Composition for fluorescent marking of biological tissue

Abstract

The present invention relates to a composition for fluorescent marking of a biological tissue, the composition containing: an amphiphilic compound having an isoprenoid chain; a phospholipid; and a fluorescent dye.

Description

Compositions for fluorescent marking of biological tissues

[0001] This invention relates to a composition for fluorescent marking of biological tissues.

[0002] Cancers of the digestive tract, such as the esophagus, stomach, and large intestine, primarily originate from the mucous membrane of the digestive tract and progress into the organ. Similarly, lung cancer primarily originates from the tracheal mucosa, and bladder cancer primarily originates from the bladder mucosa and progresses into the organ. Therefore, in the definitive diagnosis of cancers of tubular organs such as the digestive tract, trachea, and bladder, it is common practice to insert an endoscope into the tubular organ to observe the mucous membrane and take a biopsy of the affected tissue on the mucosa.

[0003] On the other hand, in surgical resection of cancerous tissue in tubular organs, open surgery, thoracotomy, or laparoscopic surgery is often performed, and in these cases, the surgeon approaches the affected area from the outside of the tubular organ rather than from the inside (inside the lumen). In this case, what can be visualized is the outer serosal membrane, not the inner mucosa of the tubular organ, and the cancerous tissue is often not directly visible. Therefore, it is common practice to mark the cancerous tissue beforehand using an endoscope inserted into the lumen of the tubular organ (preoperative marking) so that the area to be resected of the cancerous tissue can be determined from the outside of the tubular organ.

[0004] Conventional preoperative marking methods include the dot method, which involves locally injecting India ink into the submucosa directly beneath the cancerous tissue; the clip method, which involves attaching a clip equipped with a light-emitting element to the mucosa near the cancerous tissue; and the fluorescence method, which involves locally injecting a fluorescent substance such as indocyanine green into the submucosa directly beneath the cancerous tissue (for example, Non-Patent Literature 1). However, with the dot method, visibility tends to decrease due to the diffusion of the locally injected India ink, often making it difficult to identify the affected area. With the clip method, there is a risk that the affected area will become invisible if the clip falls off. Furthermore, the fluorescence method has the problem that the fluorescence weakens and visibility decreases if the time between local injection and surgery is long.

[0005] Patent Document 1 discloses a medical tissue marker composition having clusters formed by encapsulating vesicles formed by a complex of phospholipids and near-infrared fluorescent dyes, and emulsions formed by a complex of phospholipids and X-ray contrast agents in a hydrophilic solvent, and then forming and agglomerating multiple capsules with an emulsifier. However, even with this medical tissue marker composition, the long-term visibility of the fluorescent markings has not been sufficiently achieved.

[0006] Patent Document 2 discloses a medical marker clip comprising a medical marker body configured to be lockable to the inner wall of a tubular organ, a movable member containing a fluorescent dye that emits fluorescence in a predetermined wavelength range upon irradiation with excitation light, and a hinge portion connecting the medical marker body and the movable member so that the movable member can rotate relative to the medical marker body. However, there is still a risk of the clip falling off even when using this clip for preoperative marking.

[0007] Typically, several days to two weeks are required between preoperative marking and surgical excision. Therefore, there is a need for the development of localized fluorescent marking technology for biological tissue that minimizes the decrease in visibility or loss of markers during this period.

[0008] International Publication WO2012 / 173003, Japanese Patent Publication No. 2021-069802

[0009] Ozawa et al., Showa Gakushikai Journal, Vol. 80, No. 1, pp. 1-6, 2020.

[0010] The present invention aims to provide a localized fluorescent marking technology for biological tissues that facilitates the maintenance of marker visibility.

[0011] As a result of diligent research to solve the above problems, the present inventors have discovered that by using a composition containing a predetermined amphiphilic compound and a phospholipid together with a fluorescent dye for fluorescent marking, the visibility of the fluorescent marker can be maintained for a long period of time, and have completed the present invention.

[0012] In other words, the present invention encompasses the following:

[0013] [1] A composition for fluorescent marking of biological tissues, comprising an amphiphilic compound having an isoprenoid chain, a phospholipid, and a fluorescent dye. [2] The composition according to [1], wherein the amphiphilic compound having an isoprenoid chain is an amphiphilic compound represented by the following general formula (I) or a salt thereof. (In the formula, X and Y each represent a hydrogen atom or together represent an oxygen atom, n represents an integer from 0 to 2, and m represents 1 or 2.) (where R represents a single or double bond, and R represents a hydrophilic group having one or more hydroxyl groups) [3] The composition according to [1] or [2] above, which is a liquid crystal precursor composition capable of forming a non-lamellar liquid crystal in the presence of an aqueous medium. [4] The composition according to any one of [1] to [3] above, further comprising a biocompatible organic solvent. [5] The composition according to [4] above, wherein the biocompatible organic solvent is ethanol. [6] The composition according to any one of [1] to [5] above, wherein the phospholipid comprises at least one selected from the group consisting of phosphatidylcholine and phosphatidylethanolamine. [7] The composition according to any one of [1] to [6] above, wherein the phospholipid is selected from the group consisting of soybean phosphatidylcholine, egg yolk phosphatidylcholine, dioleyl phosphatidylcholine, and dioleyl phosphatidylethanolamine. [8] The composition according to any one of [1] to [7] above, further comprising an oil. [9] The composition according to [8] above, wherein the oil component is selected from the group consisting of sesame oil, soybean oil, and squalene.

[10] The composition according to any one of [1] to [9] above, wherein the fluorescent dye is a near-infrared fluorescent dye.

[11] The composition according to

[10] above, wherein the near-infrared fluorescent dye is indocyanine green.

[12] The composition according to any one of [1] to

[11] above, comprising a fluorescent dye at a concentration of 0.005 to 0.5 w / w%.

[13] The amphiphilic compound is one of the following: mono-O-(5,9,13,17-tetramethyloctadeca-4-enoyl)glycerol, mono-O-(5,9,13,17-tetramethyloctadeca-4-enoyl)sorbitan, mono-O-(5,9,13,17-tetramethyloctadecanoyl)sorbitan, mono-O-(5,9,13,17-tetramethyloctadeca-4-enoyl)isosorbide, mono-O-(5,9,13,17-tetramethyloctadecanoyl)isosorbide, mono-O-(5,9,13,17-tetramethyloctadecanoyl)propylene glycol, The composition according to any one of [1] to

[12] above, wherein at least one selected from the group consisting of mono-O-(5,9,13-trimethyltetradeca-4-enoyl)glycerol and mono-O-(5,9,13-trimethyltetradeca-4,8,12-trienoyl)glycerol.

[14] A composition according to any one of [1] to

[13] above for locally fluorescent marking the tissue of a tubular organ.

[15] A composition according to any one of [1] to

[14] above for injecting into living tissue to locally fluorescent mark the living tissue.

[16] A composition according to any one of [1] to

[14] above for applying onto the surface of living tissue to locally fluorescent mark the living tissue.

[17] A method for locally fluorescent marking affected tissue, comprising locally applying a composition according to any one of [1] to

[16] above to a site in living tissue where the location of the affected tissue can be identified.

[0014] This specification includes the disclosures of Japanese Patent Application No. 2025-052407, which forms the basis of the priority claim of this application.

[0015] The present invention provides a biological tissue fluorescent marking technology that facilitates the maintenance of marker visibility.

[0016] Figure 1 shows a photograph of a stomach fluorescence observation image using an ICG-containing precursor preparation and a ZeoClip FS (fluorescent clip). Precursor preparation No. 13 (containing 50 ppm ICG) and precursor preparation No. 11 (containing 1000 ppm ICG) were injected. A: Fluorescence observation image of the mucosal side of the stomach of an edible pig, A: Fluorescence observation image of the serosal side of the stomach of an edible pig. Figure 2 shows a photograph of the observation results of a 2-week subcutaneous implantation test of an ICG-containing precursor preparation in experimental animals. A and B are a photograph of the appearance of the subcutaneous injection site of the ICG-containing precursor preparation on the day of administration (day 0) (A: no bleeding at the injection site) and a fluorescence observation image (B: arrow indicates fluorescence at the subcutaneous injection site). Figures C and D show the appearance of the subcutaneous injection site of the ICG-containing precursor preparation 14 days after administration (C: injection mark almost disappeared) and fluorescence observation image (D: arrow indicates fluorescence at the subcutaneous injection site). Figure 3 shows photographs of the observation results of a 2-week placement test of the ICG-containing precursor preparation on the skin surface in experimental animals. A and B show the appearance of the subcutaneous application site of the ICG-containing precursor preparation on the day of application (day 0) and fluorescence observation image (B: arrow indicates fluorescence at the application site). Figures C and D show the appearance of the application site of the ICG-containing precursor preparation 14 days after application (C: no inflammation observed) and fluorescence observation image (D: arrow indicates fluorescence at the application site). Figure 4 shows photographs of the appearance of the gel produced from the fluorescent dye-containing preparation in an aqueous medium over time. A: 30 minutes later, B: 2 hours later, C: 6 hours later, D: 1 day later, E: 5 days later.

[0017] The present invention will be described in detail below.

[0018] The present invention relates to a composition using an amphiphilic compound having a fatty acid chain, particularly an amphiphilic compound having an isoprenoid chain (isoprenoid fatty acid chain), and a fluorescent dye, preferably a composition for fluorescent marking of biological tissues.

