Stable formulations of indocyanine green

Abstract

The present invention relates to an indocyanine green composition having improved stability, particularly improved storage stability. In particular, the present invention relates to an ICG composition comprising indocyanine green and histidine.

Description

[Technical field]

[0001] The present invention relates to the medical and pharmaceutical technical fields. The present invention relates to indocyanine green compositions having improved stability, especially improved storage stability. In particular, the present invention relates to indocyanine green compositions containing histidine. The present invention further relates to methods of making and using said compositions. [Background technology]

[0002] Indocyanine green (ICG) is a water-soluble tricarbocyanine fluorescent dye approved by the U.S. Food and Drug Administration for medical diagnostic use to assess liver function, cardiac output, hepatic blood flow, microcirculation in skin flaps, and in ophthalmic angiography. ICG is used for such medical applications because of its high absorbance in the spectral range (750-800 nm) through which human tissue is relatively transparent. Its extremely low toxicity also makes such medical applications possible. After intravenous administration, ICG is rapidly excreted in the bile by the liver and has an effective half-life of approximately 3-4 minutes, depending on liver function.

[0003] The above applications require that ICG be administered in the form of an aqueous solution. ICG is commercially available as a lyophilized powder that can be dissolved in sterile water and injected, often in kits. However, ICG has poor stability in aqueous environments and degrades rapidly, limiting its usefulness in some applications, such as time-sensitive surgical procedures. ICG package inserts vary among vendors, with some stating that the reconstituted ICG solution should be used immediately, others within 10 hours, and others that any unused portion should be discarded.

[0004] Studies have shown that ICG molecules in aqueous media undergo physicochemical changes, such as aggregation, especially at high concentrations. In water, ICG easily forms dimers and oligomers, whereas in human plasma, methanol, and dimethyl sulfoxide (DMSO), it maintains a monomeric state (Non-Patent Document 1). ICG also generates singlet oxygen to form dioxetanes, which can be thermally decomposed to multiple carbonyl compounds (Non-Patent Document 2). These decomposition products can reduce cell viability in vitro.

[0005] Physicochemical changes in ICG are more likely to occur when exposed to light, high temperature, the presence of oxygen, and even depending on the concentration of ICG itself (Non-Patent Document 3, Non-Patent Document 4, and Non-Patent Document 5). The type and intensity of light irradiated to ICG also affect degradation. Physicochemical changes rapidly deteriorate the optical properties of ICG, causing discoloration, a decrease in fluorescence intensity, and a shift in absorbance peak. It has been reported that when ICG is stored in water under direct light for 10 hours, the absorbance of the spectral peak decreases by 10% (Non-Patent Document 4).

[0006] To overcome the above shortcomings, many attempts have been made to improve the stability of indocyanine green in aqueous solutions.

[0007] Engel et al. have reported that sodium azide (NaN 3 It has been reported that the addition of can suppress the chemical decomposition of ICG (Non-Patent Document 2).

[0008] Mindt et al. investigated the stability of ICG samples when stored in the dark and in clear vials at various temperatures. Absorbance and fluorescence measurements showed that ICG was stable for 3 days when stored in the dark at 4°C (although 20% of the fluorescence intensity was lost during this period). Based on these studies, it has been proposed that storing reconstituted ICG solutions in the dark at 4°C may allow the use of ICG for periods longer than 1 day (Non-Patent Document 6).

[0009] Other researchers have proposed to encapsulate ICG in nanometer-sized carriers to prevent the ICG molecules from decomposing. For example, CN103301482 (Patent Document 1) discloses a three-layer polypeptide-type ICG-loaded micelle consisting of a polyleucine core, a polylysine intermediate layer, and a polyethylene glycol shell, with indocyanine green dispersed in the core. The indocyanine green is encapsulated in the core-shell structure, which effectively prevents the aggregation of ICG molecules, thereby improving the stability of ICG.

[0010] Kirchherr et al. have disclosed the encapsulation of ICG in a micellar system of Solutol HS15 (polyoxyethylene esters of 12-hydroxystearic acid). The formulation showed reduced aggregation behavior compared to free ICG in aqueous solution and high stability in aqueous solution over a period of 4 weeks (Non-Patent Document 7).

[0011] US 6,944,493 (Patent Document 2) discloses an indocyanine green composition with a high indocyanine green concentration and improved stability. The invention is based on an indocyanine green liposome formulation that is stable for more than 24 hours after reconstitution. All formulations disclosed in the document are reconstituted with a diluent containing ethanol, polysorbate 80, etc.

[0012] WO2016 / 123864 (Patent Document 3) discloses a composition in which ICG is embedded in the lipid membrane of lipid nanoparticles. The fluorescence intensity of the nanoparticles is 4 to 5 times that of free ICG. After storage at 4°C for 0 to 300 days, the nanoparticles have a fluorescence intensity 4 to 100,000 times that of free ICG in an aqueous solution.

[0013] Dedora et al. have described complexation of ICG with highly soluble methyl β-CyD and FDA-approved sulfobutyl ether β-CyD (Captisol®) in aqueous solution. Such complexes reduce ICG aggregation and provide sustained fluorescence enhancement over 24 hours that is more than two-fold higher than free ICG in water. However, ICG complexed with methyl β-CyD has been shown to severely reduce the viability of some fibroblast cells (8).

[0014] The encapsulation or complexation of ICG using the above-mentioned systems may have disadvantages such as poor compatibility with cells or may reduce the ability of ICG molecules to interact with plasma proteins after entering the blood, which may result in a prolonged excretion kinetics from the vascular system.

[0015] US 2010 / 0181535 (Patent Document 4) discloses a method for enhancing the fluorescence intensity of a fluorescent dye by mixing the fluorescent dye with a redox buffer. The fluorescent dye may be selected from rhodamines including indocyanine green and / or carborhodamines and / or oxazine dyes and / or cyanine dyes. The redox buffer may contain carotenoids (particularly tocopherols), thiols (particularly glutathione, N-acetyl-cysteine ​​and dihydrolipoic acid), amino acids (particularly tryptophan and / or tyrosine and / or histidine and / or cysteine ​​and / or methionine) and / or peptides or proteins containing them. The authors of this document showed that the use of ascorbic acid or 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox®) and methyl viologen can extend the duration of the dark state of a cyanine dye when irradiated with a laser signal by a fluorescence detection device. The authors also cite an additional advantage of using the redox buffer described in this document, which is that the shelf life of a fluorescent dye solution containing the redox buffer can be increased by 10% to 20% compared to a solution without the redox buffer. However, the document does not describe the long-term storage stability of this composition.

