthalmic angiography, or to visualize cancerous tissue. These medical uses of ICG are based on its high optical absorption in the spectral region (750-800nm) where human tissue is relatively transparent. These medical uses are possible due to its very low toxicity.

[0006] For the above applications, ICG must be administered via intravenous route 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 as pure as possible and have constant physical properties, including purity, solubility and stability.

[0007] Currently, ICG is available in the form of lyophilized powder, which can be dissolved in sterile water for injection and is usually provided in the form of a kit. However, this air- and light-sensitive indocyanine green has poor stability in aqueous environments, as well as in the solid state. Poor stability in aqueous environments limits its effectiveness in certain applications, including time-sensitive surgical procedures. For this reason, the instructions of ICG vendors range from immediate use of reconstituted ICG solution to use within six hours before the patient takes the drug, and any unused portion is discarded. The lack of stability in solid form also brings difficulties to ICG manufacturing and long-term storage.

[0008] Furthermore, although currently available ICG compositions provide adequate levels of ICG for the above-approved applications, the solubility of ICG in water for injection decreases when the concentration exceeds 5 mg / ml. The USP monograph for ICG mentions the possibility of adding up to 5% w / w sodium iodide, which would improve the solubility of ICG in solution. However, iodine is a potent allergen. The risk of anaphylactic shock induced by intravenous administration of indocyanine green preparations containing up to 5% iodine cannot be ignored.

[0009] Therefore, if the characteristics of ICG, particularly long-term storage stability, solubility, and purity, can be improved, the clinical benefit of ICG will be enhanced. Such improvements in the characteristics of ICG will provide safer therapeutic / diagnostic methods for patients in need.

[0010] Several methods have been described in the prior art in an attempt to prepare ICG with improved properties.

[0011] For example, WO9507888 discloses a method for preparing high-purity substituted benz[e]indoles, in particular ICG, which comprises preparing an arylhydrazine, reacting it with a ketone in a Fischer indole synthesis, and reacting the resulting benz[e]indole with a free radical followed by a purification step.

[0012] US5,750,722 describes the preparation of high-purity indocyanine green by reacting 1,1,2-trimethyl-1H-benz[e]indole with 1,4-butanesultone and then treating with N-((2E,4E)-5-(phenylamino)penta-2,4-dienylidene)aniline hydrochloride in the presence of triethylamine and sodium acetate. The obtained indocyanine green is free of iodide ions and has a residual impurity content of less than 0.5%. After freeze-drying, the obtained solid product exhibits limited stability.

[0013] WO2017 / 093889A1 describes a method for preparing high-purity indocyanine green, comprising the steps of 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, then reacting 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; finally extracting the obtained compound with an ester solvent. The ICG obtained has a high purity of about 99.0% and is in the form of an amorphous powder. When reproducing the method disclosed in the document, ICG is obtained in a substantially amorphous form, which is also characterized by limited stability.

[0014] US2019 / 0337896A1 describes a method for preparing indocyanine green by reacting 1,1,2-trimethyl-1H-benz[e]indole with 1,4-butanesulfonic acid lactone and then treating with N-((2E,4E)-5-(phenylamino)penta-2,4-dienyl)aniline hydrochloride in the presence of triethylamine and sodium iodide. The method also includes the steps of purifying the intermediate and the final ICG product. The purity of the indocyanine green obtained by this method is greater than 99.5%, containing less than 0.5% impurities. Analysis of the product by X-ray diffraction shows that it is mainly an amorphous product. There are two unclear peaks at 2θ of 3.30 and 4.86 degrees in the PXRD diagram, which cannot be ultimately attributed to the crystalline form of ICG. In addition, when the method disclosed in the document was reproduced, ICG in a substantially amorphous form was obtained, and rapid degradation of the solid form of ICG was observed, as confirmed in the experimental part of the present application.

[0015] CN104130178 and CN106631979 disclose methods for preparing ICG similar to those described in US2019 / 0337896A1. The obtained ICG has a purity greater than 98.0%.

[0016] None of these documents discloses the crystalline form of ICG or its preparation method.

[0017] In fact, compared with amorphous forms, crystalline forms generally have lower impurity concentrations, and more consistent and uniform product quality, for example, more consistent physical properties, such as color and long-term stability. The crystal form of the API ensures the reproducibility of quality control results in terms of physical properties between batches, thereby making the API more reliable. The crystalline product is also easy to handle and produce on an industrial scale. Therefore, providing an ICG compound that is substantially in crystalline form is an important advantage.

[0018] The invention provides a crystalline indocyanine green compound and a preparation method thereof.

[0019] The applicants have developed a new process for the preparation of ICG which directly leads to a crystalline form of indocyanine green unknown in the art.

[0020] The method of the present invention provides easier processing convenience as it produces crystalline ICG of unique polymorphism in a reproducible manner.The crystalline form of ICG offers many advantages in terms of efficiency and economy for industrial scale applications.

[0021] More particularly, the discovery of this crystalline form of ICG provides a new opportunity to improve the properties of ICG in the solid state as well as in intravenous formulations. In particular, the applicant has found that crystalline indocyanine green, as described in detail later herein, exhibits significantly improved properties, especially in terms of purity and long-term stability, compared to the prior art essentially amorphous indocyanine green. Summary of the invention

[0022] According to a first aspect, the present invention relates to indocyanine green in crystalline form, characterized in that the powder X-ray diffraction pattern exhibits peaks at least at 20.66, 14.38, 12.81, 4.22 and 3.83 degrees ± 0.2 degrees 2θ.

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

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

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

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

[0027] Advantageously, the crystalline form of ICG of the present invention has the appearance of reddish crystals.

[0028] The present invention also relates to a pharmaceutical composition comprising the indocyanine green described above and in detail below and a pharmaceutically acceptable carrier.

[0029] Preferably, the pharmaceutically acceptable carrier comprises at least one diluent selected from water for injection, in particular sterile water for injection (SWFI) and / or bacteriostatic water for injection (BWFI), ethanol, methanol, isopropanol, n-propanol, butanol, glycerol, propylene glycol and 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] According to one variant, the pharmaceutical composition comprises the indocyanine green of the invention and at least one compound chosen from ascorbic acid, its salts or mixtures thereof.

[0032] According to another variant, the pharmaceutical composition comprises the indocyanine green of the invention and histidine.

[0033] According to another variant, the pharmaceutical composition comprises the indocyanine green of the invention, histidine, ethylenediaminetetraacetic acid (EDTA) and / or its salts and sodium chloride.

[0034] The present invention also relates to indocyanine green as described above and in detail below, or to a pharmaceutical composition comprising the same, and its use as a medicament or diagnostic agent.

[0035] Preferably, indocyanine green or a pharmaceutical composition comprising the same is used to obtain angiographic images of tissues in a patient's body, to determine cardiac output, to determine liver function and liver blood flow, to detect sentinel lymph nodes, to assess and / or predict flap viability, to diagnose and treat age-related macular degeneration, and / or to diagnose and treat choroidal neovascularization and tumors.

[0036] According to a second aspect, the present invention relates to a method for preparing indocyanine green, preferably a method for preparing 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]hept-2,4,6-trienyl]-1,1-dimethylbenz[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'-dibenzoindoletricarbonylcyanine sodium salt of formula (VII) by treatment with sodium chloride:

[0037]

[0038] Advantageously, the method for preparing indocyanine green further comprises a step e) of purifying the indocyanine green after step d), wherein the purification step e) comprises at least:

[0039] e1) suspending, dispersing or dissolving indocyanine green of formula (VII) in a solvent,

[0040] e2) heating the composition obtained in e1),

[0041] e3) cooling the solution obtained in e2),

[0042] e4) recovering indocyanine green in crystalline form.