[0019] The composition according to the present invention comprises a fluorescent dye for fluorescent marking. In a preferred embodiment, the fluorescent dye used in the present invention is a near-infrared fluorescent dye. In the present invention, a near-infrared fluorescent dye means a dye that emits fluorescence that is near-infrared light (having a wavelength of 700 nm to 2500 nm) when irradiated with near-infrared light (having a wavelength of 700 nm to 2500 nm). Near-infrared light has excellent tissue penetration and is suitable for biological tissue imaging. Examples of near-infrared fluorescent dyes used in the present invention include, but are not limited to, cyanine dyes, such as indocyanine green (ICG) and its derivatives and salts thereof. Examples of ICG derivatives include, but are not limited to, PEG-modified ICG, non-sulfonated ICG, sulfonated ICG, N-hydroxysuccinimide-modified ICG, maleimide-modified ICG, amine-modified ICG, hydrazide-modified ICG, azide-modified ICG, and derivative compounds in which the linear unsaturated hydrocarbon chain of ICG is replaced with another linear unsaturated hydrocarbon chain. Near-infrared fluorescent dyes are particularly suitable for local fluorescent marking of tissues in tubular organs, especially for local fluorescent marking of biological tissues in endoscopic surgery such as laparoscopic surgery. Alternatively, the fluorescent dye used in the present invention may be a fluorescent dye other than a near-infrared fluorescent dye, or a fluorescent dye precursor (a substance that is metabolized or modified in vivo to produce a fluorescent dye), such as coumarin, rhodamine, xanthene, porphyrin, fluorescein, 5-aminolevulinic acid (5-ALA), or salts thereof. The fluorescent dye used in the present invention (preferably a near-infrared fluorescent dye) may be water-soluble (hydrophilic) or lipid-soluble (hydrophobic). In one embodiment, the fluorescent dye used in the present invention (preferably a near-infrared fluorescent dye) may have a hydrophilic part and a hydrophobic part (hydrophobic skeleton) in its molecule, and may have a relatively high lipid affinity due to such an amphiphilic structure. Examples of fluorescent dyes having an amphiphilic structure include indocyanine green (ICG) and its derivatives and salts thereof.When a fluorescent dye having such an amphiphilic structure with relatively high lipid affinity is used in the present invention, the fluorescent dye is more firmly retained within the non-lamellar liquid crystal structure, leakage into the aqueous medium in a short period of time is further suppressed, and stable local fluorescent marking over a longer period of time is possible at the application site.

[0020] The composition according to the present invention may contain a fluorescent dye at a concentration of, for example, 0.001 w / w% or more. The composition according to the present invention may contain a fluorescent dye at a concentration of, for example, less than 1 w / w%. In a preferred embodiment, the composition according to the present invention contains a fluorescent dye at a concentration of 0.005 w / w% to 0.5 w / w%. In a more preferred embodiment, the composition according to the present invention contains a fluorescent dye at a concentration of 0.01 w / w% to 0.2 w / w%. In one embodiment, the composition according to the present invention contains a fluorescent dye at a concentration of 0.05 w / w% to 0.1 w / w%.

[0021] In relation to the present invention, the concentration or proportion (%) of a component in a composition means weight percent (w / w%) unless otherwise specified. Unless otherwise specified, the concentration or proportion (w / w%) of a specific component in a composition according to the present invention is calculated as the percentage of the weight of that specific component relative to the total weight of the composition according to the present invention.

[0022] The composition according to the present invention contains, in addition to a fluorescent dye, an amphiphilic compound having an isoprenoid chain (isoprenoid fatty acid chain) (hereinafter also referred to as "isoprenoid lipid"). The isoprenoid lipid used in the present invention is preferably a low-molecular-weight amphiphilic compound. Here, "low-molecular-weight" means having a molecular weight of about 20 to 10,000. The molecular weight of the isoprenoid lipid used in the present invention is preferably 50 to 5,000, more preferably 100 to 2,500, and even more preferably 200 to 1,000. The isoprenoid lipid used in the present invention may or may not be a non-lamellar liquid crystal forming lipid. In the present invention, "non-lamellar liquid crystal forming lipid" refers to a lipid compound that can form a non-lamellar liquid crystal structure in an aqueous medium (aqueous phase) on its own.

[0023] In one embodiment, the isoprenoid lipid used in the present invention is an amphiphilic compound represented by the following general formula (I). The present invention provides a composition for fluorescent marking of biological tissues, comprising an amphiphilic compound represented by the following general formula (I), a phospholipid, and a fluorescent dye.

[0024] In general formula (I), X and Y each represent a hydrogen atom or together represent an oxygen atom. In general formula (I), n represents an integer from 0 to 2 (0, 1, or 2; preferably 1 or 2), and m represents 1 or 2. In the amphiphilic compound represented by general formula (I), the combination of n and m may be any of the following: n=0, m=1; n=0, m=2; n=1, m=1; n=1, m=2; n=2, m=1; or n=2, m=2.

[0025] In the formula: The symbol represents a single bond or a double bond.

[0026] In general formula (I), R represents a hydrophilic group having one or more (e.g., two, three, four, or five) hydroxyl groups. R in general formula (I) may also be a residue obtained by removing one hydroxyl group (OH) from a polyol. R in general formula (I) may have an ether linkage and / or a cyclic structure. In general formula (I), R is a hydrophilic group obtained by removing one hydroxyl group (OH) from any one selected from the group consisting of glycerol, erythritol, pentaerythritol, diglycerol, glyceric acid, triglycerol, xylose, sorbitol, ascorbic acid, glucose, galactose, mannose, dipentaerythritol, maltose, mannitol, xylitol, sorbitan, isosorbide, and glycol (for example, propylene glycol, ethylene glycol, diethylene glycol, butylene glycol such as 1,3-butylene glycol, and isoprene glycol, but not limited to these). Isoprene glycol is also known as 3-methyl-1,3-butanediol. In one embodiment, R in general formula (I) is more preferably a hydrophilic group obtained by removing one hydroxyl group (OH) from glycerol, glyceric acid, sorbitan, isosorbide, or glycol (e.g., propylene glycol, ethylene glycol, butylene glycol such as 1,3-butylene glycol, or isoprene glycol). In one embodiment, R in general formula (I) may be a hydrophilic group having one hydroxyl group, obtained by removing one hydroxyl group (OH) from a glycol that does not have an ether bond. In one embodiment, R in general formula (I) may be a hydrophilic group obtained by removing one hydroxyl group (OH) from glyceric acid, or a group obtained by removing the OH (hydroxyl group) contained in the carboxyl group of glyceric acid.

[0027] Furthermore, in relation to the present invention, in the chemical formula representing an amphiphilic compound: This means that the amphiphilic compound is either the E (cis) or Z (trans) geometric isomer, or a mixture thereof.

[0028] Examples of amphiphilic compounds represented by general formula (I) include the amphiphilic compounds represented by general formula (II) shown below.

[0029] In general formula (II), X and Y each represent a hydrogen atom or together represent an oxygen atom, n represents an integer from 0 to 2 (0, 1, or 2; preferably 1 or 2), and m represents 1 or 2. The combinations of n and m may be any of the following: n=0, m=1; n=0, m=2; n=1, m=1; n=1, m=2; n=2, m=1; or n=2, m=2. In one embodiment, in general formula (II), X and Y together represent an oxygen atom, and n=2, m=2; or n=2, m=1.

[0030] R in general formula (II) represents a hydrophilic group having one or two or more hydroxyl groups. R in general formula (II) may be the same as R in general formula (I). Examples of R in general formula (II) include, but are not limited to, hydrophilic groups obtained by removing one hydroxyl group (OH) from any one selected from the group consisting of glycerol, erythritol, pentaerythritol, diglycerol, glyceric acid, triglycerol, xylose, sorbitol, ascorbic acid, glucose, galactose, mannose, dipentaerythritol, maltose, mannitol, xylitol, sorbitan, isosorbide, and glycol (examples include, but are not limited to, propylene glycol, ethylene glycol, diethylene glycol, butylene glycol such as 1,3-butylene glycol, and isoprene glycol). In one embodiment, R in general formula (II) is more preferably a hydrophilic group obtained by removing one hydroxyl group (OH) from glycerol, glyceric acid, sorbitan, isosorbide, or glycol (for example, propylene glycol, ethylene glycol, butylene glycol such as 1,3-butylene glycol, isoprene glycol, and the like). In one embodiment, R in general formula (II) may be, for example, a hydrophilic group having one hydroxyl group obtained by removing one hydroxyl group (OH) from a glycol having no ether bond. In one embodiment, when R in general formula (II) is a hydrophilic group obtained by removing one hydroxyl group (OH) from glyceric acid, the group may be a group obtained by removing the OH (hydroxyl group) contained in the carboxyl group of glyceric acid.

[0031] Another example of the amphiphilic compound represented by general formula (I) is an amphiphilic compound represented by the following general formula (III).

[0032] In general formula (III), X and Y each represent a hydrogen atom, or together represent an oxygen atom; n represents an integer of 0 to 2 (0, 1 or 2; preferably 1 or 2), and m represents 1 or 2. The combination of n and m may be any of n=0, m=1; n=0, m=2; n=1, m=1; n=1, m=2; n=2, m=1; or n=2, m=2. In one embodiment, in general formula (III), X and Y together represent an oxygen atom, and n=2, m=2; or n=2, m=1.

[0033] R in general formula (III) represents a hydrophilic group having one or two or more hydroxyl groups. R in general formula (III) may be the same as R in general formula (I). R in general formula (III) includes, but is not limited to, for example, hydrophilic groups obtained by removing one hydroxyl group (OH) from any one selected from the group consisting of glycerol, erythritol, pentaerythritol, diglycerol, glyceric acid, triglycerol, xylose, sorbitol, ascorbic acid, glucose, galactose, mannose, dipentaerythritol, maltose, mannitol, xylitol, sorbitan, isosorbide, and glycols (examples include, but are not limited to, propylene glycol, ethylene glycol, diethylene glycol, butylene glycols such as 1,3-butylene glycol, and isoprene glycol). In one embodiment, R in general formula (III) is more preferably a hydrophilic group obtained by removing one hydroxyl group (OH) from glycerol, glyceric acid, sorbitan, isosorbide, or a glycol (for example, propylene glycol, ethylene glycol, butylene glycols such as 1,3-butylene glycol, or isoprene glycol). In one embodiment, R in general formula (III) may be, for example, a hydrophilic group having one hydroxyl group obtained by removing one hydroxyl group (OH) from a glycol having no ether bond. In one embodiment, with respect to R in general formula (III), the hydrophilic group obtained by removing one hydroxyl group (OH) from glyceric acid may be a group obtained by removing the OH (hydroxyl group) contained in the carboxyl group of glyceric acid.

[0034] Another example of an amphiphilic compound represented by general formula (I) is the amphiphilic compound represented by the following general formula (IV).

[0035] In general formula (IV), X and Y each represent a hydrogen atom or together represent an oxygen atom, n represents an integer from 0 to 2 (0, 1, or 2; preferably 1 or 2), and m represents 1 or 2. The combinations of n and m may be any of the following: n=0, m=1; n=0, m=2; n=1, m=1; n=1, m=2; n=2, m=1; or n=2, m=2. In one embodiment, in general formula (IV), X and Y together represent an oxygen atom, and n=2, m=2; or n=2, m=1.