[0016] WO2020 / 240514 (Patent Document 5) discloses an indocyanine green formulation containing disodium hydrogen phosphate, sodium dihydrogen phosphate, and sodium chloride. The composition may optionally contain other pharma- ceutically acceptable excipients such as sodium chloride, citrate, sodium carbonate, sodium bicarbonate, tartrate, benzoate, acetate, boric acid, lactic acid, glutaric acid, malic acid, succinic acid, carbonic acid, alkali or alkaline earth salts of these acids, and buffers of amino acids such as arginine, alanine, histidine, glycine, and lysine. The solution is stable for up to 24 hours after reconstitution with an aqueous diluent.

[0017] Although the above prior arts all report improved stability of ICG in aqueous solution, they do not provide an aqueous solution of ICG that is stable for more than 1-3 days. Thus, there is a demand for an aqueous composition of ICG that exhibits greater stability than the prior art formulations.

[0018] Some of the prior art formulations are prepared with expensive equipment and / or require long manufacturing process times and / or require special excipients, and some of these formulations contain excipients such as alcohol and polysorbates that make the injection of the ICG composition painful and difficult. [Prior art documents] [Patent documents]

[0019] [Patent Document 1] Chinese Patent No. 103301482 [Patent Document 2] U.S. Patent No. 6,944,493 [Patent Document 3] International Publication No. 2016 / 123864 [Patent Document 4] US Patent Application Publication No. 2010 / 181535 [Patent Document 5] International Publication No. 2020 / 240514 [Non-patent literature]

[0020] [Non-Patent Document 1] Holzer W, Mauerer M, Penzkofer A, Szeimies RM, Abels C, Landthaler M, Baumler W. Photostability and thermal stability of indocyanine green. J Photochem Photobiol B. 1998 Dec; 47(2-3):155-64. [Non-Patent Document 2] Engel E, Schraml R, Maisch T, Kobuch K, Konig B, Szeimies RM, Hillenkamp J, Baumler W, Vasold R. Light-induced decomposition of indocyanine green. Invest Ophthalmol Vis Sci. 2008 May; 49(5):1777-83. [Non-Patent Document 3] Saxena V, Sadoqi M, Shao J. Degradation kinetics of indocyanine green in aqueous solution. J Pharm Sci. 2003 Oct; 92(10):2090-7. [Non-Patent Document 4] Gathje J, Steuer RR, Nicholes KR. Stability studies on indocyanine green dye. J Appl Physiol. 1970 Aug; 29(2):181-5. [Non-Patent Document 5] Barbier f, Deweerdt ga. Chromatography and IR spectrography of indocyanine green. Clin Chim Acta. 1964 Dec; 10:549-54. [Non-Patent Document 6] Mindt S, Karampinis I, John M, Neumaier M, Nowak K. Stability and degradation of indocyanine green in plasma, aqueous solution and whole blood. Photochem Photobiol Sci. 2018 Sep 12; 17(9):1189-1196. [Non-Patent Document 7] Kirchherr AK, Briel A, Mader K. Stabilization of indocyanine green by encapsulation within micellar systems. Mol Pharm. 2009 Mar-Apr;6(2):480-91. [Non-Patent Document 8] DeDora DJ, Suhrland C, Goenka S, Mullick Chowdhury S, Lalwani G, Mujica-Parodi LR, Sitharaman B. Sulfobutyl ether β-cyclodextrin (Captisol((R)) ) and methyl β-cyclodextrin enhance and stabilize fluorescence of aqueous indocyanine green. J Biomed Mater Res B Appl Biomater. 2016 Oct; 104(7):1457-64. Summary of the Invention [Problem to be solved by the invention]

[0021] In particular, there is a need for aqueous compositions of ICG that are stable at room temperature for periods of one or several months.

[0022] There is also a demand for an ICG composition for intravenous administration that is compatible with target tissues, is non-toxic, and is rapidly metabolically eliminated.

[0023] Another object of the present invention is to provide an ICG composition which is simple to formulate, has readily available raw materials, and has a sufficient ICG concentration for use in applications similar to those of prior art ICG formulations which have a short shelf life.

[0024] It has surprisingly been found that formulations of ICG containing histidine exhibit improved stability compared to prior art compositions.

[0025] The formulations of the present invention contain ICG at concentrations comparable to or greater than those of the prior art. They can be formulated as lyophilized powders or aqueous solutions. They have improved storage stability and shelf life compared to the prior art.

[0026] Another object of the present invention was to provide a formulation which is stable, cost effective and easy to manufacture. Yet another object of the present invention was to provide a formulation which does not cause discomfort to the patient upon administration. [Means for solving the problem]

[0027] The present invention relates to a composition comprising indocyanine green (a) and histidine (b), wherein the amount of indocyanine green content in the composition is reduced by 10% or less when stored at room temperature for one month or more, as measured by HPLC area ratio (%) at 240 nm.

[0028] Advantageously, the content of ICG (Indocyanine Green) in the composition of the invention before storage, measured as HPLC area percentage (%) at 240 nm, is greater than or equal to 90%.

[0029] Preferably, the total amount of impurities in the ICG composition before storage, measured as an area percentage (%) at 240 nm by HPLC, is 10% or less.

[0030] Preferably, the increase in the amount of impurities in the composition of the present invention when stored at room temperature for one month or more, as measured as a change in area ratio (%) at 240 nm by HPLC, is 10% or less.

[0031] The present invention also relates to a composition comprising indocyanine green (a) and histidine (b), wherein the weight ratio of histidine (b) / indocyanine green (a) is greater than 0.05.