[0043] Advantageously, the solvent of step e1) is selected from water, acetone, methanol, ethanol, isopropanol, n-propanol, n-butanol and mixtures thereof.

[0044] Advantageously, the composition in step e2) is heated at a temperature ranging from 40 to 80°C, preferably from 40 to 65°C.

[0045] Advantageously, in step e3), the solution is cooled to a temperature comprised between 10 and 25°C.

[0046] Advantageously, the cooling in step e3) is carried out without stirring or agitation.

[0047] The present invention also relates to indocyanine green obtained, obtainable or directly obtained by the above process, in particular wherein in step d), the sodium salt of formula (VII) is treated with sodium chloride. DETAILED DESCRIPTION OF THE INVENTION

[0049] The term "consisting essentially of" followed by one or more features refers to those that may be included in the methods or materials of the invention other than the components or steps explicitly listed, components or steps that do not substantially affect the nature and characteristics of the invention.

[0050] Unless expressly stated otherwise, the expression "comprising between X and Y" includes the boundaries. The expression means that the target range includes the X and Y values, as well as all values ​​from X to Y.

[0051] Throughout the application, when reference is made to "indocyanine green" or ICG, it refers to the compound having formula (VII) described below.

[0052] References in the specification to methods of treatment are to be construed as the compounds, pharmaceutical compositions and medicaments of the present invention being used in methods of treating the human and / or animal body by therapy or diagnosis.

[0053] Crystalline form of indocyanine green

[0054] According to a first aspect, the present invention provides a crystalline form of indocyanine green.

[0055] The crystalline form of ICG according to the present invention is characterized by a powder X-ray diffraction pattern showing major peaks at least at 20.66, 14.38, 12.81, 4.22 and 3.83 degrees ± 0.2 degrees 2θ.

[0056] In a preferred embodiment, the ICG crystalline form of the invention is characterized by a powder X-ray diffraction pattern exhibiting at least major peaks at 20.66, 14.38, 12.81, 4.22 and 3.83 2θ, and additional peaks at 23.26, 20.24, 17.72 and 5.30 degrees ± 0.2 degrees 2θ.

[0057] In a more preferred embodiment, the ICG crystalline form of the present invention is characterized by a powder X-ray diffraction pattern exhibiting major peaks at least at 20.66, 14.38, 12.81, 4.22 and 3.83 2θ, and additional peaks at 23.26, 20.24, 19.24, 18.22, 18.08, 17.72, 7.69, 6.37, 5.30 and 5.01 degrees 2θ ± 0.2 degrees.

[0058] In a most preferred embodiment, the ICG crystalline form according to the invention is characterized by the following angular positions in the X-ray powder diffraction pattern expressed as scattering angle 2θ, reticular spacing d, intensity and relative intensity (expressed as a percentage of the most intense peak):

[0059]

[0060]

[0061] It should be noted that the intensities and relative intensity values ​​of the above mentioned peaks may vary by + / - 15%.

[0062] Advantageously, the ICG according to the present invention has Figure 2 The powder X-ray diffraction pattern shown characterizes the crystalline form.

[0063] Preferably, the ICG according to the present invention has the appearance of reddish crystals, whereas the amorphous ICG of the prior art has the appearance of a green powder. More precisely, the color of the ICG crystals according to the present invention can be defined as a golden red sparkle color. This characteristic can be evaluated with the naked eye or by methods known to those skilled in the art such as spectrophotometry.

[0064] Preparation method of crystalline indocyanine green

[0065] According to a second aspect, the present invention relates to a method for preparing crystalline indocyanine green, said method comprising at least one step of salifying the acid form of indocyanine green with sodium chloride to obtain the sodium salt of indocyanine green, commonly referred to as indocyanine green or ICG.

[0066] For the purposes of the present invention, the expression "acid indocyanine green" corresponds to the compound of the following formula (VI) defined as 4-[2-[7-[1,1-dimethyl-3-(4-sulfobutyl)benz[e]indol-3-ium-2-yl]hept-2,4,6-trienyl]-1,1-dimethylbenz[e]indol-3-yl]butane-1-sulfonic acid.

[0067] According to a preferred embodiment, the process according to the invention further comprises at least one step e) of purifying the indocyanine green obtained in step d).

[0068] Salt-forming step d)

[0069] In particular, the process according to the present invention comprises at least step d), i.e., converting 4-[2-[7-[1,1-dimethyl-3-(4-sulfobutyl)benz[e]indol-3-ium-2-yl]hept-2,4,6-trienyl]-1,1-dimethylbenz[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'-dibenzoindolotricyanine sodium salt of formula (VI) by treating with sodium chloride or using sodium chloride according to the following scheme:

[0070]

[0071] Advantageously, in step d), the amount of sodium chloride used is such that the molar ratio of indocyanine green in acid form of formula (VI) to sodium chloride is less than or equal to 1:1, preferably the molar ratio is from 1:5 to 1:1, more preferably from 1:3 to 1:1.

[0072] Advantageously, the reaction of step d) is carried out in a solvent selected from water, methanol, ethanol, isopropanol, n-propanol, n-butanol and mixtures thereof.

[0073] According to a preferred embodiment, step d) is carried out in a mixture of water and methanol. Preferably, water and methanol are mixed in a volume ratio of 5:1 to 1:5, preferably 2:1 to 1:5, more preferably 2:1 to 1:2.

[0074] Advantageously, step d) is carried out at a temperature ranging from 25 to 65°C, preferably from 40 to 55°C.

[0075] Advantageously, after the reaction of step d) is complete, the reaction mixture is cooled to room temperature and the product of formula (VII) is isolated from the reaction medium by precipitation. Preferably, the precipitation is carried out by adding a solvent selected from ether, water, acetone and mixtures thereof.

[0076] According to a preferred embodiment, the indocyanine green of formula (VII) is isolated from the reaction medium by precipitation with acetone.

[0077] Alternatively, it is conceivable to isolate the product of formula (VII) from the reaction medium by other purification techniques such as solvent extraction or column chromatography.

[0078] Steps a), b) and c) for preparing the acid form of indocyanine green

[0079] Indocyanine green in acid form of formula (VI) can be obtained by any method known to those skilled in the art, such as the method described in WO2017 / 093889A1 or US5,750,722.

[0080] According to a preferred embodiment of the present invention, Figure 1 The reaction scheme comprising steps a), b) and c) shown in Figure 1 prepares the acid form of indocyanine green of formula (VI).

[0081] Step a) comprises reacting 1,2,2-trimethylbenz[e]indole of formula (I) with 1,4-butanesultone of formula (II) in an equimolar ratio to obtain 4-(1,1,2-trimethyl-1H-benzo[e]indolium-3-yl)butane-1-sulfonate of formula (III) according to the following scheme:

[0082]

[0083] This reaction is known to the skilled person and is described, for example, in US 5,750,722, WO 2017 / 093889 A1 and Chem. Eur. J. 2021, 27, 14535-14542.

[0084] The reaction of step a) can be carried out in a solvent selected from pentane, hexane, heptane, cyclohexane, cyclopentane, petroleum ether, xylene, toluene and mixtures thereof. According to a preferred embodiment, step a) is carried out in toluene.

[0085] 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.

[0086] Advantageously, after the reaction is complete, the reaction mixture is cooled to room temperature and the product of formula (III) is isolated from the reaction medium by precipitation. Preferably, the precipitation is carried out by adding a solvent selected from ether, water, acetone and mixtures thereof.

[0087] Step b) comprises condensing the intermediate of formula (III) obtained in step a) with glutaraldehyde dihydrochloride of formula (IV) in an equimolar ratio in the presence of acetic anhydride according to the following scheme to obtain 4-(1,1-dimethyl-2-((1E,3E,5E)-6-(N-phenylacetylamino)-hexa-1,3,5-trienyl)-1H-benzo[e]indolium-3-yl)-butane-sulfonate of formula (V):

[0088]

[0089] This reaction is described, for example, in EP2764861, US5,750,722 and US2019 / 0337896.