[0036] In general formula (IV), R represents a hydrophilic group having one or more hydroxyl groups. R in general formula (IV) may be the same as R in general formula (I). R in general formula (IV) is not limited to the following, but examples include a hydrophilic group obtained by removing one hydroxyl group (OH) from any one selected from the group consisting of glycerol, erythritol, pentaerythritol, diglycerol, glyceric acid, triglycerol, xylose, sorbitol, ascorbic acid, glucose, galactose, mannose, dipentaerythritol, maltose, mannitol, xylitol, sorbitan, isosorbide, and glycol (examples include, but are not limited to, propylene glycol, ethylene glycol, diethylene glycol, butylene glycol such as 1,3-butylene glycol, and isoprene glycol). In one embodiment, R in general formula (IV) is more preferably a hydrophilic group obtained by removing one hydroxyl group (OH) from glycerol, glyceric acid, sorbitan, isosorbide, or glycol (e.g., propylene glycol, ethylene glycol, butylene glycol such as 1,3-butylene glycol, or isoprene glycol). In one embodiment, R in general formula (IV) may be a hydrophilic group having one hydroxyl group, obtained by removing one hydroxyl group (OH) from a glycol that does not have an ether bond. In one embodiment, R in general formula (IV) may be a hydrophilic group obtained by removing one hydroxyl group (OH) from glyceric acid, or a group obtained by removing the OH (hydroxyl group) contained in the carboxyl group of glyceric acid.

[0037] In one embodiment, the amphiphilic compound represented by general formula (I) is mono-O-(5,9,13,17-tetramethyloctadeca-4-enoyl)glycerol, mono-O-(5,9,13,17-tetramethyloctadeca-4-enoyl)sorbitan, mono-O-(5,9,13,17-tetramethyloctadecanoyl)sorbitan, mono-O-(5,9,13,17-tetramethyloctadeca-4-enoyl)isosorbide, mono-O-(5,9,13,17-tetramethyloctadecanoyl)isosorbide, mono-O-(5,9,13,17-tetramethyloctadecanoyl)propylene glycol, It may be at least one selected from the group consisting of mono-O-(5,9,13-trimethyltetradeca-4-enoyl)glycerol and mono-O-(5,9,13-trimethyltetradeca-4,8,12-trienoyl)glycerol.

[0038] In one embodiment, the amphiphilic compound represented by general formula (I) may be a mixture of mono-O-(5,9,13,17-tetramethyloctadeca-4-enoyl)sorbitan and mono-O-(5,9,13,17-tetramethyloctadeca-4-enoyl)isosorbide. In one embodiment, when preparing the composition according to the present invention, a fraction containing mono-O-(5,9,13,17-tetramethyloctadeca-4-enoyl)sorbitan may be used as the amphiphilic compound represented by general formula (I), for example, a fraction containing both mono-O-(5,9,13,17-tetramethyloctadeca-4-enoyl)sorbitan and mono-O-(5,9,13,17-tetramethyloctadeca-4-enoyl)isosorbide.

[0039] In one embodiment, the amphiphilic compound represented by general formula (I) may be a mixture of mono-O-(5,9,13,17-tetramethyloctadecanoyl)sorbitan and mono-O-(5,9,13,17-tetramethyloctadecanoyl)isosorbide. In one embodiment, when preparing the composition according to the present invention, a fraction containing mono-O-(5,9,13,17-tetramethyloctadecanoyl)sorbitan may be used as the amphiphilic compound represented by general formula (I), for example, a fraction containing both mono-O-(5,9,13,17-tetramethyloctadecanoyl)sorbitan and mono-O-(5,9,13,17-tetramethyloctadecanoyl)isosorbide.

[0040] In preferred embodiments, the isoprenoid lipids used in the present invention, such as the amphiphilic compound represented by general formula (I), exhibit low viscosity. Specifically, the isoprenoid lipids, such as the amphiphilic compound represented by general formula (I), have a viscosity of preferably 15.0 Pa·s or less, more preferably 11.0 Pa·s or less, and even more preferably 6.0 Pa·s or less, measured at 25°C. This viscosity can be measured, for example, at a temperature of 25°C using a viscosity and viscoelasticity analyzer (Gemini II, Malvern).

[0041] The composition according to the present invention may contain a salt of an amphiphilic compound represented by general formula (I). The salt of the amphiphilic compound represented by general formula (I) according to the present invention may be any salt, including salts of alkali metals such as sodium, potassium, calcium, and magnesium, or alkaline earth metals, but sodium salts and potassium salts are preferred. The salt of the amphiphilic compound represented by general formula (I) according to the present invention is preferably a pharmaceutically acceptable salt.

[0042] The composition according to the present invention may contain one or more isoprenoid lipids. The composition according to the present invention may contain one or more amphiphilic compounds represented by general formula (I). When the composition according to the present invention contains multiple isoprenoid lipids, the weight ratio of those isoprenoid lipid species is not particularly limited. When the composition according to the present invention contains multiple amphiphilic compounds represented by general formula (I), the weight ratio of those amphiphilic compound species is not particularly limited. In one embodiment, the composition according to the present invention may contain mono-O-(5,9,13,17-tetramethyloctadeca-4-enoyl)sorbitan and mono-O-(5,9,13,17-tetramethyloctadeca-4-enoyl)isosorbide in a weight ratio of 50:50 to 99:1, preferably 60:40 to 90:10, 70:30 to 90:10, or 80:20 to 90:10, for example, 8:2 or 85:15. In one embodiment, the composition according to the present invention may contain mono-O-(5,9,13,17-tetramethyloctadecanoyl)sorbitan and mono-O-(5,9,13,17-tetramethyloctadecanoyl)isosorbide in a weight ratio of 50:50 to 99:1, preferably 60:40 to 90:10, 70:30 to 90:10, or 80:20 to 90:10, for example, 8:2 or 85:15.

[0043] The composition according to the present invention includes a fluorescent dye and an isoprenoid-type lipid (for example, an amphiphilic compound represented by general formula (I)), in addition to a phospholipid. The phospholipid used in the present invention is not limited to the following, but examples include one or more phospholipids selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerin, phosphatidic acid, and sphingomyelin, and their salts, or a phospholipid preparation or fraction containing the same. Examples of phosphatidylcholine include, but are not limited to, soy phosphatidylcholine (SPC; also called soy lecithin), dioleyl phosphatidylcholine (DOPC), dimyristoyl phosphatidylcholine (DMPC), dipalmitoyl phosphatidylcholine (DPPC), and egg yolk phosphatidylcholine (EPC; also called egg yolk lecithin). Examples of phosphatidylethanolamines include, but are not limited to, dioleylphosphatidylethanolamine (DOPE). Examples of phosphatidylglycerin include dioleylphosphatidylglycerin, and examples of phosphatidylglycerin salts include, but are not limited to, dioleylphosphatidylglycerin sodium (DOPG-Na). In one embodiment, the phospholipid used in the present invention comprises at least one selected from the group consisting of phosphatidylcholine and phosphatidylethanolamine. The phospholipid used in the present invention may be a synthetic product or of natural origin. The composition according to the present invention may contain one or more phospholipids.

[0044] The weight ratio of isoprenoid lipids to phospholipids in the composition according to the present invention is not limited to the following, but isoprenoid lipids:phospholipids = 90:10 to 10:90, for example, 80:20 to 20:80. In one embodiment, in the composition according to the present invention containing isoprenoid lipids, phospholipids and oil (described later), isoprenoid lipids:phospholipids (weight ratio) = 60:40 to 30:70, and more preferably 40:60 to 30:70. In one embodiment, isoprenoid lipids:phospholipids (weight ratio) = 80:20, 60:40, 50:50, 40:60, or 30:70.

[0045] The weight ratio of the amphiphilic compound represented by general formula (I) to the phospholipid in the composition according to the present invention is not limited to the following, but it is preferably 90:10 to 10:90, for example, 80:20 to 20:80. In one embodiment, in the composition according to the present invention comprising the amphiphilic compound represented by general formula (I), a phospholipid, and an oil (described later), the weight ratio of the amphiphilic compound to the phospholipid is preferably 60:40 to 30:70, and more preferably 40:60 to 30:70. In one embodiment, the weight ratio of the amphiphilic compound to the phospholipid may be 80:20, 60:40, 50:50, 40:60, or 30:70.

[0046] The weight ratio of isoprenoid lipids (for example, amphiphilic compounds represented by general formula (I)) to phospholipids is calculated using the total amount (weight) of isoprenoid lipids when multiple types are used, and the total amount (weight) of phospholipids when multiple types are used. In this specification, the terms "weight" and "mass" are used interchangeably.

[0047] The composition according to the present invention preferably further contains a biocompatible organic solvent. In the present invention, "biocompatible" refers to the property of being unlikely to cause adverse reactions in the body (low toxicity) or not when administered into the tissues of animal organs. The biocompatible organic solvent in the present invention may be a pharmaceutically acceptable organic solvent. Examples of biocompatible organic solvents in the present invention include, but are not limited to, alcohols (monohydric or dihydric or higher polyhydric alcohols) such as ethanol (e.g., anhydrous ethanol), propylene glycol, 1,3-butylene glycol, isopropanol, and glycerin, ethers such as diethyl ether and polyethylene glycol, dimethyl sulfoxide (DMSO), N-methylpyrrolidone (NMP), and dimethylacetamide (DMA), and any combination thereof. The composition according to the present invention may contain a biocompatible organic solvent at a concentration of, for example, 1 w / w% or more, preferably 5 w / w% or more, for example 8 w / w% or more, but is not limited to the following. In one embodiment, the composition according to the present invention contains a biocompatible organic solvent at a concentration of 1 w / w% to 50 w / w%, 1 w / w% to 40 w / w%, 5 w / w% to 20 w / w%, or 8 w / w% to 10 w / w%.