[0032] The present invention also relates to a kit comprising, in separate compartments, at least 1) a composition comprising indocyanine green (a) and histidine (b), wherein the weight ratio of histidine (b) / indocyanine green (a) is greater than 0.05, and 2) an aqueous diluent.

[0033] The present invention also relates to a kit comprising, in separate compartments, an aqueous solution comprising at least 1) indocyanine green (a) and 2) at least histidine (b), wherein the weight ratio of histidine (b) / indocyanine green (a) in the kit is greater than 0.05.

[0034] The present invention also relates to a composition comprising indocyanine green (a) and histidine (b), the weight ratio of histidine (b) / indocyanine green (a) being greater than 0.05, for use as a pharmaceutical or diagnostic agent.

[0035] The present invention also relates to a method of diagnosis and / or treatment in a patient comprising the administration, in particular by intravenous route, of an effective amount of a composition comprising indocyanine green (a) and histidine (b), wherein the weight ratio of histidine (b) / indocyanine green (a) is greater than 0.05.

[0036] Advantageously, the histidine (b) / indocyanine green (a) weight ratio ranges from 0.05 to 50.

[0037] According to one embodiment, the weight ratio of histidine (b) / indocyanine green (a) is in the range of 0.05-50, preferably 0.05-40, more preferably 0.10-30, and even more preferably 0.10-20.

[0038] Advantageously, the indocyanine green composition according to the invention further comprises at least one compound (c) chosen from ethylenediaminetetraacetic acid (EDTA) or its salts, cysteine, sodium chloride, dithiothreitol (DTT) and mixtures thereof.

[0039] Preferably, the weight ratio of compound (c) to indocyanine green (a) ranges from 1:500 to 100:1, preferably from 1:100 to 10:1, more preferably from 1:100 to 4:1.

[0040] Preferably, the weight ratio of compound (c) to histidine (b) ranges from 1:100 to 100:1, preferably from 1:10 to 10:1.

[0041] In one embodiment, the composition further comprises up to 5% by weight of sodium iodide relative to the weight of indocyanine green.

[0042] In one preferred embodiment, the indocyanine green composition according to the present invention is an aqueous composition.

[0043] In a further preferred embodiment, the indocyanine green composition according to the present invention is an aqueous composition filled in a vial or ampoule.

[0044] Advantageously, the concentration of indocyanine green in said aqueous composition is between 0.1 and 50 mg / ml, preferably between 0.1 and 30 mg / ml.

[0045] According to one embodiment, the aqueous composition is reconstituted from a kit comprising at least 1) a composition comprising indocyanine green (a) and histidine (b), wherein the weight ratio of histidine (b) / indocyanine green (a) is greater than 0.05, and 2) an aqueous diluent in separate compartments.

[0046] According to another embodiment, the aqueous composition is reconstituted from a kit comprising in separate compartments an aqueous solution comprising at least 1) indocyanine green (a) and 2) histidine (b), wherein the weight ratio of histidine (b) / indocyanine green (a) is greater than 0.05.

[0047] Advantageously, the concentration of indocyanine green in the reconstituted aqueous composition from any of the kits described above and detailed herein is comprised between 0.1 and 50 mg / ml, preferably between 0.1 and 30 mg / ml.

[0048] In other embodiments, the composition is in the form of a lyophilized powder. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0049] When one or more components or processes / steps are listed in the "..." portion of the phrase "consisting essentially of...", this means that the method or material of the invention may contain other components or processes / steps in addition to the specified components or processes / steps, which do not significantly affect the characteristics or properties of the invention.

[0050] The term "X to Y" is inclusive unless otherwise specified. It means that the range includes all values ​​from X to Y in addition to the values ​​X and Y themselves.

[0051] Reference herein to any method of treatment should be interpreted as a description of the use of the compounds, pharmaceutical compositions and medicaments of the invention in therapeutic or diagnostic methods of treatment of the human (or animal) body.

[0052] (Stability of the indocyanine green composition according to the present invention) The present invention aims to provide indocyanine green compositions that have improved stability compared to prior art compositions.

[0053] The terms "stable composition" and "stability" refer to a composition in which the content of indocyanine green remains stable even after a certain period of storage (e.g., 1 week, 1 month, 2 months, 6 months, 1 year, 2 years, etc.).

[0054] The stability of the composition is evaluated by measuring the change in indocyanine green content in the composition after long-term storage.

[0055] Preferably, the compositions of the present invention are advantageous in that they exhibit substantially no signs of aggregation, decomposition, or precipitation upon extended storage, and substantially no or minimal formation of impurities due to chemical modification of the indocyanine green. The compositions of the present invention are also advantageous in that they do not experience significant discoloration or significant loss in fluorescence intensity or peak absorbance after extended storage.

[0056] In particular, the present invention provides compositions containing indocyanine green that have improved stability compared to currently available formulations of indocyanine green.

[0057] Advantageously, the compositions of the present invention are at least 10 times more stable than currently approved formulations of indocyanine green, more preferably at least 30 times more stable, more preferably at least 50 times more stable, and even more preferably up to 100 times more stable.

[0058] In one embodiment, the composition of the present invention is stable when stored at room temperature for at least 1 month, preferably at least 3 months, more preferably at least 6 months, even more preferably at least 12 months, and most preferably at least 2 years.

[0059] "Room temperature" means room temperature controlled at 20 to 25°C (15 to 30°C with some variation) in the United States Pharmacopoeia, and 15 to 25°C in the European Pharmacopoeia.

[0060] Preferably, the composition of the present invention is stable at room temperature for 1 month to 3 years, preferably 3 months to 3 years, more preferably 1 year to 3 years, and even more preferably 1 year to 2 years.

[0061] Advantageously, the compositions according to the invention are stable for at least 1 month, preferably at least 3 months, more preferably at least 6 months and most preferably at least 2 years when stored in a container that protects against natural light and / or ultraviolet light and / or fluorescent light.

[0062] A container capable of protecting the composition from natural light and / or ultraviolet light and / or fluorescent light is one having walls with a maximum light transmittance of at least 50% at any wavelength. Typically, the container can be an opaque or amber colored container and / or an opaque secondary packaging member (e.g., carton, overwrap, etc.).