[0090] Alternatively, the reaction of step b) may be carried out in the presence of maleic anhydride instead of acetic anhydride.

[0091] Preferably, the reaction of step b) is carried out at a temperature of 50°C to 170°C, preferably 60°C to 150°C, more preferably 70°C to 130°C.

[0092] 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 medium by precipitation. Preferably, the precipitation is carried out by adding a solvent selected from ether, water, acetone and mixtures thereof.

[0093] Step c) comprises reacting the intermediate of formula (V) obtained in step b) with 4-(1,1,2-trimethyl-1H-benzo[e]indol-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-yl]heptyl-2,4,6-trimethylene]-1,1-dimethylbenz[e]indol-3-yl]butane-1-sulfonic acid of formula (VI) according to the following reaction scheme:

[0094]

[0095] This reaction is described, for example, in EP2764861, US5,750,722 and US2019 / 0337896.

[0096] The reaction of step c) can be carried out in a solvent selected from water, methanol, ethanol, isopropanol, butanol and mixtures thereof. According to a preferred embodiment, the reaction of step c) is carried out in a mixture of water and methanol. Preferably, water and methanol are mixed in a volume ratio of 1:1 to 1:3.

[0097] Preferably, the base in step c) is selected from triethylamine, pyridine, ammonia, sodium carbonate, sodium bicarbonate and sodium hydroxide. More preferably, the base is triethylamine.

[0098] Preferably, the reaction of step c) is carried out at a temperature of 20°C to 40°C, preferably at 25°C.

[0099] Advantageously, the intermediates of formula (III), (V) and (VI) can be purified after steps a), b) and c), respectively. The purification of these intermediates can be carried out by one or more conventional purification steps such as precipitation, crystallization, solvent extraction. For example, the purification of these intermediates can be carried out according to the method disclosed in the embodiments of application US2019 / 0337896.

[0100] Purification step e)

[0101] According to a preferred embodiment, the method for preparing indocyanine green according to the present invention further comprises a step e) of purifying the indocyanine green after step d).

[0102] According to a preferred embodiment, the purification step e) is carried out by recrystallization of the indocyanine green of formula (VII).

[0103] Preferably, the purification step e) comprises at least the following steps:

[0104] e1) suspending, dispersing or dissolving indocyanine green of formula (VII) in a solvent,

[0105] e2) heating the composition obtained in step e1),

[0106] e3) cooling the solution obtained in step e2),

[0107] e4) recovering indocyanine green in crystalline form.

[0108] Advantageously, the solvent in step e1) is selected from water, acetone, methanol, ethanol, isopropanol, n-propanol, n-butanol and mixtures thereof.

[0109] Preferably, the solvent in step e1) is a mixture of water and acetone.

[0110] More preferably, in step e1), water and acetone are used in a ratio of 10:1 to 1:10 v / v, preferably 4:1 to 1:6 v / v, more preferably 1:1 to 1:5 v / v.

[0111] Advantageously, when the composition obtained in step e1) is a suspension or dispersion, the composition is heated in step e2) to a temperature allowing the indocyanine green to dissolve.

[0112] 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 to 65°C.

[0113] Advantageously, in step e3), the solution is cooled or allowed to cool to a temperature between 10°C and 25°C, preferably between 15°C and 25°C.

[0114] "Cooling" means cooling the reaction medium to ambient temperature, or actively cooling the reaction medium to accelerate the temperature drop, such as by immersing the reactor in an environment cooler than ambient temperature.

[0115] According to a preferred embodiment, the solution is then kept at this temperature for a sufficient time to form ICG crystals. Preferably, the solution is kept at a temperature between 10°C and 25°C, preferably between 15°C and 25°C, for a period of 10 to 120 hours, preferably 24 to 96 hours, more preferably 48 to 80 hours.

[0116] Optionally, the solution is then further cooled to a temperature lower than or equal to 10°C, preferably at a temperature between -5 and 10°C, more preferably between 0 and 10°C.

[0117] According to a preferred embodiment, step e3) is performed without stirring or agitating the solution.

[0118] "Step e3) is carried out without stirring or agitating the solution" means that all substeps, i.e. cooling to a temperature of 10 to 25°C, keeping the solution at this temperature for a certain time, and optionally cooling to a temperature below 10°C, are carried out without stirring or agitating the reaction medium.

[0119] Advantageously, in step e4), the crystalline indocyanine green is separated from the reaction medium by filtration.

[0120] 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.

[0121] Advantageously, washing is carried out with a solvent selected from the group consisting of acidic aqueous solutions, acetone, acetonitrile and mixtures thereof.

[0122] Preferably, washing is performed at least once with acetone and at least once with acetonitrile.

[0123] More preferably, washing is performed once to three times with acetone and once to three times with acetonitrile.

[0124] Properties of the obtained indocyanine green

[0125] According to a third aspect, the present invention relates to an ICG compound obtained or obtainable by a process comprising at least one step of converting 4-[2-[7-[1,1-dimethyl-3-(4-sulfobutyl)benzo[e]indol-3-ium-2-yl]hept-2,4,6-trienyl]-1,1-dimethylbenz[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'-dibenzoindoletricarbonylcyanine sodium salt of formula (VII) by treatment with sodium chloride.

[0126] The applicant has surprisingly found that the ICG obtained or obtainable by this method is crystalline. In particular, the ICG obtained or obtainable by this method has a specific crystalline form as described above.

[0127] Furthermore, the applicant has surprisingly found that the ICG obtained or obtainable by the method exhibits a higher purity and / or physical stability and / or solubility than the amorphous form of ICG obtained by the prior art methods. Furthermore, the method gives highly reproducible results, so that a constant quality of ICG can be obtained.

[0128] These improved properties and new crystalline forms of ICG are a direct result of the specific process for preparing ICG according to the present invention.

[0129] Purity of ICG

[0130] In particular, the process of the present invention enables the preparation of ICG compounds with higher purity than ICG obtained by conventional processes described in the prior art using sodium iodide or organic sodium salts as salt-forming agents.

[0131] In particular, the indocyanine green of formula (VII) obtained by the process of the present invention is substantially pure and has a purity greater than 99.0%, preferably greater than 99.5%, more preferably greater than 99.7%, and most preferably greater than 99.8% as determined by HPLC.

[0132] As used herein, the term "purity" refers to the degree to which the indocyanine green is free of undesired or adulterated chemical entities.

[0133] According to the present invention, the purity and impurities in the obtained indocyanine green were evaluated by HPLC at 240 nm in area %.

[0134] Preferably, the indocyanine green obtained by implementing the process of the present invention comprises less than 1.0%, preferably less than 0.5% of impurities measured by HPLC.

[0135] Preferably, the indocyanine green obtained by carrying out the process of the present invention comprises less than 0.50%, preferably less than 0.40%, more preferably less than 0.20%, most preferably less than 0.15% of each impurity as measured by HPLC.

[0136] The impurities contained in the indocyanine green can be any reaction byproducts or intermediates produced by the above method. For example, the impurities can include N-phenylacetamide, 4-(1,1,2-trimethyl-1H-benzo[e]indolium-3-yl)butane-1-sulfonate, 4-(1,1,2-dimethyl-2-((1E,3E,5E)-6-(N-phenylacetylamino)hex-1,3,5-trienyl)-1H-benzo[e]indolium-3-yl)butane-1-sulfonate, but are not limited thereto.

[0137] The indocyanine green obtained by carrying out the process of the invention is substantially free, preferably completely free, of iodine and / or iodide, in particular sodium iodide. The lack of iodine / iodide in the indocyanine green can be checked, for example, by ion exchange chromatography.