[0048] The composition according to the present invention may or may not contain oil. In the composition according to the present invention, the oil may be, for example, an oil mainly composed of triglycerides, a hydrocarbon oil, and / or other oils, or may contain them. An "oil mainly composed of triglycerides" contains "triglycerides" in a total amount of 70 w / w% or more, preferably 90 w / w% or more, of the lipid components. Examples of "oil mainly composed of triglycerides" include, but are not limited to, vegetable oils, animal oils, medium-chain triglyceride (MCT), and triglycerides. Examples of oils used in the compositions according to the present invention include, but are not limited to, vegetable oils such as sesame oil, soybean oil, corn oil, coconut oil, safflower oil, perilla oil, olive oil, castor oil, and cottonseed oil; animal oils such as egg yolk oil, fish oil, and lanolin; mineral oils such as medium-chain triglycerides (MCT), triglycerides, and liquid paraffin; hydrocarbon oils such as squalene and squalane; ester oils such as isopropyl myristate (IPM); cholesterol, tocopherol, tocopherol acetate, glyceryl dioleate (GDO), gelling hydrocarbons, tetrahydrofarnesylmethyl acetate, and hexahydrogeranylgeranylmethyl acetate, as well as any combination thereof. The oil is preferably a pharmaceutically acceptable oil. In this invention, the term "oil" does not include the above-mentioned isoprenoid lipids (such as amphiphilic compounds represented by general formula (I)) and phospholipids.

[0049] The composition according to the present invention may contain oil at a concentration exceeding 0 w / w%, 70 w / w% or less, preferably 60 w / w% or less, more preferably 50 w / w% or less, for example preferably 40 w / w% or less, or 30 w / w% or less. In one embodiment, the concentration of oil in the composition according to the present invention may be 0 w / w% to 70 w / w%, 5 w / w% to 70 w / w%, 0 w / w% to 50 w / w%, 5 w / w% to 50 w / w%, 0 w / w% to 40 w / w%, 5 w / w% to 40 w / w%, 0 w / w% to 30 w / w%, or 5 w / w% to 30 w / w%.

[0050] The composition according to the present invention may contain one or more isoprenoid-type lipids (for example, amphiphilic compounds represented by general formula (I)) in a total amount that is not limited to the following, but is 5 w / w% or more, preferably 10 w / w% or more, typically 10 w / w% to 80 w / w%, for example, 15 w / w% to 80 w / w%, 15 w / w% to 60 w / w%, 15 w / w% to 50 w / w%, 40 w / w% to 80 w / w%, or 40 w / w% to 60 w / w%.

[0051] The composition according to the present invention may contain the above-mentioned isoprenoid lipids, phospholipids, and oils in a total amount that is not limited to the following, but is 40 w / w% or more, preferably 50 w / w% or more, for example, 70 w / w% or more, 80 w / w% or more, 80 w / w% to 95 w / w%, 88 w / w% to 93 w / w%, or 89 w / w% to 91 w / w% of the composition according to the present invention.

[0052] The composition according to the present invention may contain, but is not limited to, an amphiphilic compound represented by general formula (I), a phospholipid, and an oil in total amounts of 40 w / w% or more, preferably 50 w / w% or more, for example, 70 w / w% or more, 80 w / w% or more, 80 w / w% to 95 w / w%, 88 w / w% to 93 w / w%, or 89 w / w% to 91 w / w%.

[0053] In one embodiment, the composition according to the present invention may contain a specific combination of isoprenoid lipids and phospholipids used in any of the formulations in Table 1 or 2 described below. In one embodiment, the composition according to the present invention may contain a specific combination of isoprenoid lipids and phospholipids used in any of the formulations in Table 1 or 2, within the range of the weight ratio of the isoprenoid lipids to phospholipids shown in Table 1 or 2. In one embodiment, the composition according to the present invention may contain a specific combination of isoprenoid lipids and oils used in any of the formulations in Table 1 or 2. In one embodiment, the composition according to the present invention may contain a specific combination of isoprenoid lipids and oils used in any of the formulations in Table 1 or 2, within the range of the weight ratio of the isoprenoid lipids to oils shown in Table 1 or 2. In one embodiment, the composition according to the present invention may contain a specific combination of phospholipids and oils used in any of the formulations in Table 1 or 2. In one embodiment, the composition according to the present invention may contain a specific combination of phospholipids and oils used in any of the formulations in Table 1 or 2, within the range of the weight ratio of the phospholipids to oils shown in Table 1 or 2. In one embodiment, the composition according to the present invention may contain a specific combination of isoprenoid lipids, phospholipids, and oils used in any of the formulations in Table 1 or 2. In one embodiment, the composition according to the present invention may contain a specific combination of isoprenoid lipids, phospholipids, and oils used in any of the formulations in Table 1 or 2, within the weight ratio range of the isoprenoid lipids, phospholipids, and oils shown in Table 1 or 2.

[0054] The composition according to the present invention may further contain a water-soluble polymer. Examples of water-soluble polymers include, but are not limited to, hydroxypropyl cellulose (HPC), hydroxyethyl cellulose, polyvinylpyrrolidone, carbopol, carrageenan, chitosan, chondroitinate, xanthan gum, hyaluronic acid (such as sodium hyaluronate), alginate (such as sodium alginate), gelatin, and dextran, as well as any combination thereof. Examples of hydroxypropyl cellulose (HPC) include five grades of HPC commercially available from Nippon Soda Co., Ltd. (Japan): HPC-SSL (molecular weight approximately 40,000, viscosity 2-2.9 mPa·s), HPC-SL (molecular weight approximately 100,000, viscosity 3-5.9 mPa·s), HPC-L (molecular weight approximately 140,000, viscosity 6-10 mPa·s), HPC-M (molecular weight approximately 620,000, viscosity 150-400 mPa·s), and HPC-H (molecular weight approximately 910,000, viscosity 1,000-4,000 mPa·s). In one embodiment, the hydroxypropyl cellulose may have a molecular weight of 1,000,000 or less, or 800,000 or less, for example, 10,000-700,000 or 10,000-80,000. The compositions according to the present invention may contain a water-soluble polymer (e.g., HPC) at a concentration of, but is not limited to, 0.1 w / w% to 3 w / w%, preferably 0.5 w / w% to 2 w / w%.

[0055] The composition according to the present invention may further contain components other than those listed above, as long as they are suitable for fluorescent marking of biological tissues. The composition according to the present invention may further contain, for example, pharmaceutically acceptable pharmaceutical additives (e.g., carriers, excipients, lubricants, disintegrants, wetting agents, buffers, flavoring agents, preservatives, colorants, fragrances, or propellants). The composition according to the present invention may or may not contain surfactants.

[0056] The composition according to the present invention is preferably a liquid crystal precursor composition. In the present invention, a liquid crystal precursor composition is a composition that does not itself form a liquid crystal such as a non-lamellar liquid crystal, but is capable of forming a liquid crystal such as a non-lamellar liquid crystal in the presence of an aqueous medium. The composition according to the present invention is preferably a liquid crystal precursor composition (non-lamellar liquid crystal precursor composition) capable of forming a non-lamellar liquid crystal in the presence of an aqueous medium. The composition according to the present invention, which is a liquid crystal precursor composition, is preferably a liquid composition. The composition according to the present invention, which is a liquid crystal precursor composition, does not form a liquid crystal such as a non-lamellar liquid crystal before application to biological tissue. The composition according to the present invention, which is a liquid crystal precursor composition, does not contain an aqueous medium, or does not contain an amount of aqueous medium sufficient to form a non-lamellar liquid crystal structure. In the present invention, "aqueous medium" means a solvent containing water, and is not limited to the following, but includes water such as water for injection, sterile water, purified water, distilled water, ion-exchanged water, ultrapure water, physiological saline, phosphate buffer, etc. The liquid crystal precursor composition according to the present invention does not contain structures such as fine particles as seen in emulsions, for example. The liquid crystal precursor composition according to the present invention is preferably a homogeneous liquid in which layer separation or solid precipitation is not observed in appearance.

[0057] The composition according to the present invention, by using an isoprenoid lipid (for example, an amphiphilic compound represented by general formula (I)) and a phospholipid in combination, enables the fluorescent dye to preferably remain at the application site for a long period of time in the biological tissue to which the composition is applied, and enables the retention of fluorescence emission by the fluorescent dye (retention of the visibility of the fluorescent marker). For this reason, the composition according to the present invention is useful as a composition for fluorescent marking of biological tissue, and is particularly useful for localized fluorescent marking of biological tissue.

[0058] The composition according to the present invention can form a non-lamellar liquid crystal (liquid crystal gel) in the presence of an aqueous medium. When the composition according to the present invention is applied (e.g., by injection, coating, dropping, spraying, etc.) to or from biological tissue (e.g., organs) or on the surface of biological tissue (e.g., organs), a non-lamellar liquid crystal (liquid crystal gel) is formed when the composition according to the present invention comes into contact with moisture such as bodily fluids (e.g., blood, tissue fluid, or lymph). The non-lamellar liquid crystal formed from the composition according to the present invention within or on the surface of biological tissue remains at the application site of the composition according to the present invention, retaining a fluorescent dye internally, and emits fluorescence upon irradiation with excitation light. As a result, the composition according to the present invention applied to biological tissue can enable localized fluorescent marking of the biological tissue.

[0059] The "non-lamellar liquid crystal" formed by the composition according to the present invention in the presence of an aqueous medium is a liquid crystal structure that is not a lamellar liquid crystal, and specifically, it may be, for example, a cubic liquid crystal, an inverse hexagonal liquid crystal (HII), an inverse micelle cubic phase (Fd3m), or a sponge phase (L3). The non-lamellar liquid crystal may contain two or more non-lamellar liquid crystal phases.

[0060] A cubic liquid crystal can be a cubic liquid crystal belonging to the crystallographic space group Ia3d (hereinafter referred to as Ia3d cubic liquid crystal), a cubic liquid crystal belonging to the crystallographic space group Pn3m (hereinafter referred to as Pn3m cubic liquid crystal), or a cubic liquid crystal belonging to the crystallographic space group Im3m (hereinafter referred to as Im3m cubic liquid crystal).

[0061] The structure of liquid crystals can be analyzed using conventional methods such as small-angle X-ray scattering (SAXS) measurements.

[0062] By checking whether the following scattering peak ratios (peak spacings) specific to each liquid crystal structure are observed as a result of SAXS measurement, the liquid crystal structure formed from the composition according to the present invention in the presence of an aqueous medium can be confirmed. Ratio of Pn3m cubic liquid crystal: √2:√3:√4:√6:√8:√9:√10:,,,, Ratio of Ia3d cubic liquid crystal: √3:√4:√7:√8:√10:√11:,,,, Ratio of Im3m cubic liquid crystal: √2:√4:√6:√8:√10:√12:√14:,,,, Ratio of Fd3m cubic liquid crystal: √3:√8:√11:√12:√16:√19:√24:√27:,,,, Ratio specific to inverse hexagonal liquid crystal: 1:√3:2:,,,,

[0063] Furthermore, by calculating the peak values ​​from the SAXS intensity distribution data according to a method well known to those skilled in the art, and then finding the ratio of their reciprocals, the space group and lattice constants can be easily determined.