[0063] Advantageously, the compositions according to the invention are stable for at least 1 month, preferably at least 3 months, more preferably at least 6 months, and most preferably at least 2 years when stored in a sealed or airtight container.

[0064] The stability of the compositions of the present invention can be assessed visually for color and / or clarity and / or by other analytical techniques.

[0065] Analytical techniques used to assess the stability of the compositions are known to those of skill in the art and include nuclear magnetic resonance (NMR), high performance liquid chromatography (HPLC), size exclusion chromatography (SEC), liquid chromatography mass spectrometry (LC-MS), dynamic light scattering (DLS), differential scanning calorimetry (DSC), UV spectroscopy, Fourier transform infrared spectroscopy (FTIR), or a combination of these techniques.

[0066] Advantageously, the stability of the composition is evaluated by measuring the change in indocyanine green content in the composition before and after long-term storage by HPLC. More advantageously, the ICG content in the composition and the change in said content are evaluated by HPLC area percentage at 240 nm.

[0067] Preferably, the stability of the composition of the present invention corresponds to a decrease in the content of indocyanine green in the composition of 10% or less when the composition is stored at room temperature for one month or more, preferably three months, more preferably six months, even more preferably twelve months, and most preferably two years or more, as measured by HPLC area ratio (%) at 240 nm.

[0068] Preferably, the stability of the composition of the present invention is evidenced by a decrease in the amount of indocyanine green content in the composition, as measured by HPLC area ratio (%) at 240 nm, of 5% or less when the composition is stored at room temperature for one month or more, preferably three months, more preferably six months, even more preferably twelve months, and most preferably two years or more.

[0069] More preferably, the stability of the composition of the present invention is evidenced by a decrease in the amount of indocyanine green content in the composition, as measured by HPLC area ratio (%) at 240 nm, of 2% or less when the composition is stored at room temperature for one month or more, preferably three months, more preferably six months, even more preferably twelve months, and most preferably two years or more.

[0070] "Reduction in content" in the sense of the present application is to be understood as the difference (%) between the measured content of ICG in the composition before storage, measured as an area percentage (%) by HPLC at 240 nm, and the measured content of ICG in the same composition after a given storage period, measured as an area percentage (%) by HPLC at 240 nm.

[0071] Preferably, the content of ICG (indocyanine green) in the composition before storage, measured as an area percentage (%) at 240 nm by HPLC, is 90% or more, preferably 95% or more, more preferably 97% or more, and most preferably 99% or more.

[0072] The stability of the indocyanine green composition of the present invention may also correspond to the total amount of impurities contained in the composition of the present invention not increasing during storage, in particular not increasing to an extent that reduces the ability of the indocyanine green composition to be used in medical and / or diagnostic applications.

[0073] For example, when the composition is stored at room temperature for more than one month and then the impurities are measured by HPLC, only small amounts of impurities are found in the composition, and the amount does not increase significantly over time.

[0074] In the context of the present invention, the term "impurities" should be understood to refer to chemicals that are neither indocyanine green nor excipients or other additives to the indocyanine green composition of the present invention.

[0075] The impurities may be process-related impurities, such as by-products or intermediates that may be formed during the manufacture of the indocyanine green, or decomposition-related impurities that arise due to chemical changes in the indocyanine green during storage, such as products that may be formed during storage due to exposure to light, temperature, humidity, reactions with oxygen, etc.

[0076] In one embodiment, when the composition of the present invention is stored at room temperature for one month or more, preferably two months, more preferably six months, and most preferably two years or more, the increase in the amount of impurities in the composition, as measured as a change in area ratio (%) at 240 nm by HPLC, is 10% or less, preferably 5% or less, and more preferably 2% or less.

[0077] In one embodiment, the total amount of impurities in the ICG composition before storage, measured as HPLC area percentage (%) at 240 nm, is 10% or less, preferably 5% or less, more preferably 1% or less.

[0078] The term "total amount of impurities" includes both process-related impurities and degradation-related impurities as discussed above.

[0079] (Indocyanine Green Composition) The present invention provides a composition comprising indocyanine green (a) and histidine (b).

[0080] Surprisingly, the inventors have found that histidine is capable of stabilizing compositions of ICG in aqueous diluents and / or avoiding and / or reducing and / or preventing degradation and / or improving and / or increasing storage stability.

[0081] Surprisingly, the inventors have found that association of histidine with several other compounds can enhance the stabilizing effect of histidine on ICG in aqueous diluents.

[0082] Advantageously, the composition according to the invention may comprise at least one co-stabilising compound (c).

[0083] (Indocyanine Green (a)) The term indocyanine green refers to indocyanine green and its pharma- ceutically acceptable salts, solvates, hydrates, acid, anhydrate and free base forms, preferably indocyanine green.

[0084] [ka]

[0085] Indocyanine green can be prepared by any method known to those skilled in the art. For example, indocyanine green can be prepared according to the methods described in WO95 / 07888 and WO2017093889. Preferably, the preparation of indocyanine green includes a purification step of the obtained product.

[0086] Advantageously, the indocyanine green used in the composition according to the invention is substantially pure, and is preferably provided as a composition having a purity of more than 90.0%, preferably more than 95.0%, more preferably more than 97.0%, even more preferably more than 99.7%, as measured by HPLC.

[0087] As used herein, the term "purity" refers to the degree to which the raw indocyanine green is free from unwanted or degrading chemicals.

[0088] In the present invention, the purity and impurities of the indocyanine green substance are evaluated by area ratio (%) at 240 nm by HPLC.

[0089] Preferably, the indocyanine green used in the compositions of the present invention is provided in a composition having less than 5.0% impurities, preferably less than 1.0%, more preferably less than 0.5% impurities as measured by HPLC.

[0090] Preferably, the indocyanine green used in the compositions of the present invention is provided in a composition having less than 0.50% of any impurity, preferably less than 0.40%, more preferably less than 0.20%, and most preferably less than 0.15%, as measured by HPLC.

[0091] The impurities contained in the indocyanine green may be any reaction by-products or intermediates generated during the manufacturing process. For example, as described in US2019 / 0337896, 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, 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, etc.