[0138] Preferably, the obtained indocyanine green contains, based on the weight of indocyanine green, less than 200ppm, preferably less than 100ppm, more preferably less than 50ppm, even more preferably less than 20ppm of metal contaminants. The metal contaminant content in indocyanine green can be measured by inductively coupled plasma mass spectrometry (ICP-MS).

[0139] “Metal contaminants” 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.

[0140] More preferably, the indocyanine green contains no lead and arsenic or contains less than 2 ppm of lead and arsenic.

[0141] Stability of ICG

[0142] The applicant has found that the crystalline indocyanine green obtained by the process of the present invention exhibits significantly improved stability compared to the indocyanine green of the prior art.

[0143] The terms "stable" and "stability" mean that the indocyanine green content in a solid and / or liquid composition, especially an aqueous composition, remains stable after storage for a certain period of time.

[0144] An "aqueous composition" is understood to mean an aqueous solution, an aqueous suspension, an aqueous colloidal suspension, an aqueous dispersion, preferably an aqueous solution.

[0145] The stability of the ICG of the present invention is evaluated by measuring the change in the indocyanine green content before and after long-term storage, wherein the indocyanine green is stored in a solid state or after being dissolved in an aqueous composition.

[0146] It should be understood that when stored in the solid state, indocyanine green is stored in the above-mentioned crystalline form (rather than lyophilized form). The crystalline indocyanine green of the present invention is advantageous because it shows substantially no signs of aggregation, degradation after long-term storage, and substantially no or little formation of impurities caused by chemical modification of indocyanine green. In addition, the advantage of the crystalline ICG of the present invention is that it has no significant discoloration or reduction in fluorescence intensity or peak absorbance after long-term storage.

[0147] Advantageously, the indocyanine green of the present invention exhibits very superior stability in the solid state compared to the indocyanine green compounds of the prior art, especially the lyophilized ICG of the prior art.

[0148] Advantageously, the crystalline indocyanine green of the present invention is stable when stored in the solid state at ambient temperature for up to one month, preferably up to 2 months, preferably up to 3 months, more preferably up to 6 months, still more preferably up to 12 months, most preferably up to 2 years.

[0149] Advantageously, the crystalline indocyanine green of the present invention is stable when stored at ambient temperature after being dissolved in an aqueous medium for up to 24 hours, preferably up to 48 hours, more preferably up to 3 days, and most preferably up to 6 days. The term "ambient temperature" refers to controlled room temperature, such as 20-25°C, with a deviation of 15-30°C, as defined in the United States Pharmacopoeia, or 15-25°C, as defined in the European Pharmacopoeia.

[0150] Advantageously, this stability of indocyanine green is observed when the indocyanine green is stored in a container that protects it from natural light and / or UV light and / or fluorescent light. Preferably, the container is tight or sealed.

[0151] 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 methods. Analytical methods for evaluating the stability of indocyanine green are known to those skilled in the art. They can include nuclear magnetic resonance (NMR), high performance liquid chromatography (HPLC), size exclusion chromatography (SEC), liquid chromatography combined with mass spectrometry (LC-MS), dynamic light scattering (DLS), differential scanning calorimetry (DSC), UV spectroscopy and Fourier transform infrared spectroscopy (FTIR) or a combination of these methods.

[0152] Advantageously, the stability of ICG is evaluated by measuring the change in ICG content by HPLC before and after long-term storage. More advantageously, the ICG content and the change in this content are evaluated by HPLC at 240 nm as % area.

[0153] Preferably, the stability of indocyanine green corresponds to a decrease of less than or equal to 10%, preferably less than or equal to 5%, more preferably less than or equal to 2%, in the indocyanine green content measured as % area at 240 nm by HPLC, when the indocyanine green is stored in the solid state at ambient temperature for up to 1 month, preferably up to 3 months, more preferably up to 6 months, still more preferably up to 12 months and most preferably up to 2 years.

[0154] Preferably, the stability of indocyanine green corresponds to a decrease of less than or equal to 10%, preferably less than or equal to 5%, more preferably less than or equal to 2% in the indocyanine green content measured as % area at 240 nm by HPLC when the indocyanine green is stored in an aqueous medium at ambient temperature for up to 24 hours, preferably up to 48 hours, more preferably up to 3 days, and most preferably up to 6 days.

[0155] The stability of the indocyanine green of the present invention in aqueous media can be greatly improved by adding one or more stabilizing compounds selected from the following: histidine, ethylenediaminetetraacetic acid (EDTA) and its salts, cysteine, sodium chloride, dithiothreitol, ascorbic acid, sodium ascorbate, disodium hydrogen phosphate, sodium dihydrogen phosphate and mixtures thereof.

[0156] In the presence of these additives, the aqueous ICG preparation is more stable and the content of indocyanine green measured by HPLC as % area at 240 nm decreases by less than or equal to 10%, preferably less than or equal to 5%, more preferably less than or equal to 2% when stored at ambient temperature for up to 1 month, preferably up to 3 months, more preferably up to 6 months, still more preferably up to 12 months, and most preferably up to 2 years. Within the meaning of the present application, "decline in content" is understood as the percentage difference between the content of indocyanine green measured by HPLC as % area at 240 nm before storage and the content of indocyanine green measured after storage for a determined time.

[0157] The stability of indocyanine green may also correspond to the fact that the total amount of impurities contained in the indocyanine green does not increase during storage, in particular does not increase to a level that reduces the ability of the indocyanine green to be used in medical and / or diagnostic applications. For example, when indocyanine green is stored in a solid state at ambient temperature for at least 1 month and then these impurities are measured by HPLC, only a small amount of impurities is observed in the composition, and this amount does not increase significantly over time.

[0158] In the context of the present invention, the term "impurity" is to be understood as a chemical entity that is not indocyanine green, or an excipient or other additive to the indocyanine green according to the present invention. Impurities may be process-related impurities, such as by-products or intermediates that may be formed during the manufacture of indocyanine green, or degradation-related impurities resulting from chemical transformations of indocyanine green during storage.

[0159] In one embodiment, the increase in impurities in the indocyanine green according to the present invention (as the area % change measured by HPLC at 240 nm) is less than or equal to 10%, preferably less than or equal to 5%, more preferably less than or equal to 2%, when the indocyanine green is stored in the solid state at ambient temperature for up to 1 month, preferably up to 2 months, more preferably up to 6 months, and most preferably up to 2 years.

[0160] Compositions comprising crystalline indocyanine green

[0161] According to a fourth aspect, the present invention relates to a pharmaceutical composition comprising the crystalline indocyanine green disclosed above and / or the indocyanine green obtained or obtainable by the above-described method.

[0162] The pharmaceutical compositions of the invention may be provided in any suitable form, such as a liquid solution or a solid powder to be reconstituted in a liquid solution for intravenous administration.

[0163] The pharmaceutical composition may further comprise at least one pharmaceutically acceptable carrier and / or at least one pharmaceutically acceptable additive.

[0164] As used herein, the term "pharmaceutically acceptable" refers to compounds, materials and compositions that are suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0165] As used herein, the term "carrier" refers to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material.

[0166] Advantageously, the pharmaceutically acceptable carrier comprises at least one diluent.

[0167] The term "diluent" includes pharmaceutically acceptable solvents which are safe and non-toxic after administration to humans or animals and can be used to prepare pharmaceutical preparations.

[0168] Suitable diluents can be any biologically acceptable liquid, and the compositions of the present invention are completely soluble therein. Water, particularly sterile water for injection (SWFI) and / or bacteriostatic water for injection (BWFI), is a preferred diluent because it does not include salts or other compounds that may affect the stability of indocyanine green. However, other diluents, such as sterile saline solution, Ringer's solution, dextrose solution, glucose solution, ethanol, methanol, isopropanol, n-propanol, butanol, glycerol, propylene glycol and polyethylene glycol and mixtures thereof, can be used. Those skilled in the art can select diluents according to desired use and relative to other compounds of the composition.

