ICG lipid derivative, and lipid microparticles each containing same

Abstract

[Problem] To provide: an ICG lipid derivative that can form lipid microparticles having excellent stability and / or capable of releasing a drug in a selective manner; and lipid microparticles each containing the ICG lipid derivative. [Solution] Provided are lipid microparticles each containing a compound represented by formula (A) or a pharmaceutically acceptable salt thereof. [In the formula, R1and R2 each independently represent -(CH2)k-CONH-R3; R3 represents a group selected from the group consisting of -(CH2)m-OPO3 --CH2-CH(CH2OCOR4)(OCOR5), a branched C14-C40 alkyl group, and a branched C14-C40 alkenyl group; R4 and R5 each independently represent a linear or branched C13-C21 alkyl group or a linear or branched C13-C21 alkenyl group; k represents an integer of 2 to 4; and m represents an integer of 2 to 4.]

Description

ICG lipid derivative and lipid microparticles containing the same

[0001] The present invention relates to an ICG lipid derivative, a lipid microparticle containing the same, and uses thereof.

[0002] Indocyanine Green (ICG), a near-infrared emitting organic dye, has the following structure: ICG is a compound having the following characteristics. ICG emits fluorescence when excited by light in the near-infrared wavelength region (near-infrared fluorescence property). ICG also has an absorption wavelength in the near-infrared region, and has the ability to generate heat (hyperthermia effect; HT effect) and generate reactive oxygen species (photodynamic effect; PDT effect) when irradiated with near-infrared light. ICG is a substance approved for ingestion, and because it has the above-mentioned effects in the near-infrared wavelength region, which has high permeability in the body, its application in various medical fields is being considered. Specifically, research and development is being conducted on applications of ICG-incorporated microparticles and molecular assemblies in molecular imaging technology, molecular probe technology, drug delivery system (DDS) technology, and the like.

[0003] For example, ICG is used in fluorescence imaging (FI) by utilizing its near-infrared fluorescence properties. By administering ICG to a subject and measuring the fluorescence signal emitted by ICG within the subject when external light is irradiated after a certain period of time, the distribution of ICG within the subject can be imaged, and this has been used, for example, to identify sentinel lymph nodes. For example, Patent Document 1 (JP 2001-299676 A) discloses a sentinel lymph node detection method utilizing the near-infrared fluorescence properties of ICG. Patent Document 2 (WO 2011 / 152046 A) discloses a fluorescent probe capable of emitting high fluorescence intensity for a long period of time, which is a liposome containing an ICG lipid derivative in which the basic skeleton of ICG is modified with an alkyl chain or phospholipid. Furthermore, Patent Document 3 (JP 2010-266295 A) discloses a fluorescent tissue marker having vesicle clusters formed by encapsulating vesicles formed by a complex of phospholipids and a near-infrared fluorescent dye such as ICG in a hydrophilic solvent, and forming and aggregating multiple capsules using an emulsifier.

[0004] Photoacoustic imaging (PAI) using ICG has also been studied. PAI is an imaging method that detects ultrasound (photoacoustic ultrasound (PA)) generated by the thermal expansion of a light absorber that has absorbed pulsed light. For example, in Non-Patent Document 1 (Akers WJ et al., ACS Nano. 2011 Jan 25;5(1):173-182, 2011), photoacoustic imaging is investigated along with fluorescence imaging, using an ICG derivative modified with ICG as a near-infrared fluorescent dye and perfluorocarbon (PFC) particles (ICG-loaded PFC particles) loaded with the ICG derivative as a probe. In this document, the ICG derivative is loaded onto a portion of the particle solely as a probe agent for imaging, and the use of the ICG derivative itself to load a drug is not considered. Furthermore, drugs such as anticancer drugs and antibacterial agents are usually water-soluble, but the ultrasound contrast agent using ICG-loaded PFC particles in the same document is an O / W emulsion of particles with perfluorocarbon as a core substance, and therefore it is practically difficult to incorporate a water-soluble drug into the emulsion.

[0005] Also known are photodynamic hyperthermal therapy (PHT), which utilizes the HT effect and PDT effect of ICG, and photodynamic hyperthermal chemotherapy (PHCT), which combines PHT with local chemotherapy. For example, Patent Document 4 (JP 2010-69001 A) discloses photothermochemotherapy using ICG as a photosensitive dye agent, utilizing its heat-generating and reactive oxygen generating properties. Furthermore, Patent Document 5 (WO 2000 / 41726 A) discloses transdermal photodynamic therapy using a photosensitizing drug such as ICG, and a liposomal delivery system comprising the photosensitizing drug as a photosensitizing drug delivery system.

[0006] Attempts have also been made to use compounds containing ICG in DDS. Patent Document 6 (WO 2013 / 051732) discloses liposomes that encapsulate a drug and contain a liposome membrane constituent bound to ICG, and states that the liposomes can be used in DDS and the like.

[0007] JP 2001-299676 A, WO 2011 / 152046 A, JP 2010-266295 A, JP 2010-69001 A, WO 2000 / 41726 A, WO 2013 / 051732 A

[0008] Akers WJ et al., ACS Nano. 2011 Jan 25;5(1):173-182, 2011

[0009] Although many microparticles containing ICG have been proposed, there remains a need for ICG lipid derivatives and microparticles containing the same that can be used for various pharmaceutical and diagnostic purposes.

[0010] The present invention is, for example, as follows.

[0011] [1] A compound represented by the following formula (A) or a pharmaceutically acceptable salt thereof: [In the formula, R １ and R ２ are each independently -(CH ２ ) ｋ -CONH-R ３ represents R ３ Ha-(CH ２ ) ｍ -OPO ３ - -CH ２ -CH(CH ２ OCOR ４ ) (OCOR ５ ), branched chain C １４ ~C ４０ Alkyl and branched C １４ ~C ４０ alkenyl; R ４ and R ５ are each independently a straight-chain or branched-chain C １３ ~C ２１ Alkyl or straight or branched chain C １３ ~C２1 represents alkenyl, k represents an integer of 2 to 4, and m represents an integer of 2 to 4.] [2] R １ and R ２ are each independently -(CH ２ ) ２ -CONH-R ３ represents R ３ Ha-(CH ２ ) ｍ -OPO ３ － -CH ２ -CH(CH ２ OCOR ４ ) (OCOR ５ ) and R ４ and R ５ are each independently a linear C １３ ~C ２１ Alkyl or linear C １３ ~C ２１ The compound according to [1], wherein m represents alkenyl, and m represents an integer of 2 to 4. [3] A compound selected from the group consisting of compounds represented by the following formula (I) and the following formula (II), or a pharmaceutically acceptable salt thereof:

[0012] [3-1] R １ and R ２ are each independently -(CH ２ ) ２ -CONH-R ３ represents R ３ is a branched chain C １４ ~C ４０ Alkyl and branched C １４ ~C ４０ [3-2] The compound according to [3-1], or a pharmaceutically acceptable salt thereof, selected from the group consisting of compounds represented by the following formula (IV) and the following formula (V):

[0013] [4] Lipid microparticles comprising a compound represented by the following formula (A) or a pharmaceutically acceptable salt thereof: [In the formula, R １ and R ２ are each independently -(CH ２ )ｋ -CONH-R ３ represents R ３ Ha-(CH ２ ) ｍ -OPO ３ - -CH ２ -CH(CH ２ OCOR ４ ) (OCOR ５ ), linear C １４ ~C ２２ Alkyl, straight chain C １４ ~C ２２ Alkenyl, branched C １４ ~C ４０ Alkyl and branched C １４ ~C ４０ alkenyl; R ４ and R ５ are each independently a straight-chain or branched-chain C １３ ~C ２１ Alkyl or straight or branched chain C １３ ~C ２1 represents alkenyl, k represents an integer of 2 to 4, and m represents an integer of 2 to 4.] [4-1] R １ and R ２ are each independently -(CH ２ ) ２ -CONH-R ３ represents R ３ Ha-(CH ２ ) ｍ -OPO ３ － -CH ２ -CH(CH ２ OCOR ４ ) (OCOR ５ ) and R ４ and R ５ are each independently a linear C １３ ~C ２１ Alkyl or linear C １３ ~C ２１ The lipid microparticle according to [4], wherein R represents alkenyl, and m represents an integer of 2 to 4. [4-2] R １ and R ２ are each independently -(CH ２ ) ２ -CONH-R３ represents R ３ is a straight chain C １４ ~C ２２ Alkyl, straight chain C １４ ~C ２２ Alkenyl, branched C １４ ~C ４０ Alkyl and branched C １４ ~C ４０ The lipid microparticle according to [4], wherein R is selected from the group consisting of alkenyl. １ and R ２ are each independently -(CH ２ ) ２ -CONH-R ３ represents R ３ is a branched chain C １４ ~C ４０ Alkyl and branched C １４ ~C ４０ [4-4] The lipid microparticle according to [4], comprising a compound selected from the group consisting of compounds represented by the following formulas (I) to (V) or a pharmaceutically acceptable salt thereof: [5] The lipid microparticle according to [4], which contains a compound selected from the group consisting of compounds represented by the following formulas (I) to (III) or a pharmaceutically acceptable salt thereof:

[0014] [6] The lipid microparticles according to any one of [4], [4-1] to [4-4], and [5], further immobilizing or carrying at least one type of drug. [7] The lipid microparticles according to any one of [4] to [6] and [4-1] to [4-4], wherein the drug comprises an anticancer drug. [8] The lipid microparticles according to any one of [4] to [7] and [4-1] to [4-4], wherein the lipid microparticles further comprise at least one lipid selected from the group consisting of neutral lipids, polyethylene glycol-modified lipids, and sterols. [9] The lipid microparticles according to any one of [4] to [8] and [4-1] to [4-4], wherein the average particle diameter is 30 to 200 nm.

[10] The lipid microparticles according to any one of [4] to [9] and [4-1] to [4-4], wherein the lipid microparticles are polymeric micelles or liposomes. [10-1] The lipid microparticles according to

[10] , wherein the lipid microparticles are liposomes.

[11] A pharmaceutical composition or diagnostic composition comprising the compound according to any one of [1] to [3], [3-1], or [3-2] or a pharmaceutically acceptable salt thereof, or the lipid microparticle according to any one of [4] to

[10] , [4-1] to [4-4], or [10-1]. [11-1] The pharmaceutical composition according to

[11] , for treating cancer.

[12] The pharmaceutical composition or diagnostic composition according to

[11] , for use in at least one selected from photothermal therapy, photodynamic therapy, and fluorescence imaging.

[0015]

[13] A method for producing a drug-encapsulating liposome, comprising: a step of preparing liposomes from one or more lipids; a step of replacing the liposome external phase to obtain a liposome dispersion containing liposomes having an ammonium sulfate ion gradient and / or a pH gradient between the internal phase and the external phase; and a step of mixing the liposome dispersion with a drug to encapsulate the drug in the liposomes of the liposome dispersion; wherein the one or more lipids are represented by the following formula (A): [In the formula, R １ and R ２ are each independently -(CH ２ ) ｋ -CONH-R ３ represents R ３ Ha-(CH ２ ) ｍ -OPO ３- -CH ２ -CH(CH ２ OCOR ４ ) (OCOR ５ ), linear C １４ ~C ２２ Alkyl, straight chain C １４ ~C ２２ Alkenyl, branched C １４ ~C ４０ Alkyl and branched C １４ ~C ４０ alkenyl, R ４ and R ５ are each independently a straight-chain or branched-chain C １３ ~C ２１ Alkyl or straight or branched chain C １３ ~C ２1

[14] Use of the lipid complex according to any one of [4] to

[10] , [4-1] to [4-4], or [10-1] in the manufacture of a pharmaceutical composition or diagnostic composition.

[15] A method for treating cancer, comprising administering to a subject the pharmaceutical composition according to

[11] .

[16] A photothermal therapy, photodynamic therapy, or fluorescence imaging method, comprising administering to a subject the pharmaceutical composition or diagnostic composition according to

[11] .

[17] Use of the lipid complex according to [4] to

[10] , [4-1] to [4-4], or [10-1] or the pharmaceutical composition according to

[11] for use in the treatment of cancer.

[18] Use of the lipid complex according to [4] to

[10] , [4-1] to [4-4], or [10-1] or the pharmaceutical or diagnostic composition according to

[11] for use in at least one selected from photothermal therapy, photodynamic therapy, and fluorescence imaging.

[0016] The ICG lipid derivatives of the present invention have one or more of the following effects. (1) The ICG lipid derivatives of the present invention can form lipid microparticles (particularly liposomes) with excellent stability (particularly stability in blood). (2) The ICG lipid derivatives of the present invention can form lipid microparticles (particularly liposomes) with encapsulated drugs that have excellent stability (particularly stability in blood). In some embodiments, lipid microparticles with encapsulated drugs can be used as drug carriers for DDS. (3) Lipid microparticles (particularly liposomes) composed of the ICG lipid derivatives of the present invention have excellent drug encapsulation efficiency. (4) Lipid microparticles (particularly liposomes) composed of the ICG lipid derivatives of the present invention can easily encapsulate drugs. In some embodiments, drug-encapsulated liposomes with excellent stability can be obtained even when using a method in which empty liposomes are prepared from the ICG lipid derivative and then the drug is encapsulated into the liposomes from outside the liposomes. (5) Lipid microparticles (particularly liposomes) composed of the ICG lipid derivative of the present invention can selectively release encapsulated drugs to the outside of the microparticles upon irradiation with near-infrared light. (6) Lipid microparticles (particularly liposomes) containing the ICG lipid derivative of the present invention can efficiently generate heat and / or active oxygen upon irradiation with near-infrared light, causing an increase in the temperature of the dispersion medium. Therefore, lipid microparticles containing the ICG lipid derivative of the present invention can be used in photothermal therapy and / or photodynamic therapy. In some embodiments, lipid microparticles containing the ICG lipid derivative of the present invention can function as temperature-sensitive liposomes. (7) Lipid microparticles (particularly liposomes) containing the ICG lipid derivative of the present invention emit fluorescence upon irradiation with excitation light. Therefore, in some embodiments, lipid microparticles containing the ICG lipid derivative of the present invention can be used for fluorescence imaging. For example, lipid microparticles containing the ICG lipid derivative of the present invention can function as probes for fluorescence imaging. (8) In some embodiments, pharmaceutical or diagnostic compositions containing the ICG lipid derivative or lipid microparticles of the present invention are provided.

[0017] FIG. 1 shows the ICG-diSA produced in Example A1. １ 2 is a diagram showing the 1 H NMR chart of ICG-diDOPE prepared in Example A2. １3 shows the H NMR chart of ICG-diDSPE produced in Example A3. １ 4 is a diagram showing the H NMR chart of ICG-DOPE produced in Comparative Example A1. １1H NMR chart. This figure shows the absorption spectra of the ICG lipid derivatives and ICG produced in the examples and comparative examples. FIG. 6 shows the release behavior of doxorubicin (DOX) from liposomes upon LED light irradiation. The left figure shows the doxorubicin release rate (%) from ICGLip73-DOX of Example B7 and Lip73-DOX of Comparative Example B3 upon LED light (near-infrared light) irradiation, and the right figure shows the doxorubicin release rate (%) from ICGLip82-DOX of Example B8 and Lip82-DOX of Comparative Example B4 upon LED light (near-infrared light) irradiation. FIG. 7A shows the release behavior of cisplatin from liposomes upon LED light irradiation. The left graph shows the cisplatin release rate (%) from ICG Lip73-Cis of Example C1 and Lip73-Cis of Comparative Example C1 upon irradiation with LED light (near-infrared light), and the right graph shows the cisplatin release rate (%) from ICG Lip82-Cis of Example C2 and Lip82-Cis of Comparative Example C2 upon irradiation with LED light (near-infrared light). Figure 7B shows the temperature change over time of the liposome dispersions (ICCG Lip73-Cis, Lip73-Cis, ICG Lip82-Cis, Lip82-Cis) of Examples C1 and C2 and Comparative Examples C1 and C2 upon irradiation with LED (near-infrared light). Figure 8 shows the temperature of solid tumor sites in mice administered with saline (saline) or liposome formulations (ICGLip, Lip-DOX, ICG Lip-DOX), measured before and after LED light irradiation using a thermography camera (E5, FLIR Systems). Figure 9 shows the effect of cancer treatment (change in solid tumor volume (relative ratio from the day of drug administration)) by administration of saline (saline) or liposome formulations (ICGLip, Lip-DOX, ICG Lip-DOX) and / or LED light irradiation (+LED). Figure 10 shows the change in body weight of mice administered with saline (saline) or liposome formulations (ICGLip, Lip-DOX, ICG Lip-DOX) and / or LED light irradiation (+LED). FIG. 11 shows the temperatures at the solid tumor site in mice administered with saline or liposome preparations (ICGLip, Lip-Cis, ICG Lip-Cis), measured using a thermography camera (E5, FLIR Systems) before and after LED light irradiation.Figure 12 shows the effect of cancer treatment (change in tumor volume of solid tumors (relative ratio from the day of drug administration)) by administration of saline (saline) or liposome preparations (ICGLip, Lip-Cis, ICG Lip-Cis) and / or LED light irradiation (+LED). Figure 13 shows the change in mouse body weight by administration of saline (saline) or liposome preparations (ICGLip, Lip-Cis, ICG Lip-Cis) and / or LED light irradiation (+LED).