[0092] Preferably, the indocyanine green is provided as a composition having a metal contaminant content of 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 of metal based on the weight of indocyanine green. The metal contaminant content in the indocyanine green can be measured by inductively coupled plasma mass spectrometry (ICP-MS).

[0093] "Metal contaminants" is 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.

[0094] More preferably, the indocyanine green is free of lead and arsenic or contains less than 2 ppm of lead and arsenic.

[0095] The indocyanine green used in the composition of the present invention is provided as a composition which may further contain sodium iodide, an additive which is often present in commercially available indocyanine green compositions. When present, the iodide content is preferably kept below 5% by weight based on the weight of indocyanine green. The sodium iodide content in the indocyanine green can be measured by potentiometry.

[0096] The indocyanine green used in the compositions of the present invention may be provided in any suitable form, such as a lyophilisate or crystalline powder.

[0097] (Histidine (b)) By "histidine" is meant for the purposes of the present invention the L or D-enantiomer of the amino acid histidine. Advantageously, histidine means the L-enantiomer of the amino acid histidine.

[0098] The present inventors have surprisingly found that association of indocyanine green (a) with histidine (b) improves the stability of the indocyanine green composition in an aqueous medium.

[0099] In a preferred embodiment, the weight ratio of histidine (b) to indocyanine green (a) is greater than 0.05. Advantageously, the weight ratio of histidine (b) to indocyanine green (a) ranges from 0.05 to 50, preferably from 0.10 to 40, more preferably from 0.10 to 30, and even more preferably from 0.10 to 20. It has been observed that if the weight ratio of histidine (b) to indocyanine green (a) is too high, histidine precipitates during storage.

[0100] In a preferred embodiment of the invention, the composition consists essentially of indocyanine green (a) and histidine (b).

[0101] In another preferred embodiment of the invention, the composition consists essentially of indocyanine green (a), histidine (b) and iodide.

[0102] According to another aspect, the present invention relates to the use of histidine for improving the storage stability of an ICG composition, wherein the weight ratio of histidine (b) to indocyanine green (a) in the composition is greater than 0.05, preferably between 0.05 and 50, preferably between 0.10 and 40, more preferably between 0.10 and 30, even more preferably between 0.10 and 20.

[0103] (Co-stabilizing compound (c)) In another embodiment of the invention, the composition may further comprise one or more compounds (c).

[0104] Advantageously, compound (c) may be chosen from ethylenediaminetetraacetic acid (EDTA) and its salts, cysteine, sodium chloride, dithiothreitol (DTT) and mixtures thereof.

[0105] By "mixtures thereof" is meant any mixture of two or more of the above compounds in any proportion.

[0106] Salts of ethylenediaminetetraacetic acid include, but are not limited to, disodium EDTA, calcium sodium EDTA, tetrasodium EDTA, dicalcium EDTA and mixtures thereof. In a preferred embodiment, the salt of EDTA is selected from tetrasodium EDTA and calcium sodium EDTA or mixtures thereof. These salts of ethylenediaminetetraacetic acid may also be in the form of hydrated salts.

[0107] By "cysteine" is meant for the purposes of the present invention the L- or D-enantiomer of the amino acid cysteine. Advantageously, cysteine ​​means the L-enantiomer of the amino acid cysteine.

[0108] The present inventors have found that by associating compound (b) and compound (c) with indocyanine green (a), the stability of the indocyanine green composition in an aqueous medium is improved.

[0109] Preferably, the weight ratio of compound (c) to indocyanine green (a) ranges from 1:500 to 100:1, preferably from 1:500 to 10:1, more preferably from 1:100 to 10:1, most preferably from 1:100 to 4:1.

[0110] Preferably, the weight ratio of compound (c) to compound (b) is in the range of 1:100 to 100:1, preferably 1:10 to 10:1.

[0111] The co-stabilizing compound may be provided in any form, such as a solid, liquid, or semi-solid, and may be in the form of an aqueous solution.

[0112] In a preferred embodiment of the invention, the composition consists essentially of indocyanine green (a), histidine (b), and a co-stabilizing compound (c) selected from ethylenediaminetetraacetic acid (EDTA) and its salts, cysteine, sodium chloride, dithiothreitol (DTT), and mixtures thereof.

[0113] In another preferred embodiment of the invention, the composition consists essentially of indocyanine green (a), histidine (b), iodide, and a co-stabilizing compound (c) selected from ethylenediaminetetraacetic acid (EDTA) and its salts, cysteine, sodium chloride, dithiothreitol (DTT), and mixtures thereof.

[0114] (Other additives) The composition of the present invention may further contain one or more pharma- ceutically acceptable excipients.

[0115] As used herein, the term "pharmacologically acceptable" refers to compounds, substances and compositions that are suitable for use in contact with the tissues of humans or animals without excessive toxicity, irritation or other problems or complications commensurate with a reasonable benefit-risk ratio.

[0116] The term "additives" includes components present in the composition other than components (a), (b) and (c) as defined above or sodium iodide. Examples of such additives include buffering agents, pH adjusting agents, tonicity adjusting agents, surfactants, preservatives, tonicifying agents, antimicrobial agents, wetting agents, emulsifying agents, etc.

[0117] For example, the compositions of the present invention may contain one or more additives selected from the group including: sodium hydroxide; potassium hydroxide; methylparaben; propylparaben; sodium acetate; sodium citrate; sodium carbonate; ammonium carbonate; sodium bicarbonate; benzoates or benzoates; acetates or acetates; boric acid; lactic acid; glutaric acid; malic acid; succinic acid; carbonic acid; alkali or alkaline earth salts of these acids; meglumine; hydrochloric acid; cyclodextrin derivatives; sorbitan monolaurate; triethanolamine acetate; triethanolamine oleate; citric acid; lactic acid; phosphoric acid; sulfuric acid; arginine; alanine; glycine; and lysine.

[0118] These additives are commonly available to one of ordinary skill in the art and may be in any form, such as solid, liquid, or semi-solid.