[0169] According to a preferred embodiment, the diluent is selected from water for injection, bacteriostatic water for injection, sterile saline solution, Ringer's solution, dextrose solution and glucose solution.

[0170] In a preferred embodiment, the diluent is water for injection, in particular sterile water for injection (SWFI) and / or bacteriostatic water for injection (BWFI).

[0171] The term "additive" includes components of the pharmaceutical composition other than indocyanine green. Examples of such additives include buffers, pH adjusters, isotonic agents, surfactants, preservatives, tonicity adjusters, antimicrobial agents, wetting agents and emulsifiers. These additives are generally available to those of ordinary skill in the art and may be in any form, such as solid, liquid or semisolid.

[0172] According to one embodiment, the pharmaceutical composition comprising indocyanine green does not comprise sodium iodide.

[0173] According to a preferred embodiment, the pharmaceutical composition containing indocyanine green of the present invention further comprises at least one stabilizing 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.

[0174] These stabilizing compounds are described, for example, in applications PCT / EP2022 / 056443, PCT / EP2022 / 056445 and PCT / EP2022 / 056446. The addition of these compounds can further improve the long-term stability of aqueous pharmaceutical compositions comprising indocyanine green according to the invention.

[0175] Preferably, according to one variant, the pharmaceutical composition comprises, preferably consists of, the indocyanine green of the invention and at least one compound chosen from ascorbic acid, its salts or mixtures thereof.

[0176] Preferably, according to the second variant, the pharmaceutical composition comprises, preferably consists of, the indocyanine green of the invention and histidine.

[0177] Preferably, according to the third variant, the pharmaceutical composition comprises, preferably consists of, the indocyanine green of the invention, histidine, ethylenediaminetetraacetic acid (EDTA) and / or its salts and sodium chloride.

[0178] Preferably, the amount of the additive in the composition of the present invention is within a range that does not substantially negatively affect the activity of indocyanine green. Preferably, the composition of the present invention contains the minimum number and amount of additives required to provide a stable and effective composition. The technician can adjust the amount of the additive according to the solubility of the additive in the carrier. The pharmaceutical composition of the present invention can be prepared using any known method.

[0179] The concentration of indocyanine green in the composition may be any concentration suitable for use in medical treatment or diagnosis, in particular for producing angiographic images of satisfactory quality.

[0180] According to a preferred embodiment, the pharmaceutical composition of the present invention is in the form of a kit, which comprises the indocyanine green of the present invention and optionally at least one additive in a first part, and comprises the carrier as described above in a second part.

[0181] According to another preferred embodiment, the pharmaceutical composition of the present invention is in the form of a kit, which comprises the indocyanine green of the present invention in a first part and a carrier and optionally at least one additive as described above in a second part.

[0182] All preferred indocyanine green and pharmaceutical composition embodiments described above also apply to any of these kit variations.

[0183] Use of indocyanine green of the present invention

[0184] The crystalline indocyanine green of the present invention, in particular the pharmaceutical composition comprising it, can be used in a method of diagnosis and / or treatment of a patient (as a therapeutic agent). The method comprises administering an effective amount of an aqueous composition of indocyanine green. This administration can be carried out by enteral route, parenteral route, in particular intravenous injection or by topical application. Preferably, administration is carried out by intravenous injection.

[0185] The term "effective amount" refers to that amount of indocyanine green that will elicit the biological or medical response of a tissue, system, animal or human that is being sought, for example, by a researcher or clinician.

[0186] For example, the crystalline indocyanine green according to the present invention, especially the pharmaceutical composition comprising it, can be used for thermotherapy, selective photocoagulation enhanced by indocyanine green, photodynamic therapy (PDT), photodynamic and thermotherapy (PHT). More particularly, as a PDT application, the indocyanine green according to the present invention can be used for infection treatment, acne treatment, macular surgery, cancer treatment, etc. The application of the indocyanine green of the present invention can be related to other therapies such as immunotherapy, radiotherapy, ultrasound and chemotherapy.

[0187] The crystalline indocyanine green of the present invention can also be used to obtain angiographic images of tissues in a patient's body, for determining cardiac output, for determining liver function and liver blood flow, for detecting sentinel lymph nodes, for evaluating and / or predicting flap viability, for diagnosing and treating age-related macular degeneration, related choroidal neovascularization and tumors. Preferably, the crystalline indocyanine green of the present invention is used to obtain angiographic images of tissues, for determining cardiac output, for determining liver function and liver blood flow, for detecting sentinel lymph nodes and / or for evaluating and predicting flap viability.

[0188] For diagnostic applications, given that the peak absorption and emission of indocyanine green are in the range of 800-850 nm, the amount of indocyanine green composition administered to the patient should be sufficient to allow the dye to fluoresce when illuminated at the appropriate wavelength. The same criteria apply to treatments using ICG solutions; sufficient dye should be administered to make the treatment effective. The amount of indocyanine green administered can be readily determined by one skilled in the art and should be at least the concentration currently accepted for use in ophthalmic angiography, e.g.,

[0189] For diagnostics, 2 mL of a 20 mg / mL indocyanine green solution. As known to the skilled artisan, higher dye concentrations may be advantageously used in any of these diagnostic and therapeutic methods.

[0190] Compared to prior art indocyanine green preparations, the improved stability indocyanine green of the present invention provides improved diagnosis / treatment for patients using the same amount of injected compound. This is possible because the improved stability of indocyanine green will provide a stronger response to fluorescent illumination. BRIEF DESCRIPTION OF THE DRAWINGS

[0191] Figure 1 Reaction scheme to obtain indocyanine green of formula (VI) in acid form is shown.

[0192] Figure 2 The figure shows the X-ray diffraction pattern of indocyanine green prepared by the method of the present invention (Example 1).

[0193] Figure 3 Shown is an X-ray diffraction pattern of indocyanine green prepared according to a comparative method including a salt-forming step using sodium iodide (Example 2).

[0194] Figure 4 Shown is an X-ray diffraction pattern of indocyanine green prepared according to a comparative method including a salt-forming step using sodium acetate (Example 3).

[0195] Figure 5 The X-ray diffraction pattern of indocyanine green prepared according to the comparative method of Example 4 is shown. Example

[0196] I-Materials and Methods:

[0197] I-1. Reagents and chemicals:

[0198] 1,2,2-Trimethylbenz[e]indole (CAS No 41732-24-7) was provided by Matrix Fine Chemical (HPLC purity > 99.0%). 1,4-Butanesultone (CAS No 1633-83-6) was provided by Matrix Fine Chemical (HPLC purity > 99.9%). Pentadienaldehyde diphenylamine hydrochloride (CAS No 1497-49-0) was provided by Matrix Fine Chemical.

[0199] Other reagents and solvents (acetone, methanol, triethylamine, sodium chloride...) were purchased from commercial suppliers such as SigmaAldrich and TCI.

[0200] I-2. Chromatographic conditions:

[0201] In order to study the purity and stability, the content of indocyanine green in the above examples was determined by high pressure liquid chromatography (HPLC). The purity of indocyanine green was expressed as % area measured by HPLC at 240 nm. The analysis conditions are given in Table 1 below:

[0202] Table I: HPLC analysis conditions

[0203] Chromatographic columns AGILENT Poroshell 120EC-C8(150x4.6-2.7μm) Column temperature 40℃ Flow rate 1mL / min Detector UV-Vis at 240 nm and MS ESI spectrometer Injection volume 5μL Thinner Methanol

[0204] Elution was performed by gradient. The mobile phase consisted of 100 mM ammonium formate buffer at pH 4.5 (eluent A) and acetonitrile (eluent B). The composition of the mobile phase was continuously changed during elution according to Table II below:

[0205] Table II: Gradient elution during HPLC characterization

[0206] Time (min) Eluent A% Eluent B% 0 82.5 17.5 5 82.5 17.5 40 30 70 45 30 70 45.1 10 90 50 10 90 50.1 82.5 17.5 60 82.5 17.5

[0207] Impurities that may be detected by the HPLC method in the context of the present invention are summarized below.