[0018] The present invention will be described in detail below with reference to embodiments and examples, but the present invention is not limited to the embodiments and examples shown below and can be modified as desired without departing from the spirit of the present invention. All documents and publications mentioned in this specification are incorporated herein by reference in their entirety, regardless of their purpose.

[0019] The meanings of terms used in this specification are explained below, and the present invention is described in detail. "Microparticles" refer to particles having an average particle size of nano- to micron-size (1 nm to 1 μm or μm) and are also known as nanospheres, microspheres, nanocapsules, microcapsules, etc. Microparticles can be, for example, the dispersed phase in an emulsion or the internal phase in a suspension. "Lipid microparticles" refer to microparticles containing multiple lipids physically bound to each other by intermolecular forces. Typically, "lipid microparticles" are microparticles consisting of membranous molecular assemblies formed by lipid association. "Microparticle carriers" or "lipid microparticle carriers" refer to microparticles or lipid microparticles capable of immobilizing or carrying chemical substances such as drugs. In one embodiment, "microparticle carriers" or "lipid microparticle carriers" refer to structures that are biocompatible and sufficiently resistant to chemical and / or physical destruction for a period of time sufficient to reach the desired target. "Immobilization" refers to immobilization on the outer surface and / or interior. "Loading" refers to mobilization of drugs or other substances on the outer surface and / or interior of microparticles. "Liposome" refers to a closed vesicle formed by two molecular membrane layers. Typically, liposomes are composed of a single or multiple lipid bilayers. "Polymer micelle" refers to a closed vesicle formed by one molecular membrane layer. "Alkyl" refers to a linear, cyclic, or branched saturated aliphatic hydrocarbon group having a specified number of carbon atoms. "Alkenyl" refers to a linear or branched hydrocarbon group having a specified number of carbon atoms and at least one carbon-carbon double bond. Examples include, but are not limited to, monoenes, dienes, trienes, and tetraenes.

[0020] 1. ICG Lipid Derivatives One aspect of the present invention relates to a compound represented by the following formula (A) or a pharmaceutically acceptable salt thereof:

[0021] The compound represented by formula (A) is an R １ and R ２and can function as a lipid. The compound represented by formula (A) has a basic skeleton of ICG, and thus can exhibit near-infrared fluorescence properties, a heat-generating effect upon irradiation with near-infrared light (HT effect), and an effect of generating active oxygen (PDT effect). Hereinafter, the compound represented by formula (A) or a salt thereof will also be referred to as an "ICG-derivative lipid." For example, an ICG lipid derivative exhibits absorption and fluorescence in the near-infrared light region of 700 to 1300 nm, particularly about 700 to 900 nm, and has a molar absorption coefficient of 1.6 x 10 in any wavelength region of 700 to 1300 nm (preferably 700 to 900 nm). ５ [L / mol / cm] or more.

[0022] In formula (A), R １ and R ２ are each independently -(CH ２ ) ｋ -CONH-R ３ Represents R １ and R ２ may be the same or different. ３ Ha-(CH ２ ) ｍ -OPO ３ - -CH ２ -CH(CH ２ OCOR ４ ) (OCOR ５ ), linear C １４ ~C ２２ Alkyl, straight chain C １４ ~C ２２ Alkenyl, branched C １４ ~C ４０ Alkyl and branched C １４ ~C ４０ It should be noted that in this specification, "-OPO ３ - "-" indicates the following structure (* indicates the linking portion): R ４ and R ５ are each independently a straight-chain or branched-chain C １３ ~C ２１ Alkyl or straight or branched chain C １３ ~C ２1 represents alkenyl.４ and R ５ may be the same or different. In formula (A), k represents an integer of 2 to 4. k is preferably 2 or 3, and more preferably 2. In formula (A), m represents an integer of 2 to 4. m is preferably 2 or 3, and more preferably 2.

[0023] In some embodiments, the compound represented by formula (A) or a pharmaceutically acceptable salt thereof is a compound represented by formula (A) wherein R １ and R ２ are each independently -(CH ２ ) ｋ -CONH-R ３ represents R ３ Ha-(CH ２ ) ｍ -OPO ３ - -CH ２ -CH(CH ２ OCOR ４ ) (OCOR ５ ), branched chain C １４ ~C ４０ Alkyl and branched C １４ ~C ４０ alkenyl, R ４ and R ５ are each independently a straight-chain or branched-chain C １３ ~C ２１ Alkyl or straight or branched chain C １３ ~C ２1 represents alkenyl, k represents an integer of 2 to 4 (preferably 2 or 3, more preferably 2), and m represents an integer of 2 to 4 (preferably 2 or 3, more preferably 2), or a pharmaceutically acceptable salt thereof. The compound of this embodiment is preferable in that it is excellent in at least one of biocompatibility, ease of synthesis, affinity to lipid microparticles, chemical stability, physical stability of lipid microparticles (inhibition of aggregation), stability of lipid microparticle carrier when a drug is encapsulated in the lipid microparticles, drug encapsulation rate, and reactivity of lipid microparticles when irradiated with near-infrared light (heat generation / drug release).

[0024] In some embodiments, the compound represented by formula (A) or a pharmaceutically acceptable salt thereof is a compound represented by formula (A) wherein R １ and R ２ are each independently -(CH ２ ) ｋ -CONH-R ３ represents R ３ Ha-(CH ２ ) ｍ -OPO ３ - -CH ２ -CH(CH ２ OCOR ４ ) (OCOR ５ ), linear C １４ ~C ２２ Alkyl and branched C １４ ~C ４０ R represents a group selected from the group consisting of alkyl ４ and R ５ are each independently a straight-chain or branched-chain C １３ ~C ２１ Alkyl or straight or branched chain C １３ ~C ２1 represents alkenyl, k represents an integer of 2 to 4 (preferably 2 or 3, more preferably 2), and m represents an integer of 2 to 4 (preferably 2 or 3, more preferably 2), or a pharmaceutically acceptable salt thereof. The compound of this embodiment is preferable in that it is excellent in at least one of biocompatibility, ease of synthesis, affinity to lipid microparticles, chemical stability, physical stability of lipid microparticles (inhibition of aggregation), stability of lipid microparticle carrier when a drug is encapsulated in the lipid microparticles, drug encapsulation rate, and reactivity of lipid microparticles when irradiated with near-infrared light (heat generation / drug release).

[0025] In some embodiments, the compound represented by formula (A) or a pharmaceutically acceptable salt thereof is a compound represented by formula (A) wherein R １ and R ２ are each independently -(CH ２ ) ｋ -CONH-R ３ represents R ３ Ha-(CH ２ ) ｍ -OPO ３- -CH ２ -CH(CH ２ OCOR ４ ) (OCOR ５ ), and branched chain C １４ ~C ４０ R represents a group selected from the group consisting of alkyl ４ and R ５ are each independently a straight-chain or branched-chain C １３ ~C ２１ Alkyl or straight or branched chain C １３ ~C ２1 represents alkenyl, k represents an integer of 2 to 4 (preferably 2 or 3, more preferably 2), and m represents an integer of 2 to 4 (preferably 2 or 3, more preferably 2), or a pharmaceutically acceptable salt thereof. The compound of this embodiment is preferable in that it is excellent in at least one of biocompatibility, ease of synthesis, affinity to lipid microparticles, chemical stability, physical stability of lipid microparticles (inhibition of aggregation), stability of lipid microparticle carrier when a drug is encapsulated in the lipid microparticles, drug encapsulation rate, and reactivity of lipid microparticles when irradiated with near-infrared light (heat generation / drug release).