[0119] Preferably, the amount of additives in the composition of the present invention is within a range that does not significantly adversely affect the activity of the indocyanine green. Preferably, the composition of the present invention contains the minimum number and amount of additives necessary to provide a stable and effective composition.

[0120] (Form of the composition according to the present invention) The compositions of the present invention may be provided in any suitable form, but are preferably provided in the form of an aqueous composition or a lyophilized solid composition.

[0121] (aqueous composition) In a first embodiment, the composition according to the invention is in the form of an aqueous composition.

[0122] An "aqueous composition" is to be understood as meaning an aqueous solution, aqueous suspension, colloidal aqueous suspension, aqueous dispersion, preferably an aqueous solution.

[0123] The term "aqueous solution" refers to the aforementioned indocyanine green composition in which the components are dissolved in an aqueous solvent.

[0124] Advantageously, according to this variant, the composition comprises an aqueous diluent, indocyanine green (a), histidine (b), optionally one or more compounds (c) as defined above, and optionally one or more pharma- ceutically acceptable excipients.

[0125] The aqueous diluent is a pharma- ceutically acceptable solvent, safe and non-toxic for administration to humans or animals, and useful for preparing pharmaceutical formulations.

[0126] The aqueous diluent comprises water and may comprise one or more pharma- ceutically acceptable additives. In some variations of the methods disclosed herein, histidine (b) as defined above, and optionally one or more compounds (c) as defined above, are introduced into the aqueous diluent before mixing with ICG. In such embodiments, histidine (b) and optionally one or more compounds (c) can be considered as part of the aqueous diluent.

[0127] The suitable aqueous diluent may be any liquid that is biologically acceptable and in which the composition of the present invention can be completely dissolved. Water, particularly sterile water for injection (SWFI) and / or bacteriostatic water for injection (BWFI), is the preferred diluent since it does not contain salts or other compounds that may affect the stability of the ICG. However, other types of aqueous diluents may be used, such as sterile saline, Ringer's solution, dextrose solution, glucose solution, etc.

[0128] In one embodiment, the aqueous 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.

[0129] A person skilled in the art can select the diluent depending on the desired application and in relation to the other compounds of the composition, and can also adjust the amount of the excipient depending on its solubility in the aqueous solution.

[0130] In a preferred embodiment, the aqueous diluent is water for injection, in particular sterile water for injection (SWFI) and / or bacteriostatic water for injection (BWFI).

[0131] The aqueous diluent may further contain buffers, bacteriostats, and solutes that render the composition isotonic with the blood of the intended recipient.

[0132] In one embodiment, the aqueous diluent is not a buffer solution and does not contain a buffering agent.

[0133] Advantageously, the osmotic pressure of the aqueous solution is 1 mOsM or more, more advantageously, the osmotic pressure of the aqueous solution is 1 to 300 mOsM, more preferably, 1 to 200 mOsM.

[0134] The concentration of indocyanine green in the aqueous compositions of the present invention may be any concentration suitable for use in medical treatment or diagnosis, particularly any concentration suitable for producing angiographic images of satisfactory quality.

[0135] In a preferred embodiment, the concentration of indocyanine green in the aqueous composition according to the present invention is 0.1 to 50 mg / ml, preferably 0.1 to 30 mg / ml.

[0136] If the concentration of the ICG solution is less than 0.1 mg / ml, it will not be fluorescent enough for satisfactory detection in analytical instruments.

[0137] In a preferred embodiment, the concentration of indocyanine green in the aqueous composition according to the present invention is 0.1 to 25 mg / ml, preferably 1 to 25 mg / ml, and more preferably 1 to 10 mg / ml.

[0138] When one or more components of the composition of the present invention, such as histidine (b) or optionally compound (c), are added as an aqueous solution, the amount of aqueous diluent can be adjusted to obtain the desired concentration of ICG.

[0139] In one embodiment of the invention, the aqueous composition consists essentially of an aqueous diluent, indocyanine green (a), and histidine (b).

[0140] In another embodiment, the aqueous composition consists essentially of an aqueous diluent, indocyanine green (a), histidine (b) and sodium iodide.

[0141] In another embodiment, the aqueous composition consists essentially of an aqueous diluent, indocyanine green (a), histidine (b), and a co-stabilizing compound (c) selected from ethylenediaminetetraacetic acid (EDTA) and its salts, cysteine, sodium chloride, dithiothreitol (DTT), and mixtures thereof.

[0142] In another embodiment, the aqueous composition consists essentially of an aqueous diluent, indocyanine green (a), histidine (b), and a co-stabilizing compound (c) selected from ethylenediaminetetraacetic acid (EDTA) and its salts, cysteine, sodium chloride, dithiothreitol (DTT) and mixtures thereof, and sodium iodide.

[0143] In these embodiments, the preferred ratios of the compounds (ICG (a), histidine (b) and co-stabilizing compound (c)) in the aqueous composition are the same as those presented in the "Compositions of Indocyanine Green" section above.

[0144] (lyophilized solid composition) In another preferred embodiment, the composition according to the invention is in the form of a lyophilized composition or a lyophilized powder.

[0145] The terms "lyophilized composition", "lyophilized powder" and "lyophilisate" refer to a solid composition according to the invention obtained by lyophilization of an aqueous solution.

[0146] The term "lyophilization" refers to a process in which the composition in an aqueous solvent is first frozen and then the frozen solvent is removed by sublimation in a vacuum environment.

[0147] When the compositions of the present invention are provided as lyophilizates, they can be converted to infusion solutions using the aqueous diluents described above prior to administration, and the reconstituted aqueous solutions (which are also compositions of the present invention) are stable at room temperature for at least one month, preferably at least two months, more preferably at least six months, and most preferably at least two years.

[0148] All the limitations regarding ICG concentration, pH, osmolality and stability mentioned above in relation to the aqueous diluent also apply to the aqueous solution resulting from reconstitution of the lyophilized composition according to the invention.

[0149] In one embodiment, the lyophilized composition of the present invention has a short reconstitution time of less than 2 minutes, preferably less than 1 minute, making it suitable for intravenous administration after dilution with an aqueous diluent.

[0150] By "reconstitution time" is meant the time required for the lyophilized composition to be rehydrated with an aqueous solution to a clear, particle-free solution.