[0208]

[0209]

[0210] I-3.X-ray powder diffraction:

[0211] X-ray powder diffraction data were obtained using a Panalytical Empyrean S3 (DY2626) powder X-ray diffractometer equipped with a PIXcel 1D-Medipix3 detector by using methods known in the art. and polypropylene. The artifact can be generated at 2θ = 5.5°. Scanning parameters include: radiation source: copper (1.54A), X-ray tube power: 40kV / 40mA, range: 2.0-50.0 degrees 2θ; scanning mode: continuous scanning; step size: 0.026 degrees; number of scans: 5; acquisition time 20.40 seconds. The results are in scattering angle 2θ (°), d-interval Peak intensities and relative intensities expressed as a percentage of the most intense peak are presented. All peak positions are within 0.2 degrees 2θ.

[0212] I-4.NMR spectrum:

[0213] NMR spectra were recorded on a Bruker Advanced NanoBay 300 MHZ using manual shims. 1H chemical shifts (δ) are reported in ppm relative to TMS. Proton NMR information is given in the following form: multiplicity (s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet), coupling constant J, integral.

[0214] I-5. Determination of sodium content by atomic absorption method:

[0215] The sodium content in the above examples was determined using an atomic absorption spectrometer Agilent AA 240FS coupled with SpectraA software. The flame composition was air / acetylene and the sample was dissolved in acidic water.

[0216] II-Example 1 (according to the invention):

[0217] II-1. Synthesis of 4-(1,1,2-trimethyl-1H-benzo[e]indolium-3-yl)butane-1-sulfonate (step a):

[0218] A mixture of 1,1,2-trimethylbenz[e]indole (20.0 g, 95.6 mmol, 1 eq) and 1,4-butanesultone (19.52 g, 142.4 mmol, 1.5 eq) in toluene (40 mL) was stirred vigorously at 140° C. for 12 hours. After cooling to room temperature, ether (400 mL) was added. The solid was collected by filtration and dried. A blue / grey powder was obtained with a yield of 97%.

[0219] 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).

[0220] 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.

[0221] HPLC retention time = 7.6 minutes (relative retention time to ICG = 0.26).

[0222] II-2.4-(1,1-dimethyl-2-((1E,3E,5E)-6-(N-phenylacetylamino)-hexa-1,3,5-trienyl)- Synthesis of 1H-benzo[e]indolium-3-yl)-butane-sulfonate (step b):

[0223] 4-(1,1,2-trimethyl-1H-benzo[e]indolium-3-yl)butane-1-sulfonate (2.08 g, 6.0 mmol) and pentadienal diphenylamine hydrochloride (1.88 g, 6.6 mmol) were mixed in a 50 ml four-necked flask and then stirred at 120° C. for 1 hour in 20 ml of acetic anhydride. The mixture was cooled to room temperature and stirred for 1 hour, and then the crystals were separated by filtration. The resulting crystals were suspended in acetone and washed, separated by filtration, and air-dried. A lavender powder was isolated with a yield of 56%.

[0224] 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).

[0225] HPLC retention time = 21.8 minutes (relative retention time to ICG = 0.76).

[0226] II-3. Synthesis of Indocyanine Green (Steps c and d)

[0227] Step c): 28.646 g (82.92 mmol, 0.9 eq) of 4-(1,1,2-trimethyl-1H-benzo[e]indolium-3-yl)butane-1-sulfonate and 50.000 g (92.13 mmol, 1.0 eq) 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 were added to a 2L double jacketed borosilicate reactor equipped with a mechanical stirrer and diluted with distilled water (75.0 mL) and MeOH (125.0 mL). The mixture was stirred at room temperature at 150 rpm for 5 minutes. 12.8 mL of triethylamine (92.13 mmol, 1.00 eq) was added once and the mixture turned green. The mixture was stirred at 25 °C for 3 h and warmed to 50 °C.

[0228] Step d): A solution of 8.615 g of sodium chloride (147.41 mmol, 1.70 equivalents) in 50.0 mL of distilled water was added at once. When the mixture thickened, 1.5 L of acetone was slowly added while keeping the reaction mixture above 40 ° C. The mixture was stirred at 200 rpm for another 1 hour. The reaction was cooled to 25 ° C, stirred overnight at 200 rpm, then filtered and washed three times with 250 mL of acetone. The solid was dried in vacuo at 40 ° C to obtain 64.3 g of crude indocyanine green as a green powder.

[0229] 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%).

[0230] Sodium content = 3.90%.

[0231] II-4. Purification of indocyanine green (step e):

[0232] Crude indocyanine green (50.0 g) and 350.0 mL of distilled water were added to a 2 L double-jacketed borosilicate reactor equipped with a mechanical stirrer. The mixture was warmed to 50 ° C, thickening was observed at 35-37 ° C, and 1.5 L of acetone was added in batches while keeping the mass temperature above 40 ° C (green uniform liquid mixture). After stirring at 50 ° C for 1 hour, the stirring system was turned off and the mixture was cooled to room temperature without any stirring. Red-gold crystals appeared on the surface of the liquid. After 60 hours at room temperature, the mixture was cooled to 5 ° C for 16 hours without any stirring, filtered and washed three times with 5 volumes of acetone, and then washed with 5 volumes of acetonitrile. The filter cake was dried at 70 ° C under vacuum (50 mbar) for 72 hours. Indocyanine green was obtained as a red-gold flashing solid with a total separation yield of 64% (34.3 g, 68% separation and purification yield).

[0233] Purity = 99.95% (HPLC profile: ICG 99.95%; each impurity < 0.05%).

[0234] Sodium content = 2.82% (corrected for water content by heat balance analysis).

[0235] 1H 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), 6.6 0(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).

[0236] The obtained PXRD pattern is described in Figure 2 In FIG. 5 , a crystalline form of indocyanine green is shown, which is characterized by the following peaks:

[0237] Table III: Peaks of the PXRD pattern of crystalline indocyanine green according to the present invention

[0238]

[0239] The intensities and relative intensity values ​​of the above peaks may vary within + / - 15%.

[0240] Solid State Stability: 5.0 g of crystalline indocyanine green powder was stored in 20 mL amber Type II glass and protected from light. The results are summarized in Table IV.

[0241] Table IV: Storage stability of ICG of the present invention obtained by the method of Example 1 in the solid state

[0242] Storage time t0 t = 3 months t = 6 months ICG Impurities (HPLC) 99.95％ 99.95％ 99.95％ Total impurities (HPLC) <0.05％ <0.05％ <0.05％

[0243] III-Example 2 (Comparative):

[0244] The scheme for preparing indocyanine green disclosed in the examples of prior art US2019 / 0337896A1 is reproduced.

[0245] III-1. Synthesis of indocyanine green:

[0246] To a 2L double jacketed borosilicate reactor equipped with a mechanical stirrer was added 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 in 20.0 mL of methanol. 4-(1,1,2-trimethyl-1H-benzo[e]indolium-3-yl)butane-1-sulfonate (12.73 g, 0.037 mol, 1.0 equiv) and triethylamine (1.0 mL, 0.007 mol, 0.20 equiv) were added sequentially to the reaction mixture (fluidization was observed). After stirring at 60-65°C for 1.5 hours, no green color was observed, indicating that the reaction had not started. To the mixture was added 4.0 mL (0.028 mol, 0.8 eq.) of triethylamine: a green color appeared instantly.