[0064] On the other hand, in SAXS measurements of the sponge phase (L3 phase), a broad scattering peak is observed.

[0065] The scattering vector value q1 [nm] of the peak located at the smallest angle. －１ By analyzing the scattering vector values ​​of peaks in measurement samples, including [specific example], it is possible to determine not only the type of liquid crystal phase but also its interplanar spacing and lattice constant.

[0066] The compositions according to the present invention are extremely useful for locally fluorescently marking biological tissues. In particular, the compositions according to the present invention are extremely useful for locally fluorescently marking tissues of tubular organs (more preferably the gastrointestinal tract), such as the gastrointestinal tract, trachea, bladder, urethra, and blood vessels, from the inside of the tubular organ (intraluminal; mucosal side). For example, the compositions according to the present invention are extremely useful for fluorescently marking cancerous tissue that has developed inside (intraluminally) biological tissues, particularly tubular organs (more preferably the gastrointestinal tract), such as the gastrointestinal tract, trachea, bladder, urethra, and blood vessels, for the purpose of preoperative marking for surgical resection.

[0067] In one embodiment, the composition according to the present invention may be a composition useful for biotissue fluorescent marking, for injection into biological tissue to locally fluorescently mark the biological tissue. In one embodiment, the composition according to the present invention may be a composition useful for biotissue fluorescent marking, for application to the surface of biological tissue to locally fluorescently mark the biological tissue.

[0068] The present invention also provides a method for locally fluorescently marking biological tissue, which includes locally applying the composition according to the present invention to the tissue of any organ, such as the digestive tract, trachea, bladder, urethra, blood vessels, and other tubular organs. This method may be a method for locally fluorescently marking biological tissue, which includes applying (administering) the composition according to the present invention to the tissue of any organ, such as the digestive tract, trachea, bladder, urethra, blood vessels, and other tubular organs, either inside or on the tissue surface. Application to biological tissue can be carried out by, for example, injection, coating, dropping, spraying, etc. Application to biological tissue can be carried out by, for example, injection using a syringe, but is not limited thereto. Injection of the composition according to the present invention using a syringe can be carried out by, for example, a needle (injection needle) of 16 to 30 G (gauge), preferably 18 to 27 G, more preferably 20 to 25 G, and even more preferably 23 to 25 G (e.g., 25 G), but is not limited thereto. Application to the surface of biological tissue can be carried out by, for example, coating, dropping, spraying, etc. In a preferred embodiment, the composition according to the present invention can be applied to biological tissue under the guidance of an endoscope, such as a laparoscope.

[0069] The method of the present invention may further include detecting fluorescence derived from a fluorescent dye contained in the composition according to the present invention. The method of the present invention may further include irradiating a local application site (administration site) with excitation light of the fluorescent dye contained in the composition according to the present invention. The biological tissue to which the composition according to the present invention is applied may be affected tissue such as cancerous tissue. The biological tissue to which the composition according to the present invention is applied may be affected tissue that requires or is scheduled to receive medical treatment (e.g., surgical excision and / or radiation therapy).

[0070] In particular, the present invention provides a method for locally fluorescently marking affected tissue, comprising locally applying the composition according to the present invention to a site where the location of affected tissue (e.g., cancerous tissue) can be identified in any organ, including tubular organs such as the digestive tract, trachea, bladder, urethra, and blood vessels. In a preferred embodiment, the present invention also provides a method for locally fluorescently marking affected tissue in a tubular organ, comprising locally applying the composition according to the present invention to a site where the location of affected tissue (e.g., cancerous tissue) can be identified inside (in the lumen) of a tubular organ such as the digestive tract, trachea, bladder, urethra, and blood vessels. The present invention also provides a method for locally fluorescently marking affected tissue in an organ other than a tubular organ (e.g., heart, brain, liver, skin, muscle, bone, etc.), comprising locally applying the composition according to the present invention to a site where the location of affected tissue (e.g., cancerous tissue) can be identified. The site where the location of affected tissue in an organ can be identified may be, for example, inside the affected tissue, on the surface of the affected tissue, or directly below and / or around the affected tissue. In one embodiment, the composition according to the present invention is locally applied from the inside of a tubular organ to a site where the location of the affected tissue (e.g., cancerous tissue) in the organ can be identified. In a preferred embodiment, the present invention also provides a method for locally fluorescently marking affected tissue in a tubular organ, such as the digestive tract, trachea, bladder, urethra, or blood vessel, which includes locally applying the composition according to the present invention endoscopically to a site where the location of the affected tissue (e.g., cancerous tissue) inside the tubular organ can be identified (e.g., within the affected tissue, on the surface of the affected tissue, or directly beneath and / or surrounding the affected tissue). The method of the present invention may further include detecting fluorescence derived from a fluorescent dye contained in the composition according to the present invention, preferably from the outside (serous side) of the tubular organ. By detecting the fluorescence, the location of the affected tissue in the organ can be accurately identified. In one embodiment of the present invention, using the composition according to the present invention, cancerous tissue that has developed inside (in the lumen) of tubular organs such as the digestive tract, trachea, bladder, urethra, and blood vessels (more preferably the digestive tract) (typically on the mucosa) can be locally fluorescently marked, typically under endoscopy. Subsequently, fluorescence detection can be performed from the serosal side of the cancerous tubular organ, and the location of the cancerous tissue can be identified by fluorescence observation.

[0071] Fluorescence derived from a fluorescent dye can be detected by irradiating a biological tissue to which the composition according to the present invention has been applied with light matching the excitation wavelength (absorption wavelength) of the fluorescent dye used, and detecting the fluorescence emitted from the fluorescent dye at the fluorescence wavelength. For example, when using indocyanine green (ICG) as the fluorescent dye, a wavelength of around 730 to 810 nm can be used as the excitation wavelength (absorption wavelength), and a wavelength of around 820 to 920 nm can be used as the fluorescence wavelength.

[0072] As described above, the present invention also provides a method for treating a disease (e.g., cancer), which includes locally applying the composition according to the present invention to a site in an organ where the location of the affected tissue (e.g., cancerous tissue) can be identified, thereby locally fluorescently marking the affected tissue, detecting the fluorescence derived from the fluorescent dye contained in the composition according to the present invention to identify the location of the affected tissue, and further performing a medical procedure on the identified affected tissue (e.g., surgical resection and / or radiation therapy). Surgical resection can be performed by conventional methods, and may be a highly invasive surgery such as abdominal surgery or thoracotomy, or a relatively less invasive surgery such as endoscopic surgery including laparoscopic surgery. Radiation therapy can be performed by conventional methods, and the treatment schedule, such as radiation dose and treatment period, can be appropriately determined by a person skilled in the art depending on the type and condition of the disease (e.g., cancer).

[0073] The biological tissues or affected tissues to which the compositions according to the present invention are applied include, but are not limited to, tissues of tubular organs such as the digestive tract, trachea, bladder, urethra, and blood vessels. Examples of the digestive tract include the oral cavity, esophagus, stomach, small intestine, large intestine, or anus. The compositions according to the present invention are preferably applied to biological tissues, affected tissues, or organs within the body, but may also be applied to biological tissues, affected tissues, or organs that have been removed from the body (for example, isolated from the body).

[0074] The composition according to the present invention may be applied to the living tissue of any subject. The subject may be, but is not limited to, humans, primates such as gorillas, chimpanzees, and orangutans, rodents such as mice, rats, and hamsters, livestock such as horses, cattle, sheep, goats, and camels, and mammals such as dogs, cats, and rabbits, and preferably humans. In one embodiment, the subject to which the composition according to the present invention is applied is a patient who is scheduled to undergo surgical resection of affected tissue, preferably a patient who is scheduled to undergo surgical resection of cancerous tissue in a tubular organ such as the digestive tract.

[0075] The composition according to the present invention can be applied to living tissue by any method such as injection (local injection), application, dropping, or spraying. In one embodiment, the composition according to the present invention can be applied to the affected tissue (e.g., cancerous tissue) by injection into the affected tissue, on the surface of the affected tissue, or directly beneath and / or around the affected tissue. The composition according to the present invention should be applied to living tissue, such as affected tissue, in an amount effective for fluorescent marking. In one embodiment, the composition according to the present invention can be applied to living tissue to be fluorescently marked, such as affected tissue, in an amount of 10 μL to 500 μL, for example, 50 μL to 200 μL per application site, but the amount applied is not limited to this range.

[0076] Fluorescent marking of biological tissue using the composition according to the present invention can stably maintain localized fluorescence emission for a long period of time after application to the biological tissue. In a preferred embodiment, fluorescent marking of biological tissue using the composition according to the present invention does not cause the fluorescent dye to diffuse and disappear from the application site for 5 days or more, preferably 7 days or more, more preferably 10 days or more, even more preferably 14 days or more, for example 28 days or more, after application to the biological tissue, and a high level of fluorescence can be detected at the application site.

[0077] The present invention will be described in more detail below using examples. However, the technical scope of the present invention is not limited to these examples.