[0026] In some embodiments, the compound of formula (A) or a pharmaceutically acceptable salt thereof is a compound of formula (A) wherein R １ and R ２ are each independently -(CH ２ ) ｋ -CONH-R ３ represents R ３ Ha-(CH ２ ) ｍ -OPO ３ - -CH ２ -CH(CH ２ OCOR ４ ) (OCOR ５ ) and R ４ and R ５ are each independently a straight-chain or branched-chain C １３ ~C ２１ Alkyl or straight or branched chain C １２ ~C ２２ Alkenyl (preferably straight chain C １３ ~C ２１Alkyl or linear C １３ ~C ２１ k represents an integer of 2 to 4 (preferably 2 or 3, more preferably 2), and m represents an integer of 2 to 4 (preferably 2 or 3, more preferably 2), or a pharmaceutically acceptable salt thereof. The compound of this embodiment is preferable in that it is excellent in at least one of biocompatibility, ease of synthesis, affinity to lipid microparticles, chemical stability, physical stability of lipid microparticles (inhibition of aggregation), stability of lipid microparticle carrier when a drug is encapsulated in the lipid microparticles, drug encapsulation rate, and reactivity of lipid microparticles when irradiated with near-infrared light (heat generation / drug release).

[0027] In some embodiments, the compound of formula (A) or a pharmaceutically acceptable salt thereof is a compound of formula (A) wherein R １ and R ２ are each independently -(CH ２ ) ２ -CONH-R ３ represents R ３ Ha-(CH ２ ) ｍ -OPO ３ - -CH ２ -CH(CH ２ OCOR ４ ) (OCOR ５ ) and R ４ and R ５ are each independently a linear C １３ ~C ２１ Alkyl or linear C １３ ~C ２１ represents alkenyl, and m represents an integer of 2 to 4 (preferably 2 or 3, more preferably 2), or a pharmaceutically acceptable salt thereof. The compound of this embodiment is preferable in that it is excellent in at least one of biocompatibility, ease of synthesis, affinity to lipid microparticles, chemical stability, physical stability of lipid microparticles (inhibition of aggregation), stability of lipid microparticle carriers when drugs are encapsulated in lipid microparticles, drug encapsulation rate, and reactivity of lipid microparticles when irradiated with near-infrared light (heat generation / drug release).

[0028] In one embodiment, the compound represented by formula (A) or a pharmaceutically acceptable salt thereof is a compound represented by the following formula (B) or a pharmaceutically acceptable salt thereof: In formula (B), R ４ and R ５ are each independently a straight-chain or branched-chain C １３ ~C ２１ Alkyl or straight or branched chain C １２ ~C ２２ represents alkenyl, each k independently represents an integer of 2 to 4 (preferably 2 or 3, more preferably 2), and each m independently represents an integer of 2 to 4 (preferably 2 or 3, more preferably 2). The compound of this embodiment is preferable in that it is excellent in at least one of biocompatibility, ease of synthesis, affinity to lipid microparticles, chemical stability, physical stability of lipid microparticles (inhibition of aggregation), stability and drug encapsulation rate when drugs are encapsulated in lipid microparticles, and reactivity of lipid microparticles upon irradiation with near-infrared light (heat generation and drug release).

[0029] In one embodiment, in the above formula (B), R ４ and R ５ are each independently a straight chain C １３ ~C ２１ Alkyl or linear C １２ ~C ２２ represents alkenyl, each k independently represents an integer of 2 to 4 (preferably 2 or 3, more preferably 2), and each m independently represents an integer of 2 to 4 (preferably 2 or 3, more preferably 2). The compound of this embodiment is more preferable in that it is excellent in at least one of biocompatibility, ease of synthesis, affinity to lipid microparticles, chemical stability, physical stability of lipid microparticles (inhibition of aggregation), stability of lipid microparticle carrier when a drug is encapsulated in the lipid microparticles, drug encapsulation rate, and reactivity of lipid microparticles when irradiated with near-infrared light (heat generation / drug release).

[0030] In some embodiments, in the above formula (B), R ４ and R ５ are identical.

[0031] In some embodiments, the compound of formula (A) or a pharmaceutically acceptable salt thereof is a compound of formula (A) wherein R １ and R ２ are each independently -(CH ２ ) ｋ -CONH-R ３ represents R ３ is a straight chain C １４ ~C ２２ Alkyl, straight chain C １４ ~C ２２ Alkenyl, branched C １４ ~C ４０ Alkyl and branched C １４ ~C ４０ represents a group selected from the group consisting of alkenyl, k represents an integer of 2 to 4 (preferably 2 or 3, more preferably 2), and m represents an integer of 2 to 4 (preferably 2 or 3, more preferably 2), or a pharmaceutically acceptable salt thereof. The compound of this embodiment is preferable in that it is excellent in at least one of biocompatibility, ease of synthesis, affinity to lipid microparticles, chemical stability, physical stability of lipid microparticles (inhibition of aggregation), stability of lipid microparticle carrier when a drug is encapsulated in the lipid microparticles, drug encapsulation rate, and reactivity of lipid microparticles when irradiated with near-infrared light (heat generation / drug release).

[0032] In some embodiments, the compound of formula (A) or a pharmaceutically acceptable salt thereof is a compound of formula (A) wherein R １ and R ２ are each independently -(CH ２ ) ｋ -CONH-R ３ represents R ３ is a branched chain C １４ ~C ４０ Alkyl and branched C １４ ~C ４０represents a group selected from the group consisting of alkenyl, and k represents an integer of 2 to 4 (preferably 2 or 3, more preferably 2), or a pharmaceutically acceptable salt thereof. The compound of this embodiment is preferable in that it is excellent in at least one of biocompatibility, ease of synthesis, affinity to lipid microparticles, chemical stability, physical stability of lipid microparticles (inhibition of aggregation), stability of lipid microparticle carriers when drugs are encapsulated in lipid microparticles, drug encapsulation rate, and reactivity of lipid microparticles when irradiated with near-infrared light (heat generation / drug release).

[0033] In some embodiments, the compound of formula (A) or a pharmaceutically acceptable salt thereof is a compound of formula (A) wherein R １ and R ２ are each independently -(CH ２ ) ２ -CONH-R ３ represents R ３ is a branched chain C １４ ~C ４０ Alkyl and branched C １４ ~C ４０ The compound of this embodiment is a compound or a pharmaceutically acceptable salt thereof, which represents a group selected from the group consisting of alkenyl. The compound of this embodiment is preferable in that it is excellent in at least one of biocompatibility, ease of synthesis, affinity to lipid microparticles, chemical stability, physical stability of lipid microparticles (suppression of aggregation), stability of lipid microparticle carrier when drug is encapsulated in lipid microparticles, encapsulation rate of drug, reactivity of lipid microparticles when irradiated with near-infrared light (heat generation, drug release), etc.

[0034] In some embodiments, the compound represented by formula (A) or a pharmaceutically acceptable salt thereof is a compound represented by formula (C) below or a pharmaceutically acceptable salt thereof: In formula (C), R ３ are each independently a straight chain C １４ ~C ２２ Alkyl, straight chain C １４ ~C ２２ Alkenyl, branched C １４ ~C ４０ Alkyl and branched C １４ ~C ４０alkenyl; and k's each independently represent an integer of 2 to 4 (preferably 2 or 3, more preferably 2).

[0035] In one embodiment, in formula (C), R ３ are each independently a branched chain C １４ ~C ４０ Alkyl and branched C １４ ~C ４０ alkenyl; and k's each independently represent an integer of 2 to 4 (preferably 2 or 3, more preferably 2).

[0036] In one embodiment, in formula (C), R ３ are each independently a branched chain C １４ ~C ４０ Each k independently represents an integer of 2 to 4 (preferably 2 or 3, more preferably 2).

[0037] In the above-described embodiment, in formula (A) or formula (B), R ４ or R ５ Linear C as １３ ~C ２１ The alkyl is, for example, a straight chain C １３ ~C １９ alkyl, for example, linear C １５ ~C １７ In the above-described embodiment, in formula (A) or formula (B), R ４ or R ５ Branched chain C as １３ ~C ２１ The alkyl may be, for example, a branched C １３ ~C １９ alkyl, for example branched chain C １５ ~C １７ In the above-mentioned embodiment, in formula (A) or formula (B), R ４ or R ５ Linear C as １３ ~C ２１ Alkenyl is, for example, a straight chain C １３ ~C １９ Alkenyl, for example, straight chain C １５ ~C１７ In the above-mentioned embodiment, in formula (A) or formula (B), R ４ or R ５ Branched chain C as １３ ~C ２１ Alkenyl is, for example, a branched C １３ ~C １９ alkenyl, for example, branched C １５ ~C １７ Alkenyl, for example, 1,3-dimethyl-8-heptadecenyl.