[0247] The mixture was cooled to 10-15°C and a solution of 5.52 g (0.037 mol, 1.0 eq.) of sodium iodide in 100.0 mL of methanol was added. 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 obtain a green viscous residue. 400 mL of acetone was poured into the reactor and the heterogeneous mixture 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.

[0248] III-2. Purification of indocyanine green:

[0249] 23.5 g of crude indocyanine green was diluted with 94 mL (4 volumes) of methanol and 141 mL of isopropanol (6 volumes). The mixture was heated at 60-70° C. for 1 hour, filtered at 50° C., and the filter cake was washed with 45 mL of isopropanol to obtain 21.2 g of pure indocyanine green as a green powder with a separation and purification yield of 90%.

[0250] Purity = 99.21% (HPLC spectrum ICG 99.21%; impurity A < 0.30%; impurity C < 0.07%; impurity M < 0.07%; impurity L < 0.06%; total unknown impurities = 0.29%).

[0251] Sodium content = 3.27%.

[0252] The obtained PXRD pattern is described in Figure 3 , showing mainly amorphous product with two ill-defined peaks at 3.30 and 4.86 degrees 2θ.

[0253] Solid State Stability: 5.0 g of indocyanine green powder was stored in 20 mL of amber Type II glass and protected from light. The results are summarized in Table V.

[0254] Table V: Storage stability of comparative ICG obtained by the method of Example 2 in solid state

[0255]

[0256]

[0257] IV-Example 3 (Comparative):

[0258] The method for preparing indocyanine green disclosed in the example of prior art WO2017093889A1 is reproduced.

[0259] IV-1. Synthesis of Indocyanine Green:

[0260] In a 2 L double jacketed borosilicate reactor equipped with a mechanical stirrer, 4-(1,1,2-trimethyl-1H-benzo[e]indolium-3-yl)butane-1-sulfonate (56.17 g, 0.163 mol, 1.89 equiv) was added and diluted in 140.0 mL of methanol. 35.10 g of sodium acetate (0.428 mol, 4.97 equiv) was added and the reaction was stirred at room temperature for 5 minutes. The mixture was heated at 65°C and a solution of 25.0 g (0.086 mol, 1.0 equiv) of N-phenyl-N-((1E,3E,5E)-5-(phenylimino)penta-1,3-dienyl)acetamide in 25.0 mL of methanol was added. A green color appeared. The reaction mixture was stirred at 65°C for 2 hours, 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 (yield = 100%) of crude indocyanine green as a green sticky solid.

[0261] IV-2. Purification of indocyanine green:

[0262] 60.0 g of crude indocyanine green was diluted in 420 mL of distilled water and treated with 1.2 g (2% w / w) of sodium iodide. The mixture was heated at 55-60°C and 1.8 L of acetone was added while keeping the reaction mixture above 45°. The reactants were heated at 65°C for 1 hour, cooled to room temperature and stirred at room temperature for one week. The observed green solid was filtered and washed with acetone to obtain 30.9 g of indocyanine green as a green / red powder with an isolated yield of 52%.

[0263] Purity = 98.60% (HPLC spectrum: ICG 98.60%; impurity E < 0.16%; impurity L < 0.11%, impurity M < 0.17%).

[0264] Sodium content = 7.04%.

[0265] The obtained PXRD pattern is described in Figure 4 In the figure, it is mainly an amorphous product. The figure also shows some very weak and unclear peaks, which can be attributed to a few crystalline forms. However, these peaks are different from the peaks observed for the product obtained in Example 1, and the observed peaks are summarized in Table VI.

[0266] Table VI: Peaks of the PXRD pattern of comparative indocyanine green obtained by the method of Example 3

[0267]

[0268]

[0269] Solid State Stability: Store 5.0 g of indocyanine green powder in 20 mL of amber Type II glass in the dark.

[0270] Table VII: Storage stability of comparative ICG obtained by the method of Example 3 in solid state

[0271] Storage time t0 t = 1 month t = 5 months ICG purity (HPLC) 98.60％ 98.39％ 97.95％ Total impurities (HPLC) 1.40％ 1.61％ 2.05％

[0272] V-Example 4 (comparative):

[0273] The scheme for preparing indocyanine green disclosed in the example of prior art WO9507888A1 was reproduced.

[0274] V-1. Synthesis of indocyanine green:

[0275] In a 0.5 L double jacketed borosilicate reactor equipped with a mechanical stirrer was added 4-(1,1-dimethyl-2-((1E,3E,5E)-6-(N-phenylacetamido)-hexa-1,3,5-trienyl)-1H-benzo[e]indolium-3-yl)-butane-sulfonate (10.0 g, 0.184 mol, 1.00 equiv) and EtOH (58 mL). The mixture was stirred for 5 minutes, and 4-(1,1,2-trimethyl-1H-benzo[e]indolium-3-yl)butane-1-sulfonate (6.36 g, 0.184 mol, 1.00 equiv) and triethylamine (2.6 mL, 0.184 mol, 1.00 equiv) were added. The reaction mixture turned green and was heated at 78° C. for 15 minutes and cooled to 20° C. with stirring. A solution of 1.52 g AcONa (0.184 mol, 1.0 equivalent) in 105 mL EtOH was added, and the mixture was stirred at 20° C. for 30 minutes. The mixture was filtered, washed twice with acetone (50 mL), and dried under reduced pressure (50 mbar) at 40° C. to give 11 g of a red-green solid. The purity of the crude product was controlled by HPLC analysis (typical HPLC spectrum ICG 92.62%; impurity A 4.82%; impurity C 1.07%).

[0276] V-2. Purification of indocyanine green:

[0277] The red-green ICG crude product (11 g) was diluted in 110 mL of acetone, heated to reflux for 30 minutes, cooled at room temperature, filtered, and washed with 20 mL of acetone. This process was repeated 2 times to obtain 10.3 g of purified ICG as a red-green solid (isolated yield 72%).

[0278] Purity = 95.98% (HPLC spectrum: 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 < 0.17%).

[0279] Sodium content = 2.43%.

[0280] The obtained PXRD pattern is as follows Figure 5 As shown, it appears as an amorphous product.

[0281] Solid state stability: 5.0 g of indocyanine green powder was stored in 20 mL of amber type II glass and protected from light. The results are listed in Table VIII.

[0282] Table VIII: Storage stability of comparative ICG obtained by the method of Example 4 in solid state

[0283] Storage time t0 t = 4 months ICG purity (HPLC) 95.98％ 94.11％ Total impurities (HPLC) 4.02％ 5.89％

[0284] VI-Example 5: Comparison of salt-forming reagents

[0285] The procedure of Example 1 was repeated by replacing NaCl with several other salt-forming agents. The salt-forming agents tested were sodium bromide (NaBr), sodium acetate (NaOAc), sodium ascorbate (NaOAsc), sodium ethoxide (NaOEt).

[0286] VI-1. Synthesis of indocyanine green:

[0287] In a 500 mL three-necked round bottom flask were added 4-(1,1-dimethyl-2-((1E,3E,5E)-6-(N-phenylacetamido)-hexa-1,3,5-trienyl)-1H-benzo[e]indolium-3-yl)-butane-sulfonate (5.73 g, 16.59 mmol, 0.90 equiv) and 4-(1,1,2-trimethyl-1H-benzo[e]indolium-3-yl)butane-1-sulfonate (10.0 g, 18.43 mmol, 1.00 equiv). H2O (15.0 mL, 1.5 volumes) and MeOH (25.0 mL, 2.5 volumes) were added to the reaction medium. Et3N (2.6 mL, 18.43 mmol, 1.00 equiv) was added to the mixture and stirred at 25°C for 2 hours and then heated to 50°C. A solution of the appropriate sodium salt (31.33 mmol, 1.70 equiv) in 10 mL of H2O was added while keeping the reaction mixture above 45°C. The mixture was kept at 50°C until the reaction medium was observed to thicken. Acetone (300 mL, 30 vol) was added in portions while keeping the temperature above 40°C, the mixture was kept at 50°C for 1 h30, and then cooled to 25°C. The solid was filtered on sintered glass (POR 3), washed with 3x50 mL (5 vol) of acetone, and dried under vacuum at 40°C for 16 h to give crude indocyanine green as a green powder.