[0078] [Example 1] Synthesis of amphiphilic compounds (1) Synthesis of mono-O-(5,9,13,17-tetramethyloctadeca-4-enoyl)sorbitan

[0079]

[0080] 70.5 g (200 mmol) of methyl 5,9,13,17-tetramethyloctadeca-4-enoate and 54.7 g (300 mmol, sorbitan M-90, Sanko Chemical Industry Co., Ltd., containing 90% solids and 10% water, solids content: sorbitan 79-84%, isosorbide 15-18%) of a 90% by weight aqueous solution of sorbitan were added to a reaction vessel at room temperature, and the mixture was stirred at 120°C and 8 kPa for 1 hour. The pressure was released with nitrogen, and 2.2 g (40 mmol) of sodium methoxide and 0.04 g of sodium phosphinate monohydrate were added, followed by stirring at 160°C and 8 kPa for 1 hour. The pressure was released with nitrogen, and 1.1 g (20 mmol) of sodium methoxide was added, followed by stirring at 160°C and 8 kPa for another hour. The pressure was released with nitrogen, and 1.1 g (20 mmol) of sodium methoxide was added again. The mixture was then stirred at 160°C and 8 kPa for 1.5 hours. After cooling to 60°C, 200 mL of ethyl acetate and 200 mL of 0.5 M hydrochloric acid were added while stirring. 600 mL of ethyl acetate was added to the resulting reaction mixture and extracted. The extract was sequentially washed with saturated sodium bicarbonate solution and saturated saline solution, dried over magnesium sulfate, filtered, and concentrated. The resulting residue was purified by silica gel column chromatography (mobile phase: ethyl acetate / hexane mixture) to obtain 36.1 g (yield 37%) of the compound in question as a light brown transparent liquid. The obtained fraction contained mono-O-(5,9,13,17-tetramethyloctadeca-4-enoyl)sorbitan and mono-O-(5,9,13,17-tetramethyloctadeca-4-enoyl)isosorbide in a ratio of approximately 8:2 (by weight) (calculated from the TIC area value obtained by GC-MS measurement in ion mode EI+). The obtained fraction also contained a small amount of sorbitan-derived diester (estimated by GC-MS measurement and TLC analysis). Regarding the obtained fraction, １ The results of the H-NMR measurements are as follows:

[0081] １ H-NMR spectrum (300 MHz, CDCl) ３ ​, TMS) δ: 0.7-0.9 (m, 12H), 0.9-1.8 (m, 22H), 1.85-2.0 (m, 2H), 2.0-2.5 (m, 4H), 3.5-4.9 (m, 6.4H), 5.04 (m, 1H), 5.0-5.2 (m, 0.6H)

[0082] Mono-O-(5,9,13,17-tetramethyloctadeca-4-enoyl)sorbitan is also called C22 sorbitan ester.

[0083] The obtained fraction was used as mono-O-(5,9,13,17-tetramethyloctadeca-4-enoyl)sorbitan fraction (C22 sorbitan ester fraction) in the examples described later.

[0084] Furthermore, 13.00 g of the C22 sorbitan ester fraction was purified using silica gel column (mobile phase: ethyl acetate / hexane mixture) to remove low-polarity components such as sorbitan-derived diesters and mono-O-(5,9,13,17-tetramethyloctadeca-4-enoyl)isosorbide from the C22 sorbitan ester fraction, yielding 6.78 g of a purified fraction as a slightly yellowish transparent liquid. Its purity was over 99% mono-O-(5,9,13,17-tetramethyloctadeca-4-enoyl)sorbitan (calculated from the TIC area value measured by GC-MS in ion mode EI+). In addition, it contained almost no low-polarity components such as sorbitan-derived diesters (estimated by GC-MS measurement and TLC analysis). Regarding the obtained compound, １ The results of the H-NMR measurements are as follows:

[0085] １ H-NMR spectrum (300 MHz, CDCl) ３ , TMS) δ: 0.8-0.9 (m, 12H), 0.9-1.7 (m, 22H), 1.9-2.0 (m, 2H), 2.2-2.5 (m, 4H), 2.93 (brs, OH), 3.6-4.5 (m, 7.5H), 5.06 (brd, J=5.0Hz, 1H), 4.9-5.2 (m, 0.5H)

[0086] ​The purified fraction obtained as described above is referred to as high-purity mono O-(5,9,13,17-tetramethyloctadec-4-enoyl)sorbitan or high-purity C22 sorbitan ester.

[0087] (2) Synthesis of mono O-(5,9,13,17-tetramethyloctadecanoyl)sorbitan

[0088]

[0089] Under a nitrogen atmosphere, 0.70 g of 5% palladium on carbon was added to a solution of 5.82 g (12.0 mmol) of the aforementioned C22 sorbitan ester fraction in 17.4 mL of ethyl acetate. After replacing nitrogen in the system with hydrogen, the mixture was stirred at room temperature for 2 days under a normal-pressure hydrogen atmosphere. After replacing hydrogen in the system with nitrogen, 5% palladium on carbon was filtered off. The filtrate was purified by silica gel column chromatography (mobile phase: ethyl acetate), to obtain 5.69 g (yield 97%) of a fraction containing the title compound as a colorless transparent liquid. The obtained fraction contained mono O-(5,9,13,17-tetramethyloctadecanoyl)sorbitan and mono O-(5,9,13,17-tetramethyloctadecanoyl)isosorbide at a ratio (weight ratio) of about 8:2 (calculated from the area value of TIC obtained by GC-MS measurement in ion mode EI+). The obtained fraction further contained a small amount of sorbitan-derived diester (estimated by GC-MS measurement and TLC analysis). For the obtained fraction, １ The results of ¹H-NMR measurement are as follows.

[0090] １ ¹H-NMR spectrum (300 MHz, CDCl ３ , TMS) δ: 0.7-0.9 (m, 15H), 0.9-1.7 (m, 26H), 2.2-2.4 (m, 2H), 3.5-4.9 (m, 6.4H), 5.0-5.2 (m, 0.6H)

[0091] Mono O-(5,9,13,17-tetramethyloctadecanoyl)sorbitan is also referred to as saturated C22 sorbitan ester.

[0092] The obtained fraction was used as mono-O-(5,9,13,17-tetramethyloctadecanoyl)sorbitan fraction (saturated C22 sorbitan ester fraction) in the examples described later.

[0093] (3) Synthesis of mono-O-(5,9,13,17-tetramethyloctadecanoyl)propylene glycol

[0094]

[0095] Under a nitrogen atmosphere, 0.48 g of 5% palladium carbon was added to a solution of 3.96 g (10.0 mmol) of mono-O-(5,9,13,17-tetramethyloctadeca-4-enoyl)propylene glycol in ethyl acetate (12 mL). After replacing the nitrogen in the system with hydrogen, the mixture was stirred at room temperature under atmospheric pressure and hydrogen for 48 hours. After replacing the hydrogen in the system with nitrogen, the 5% palladium carbon was filtered off. The filtrate was purified by silica gel column chromatography (ethyl acetate) to obtain 3.94 g (99% yield) of the title compound as a colorless, transparent liquid. Regarding the obtained compound, １ The results of the H-NMR measurement are as follows:

[0096] １ H-NMR spectrum (300 MHz, CDCl) ３ , TMS) δ: 0.75-0.9 (m, 15H), 0.95-1.7 (m, 29H), 2.28 (brs, OH), 2.30 (m, 2H), 3.54-3.67 (m, 0.62H), 3.88-4.1 (m, 2.07H), 4.97 (m, 0.31H)

[0097] Mono-O-(5,9,13,17-tetramethyloctadecanoyl)propylene glycol is also called saturated C22 propylene glycol ester.

[0098] (4) Synthesis of mono-O-(5,9,13-trimethyltetradeca-4-enoyl)glycerol

[0099]

[0100] ​To a solution of 0.65 g (7.1 mmol) of glycerol and 0.59 g (4.3 mmol) of potassium carbonate in 3.5 mL of dry N,N-dimethylformamide, 1.0 g (3.5 mmol) of methyl 5,9,13-trimethyltetradeca-4-enoate (methyl tetrahydrofarnesyl acetate) was slowly added dropwise at 80°C. After stirring at 100°C for 18 hours, 1 M hydrochloric acid was added to the reaction mixture, and it was extracted with ether. The extract was sequentially washed with saturated sodium bicarbonate solution and saturated saline solution, dried over anhydrous sodium sulfate, filtered, and concentrated. The resulting residue was purified by silica gel column chromatography (mobile phase: ethyl acetate / hexane mixture) to obtain the title compound as a colorless, transparent liquid. Regarding the obtained compound, １ The results of the H-NMR and viscosity measurements are as follows:

[0101] １ H-NMR spectrum (300 MHz, CDCl) ３ , TMS) δ: 0.80-0.90 (m, 9H), 1.00-1.70 (m, 15H), 1.97 (td, J = 7.8, 17.0Hz, 2H), 2.13 (t, J = 6.1Hz, 1H, OH), 2.25-2.45 (m, 4H), 2.55 (d, J = 5.2Hz, 1H, OH), 3.50-4.00 (m, 3H), 4.10-4.25 (m, 2H), 5.08 (t, J = 6.7Hz, 1H) Viscosity: 0.48 Pa・s (shear rate 92 1 / s)

[0102] Mono-O-(5,9,13-trimethyltetradeca-4-enoyl)glycerol is also known as C17 glycerol ester.

[0103] (5) Synthesis of mono-O-(5,9,13,17-tetramethyloctadeca-4-enoyl)glycerol

[0104]

[0105] ​Under reduced pressure of 60-70 mmHg and a nitrogen stream, 23.5 g (255 mmol) of glycerol and 0.55 g (4.0 mmol) of potassium carbonate were dissolved in 48 mL of dry N,N-dimethylformamide. 28.2 g (80.0 mmol) of methyl 5,9,13,17-tetramethyloctadeca-4-enoate was gradually added dropwise at 80°C, and the mixture was stirred at the same temperature for 3 hours. The resulting reaction solution was diluted with ethyl acetate / hexane mixed solvent (1:1,200 mL), washed with water, saturated sodium bicarbonate solution, and saturated brine (twice), and then dried over magnesium sulfate. After filtration and concentration, the resulting residue was purified by silica gel column chromatography (mobile phase: hexane / ethyl acetate = 100:0-30:70) to obtain 13.3 g (40% yield) of the title compound as a slightly yellowish transparent liquid. Regarding the obtained compound, １ The results of the H-NMR measurement are as follows:

[0106] １ H-NMR spectrum (300 MHz, CDCl) ３ , TMS) δ: 0.80-0.95 (m, 12H), 1.00-1.70 (m, 22H), 1.85-2.15 (m, 2H), 2.15-2.55 (m, 4H), 3 .53-3.78 (m, 3H), 3.80-4.00 (m, 1H), 4.10-4.25 (m, 2H), 5.08 (dd, J=6.9Hz, J=6.9Hz, 1H)

[0107] Mono-O-(5,9,13,17-tetramethyloctadeca-4-enoyl)glycerol is also called C22 glycerol ester.