[0038] In the above-described embodiment, in formula (A) or formula (C), R ３ Linear C as １４ ~C ２２ The alkyl is, for example, a straight chain C １４ ~C ２０ alkyl, for example, linear C １６ ~C １８ In the above-described embodiment, in formula (A) or formula (C), R ３ Linear C as １４ ~C ２２ Alkenyl is, for example, a straight chain C １４ ~C ２０ Alkenyl, for example, straight chain C １６ ~C １８ In the above-mentioned embodiment, in formula (A) or formula (C), R ３ Branched chain C as １４ ~C ４０ The alkyl may be, for example, a branched C ２０ ~C ４０ alkyl, for example branched chain C 28 ~C 40 In the above-mentioned embodiment, in formula (A) or formula (C), R ３ Branched chain C as １４ ~C ４０ Alkenyl is, for example, a branched C ２０ ~C ４０ alkenyl, for example, branched C 28 ~C 40In the above-described embodiment, in formula (A) or formula (C), R ３ Ha-CHR ３１ R ３２ represents R ３１ and R ３２ are each independently a straight chain C ７ ~C １９ (Preferably C １０ ~C １９ , more preferably C １４ ~C １９ ) alkyl; and k's each independently represent an integer of 2 to 4 (preferably 2 or 3, more preferably 2).

[0039] Examples of compounds according to the present embodiment are shown below.

[0040] One embodiment of the present invention is a compound represented by any one of the above formula (I) (ICG-diDSPE), formula (II) (ICG-diDOPE), formula (III) (ICG-diSA), or formula (IV), or a pharmaceutically acceptable salt thereof. A particular embodiment is a compound represented by any one of the above formula (I) (ICG-diDSPE), or formula (II) (ICG-diDOPE), or a pharmaceutically acceptable salt thereof. A particular embodiment is a compound represented by any one of the above formula (I) (ICG-diDSPE), formula (II) (ICG-diDOPE), or formula (III) (ICG-diSA), or a pharmaceutically acceptable salt thereof.

[0041] "Pharmaceutically acceptable salts" refers to salts that are suitable for use in contact with human and animal tissues without undue toxicity, irritation, or allergic response, within the scope of sound medical judgment. Pharmaceutically acceptable salts are well known in the art. For example, SM Berge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences 66: 1-19, 1977. Salts can be prepared in situ during the final isolation and purification of the compound, or separately by reacting the free base with a suitable organic acid. Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, Included are lactate, laurate, lauryl sulfate, malate, maleate, monomaleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, trifluoroacetate, undecanoate, valerate, etc. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, etc., as well as non-toxic ammonium, quaternary ammonium, and amine cations, including, but not limited to, ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, and ethylamine.

[0042] In the present invention, the compound represented by formula (A) may exist as stereoisomers such as geometric isomers and optical isomers, tautomers, etc., and the compound of the present invention includes all possible isomers and mixtures thereof. In addition, the compound represented by formula (A) or a salt thereof may be in the form of a hydrate or solvate, and the hydrate or solvate is also included in the compound of the present invention.

[0043] (Method for producing ICG-derivative lipid) A method for producing the ICG-derivative lipid of the present invention will be described. The compound of formula (A) can be synthesized, for example, by referring to the methods described in J.Am.Chem.Soc. 2003, 125, 7766, WO 2011 / 152046 and WO 2013 / 051732. For example, it can be synthesized according to Scheme 1 above.

[0044] (Wherein, k and R ３ are the same as those in formula (A).

[0045] (Step 1) 1,1,2-trimethyl[1H]-benz[e]indole (a1) and halogen-substituted carboxylic acid X—(CH ２ ) ｋ—COOH (X is a halogen atom, preferably bromine (Br)) to obtain a carboxyalkyl-substituted indole derivative (a2). Examples of solvents include dichlorobenzene, dichloromethane, and toluene. (Step 2) Glutaconaldehyde dianilide (a3) ​​is reacted with acetic anhydride, preferably in the presence of a base (e.g., N,N-diisopropylethylamine (DIPEA)), to obtain compound (a4). Examples of solvents include dichloromethane. (Step 3: Synthesis of Dicarboxylic Acid) Next, the carboxyalkyl-substituted indole derivative (a2) is reacted with compound (a4) in the presence of a base to obtain indocyanine green dicarboxylic acid compound (a5) in which a carboxyalkyl-substituted indole derivative is linked to both ends of the hexatriene chain. Examples of bases include sodium acetate and pyridine. Examples of solvents include methanol and tetrahydrofuran. In addition, by reacting (a4) with one equivalent of a carboxyalkyl-substituted indole derivative (a2) to link the carboxyalkyl-substituted indole derivative to one end of the hexatriene chain, and then further adding a carboxyalkyl-substituted indole derivative (a2) having a different number of carbon atoms (k) in the alkyl chain, it is possible to obtain the alkyl chain -(CH ２ ) ｋ - It is possible to obtain compounds in which the left and right sides are different.

[0046] (Step 4: Amidation) A dicarboxylic acid compound (a5) and an amine R ３ -NH ２ and amidation is carried out to obtain the target compound of formula (A). More specifically, ３ -NH ２ The compound of formula (B) can be prepared by using the compound shown in the following (a6) as the compound of formula (B). (In the formula, m, R ４ , R ５ has the same meaning as formula (B). ３ -NH ２ As R ３ is a straight chain C １４ ~C ２２Alkyl, straight chain C １４ ~C ２２ Alkenyl, branched C １４ ~C ４０ Alkyl or branched C １４ ~C ４０ The use of alkenyl provides the compound of formula (C) above. ３ -NH ２ The amidation reaction may be carried out by a conventional method, or a commercially available product may be used. The amidation reaction may be carried out according to a conventional means, and is preferably carried out in the presence of a benzotriazole such as 1-hydroxybenzotriazole (HOBt), N-hydroxysuccinimide (HOSu), 1-hydroxybenzotriazole (HOAt), or 1-[bis(dimethylamino)methylene]-1H-benzotriazolium 3-oxide hexafluorophosphate (HBTU), or a condensing agent such as 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide (EDC) hydrochloride or N,N'-dicyclohexylcarbodiimide (DCC). A base may be added to the reaction as necessary, and examples of the base include N-methylmorpholine (NMM), triethylamine (TEA), N,N-diisopropylethylamine (DIPEA), and 4-(dimethylamino)pyridine (DMAP). Examples of the solvent include dimethylformamide, tetrahydrofuran (THF), dichloromethane, and acetonitrile. ３ In order to obtain a compound having a different R, in the above (Step 3), one equivalent of the carboxyalkyl-substituted indole derivative (a2) is reacted with (a4) to link the carboxyalkyl-substituted indole derivative to one end of the hexatriene chain, and then R ３ -NH ２ is reacted to form R ３ -NH ２ After synthesizing the compound with R ３ R is different ３ -NH ２ Just react the following.

[0047] In synthesizing compounds of the present invention, unless the preparation of starting materials is specifically described, those compounds are known or can be prepared by analogous methods known in the art or as described in the Examples below. Those of skill in the art will recognize that the above schemes are merely representative of methods for preparing compounds of the present invention, and other well-known methods can similarly be used. Compounds of the present invention may possess one or more asymmetric centers; therefore, such compounds can occur as individual (R)- or (S)-stereoisomers, or as mixtures thereof. Unless otherwise specified, reference to a particular compound herein is intended to include both individual enantiomers and racemic mixtures thereof. Methods for the determination of stereochemistry and the separation of stereoisomers are well known to those of skill in the art.

[0048] 2. Lipid Microparticles One aspect of the present invention provides lipid microparticles (also referred to herein simply as "lipid microparticles") containing the ICG-derivative lipid of the above-described embodiment. Since the ICG-derivative lipid has an ICG structure as part of its structure, it can itself be a lipid microparticle carrying ICG. Therefore, in some embodiments, the lipid microparticle is composed of the above-described ICG-derivative lipid.