[0151] Once the composition has been reconstituted in an aqueous solution for medical or diagnostic use, it is preferably stored in a container that protects it from light and oxygen.

[0152] (kit) In one preferred embodiment, the invention relates to a kit comprising in a first part the composition according to the invention in the form of a lyophilized powder and in a second part an aqueous diluent.

[0153] According to another preferred embodiment, the present invention relates to a kit comprising an aqueous solution comprising indocyanine green (a) in a first part and histidine (b) in a second part, preferably comprising a co-stabilizing compound (c) as defined above, and optionally one or more additives as described above.

[0154] According to this embodiment, the indocyanine green is preferably in the form of a lyophilized powder.

[0155] All preferred embodiments described above for the ICG composition and aqueous diluent apply to either kit variant.

[0156] In one embodiment, the kit of the present invention is composed of each component (powder (power) and diluent) in such amounts that the ICG concentration of an aqueous solution reconstituted from the kit is within the range of 0.1 to 50 mg / ml, preferably 0.1 to 30 mg / ml, more preferably 0.1 to 25 mg / ml, even more preferably 1 to 25 mg / ml, and most preferably 1 to 10 mg / ml.

[0157] In the above preferred embodiment, the kit comprises at least two separate compartments in which the lyophilized powder and the aqueous diluent are stored separately.

[0158] The kit may consist of at least two separate vials packaged with or without a syringe, or may consist of a pre-filled syringe having two compartments, such as a dual chamber bypass syringe.

[0159] Such a kit allows for mixing of the powder and the aqueous solution prior to administration.

[0160] Either of the kits allows for the immediate reconstitution of a stable aqueous solution of indocyanine green as described. They have the advantage that each component of the composition required for injection is available in the appropriate amount and under optimal storage conditions to obtain the desired concentration. In other words, the aqueous composition can be reconstituted without the need for dilution with a diluent other than that included in the kit. This packaging as a kit can avoid administration errors and the introduction of contaminants.

[0161] (Method of preparing the composition according to the present invention) The composition according to the present invention may be prepared by any known method and is not limited to any particular method.

[0162] For example, the composition can be prepared by mixing indocyanine green (a) with histidine (b), and optionally one or more of the compounds described above (c), and optionally one or more pharma- ceutically acceptable excipients.

[0163] Preferably, the components of the composition of the present invention are mixed in the aqueous solvent described above. The components constituting the composition of the present invention may be added in various orders. For example, indocyanine green may be dissolved in the aqueous diluent, and then histidine (b) and optionally co-stabilizing compound (c) and additives may be added. Compounds (b) and (c) may be mixed together and then added to the dissolved indocyanine green. Alternatively, indocyanine green may be added to an aqueous solution in which histidine (b) and optionally compound (c) have been dissolved in advance.

[0164] Preferably, mixing is carried out under an inert atmosphere, such as under an argon or nitrogen atmosphere.The aqueous diluent and / or liquid components are preferably degassed prior to use.

[0165] When the composition of the present invention is provided as a lyophilisate, lyophilisation can be carried out using standard equipment for lyophilisation or vacuum drying. The lyophilisation cycle can be modified by those skilled in the art to suit the equipment and facilities used.

[0166] Preferably, said pre-lyophilized aqueous solution is an aqueous solution of indocyanine green as described above. The pre-lyophilized aqueous solution according to the invention may be sterilized before lyophilization. Typically, this sterilization is carried out by filtering the solution through a suitable membrane.

[0167] Preferably, the aqueous solution is lyophilized shortly after preparation to prevent degradation of the composition. The lyophilized product can be used even after storage for several months. Preferably, the lyophilized product is stored under conditions protected from air and / or moisture and / or light.

[0168] In addition, when the indocyanine green composition according to the present invention is provided as an aqueous composition, it may be reconstituted using the lyophilized composition and the aqueous diluent of the above-mentioned kit. When reconstituting, the lyophilized composition may be introduced into a vial, and then the aqueous diluent may be added thereto.

[0169] Advantageously, the composition prepared in lyophilized or aqueous form is immediately after preparation placed in a light-protected, airtight, sealed container, which may be, for example, a bottle, an ampoule, a vial, a syringe, a tube, etc. The container may be, for example, made of glass, polymer, metal, etc. The container may be a single-dose or multi-dose container.

[0170] In a preferred embodiment of the present invention, the container is preferably a colored glass vial or a colored glass ampoule. Advantageously, the container is selected from a colored glass vial with a vacuum headspace or a colored glass ampoule. Preferably, the composition according to the present invention is stored in a colored glass ampoule.

[0171] Advantageously, the composition according to the invention is stored at room temperature.

[0172] (Use of the composition according to the present invention) The composition of the invention may be used in a method of diagnosis and / or (as a therapeutic agent) treatment of a patient, comprising administering an effective amount of an aqueous solution according to the invention. The administration may be by enteral route, parenteral route, in particular by intravenous injection or topical application. Preferably, the administration is by intravenous injection.

[0173] The term "effective amount" refers to an amount of indocyanine green that elicits the biological or medical response in a tissue, system, animal or human that is desired by, for example, a researcher, clinician, or the like.

[0174] Advantageously, the composition of the present invention may be used in methods of diagnosis and / or treatment in which the aqueous ICG composition is administered to a patient after it has been stored for at least 1 month, preferably at least 6 months, more preferably at least 12 months, and most preferably at least 2 years since preparation or reconstitution from one of the kits as described above.

[0175] In particular, the composition according to the present invention can be used for hyperthermia, ICG-enhanced selective photocoagulation, photodynamic therapy (PDT), and photodynamic thermotherapy (PHT). More specifically, the composition according to the present invention can be used for PDT, in the treatment of infectious diseases, acne, macular surgery, and cancer treatment. The composition according to the present invention may be used in conjunction with other therapeutic techniques, such as immunotherapy, radiation therapy, ultrasound therapy, and chemotherapy.

[0176] The compositions of the present invention may also be used to obtain angiographic images of patient tissue, measure cardiac output, measure liver function and hepatic blood flow, and diagnose and treat age-related macular degeneration and associated choroidal neovascularization and tumors.