[0288] VI-2. Purification of indocyanine green:

[0289] In a 500 mL three-necked round bottom flask, crude indocyanine green (12 g) was charged into 84 mL (7 volumes) of H2O and the mixture was warmed to 50°C. While maintaining the temperature above 40°C, 360 mL of acetone (30 volumes) were added in portions. The mixture was stirred at 50°C for 30 minutes. Stirring was stopped and the mixture was cooled to room temperature overnight. The mixture was then cooled to 0-5°C (ice bath) for 2 hours. The mixture was filtered on sintered glass (POR 3), washed with 3×60 mL of acetone (5 volumes) and dried under vacuum at 50°C. The results are listed in Table IX.

[0290] Table IX: Storage stability of solid ICG obtained by different salt-forming agents

[0291]

[0292] These results indicate that replacing NaCl in step d) of the process of the present invention with other organic or inorganic salts, such as those commonly used in the art, results in ICG that is not storage stable.

[0293] VII-Example 6: Solubility Test

[0294] Solubility tests were performed using ICG obtained by the method of the present invention (Example 1) and ICG obtained by a similar method of changing the salt-forming agent (Example 5). The obtained ICG (250 mg), sodium ascorbate (100 mg) and water for injection were added to a 100 mL round-bottom flask. The mixture was stirred at room temperature for 5 h 20 under N2 atmosphere. The solution was filtered on a 0.22 μm PA filter. The results are listed in Table X.

[0295] Table X: Solubility of ICG obtained by different salt-forming reagents

[0296] Salt-forming reagent Solubility NaCl (Example 1) Complete dissolution, no trace of solids remaining on the membrane Sodium Methanol Partially dissolved, traces of undissolved solids remain on the membrane Sodium acetate Partially dissolved, traces of undissolved solids remain on the membrane Sodium ethoxide Partially dissolved, traces of undissolved solids remain on the membrane

[0297] These results indicate that replacing NaCl in step d) of the process of the present invention with other organic salts, such as those commonly used in the art, yields ICG that is not completely soluble in aqueous solution.

Claims

1. Indocyanine green in crystalline form, characterized in that The powder X-ray diffraction pattern exhibits peaks at least at 20.66, 14.38, 12.81, 4.22 and 3.83 degrees 2θ ± 0.2 degrees 2θ.

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

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

4. Indocyanine green according to any one of claims 1 to 3, having a purity of at least 98.0%, preferably at least 99.0%, more preferably at least 99.5%, as measured by HPLC as % area at 240 nm.

5. Indocyanine green according to any one of claims 1 to 4, wherein each impurity measured by HPLC as % area at 240 nm is less than 0.50%, preferably less than 0.40%, more preferably less than 0.20%, most preferably less than 0.15%.

6. Indocyanine green according to any one of claims 1 to 5, wherein the crystalline form has the appearance of reddish crystals.

7. A pharmaceutical composition comprising the indocyanine green according to any one of claims 1 to 6 and a pharmaceutically acceptable carrier.

8. The pharmaceutical composition of claim 7, wherein the pharmaceutically 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), ethanol, methanol, isopropanol, n-propanol, butanol, glycerol, propylene glycol and polyethylene glycol and mixtures thereof, preferably water for injection.

9. A pharmaceutical composition as claimed in claim 7 or claim 8, further comprising at least one compound selected from histidine, ethylenediaminetetraacetic acid (EDTA) and its salts, cysteine, sodium chloride, dithiothreitol, ascorbic acid, sodium ascorbate, disodium hydrogen phosphate, sodium dihydrogen phosphate and mixtures thereof.

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

11. The pharmaceutical composition of claim 9, comprising histidine.

12. The pharmaceutical composition of claim 9, comprising histidine, ethylenediaminetetraacetic acid (EDTA) and / or its salt and sodium chloride.

13. Indocyanine green according to any one of claims 1 to 6 or a pharmaceutical composition according to any one of claims 7 to 12 for use as a medicament or a diagnostic agent.

14. The indocyanine green or the pharmaceutical composition for use as claimed in claim 13, for obtaining angiographic images of patient tissues, for determining cardiac output, for determining liver function and liver blood flow, for detecting sentinel lymph nodes, for assessing and / or predicting flap viability, for diagnosing and treating age-related macular degeneration, and / or for diagnosing and treating choroidal neovascularization and tumors.

15. A method for preparing the indocyanine green according to any one of claims 1 to 6, characterized in that: It comprises at least the step d) of converting 4-[2-[7-[1,1-dimethyl-3-(4-sulfobutyl)benz[e]indol-3-ium-2-yl]hept-2,4,6-trienyl]-1,1-dimethylbenz[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'-dibenzoindoletricarbonylcyanine sodium salt of formula (VII) by treating with sodium chloride:

16. The method for preparing indocyanine green according to claim 15, further comprising a step e) of purifying the indocyanine green after step d), wherein the purification step e) comprises at least: e1) suspending, dispersing or dissolving indocyanine green of formula (VII) in a solvent, e2) heating the composition obtained by e1), e3) cooling the solution obtained in e2), e4) recovering indocyanine green in crystalline form.

17. The method for preparing indocyanine green according to claim 16, wherein: The solvent in step e1) is selected from water, acetone, methanol, ethanol, isopropanol, n-propanol, n-butanol and mixtures thereof.

18. The method of claim 16 or claim 17, wherein the composition in step e2) is heated to a temperature of 40 to 80°C, preferably 40 to 65°C, and the solution in step e3) is cooled to a temperature of 10 to 25°C.

19. The method for preparing indocyanine green according to any one of claims 16 to 18, wherein the cooling in step e3) is performed without stirring or agitation.

20. A method for preparing indocyanine green, characterized in that: It comprises at least the step d) of converting 4-[2-[7-[1,1-dimethyl-3-(4-sulfobutyl)benz[e]indol-3-ium-2-yl]hept-2,4,6-trienyl]-1,1-dimethylbenz[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'-dibenzoindoletricarbonylcyanine sodium salt of formula (VII) by treating with sodium chloride:

21. The method for preparing indocyanine green according to claim 20, further comprising a step e) of purifying the indocyanine green after step d), wherein the purification step e) comprises at least: It further comprises a step e) of purifying the indocyanine green after step d), wherein the purification step e) comprises at least: e1) suspending, dispersing or dissolving indocyanine green of formula (VII) in a solvent, e2) heating the composition obtained by e1), e3) cooling the solution obtained in e2), e4) recovering indocyanine green in crystalline form.

22. The method for preparing indocyanine green according to claim 21, wherein: The solvent in step e1) is selected from water, acetone, methanol, ethanol, isopropanol, n-propanol, n-butanol and mixtures thereof.

23. The method of claim 21 or claim 22, wherein the composition in step e2) is heated to a temperature of 40 to 80°C, preferably 40 to 65°C, and the solution in step e3) is cooled to a temperature of 10 to 25°C.

24. The method for preparing indocyanine green according to any one of claims 21 to 23, wherein the cooling in step e3) is performed without stirring or agitation.

25. Indocyanine green obtained by the method of any one of claims 20 to 24.

Images