[0049] The form of the lipid microparticles is not particularly limited and may be, for example, any of polymeric micelles, liposomes, emulsions, microspheres, nanospheres, etc. In some embodiments, the lipid microparticles are polymeric micelles or liposomes formed from one or more lipids containing the ICG derivative lipid. In other embodiments, the lipid microparticles are at least one type selected from, for example, liposomes, lipid nanoparticles, O / W emulsions using lipids as interfacial materials, etc. In one embodiment, the lipid microparticles are liposomes. In some embodiments, the lipid microparticles exist in an aqueous solvent in the form of polymeric micelles or liposomes formed from one or more lipids containing the ICG derivative lipid. Examples of aqueous solvents include sterilized water; physiological saline; isotonic solutions containing adjuvants such as glucose, D-sorbitol, D-mannose, D-mannitol, and sodium chloride; and buffer solutions such as phosphate buffer, citrate buffer, and acetate buffer.

[0050] In certain embodiments, the lipid microparticles are, for example, liposomes. In one embodiment, the liposomes are composed of a lipid bilayer containing the ICG derivative lipid, and have an aqueous phase (internal phase) inside the lipid bilayer (the space of the closed vesicle formed by the lipid bilayer). The liposomes may be monolayer lipid bilayers or multilayer lipid bilayers, but from the viewpoint of stability, liposomes with a monolayer lipid bilayer structure are preferred.

[0051] The lipid microparticles of this embodiment contain, for example, 0.001 to 100 mol %, for example, 0.01 to 20 mol %, for example, 0.1 to 10 mol %, of the ICG-derivative lipid of the above-mentioned embodiment based on the total lipid contained in the lipid microparticles. The ICG-derivative lipids can be used alone or in combination of two or more.

[0052] In a representative embodiment, the lipid microparticles further comprise at least one lipid selected from the group consisting of neutral lipids, polyethylene glycol-modified lipids, and sterols. The ICG-derivative lipids of the present invention have high affinity with these lipids and can form microparticle structures with excellent stability. In some embodiments, the lipid microparticles contain, as lipid components, (I) the ICG-derivative lipid described above and (II) at least one lipid selected from the group consisting of neutral lipids, polyethylene glycol-modified lipids, and sterols, and the lipid component is contained in an amount of, for example, 50 to 100 wt %, for example, 70 to 100 wt %, for example, 90 to 100 wt %, based on the total weight of the lipid microparticles. For example, the lipid microparticles are polymeric micelles or liposomes (preferably liposomes) formed from lipids containing the ICG-derivative lipid and at least one lipid selected from the group consisting of neutral lipids, polyethylene glycol-modified lipids, and sterols.

[0053] The combination of lipid components in the lipid microparticles of this embodiment is not particularly limited, and examples include a combination of the above-mentioned ICG-derivative lipid, neutral lipid, and sterol, a combination of the above-mentioned ICG-derivative lipid, neutral lipid, polyethylene glycol-modified lipid, and sterol, etc.

[0054] Neutral lipids refer to lipids that exist at physiological pH in either uncharged or neutral zwitterionic form. Examples of neutral lipids include dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), egg phosphatidylcholine (EPC), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), diarachidoylphosphatidylcholine (DAPC), dibehenoylphosphatidylcholine (DBPC), dilignoceroylphosphatidylcholine (DLPC), dioleoylphosphatidylcholine (DOPC), sphingomyelin, ceramide, dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), and the like. The neutral lipids may be used alone or in combination of two or more. The lipid microparticles of the present embodiment may contain, for example, 0 to 99.999 mol%, for example, 50 to 99.99 mol%, for example, 60 to 99.9 mol%, of the neutral lipids based on the total lipid content of the lipid microparticles.

[0055] Polyethylene glycol-modified lipids include PEG2000-DMG (PEG2000-dimyristylglycerol), PEG2000-DPG (PEG2000-dipalmitoylglycerol), PEG2000-DSG (PEG2000-distearoylglycerol), PEG5000-DMG (PEG5000-dimyristylglycerol), PEG5000-DPG (PEG5000-dipalmitoylglycerol), PEG5000-DSG (PEG5000-distearoylglycerol), and P Examples of the PEG-dialkyloxypropyl include EG-cDMA (N-[(methoxypoly(ethylene glycol)2000)carbamyl]-1,2-dimyristyloxylpropyl-3-amine), PEG-C-DOMG (R-3-[(ω-methoxy-poly(ethylene glycol)2000)carbamoyl)]-1,2-dimyristyloxylpropyl-3-amine), polyethylene glycol (PEG)-diacylglycerol (DAG), PEG-dialkyloxypropyl (DAA), PEG-phospholipid, and PEG-ceramide (Cer). Examples of the PEG-dialkyloxypropyl include PEG-dilauryloxypropyl, PEG-dimyristyloxypropyl, PEG-dipalmityloxypropyl, and PEG-distearyloxypropyl. Examples of PEG-phospholipids include PEG2000-DSPE ((poly(ethylene glycol)2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine), MPEG2000-DSPE ((methoxypoly(ethylene glycol)2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine), and the like. The polyethylene glycol-modified lipids can be used alone or in combination of two or more. The polyethylene glycol-modified lipids may have a methoxylated end of PEG (polyethylene glycol) (MPEG; methoxy(polyethylene glycol)). The lipid microparticles of this embodiment may contain, for example, 0 to 30 mol%, for example, 0 to 20 mol%, for example, 0 to 10 mol%, of polyethylene glycol-modified lipids based on the total lipids contained in the lipid microparticles.

[0056] Sterols are alcohols having a steroid skeleton. Examples of sterols include cholesterol, dihydrocholesterol, lanosterol, β-sitosterol, campesterol, stigmasterol, brassicasterol, ergocastol, fucosterol, and 3β-[N-(N',N'-dimethylaminoethyl)carbamoyl]cholesterol (DC-Chol). Sterols can be used singly or in combination of two or more. The lipid microparticles of this embodiment may contain sterols in an amount of, for example, 0 to 90 mol%, for example, 0 to 80 mol%, for example, 0 to 50 mol%, 0 to 40 mol%, for example, 0 to 30 mol%, for example, 0 to 20 mol%, for example, 0 to 10 mol%, or for example, 0 to 5 mol%, based on the total lipid content of the lipid microparticles. The inclusion of sterols can improve blood stability. Meanwhile, thermal sensitivity tends to decrease as the amount of sterol increases. In one embodiment, the lipid microparticles do not contain sterols.

[0057] An example of the formulation of lipid components in the lipid microparticles of this embodiment includes, based on the total lipid content of the lipid microparticles, 0.01 to 20 mol% of the above-mentioned ICG-derivative lipid, 50 to 99.99 mol% of neutral lipid, and 0 to 49.99 mol% of sterol. Another example includes, based on the total lipid content of the lipid microparticles, 0.1 to 10 mol% of the above-mentioned ICG-derivative lipid, 60 to 99.9 mol% of neutral lipid, 0 to 10 mol% of polyethylene glycol-modified lipid, and 0 to 39.9 mol% of sterol. Another example includes, based on the total lipid content of the lipid microparticles, 0.1 to 10 mol% of the above-mentioned ICG-derivative lipid, 70 to 99.9 mol% of neutral lipid, 0 to 10 mol% of polyethylene glycol-modified lipid, and 0 to 10 mol% of sterol. Another example comprises, based on the total lipid content of the lipid microparticles, 0.1 to 10 mol % of the ICG-derivative lipid, 80 to 99.9 mol % of neutral lipid, and 0 to 10 mol % of polyethylene glycol-modified lipid.

[0058] In some embodiments, the lipid microparticles further comprise at least one drug. Examples of lipid microparticles formed from a lipid containing an ICG-derivative lipid and a drug include a complex between the drug and a membrane (reverse micelle) consisting of a single lipid layer (monolayer), a complex between the drug and a liposome, and a complex between the drug and a micelle (normal micelle). In some embodiments, the drug is encapsulated in lipid microparticles composed of lipids containing an ICG-derivative lipid. "Encapsulated" means that the drug is contained within the lipid layer itself or within a closed space formed by the lipid layer. In one embodiment, the drug is contained within a closed space formed by the lipid layer of the lipid microparticle. In other embodiments, the drug is present within one or more lipid layers of the lipid microparticle. In other embodiments, the drug is bound to the surface of the outer or inner lipid layer of the lipid microparticle. In certain embodiments, the drug is contained in the internal phase and / or lipid layer of a liposome composed of lipids containing an ICG-derivative lipid.