[0108] (6) Synthesis of mono-O-(5,9,13-trimethyltetradeca-4,8,12-trienoyl)glycerol

[0109] ​Under reduced pressure of 200–250 mmHg, 13.9 g (50.0 mmol) of 5,9,13-trimethyltetradeca-4,8,12-trienoic acid methyl (farnesyl acetate) was gradually added dropwise to a solution of 9.2 g (0.10 mol) of glycerol and 0.28 g (2.0 mmol) of potassium carbonate in 20 mL of dry N,N-dimethylformamide at 85°C, and the mixture was stirred at the same temperature for 3 hours. During this time, methanol produced in the reaction was removed by distillation. The resulting reaction solution was diluted with ethyl acetate / hexane mixed solvent (1:1,150 mL), washed with water, saturated sodium bicarbonate solution, and saturated brine (twice), and then dried over magnesium sulfate. After filtration and concentration, the resulting residue was purified by silica gel column chromatography (hexane / ethyl acetate = 100:0 to 0:100) to obtain 8.22 g (yield 49%) of the title compound as a colorless, transparent liquid. １ The results of the H-NMR and viscosity measurements are as follows:

[0110] １ H-NMR spectrum (270 MHz, CDCl) ３ , TMS) δ: 1.5-1.8 (m, 12H), 1.9-2.1 (m, 8H), 2.1 (brs, 1H, OH), 2.25-2.45 (m, 4H), 2.56 (brs, 1H, OH), 3.59 (dd, J = 5.6, 11.2Hz, 1H), 3.68 (dd, J = 3.6, 11.2Hz, 1H), 3.92 (m, 1H), 4.14 (dd, J = 6.0, 11.6Hz, 1H), 4.21 (dd, J = 4.8, 11.6Hz, 1H), 5.02-5.16 (m, 3H) Viscosity: 0.26 Pa・s (shear rate 92 1 / s)

[0111] The synthesized mono-O-(5,9,13-trimethyltetradeca-4,8,12-trienoyl)glycerol is also called farnesyl acetate glyceryl.

[0112] ​[Example 2] After preparation of ICG-containing formulations, gel formation test, and analysis of liquid crystal structure, indocyanine green (hereinafter also referred to as ICG), the C22 sorbitan ester fraction obtained in Example 1 as an isoprenoid lipid, soy phosphatidylcholine (abbreviated as SPC; LIPOID S100, Lipoid Co., Ltd.) as a phospholipid, sesame oil as an oil component, and ethanol (anhydrous ethanol; the same applies hereafter unless otherwise specified) as a biocompatible organic solvent were mixed according to the mixing ratios shown in Table 1. In the table, "EtOH" refers to ethanol (the same applies to Tables 1 to 3). The resulting mixture was stirred in a water bath at 40°C or below to prepare ICG-containing formulations No. 1 to 15 shown in Table 1 as homogeneous solutions.

[0113] Although ICG is extremely insoluble in ethanol alone, the coexistence of ethanol and lipids made it possible to prepare a formulation in which ICG was uniformly dissolved, even at high concentrations (e.g., 0.5%).

[0114] Furthermore, according to the mixing ratios shown in Table 2 below, ICG was mixed with isoprenoid lipids such as C22 sorbitan ester fraction, saturated C22 sorbitan ester fraction, saturated C22 propylene glycol ester, farnesyl acetate glyceryl, C17 glycerin ester, or C22 glycerin ester, phospholipids such as SPC, purified egg yolk lecithin (abbreviated as EPC; PL-100M, Kewpie Corporation), dioleyl phosphatidylcholine (DOPC; D4250, Tokyo Chemical Industry Co., Ltd.), or dioleyl phosphatidylethanolamine (DOPE; D4251, Tokyo Chemical Industry Co., Ltd.), oils such as sesame oil, soybean oil, or squalene, and ethanol. In formulation No. 16, these components were mixed with hydroxypropyl cellulose (HPC; HPC-SSL, Nippon Soda Co., Ltd.), a water-soluble polymer. The resulting mixtures were stirred in a water bath at 40°C or below to prepare homogeneous solutions of ICG-containing formulations No. 16 to 26 shown in Table 2. Note that the C22 sorbitan ester fraction, saturated C22 sorbitan ester fraction, farnesyl acetate glyceryl, C17 glycerin ester, and C22 glycerin ester are non-lamellar liquid crystal forming lipids, but saturated C22 propylene glycol ester is not a non-lamellar liquid crystal forming lipid.

[0115] Furthermore, without using either isoprenoid lipids or phospholipids, the components were mixed according to the mixing ratios shown in Table 3 below, and the resulting mixture was stirred in a water bath at 40°C or below to prepare ICG-containing formulations No. 27 to 29 as homogeneous solutions.

[0116] A gel formation test was performed on ICG-containing formulations No. 1 to 29 obtained in this manner. A portion of each formulation (approximately 100 to 300 mg) was added to excess sterile water for injection in a vial and mixed at room temperature (25°C) using a spatula and / or a vortex mixer. As a result, ICG-containing formulations No. 1 to 26 yielded a transparent to cloudy, green gel-like composition separated in the excess aqueous medium. On the other hand, ICG-containing formulation No. 27 yielded a green oily substance separated in the excess aqueous medium, while ICG-containing formulations No. 28 and 29 yielded a green suspension that was diffused throughout.

[0117] The gel-like compositions obtained from ICG-containing formulations No. 1 to 26, the green oily substance obtained from ICG-containing formulation No. 27, and the green suspensions obtained from ICG-containing formulations No. 28 and 29 were embedded directly into pinhole slits, and their liquid crystal structures were analyzed by performing small-angle X-ray scattering diffraction measurements using a small-angle X-ray scattering (SAXS) instrument (Rigaku Corporation, Nano-Viewer).

[0118] The obtained liquid crystal phase and the scattering vector value q1 [nm] of the peak located at the smallest angle. －１ The results are shown in Tables 1-3. Note that HII represents an inverse hexagonal liquid crystal, Pn3m represents an inverse cubic liquid crystal belonging to the crystallographic space group Pn3m, and L2 represents an inverse micelle phase. Gel compositions having two different types of liquid crystal phases also existed (ICG-containing formulations No. 3 and 15).

[0119] As shown in Table 1, non-lamellar liquid crystals were formed from ICG-containing formulations at a wide range of isoprenoid lipid:SPC (weight ratio) ratios using C22 sorbitan ester as the isoprenoid lipid. Furthermore, non-lamellar liquid crystals were formed from ICG-containing formulations at a wide range of isoprenoid lipid + SPC (total amount of isoprenoid lipid and SPC):sesame oil (weight ratio) ratios (100:0 to at least 30:70). It was also revealed that liquid crystal structures such as inverse cubic liquid crystals and inverse hexagonal liquid crystals, whose crystallographic space group belongs to Pn3m, appeared depending on the various blending ratios of the isoprenoid lipid, SPC, and sesame oil, and that the q1 value due to the lattice constant took on a wide range of values. ICG-containing formulations No. 1 to 15 shown in Table 1 were shown to be non-lamellar liquid crystal precursor formulations (non-lamellar liquid crystal precursor compositions).

[0120] Furthermore, as shown in Table 2, non-lamellar liquid crystals were formed from ICG-containing formulations using a variety of isoprenoid lipids, phospholipids, and oils. Even when isoprenoid lipids that are not non-lamellar liquid crystal forming lipids were used, non-lamellar liquid crystals were formed when combined with phospholipids. In addition, even when a certain amount of water-soluble polymer (HPC) was added as an additive, liquid crystal gels were successfully formed from the ICG-containing formulations. ICG-containing formulations No. 16 to 26 shown in Table 2 were also shown to be non-lamellar liquid crystal precursor formulations (non-lamellar liquid crystal precursor compositions).

[0121] On the other hand, as shown in Table 3, no gel was formed from ICG-containing formulations that did not contain isoprenoid lipids, contained SPC as a phospholipid, and contained or did not contain sesame oil (triglycerides) as an oil component; only peaks presumed to be lamellar liquid crystals were detected. Furthermore, no liquid crystal gel was formed from an ICG-containing formulation that contained C22 sorbitan ester, an isoprenoid lipid, did not contain phospholipids, and contained sesame oil (triglycerides) in a high ratio relative to the amount of isoprenoid lipids (Formulation No. 27 in Table 3).

[0122]

[0123]

[0124]

[0125] [Example 3] Fluorescence observation of ICG-containing formulations (1) To each of the 100 μL of ICG-containing formulations prepared in Example 2, the same amount (100 μL) of fresh blood collected from the ear vein of a rabbit using a syringe was dropped and lightly mixed on a well plate to form a mixture. Using a fluorescence camera (HEMS plus + MNIRC-600, manufactured by Mizuho), near-infrared light with an excitation wavelength of 730 nm was irradiated onto the mixture from a distance of 20 cm, and the presence or absence of fluorescence emission from approximately 820 nm to approximately 900 nm was observed. For this fluorescence observation, ZeoClip FS (manufactured by Zeon Medical), a medical device for fluorescent marking with a fluorescent resin part, was used as a comparative sample.

[0126] The fluorescence camera used for fluorescence observation is a device with a proven track record of use in medical settings. It emits near-infrared light at the excitation wavelength of the fluorescent dye and simultaneously receives near-infrared fluorescence with a light-receiving unit, allowing the fluorescence emission state to be displayed on a monitor.

[0127] Fluorescence observation was performed continuously from the day the ICG-containing preparation was mixed with fresh blood until two weeks later. Preparations were classified as "S" if the liquid crystal gel maintained its shape for two weeks and fluorescence equal to or greater than that of the comparison sample was observed with a fluorescence camera; "A" if the gel maintained its shape for two weeks and weaker fluorescence than the comparison sample was observed with a fluorescence camera; "B" if the gel's shape had collapsed after two weeks and weaker fluorescence than the comparison sample was observed with a fluorescence camera; and "C" if no fluorescence was visible with the fluorescence camera from the time of mixing with fresh blood. The results are shown in Table 4. However, even in the case of a "B" rating, fluorescence detection was possible at the marked locations. On the other hand, ICG-containing preparation No. 27, which did not contain phospholipids, did not show any fluorescence detection and received a "C" rating, indicating that phospholipids are necessary for fluorescence detection.

[0128] Furthermore, when the test was conducted using the same method except that water was used instead of fresh blood, the fluorescence observation results obtained when the ICG-containing preparation was mixed with water were almost the same as those obtained when fresh blood was used.