[0177] The compositions of the present invention are based on components that have undergone clinical trials and are approved for administration to humans and / or animals.

[0178] The amount of the ICG composition administered to a patient for diagnostic use should be sufficient to cause the dye to fluoresce when irradiated with a suitable wavelength, taking into account that the absorption and emission peaks of ICG are within the range of 800-850 nm. In the same manner, in the case of a treatment method using an ICG solution, a sufficient amount of dye should be administered so that the treatment can be performed efficiently. The dosage of the ICG composition can be easily determined by a person skilled in the art, and is preferably equal to or higher than the currently permitted concentration for use in ophthalmic angiography, for example, 2 ml of a 20 mg / mL ICG solution for diagnostic use. A person skilled in the art will understand that in any of the above diagnostic and treatment methods, a higher concentration of dye may be advantageous.

[0179] The increased stability of ICG in the solutions of the present invention allows for improved diagnosis / treatment of patients with the same injected dose of compound as in prior art ICG compositions, because the increased stability of ICG results in a stronger response to fluorescent illumination. EXAMPLES

[0180] I. Materials and Methods (1. Reagents and Chemicals) Indocyanine green was provided by Biophore India Pharmaceuticals and used without further purification. Cysteine ​​(CAS number: 3374-22-9) was provided by Fisher Scientific. Sodium ascorbate (CAS number: 134-03-2) and histidine (CAS number: 4998-57-6) were provided by Aldrich. Other reagents were purchased from Aldrich and used without further purification.

[0181] (2.Equipment) The stability of the formulations of the examples was measured by high pressure liquid chromatography (HPLC).The equipment used for the analysis was an Agilent Infinity1260 HPLC / MS system equipped with a diode array detector (UV-visible light), a Waters XbridgeShield RP18 (4.6x100; 5μm) column, and an ESI type mass spectrometer (solution input volume: 5μL, flow rate: 1ml / min, column temperature: 30℃, detection: 240nm).

[0182] The elution is performed by gradient elution. The mobile phase consists of a mixture of ammonium acetate / acetic acid buffer (10 mM, pH: 5.5) (eluent A) and acetonitrile (eluent B). The composition of the mobile phase was changed continuously during the elution process according to Table I below:

[0183] [Table 1]

[0184] (II- Sample Preparation) The following ICG composition was prepared as follows: Histidine and, if present, co-stabilizing compounds are weighed into a Type I clear glass vial. Then, 25 mg of indocyanine green is weighed, taking into account the tare weight of the vial. The vial is closed with a bromobutyl rubber stopper (VWS) and purged with a stream of nitrogen. Then, 10 ml of WFI, previously degassed with nitrogen, is added via syringe. The vial is gently stirred by hand until the solubility of the solute is reached. The vial is then stored at room temperature away from light.

[0185] (III - Composition containing indocyanine green (a) and histidine (b))

[0186] [Table 2]

[0187] The results reported in Table II demonstrate that the presence of histidine enhances the stability of aqueous indocyanine green compositions.

[0188] (IV - Composition Comprising Indocyanine Green (a), Histidine (b) and Co-stabilizing Compound (c))

[0189] [Table 3]

[0190] The results reported in Table III demonstrate that the association of histidine and compound (c) with indocyanine green improves the stability of aqueous indocyanine green compositions.

[0191] (V-comparative composition) (Composition containing only co-stabilizing compound (c))

[0192] [Table 4]

[0193] The results reported in Table IV show that compound (c), when used alone, improves the storage stability of aqueous indocyanine green compositions, but not to the same extent as histidine.

[0194] Compositions containing components according to the prior art

[0195] [Table 5]

[0196] The results reported in Table V show that other stabilizing compounds disclosed in the prior art do not exert sufficient potency with respect to the long-term stability of ICG.

Claims

1. A composition comprising at least indocyanine green (a) and histidine (b), wherein the weight ratio of histidine (b) / indocyanine green (a) is 0.05 or more.

2. 2. The indocyanine green composition of claim 1, wherein the weight ratio of histidine (b) / indocyanine green (a) is in the range of 0.05-50.

3. 3. The indocyanine green composition according to claim 2, wherein the weight ratio of histidine (b) / indocyanine green (a) is in the range of 0.10-40, preferably 0.10-30, more preferably 0.10-20.

4. 4. The indocyanine green composition according to any one of claims 1 to 3, further comprising at least one compound (c) selected from ethylenediaminetetraacetic acid (EDTA) and its salts, cysteine, sodium chloride, dithiothreitol (DTT) and mixtures thereof.

5. 5. The indocyanine green composition of claim 4, wherein the weight ratio of compound (c) to indocyanine green (a) is in the range of 1:500 to 100:1, preferably 1:100 to 10:1, more preferably 1:100 to 4:

1.

6. The indocyanine green composition according to claim 4 or 5, wherein the weight ratio of compound (c) to histidine (b) is in the range of 1:100 to 100:1, preferably 1:10 to 10:

1.

7. 7. The indocyanine green composition of claim 1, comprising up to 5% by weight of sodium iodide, based on the weight of indocyanine green.

8. The indocyanine green composition according to any one of claims 1 to 7, which is an aqueous composition.

9. 9. The indocyanine green composition according to claim 8, wherein the concentration of indocyanine green in the aqueous composition is comprised between 0.1 and 50 mg / ml.

10. 10. The aqueous composition according to claim 8 or claim 9, wherein the ICG content in the composition before storage is 90% or more, and the indocyanine green content is measured by HPLC area ratio (%) at 240 nm.

11. The indocyanine green composition of any one of claims 1 to 7, wherein the composition is in the form of a lyophilized powder.

12. The indocyanine green composition according to any one of claims 1 to 11 for use as a pharmaceutical or diagnostic agent.

13. A kit comprising at least 1) the composition of claim 11, and 2) an aqueous diluent in separate compartments.

14. A kit usable for preparing the composition according to any one of claims 8 to 10, comprising at least 1) indocyanine green (a) and 2) an aqueous solution containing at least histidine in separate compartments.