[0059] The drug may be either water-soluble or lipid-soluble. The drug may also be either hydrophilic or hydrophobic. In some embodiments, the drug is water-soluble. In certain embodiments, the lipid microparticles are present in an aqueous solvent, and a water-soluble drug is encapsulated within the closed space formed by the lipid layers of the lipid microparticles (e.g., the aqueous phase within the lipid microparticles, such as the internal phase of a liposome) and / or within one or more lipid layers of the lipid microparticles (in a single or multiple lipid layers). Examples of aqueous solvents include sterilized water; physiological saline; isotonic solutions containing auxiliary agents such as glucose, D-sorbitol, D-mannose, D-mannitol, sodium chloride, etc.; and buffer solutions such as phosphate buffer, citrate buffer, and acetate buffer.

[0060] Drugs may be synthetic compounds, fermentation products, peptides, proteins, steroid compounds, genes, viruses, sugars (polysaccharides), fatty acids, vitamins, coenzymes (or derivatives), etc. Furthermore, drugs used in any field, such as medicine, pesticides, and fertilizers, are also acceptable, with medicines being particularly preferred. Drugs may be, for example, any molecule or compound (e.g., active drug) that can exert a desired effect on cells, tissues, organs, or subjects. The drugs listed above are merely examples and are not limited thereto. Such drugs may be purified or partially purified, naturally occurring, synthetic, genetically engineered, or chemically modified.

[0061] In some embodiments, the drug is a therapeutic agent. In some embodiments, the drug is a small molecule compound. In some embodiments, the drug comprises at least one selected from an anticancer agent or an antibacterial agent. In one embodiment, the drug comprises an anticancer drug. In certain embodiments, examples of anticancer drugs include, but are not limited to, camptothecin derivatives such as irinotecan hydrochloride, nogitecan hydrochloride, and exatecan; taxane derivatives such as docetaxel hydrate, docetaxel, and paclitaxel; ifosfamide, nimustine hydrochloride, carboquone, cyclophosphamide, dacarbazine, thiotepa, busulfan, melphalan, ranimustine, estramustine phosphate sodium, 6-mercaptopurine riboside, enocitabine, gemcitabine hydrochloride, carmofur, cytarabine, cytarabine ocfosfate, tegafur, doxifluridine, hydroxycarbamide, and fluorouracil. Examples of anti-cancer drugs include cyclosporin, methotrexate, mercaptopurine, fludarabine phosphate, actinomycin D, aclarubicin hydrochloride, idarubicin hydrochloride, pirarubicin hydrochloride, epirubicin hydrochloride, daunorubicin hydrochloride, doxorubicin hydrochloride, epirubicin, pirarubicin, daunorubicin, doxorubicin, bleomycin hydrochloride, zinostatin stimalamer, neocarzinostatin, mitomycin C, bleomycin sulfate, hepuromycin sulfate, etoposide, vinorelbine tartrate, vincristine sulfate, vindesine sulfate, vinblastine sulfate, amrubicin hydrochloride, gefitinib, exemestane, and capecitabine. In one embodiment, the anti-cancer drug comprises doxorubicin. In the anti-cancer drug, the compound may be any salt.In certain embodiments, examples of antibacterial agents include, but are not limited to, antifungal triazole derivatives (fluconazole, itraconazole, ketoconazole, miconazole); antibacterial cephalosporins (cefazolin, cefonicid, cefotaxime, ceftazimide, cefoxime); antibacterial β-lactam derivatives (aztreopam, cefmetazole, cefoxitin); erythromycin group antibacterial agents (erythromycin, azithromycin, clarithromycin, oleandomycin); penicillins (benzylpenicillin, phenoxymethylpenicillin, cloxacillin, methicillin, nafcillin, oxacillin, carbenicillin); tetracyclines, etc. In the above antibacterial agents, the compound may be any salt.

[0062] The lipid microparticle carrier of this embodiment contains, for example, 0.01 to 50% by weight, for example, 0.1 to 30% by weight, for example, 1 to 10% by weight of the drug relative to the total weight of the lipid microparticle carrier.

[0063] The "average particle diameter" (hereinafter also referred to as "average particle size" or "particle size") of the lipid microparticles of this embodiment can be calculated by any of the volume average, number average, and Z-average methods, but the "average particle diameter" of the lipid microparticles according to the present invention refers to the Z-average particle size. The average particle diameter (Z-average particle size) of the lipid microparticles of this embodiment may be, for example, 30 to 200 nm, for example, 70 to 150 nm.

[0064] The lipid microparticles of the embodiment are excellent in stability of the microparticle structure (particularly stability in vivo), drug encapsulation ability, and / or drug release controllability. The ICG derivative lipid of the above-mentioned embodiment has an ICG skeleton that is a hydrocarbon chain (R １ and R ２ ) containing group, and can form a stable particulate structure. For example, the lipid particulate of the embodiment forms a liposome with excellent stability in vivo (particularly in blood). The ICG derivative lipid of the above-mentioned embodiment has an ICG skeleton that is modified with a hydrocarbon chain (R １ and R ２)-containing groups, enabling stable drug loading. Therefore, the lipid microparticles of the present embodiment can form a microparticle structure in which a drug is stably encapsulated. For example, the lipid microparticles of the present embodiment form drug-encapsulated liposomes that are excellent in vivo (particularly in blood) and / or in drug encapsulation efficiency. Furthermore, lipid microparticles (particularly liposomes) composed of the ICG lipid derivative of the present invention can selectively release encapsulated drugs to the outside of the lipid microparticles upon irradiation with near-infrared light. Furthermore, the inventors' studies have revealed that when the modifying group contains a polar group such as a sulfo group, stable drug loading is difficult, making it difficult to control the encapsulation and release of drugs. Since the lipid microparticles of the present invention contain the ICG-derivative lipid of the above-described embodiment, they have near-infrared fluorescence properties derived from ICG. The lipid microparticles of the present embodiment emit fluorescence upon irradiation with excitation light (e.g., near-infrared light). The lipid microparticles of the present invention contain the ICG-derivative lipid of the above-mentioned embodiment, and therefore have a heat-generating effect (hyperthermic effect; HT effect) and / or an effect of generating active oxygen (photodynamic effect; PDT effect) when irradiated with near-infrared light. In some aspects, the lipid microparticles containing the ICG-lipid derivative of the present invention function as temperature-sensitive liposomes.

[0065] (Method for Producing Lipid Microparticles) The lipid microparticle carriers of the embodiment can be produced using any method known in the art. Hereinafter, a method for producing lipid microparticle carriers will be described using an example in which the lipid microparticles are liposomes. For example, one embodiment of the production method includes the steps of (1) obtaining a liposome dispersion containing liposomes, and, if necessary, (2) mixing the liposome dispersion with a drug and encapsulating the drug in the liposomes of the liposome dispersion.

[0066] (1) Step of obtaining a liposome dispersion containing liposomes For example, the step of obtaining a liposome dispersion containing liposomes preferably includes the steps of (i) preparing liposomes from one or more lipids containing the ICG lipid derivative of the above-mentioned embodiment, and (ii) replacing or diluting the liposome external phase.

[0067] Step (i) The method for preparing liposomes is not particularly limited, and examples thereof include the lipid film method (vortex method), reverse phase evaporation, ultrasonic method, pre-vesicle method, ethanol injection method, French Press method, cholate removal method, Triton X-100 batch method, Ca fusion method, ether injection method, annealing method, freeze-thaw fusion method, etc. Various conditions for preparing liposomes (such as the amounts of membrane components and temperature) can be appropriately set depending on the method for preparing liposomes and the desired composition, particle size, etc. of the liposomes.

[0068] As an example, liposomes are prepared by the following procedure. First, one or more lipids containing an ICG lipid derivative are dissolved in a suitable organic solvent (e.g., chloroform) to prepare a lipid solution. Next, the organic solvent is removed from the lipid solution (preferably under reduced pressure) to form a lipid thin film. Subsequently, an aqueous solution is added to the lipid thin film to hydrate it, and the lipid thin film is stirred using a vortex mixer or the like to form liposomes. Examples of aqueous solutions include buffers such as Hepes buffer, phosphate buffer, citrate buffer, and phosphate-buffered saline, as well as physiological saline. Hydration is preferably performed while heating to a temperature above the phase transition temperature of the lipid bilayer membrane of the liposome. When preparing unilamellar liposomes, it is preferable to perform ultrasonic treatment, if necessary, after stirring using a vortex mixer or the like.

[0069] Step (ii) A liposome dispersion can be obtained by replacing or diluting the external phase of the obtained liposomes. The replacement or dilution of the liposome external phase may be performed once, or multiple times by combining various replacement or dilution methods. Methods for replacing the liposome external phase of the liposome preparatory solution include dialysis, centrifugation, gel filtration, and the like. Examples of the solvent (dispersion medium) used to replace and / or dilute the liposome external phase include buffers such as Hepes buffer, phosphate buffer, citrate buffer, and phosphate-buffered saline, as well as physiological saline. The pH of the solvent is not particularly limited, but is preferably 3 to 11, more preferably 4 to 10, and even more preferably 5 to 10.