[0129]

[0130] [Example 4] Fluorescence observation of ICG-containing preparations (2) Of the ICG-containing preparations that showed stronger fluorescence emission compared to the comparison sample in the fluorescence observation of Example 3, preparations No. 11 and 13 were used for fluorescence observation using stomachs extracted from edible pigs. Using a syringe with a 25G needle attached to the tip, 100 μL each of ICG-containing preparations No. 11 and 13 were injected locally into different sites on the mucosal side of a section of the stomach of an edible pig. As a comparison sample, a ZeoClip FS attached to a ZeoClip device (manufactured by Zeon Medical) was clipped to the mucosal side of the stomach. The presence or absence of fluorescence was observed from the serosal side of the stomach using a fluorescence camera for both the preparation samples of the present invention and the ZeoClip FS. The fluorescence observation images are shown in Figure 1.

[0131] When the stomach wall thickness was approximately 2 mm, the ICG-containing preparation of the present invention and the comparative sample showed comparable fluorescence intensity when viewed from the serosal side of the stomach using a fluorescence camera. However, when the stomach wall thickness increased to approximately 4 mm, fluorescence from the ICG-containing preparation of the present invention was visible from the serosal side of the stomach using a fluorescence camera, but fluorescence from the comparative sample was not visible from the serosal side of the stomach using a fluorescence camera. Since the ICG-containing preparation of the present invention can be injected into tissue, it was shown to be suitable for fluorescently marking areas in thick tissue.

[0132] [Example 5] Subcutaneous tissue implantation study in experimental animals A two-week subcutaneous tissue implantation study was conducted in experimental animals (rabbits) using ICG-containing formulation No. 11. 100 μL of ICG-containing formulation No. 11 was administered subcutaneously into the ear of experimental animals (rabbits) under aspiration anesthesia with cervofuran using a syringe fitted with a 25G needle. The presence or absence of fluorescence at the subcutaneous injection site was observed using a fluorescence camera (HEMS plus + MNIRC-600, Mizuho). The fluorescence observation image is shown in Figure 2.

[0133] Fluorescence observation was performed continuously from the day of administration to the experimental animals (rabbits) (day 0) until two weeks later. No changes were observed in the size or intensity of the luminescent area visible with the fluorescence camera until two weeks later, confirming that the fluorescent dye remained locally at the injection site.

[0134] [Example 6] Two-week skin surface implantation test in experimental animals A two-week skin surface implantation test was conducted on experimental animals (rabbits) using ICG-containing formulation No. 11. After shaving the back of the neck of the experimental animals (rabbits), ICG-containing formulation No. 11 was applied to the skin surface using a brush. The presence or absence of fluorescence at the skin application site was observed using a fluorescence camera (HEMS plus + MNIRC-600, Mizuho). The fluorescence observation image is shown in Figure 3.

[0135] Fluorescence observation was performed continuously from the day of administration to the experimental animals (rabbits) (day 0) until two weeks later. No changes were observed in the size or intensity of the luminescent area visible with the fluorescence camera until two weeks later, confirming that the fluorescent dye remained locally at the application site.

[0136] [Example 7] Verification of the operability of ICG-containing formulations as fluorescent marking materials In this example, in order to verify the operability as fluorescent marking materials, a comparative test of needle penetration (extrusion pressure) was performed on an ICG-containing formulation using glyceryl monooleate having a linear oleic acid chain as the fatty acid chain and an ICG-containing formulation using an isoprenoid type lipid.

[0137] First, instead of isoprenoid lipids, glyceryl monooleate (Likemar XO-100, NOF Corporation), which has linear oleic acid as the fatty acid chain, was used. ICG-containing formulations No. 30 and 31 were prepared as homogeneous solutions by mixing and dissolving them in the same manner as in Example 2, according to the mixing ratio shown in Table 5 below.

[0138] The prepared ICG-containing formulations No. 30 and 31 were subjected to a gel formation test in the same manner as in Example 2. As a result, in ICG-containing formulation No. 31, which contained oil, a greenish-white, cloudy gel-like composition was obtained, separated in an excess aqueous medium. On the other hand, in ICG-containing formulation No. 30, which did not contain oil, no gel-like composition was obtained, and the entire solution in the vial became emulsion-like. Since ICG-containing formulation No. 30 did not form a gel and the viscosity of the formulation itself was high, it was excluded from the evaluation of needle permeability.

[0139] The needle penetration (extrusion pressure) of ICG-containing formulation No. 31 using glyceryl monooleate, and ICG-containing formulations No. 11, 22, and 25 using isoprenoid lipids were evaluated. The needle penetration evaluation was performed at room temperature of 22°C using a 25G needle (Terumo Neoras 25G x 1RB, NN-2525R) and a syringe (Terumo Syringe 1 mL, SS-01T) as the small-diameter needles.

[0140] Specifically, a syringe with a 25G needle, filled with 0.8 mL of each ICG-containing preparation, was placed vertically on the center of an electronic balance (Mettler Toledo, ME4002T / 00) with the needle facing upwards and the plunger head positioned vertically. The outer barrel of the syringe was pressed vertically downwards for 5 to 6 seconds, and the measurement value (g) on ​​the electronic balance was read during the latter half of the pressing time (when 0.4 to 0.8 mL was dispensed), when the dispensing rate of the preparation became nearly constant. Each measurement value is shown as the dispensing pressure in Table 6 below.

[0141] ICG-containing formulation No. 31, which used glyceryl monooleate, had a very high extrusion pressure of over 2,800 g. In contrast, ICG-containing formulations No. 11 and 22, which used C22 sorbitan ester fraction or saturated C22 sorbitan ester fraction as isoprenoid lipids, had significantly lower extrusion pressures of 1,300 g and 1,700 g, respectively. Furthermore, ICG-containing formulation No. 25, which used C17 glycerol ester as isoprenoid lipids, also had a significantly lower extrusion pressure, similar to formulations No. 11 and 22, even with a lower oil content.

[0142] The results above indicate that the extrusion pressure of ICG-containing preparations using glyceryl monooleate is very high, making administration through small needles or long catheters difficult. On the other hand, ICG-containing preparations using isoprenoid lipids exhibit significantly lower extrusion pressure, regardless of the number of carbon atoms, degree of unsaturation, or type of hydrophilic group in the isoprenoid lipid chain, and possess needle passability advantageous for injection using small needles and endoscopic procedures, as demonstrated in Examples 4 and 5.

[0143]

[0144]

[0145] [Example 8] Evaluation of the persistence of fluorescence emission The release of the fluorescent dye from the gel in an aqueous medium (persistence of fluorescence emission) of ICG-containing formulation No. 11 was evaluated. At a room temperature of 22°C, 0.1 g of the formulation was gently dropped into screw tube No. 3 (9 mL transparent, Maruemu Co., Ltd.) containing 6 mL of physiological saline as the aqueous medium. Figure 4 shows photographs of the appearance 30 minutes, 2 hours, 6 hours, 1 day, and 5 days after dropping the formulation into the aqueous medium. As a result, the dropped formulation formed a gel and remained floating near the surface of the water throughout (Figure 4).

[0146] In the screw-cap tube containing ICG-containing formulation No. 11, the gel formed from the formulation retained its fluorescent color (green) even after 5 days, while the aqueous medium remained virtually colorless and transparent.

[0147] Based on these results, in the ICG-containing formulation, no substantial release of ICG from the gel was observed for at least 5 days, and fluorescence emission from the gel persisted. Furthermore, this state was maintained for at least 2 weeks. These results support the long-term marking performance demonstrated in Examples 3, 5, and 6.

[0148] All publications, patents, and patent applications cited herein shall be incorporated herein by direct reference.

Claims

1. A composition for fluorescent marking of biological tissues, comprising an amphiphilic compound having an isoprenoid chain, a phospholipid, and a fluorescent dye.

2. The composition according to claim 1, wherein the amphiphilic compound having an isoprenoid chain is an amphiphilic compound represented by the following general formula (I) or a salt thereof. (In the formula, X and Y each represent a hydrogen atom or together represent an oxygen atom, n represents an integer from 0 to 2, and m represents 1 or 2.) (where R represents a single or double bond, and R represents a hydrophilic group having one or more hydroxyl groups.) 3. The composition according to claim 1, which is a liquid crystal precursor composition capable of forming a non-lamellar liquid crystal in the presence of an aqueous medium.

4. The composition according to claim 1, further comprising a biocompatible organic solvent.

5. The composition according to claim 4, wherein the biocompatible organic solvent is ethanol.

6. The composition according to claim 1, wherein the phospholipid comprises at least one selected from the group consisting of phosphatidylcholine and phosphatidylethanolamine.

7. The composition according to claim 1, wherein the phospholipid is selected from the group consisting of soy phosphatidylcholine, egg yolk phosphatidylcholine, dioleyl phosphatidylcholine, and dioleyl phosphatidylethanolamine.

8. The composition according to claim 1, further comprising an oil component.

9. The composition according to claim 8, wherein the oil component is selected from the group consisting of sesame oil, soybean oil, and squalene.

10. The composition according to claim 1, wherein the fluorescent dye is a near-infrared fluorescent dye.

11. The composition according to claim 10, wherein the near-infrared fluorescent dye is indocyanine green.

12. The composition according to claim 1 or 11, comprising a fluorescent dye at a concentration of 0.005 to 0.5 w / w%.

13. The amphiphilic compounds are: mono-O-(5,9,13,17-tetramethyloctadeca-4-enoyl)glycerol, mono-O-(5,9,13,17-tetramethyloctadeca-4-enoyl)sorbitan, mono-O-(5,9,13,17-tetramethyloctadecanoyl)sorbitan, mono-O-(5,9,13,17-tetramethyloctadeca-4-enoyl)isosorbide, mono-O-(5,9,13,17-tetramethyloctadecanoyl)isosorbide, mono-O-(5,9,13,17-tetramethyloctadecanoyl)propylene glycol, The composition according to claim 1, wherein the composition is at least one selected from the group consisting of mono-O-(5,9,13-trimethyltetradeca-4-enoyl)glycerol and mono-O-(5,9,13-trimethyltetradeca-4,8,12-trienoyl)glycerol.

14. The composition according to claim 1 for locally fluorescently marking the tissue of a tubular organ.

15. The composition according to claim 1, for injection into biological tissue to locally fluorescently mark the biological tissue.

16. The composition according to claim 1, for application to the surface of biological tissue to locally fluorescently mark the biological tissue.

17. A method for locally fluorescently marking affected tissue, comprising applying the composition described in claim 1 to a site within living tissue where the location of the affected tissue can be identified.

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