[0070] The particle size of the liposomes can be adjusted as needed. For example, the particle size can be adjusted by extrusion filtration under high pressure using a membrane filter with a uniform pore size. The particle size can be adjusted at any time during the production of liposomes, for example, before replacing or diluting the liposome external phase, after replacing or diluting the liposome external phase, or after introducing a drug into the liposome internal phase. The particle size is preferably adjusted before introducing a drug into the liposome internal phase, and more preferably before replacing or diluting the liposome external phase.

[0071] In the case of encapsulating a drug in liposomes, it is preferable to introduce an ion gradient or pH gradient between the internal and external phases of the liposomes in step (ii) (remote loading method). Therefore, in some embodiments, step (ii) is a step of displacing the external phase of the liposomes to obtain a liposome dispersion containing liposomes having an ion gradient and / or pH gradient between the internal and external phases. The manufacturing method of this embodiment provides drug-encapsulated liposomes with excellent drug encapsulation efficiency and stability.

[0072] The pH gradient method is a technique for incorporating a compound into liposomes by utilizing the shift in the dissociation equilibrium between the molecular form and the ionic form of the drug due to the pH of the drug. When a pH gradient is introduced, the difference in pH between the liposome internal phase and the liposome external phase in the liposome dispersion is preferably 1 to 5, more preferably 2 to 3. Depending on the type of drug, the pH of either the liposome internal phase or the liposome external phase can be increased. Alternatively, the pH of the liposome internal phase and the liposome external phase may be substantially the same. The pH gradient can be adjusted using conventionally known compounds used in the pH gradient method, including, for example, amino acids such as arginine, histidine, and glycine, acids such as ascorbic acid, benzoic acid, citric acid, glutamic acid, phosphoric acid, acetic acid, propionic acid, tartaric acid, carbonic acid, lactic acid, boric acid, maleic acid, fumaric acid, malic acid, adipic acid, hydrochloric acid, and sulfuric acid, salts of the above acids such as sodium salts, potassium salts, and ammonium salts, and alkaline compounds such as trishydroxymethylaminomethane, aqueous ammonia, sodium hydroxide, potassium hydroxide, and sodium bicarbonate. An example of a compound that can be encapsulated in liposomes by the pH gradient method is doxorubicin (DOX, pKa: 8.2). For example, a liposome dispersion is prepared using a buffer solution with a pH of about 4 (e.g., citrate buffer), and then the liposome external phase is replaced with a buffer solution with a pH of about 7 (e.g., Hepes buffer), resulting in a liposome dispersion containing liposomes with a pH gradient between the internal and external phases. When DOX is added to this liposome dispersion in step (2) described below, molecular DOX becomes lipid-soluble in the external phase at pH 7, and therefore migrates to the liposome membrane rather than the aqueous phase. Furthermore, when DOX that has migrated to the liposome membrane comes into contact with the liposome internal phase at pH 4, DOX becomes ionic and dissolves in the liposome internal phase. In this way, DOX can be transported from the liposome external phase to the internal phase by shifting the dissociation equilibrium.

[0073] The ion gradient method is a technique for incorporating a drug into the liposome internal phase by utilizing an ion gradient formed between the internal and external layers of the liposome. Ions used in the ion gradient method are not particularly limited, but include ammonium sulfate, ammonium chloride, ammonium borate, ammonium formate, ammonium acetate, ammonium citrate, ammonium tartrate, ammonium succinate, and ammonium phosphate. In the ion gradient method, the ion concentration of the liposome internal phase can be appropriately selected depending on the type of drug; the higher the concentration, the better, preferably 10 mM or higher, more preferably 20 mM or higher, and even more preferably 50 mM or higher. Depending on the type of drug, the ion concentrations of either the liposome internal phase or the external phase can also be increased. On the other hand, the ion concentrations of the liposome internal phase and the external phase may not substantially differ from each other, i.e., the ion concentrations of the liposome external phase and the internal phase may be substantially the same. The ion gradient can also be adjusted by replacing or diluting the liposome external phase. Preferably, an ammonium sulfate ion gradient is used instead of the pH gradient used in the pH gradient method. Therefore, in some embodiments, step (ii) is a step of replacing the liposome external phase to obtain a liposome dispersion containing liposomes having an ammonium sulfate ion gradient and / or a pH gradient between the internal and external phases. In certain embodiments, step (ii) is a step of replacing the liposome external phase to obtain a liposome dispersion containing liposomes having an ammonium sulfate ion gradient and a pH gradient between the internal and external phases. According to this embodiment, drugs can be efficiently encapsulated in the liposome internal phase. Specifically, when an ammonium sulfate ion gradient is introduced, an ammonium sulfate aqueous solution can be used as the aqueous medium for hydration in step (i), and a buffer solution such as Hepes buffer, phosphate buffered saline, citrate buffer, or phosphate-buffered saline, or physiological saline can be used in step (ii).

[0074] The lipid concentration in the liposome dispersion is not particularly limited, but is preferably 1 to 100 mM, and more preferably 1 to 50 mM. Within these ranges, a larger number of liposome particles can be suitably formed without impairing the physical properties of the liposome dispersion.

[0075] (2) A step of mixing the liposome dispersion with a drug and encapsulating the drug in liposomes of the liposome dispersion. The resulting liposome dispersion is mixed with a drug to encapsulate the drug in liposomes (preferably in the internal phase of the liposomes) of the liposome dispersion. This allows liposomes with the drug encapsulated therein to be obtained. Preferably, the method includes a step of increasing the membrane permeability of the liposomes in the mixture of the liposome dispersion and the drug after mixing the liposome dispersion with the drug. This allows the encapsulation of the drug in the liposomes to be completed in a shorter time. Methods for increasing the membrane permeability of the liposomes in the mixture include, for example, heating the mixture or adding a membrane fluidizer to the mixture. When heating the mixture, generally, increasing the temperature to a higher temperature allows the drug to be introduced into the internal phase of the liposomes more efficiently. The temperature to which the temperature is increased is not particularly limited, and it is preferable to set the temperature taking into consideration the thermal stability of the drug and the liposome membrane components used. For example, it is preferably set to room temperature, or more preferably to a temperature equal to or higher than the phase transition temperature of the lipid bilayer membrane of the liposome.

[0076] 3. Compositions A further aspect of the present invention provides a composition comprising the lipid microparticles described above. In some embodiments, the composition contains the lipid microparticles described above, and optionally a pharmaceutically acceptable vehicle and other additives.

[0077] Pharmaceutically acceptable vehicles include sterile water; physiological saline; isotonic solutions containing adjuvants such as glucose, D-sorbitol, D-mannose, D-mannitol, and sodium chloride; and buffers such as phosphate buffer, citrate buffer, and acetate buffer. In some embodiments, the pharmaceutically acceptable vehicle is an aqueous solvent. Other additives may be added, for example, solubilizers such as alcohols (e.g., ethanol, propylene glycol, and polyethylene glycol), stabilizers, antioxidants, preservatives, excipients commonly used in pharmaceutical manufacturing, fillers, extenders, binders, wetting agents, disintegrants, lubricants, surfactants, dispersants, preservatives, flavoring agents, and soothing agents. Furthermore, other additives may be added, for example, sugars (e.g., sucrose, glucose, sorbitol, and lactose); amino acids (e.g., glutamine, glutamic acid, sodium glutamate, and histidine); and acid salts (e.g., citric acid, phosphoric acid, acetic acid, lactic acid, carbonic acid, and tartaric acid).

[0078] The composition includes those in a solid state and a liquid state. When the composition is in a solid state, it can be made into a liquid state by dissolving or suspending it in a predetermined pharmaceutically acceptable medium as described below. Furthermore, when the composition is a frozen solid, it can be made into a liquid state by melting it by leaving it at room temperature, etc. In some embodiments, the composition may be in a powder state from which the solvent has been removed by, for example, lyophilization, or may be in a liquid state. A composition according to one embodiment of the present invention is a powder composition containing the lipid microparticles of the above-mentioned embodiment. The powder composition may be prepared by removing the solvent from a liquid composition (dispersion) by, for example, filtration, centrifugation, etc., or may be prepared by lyophilizing the dispersion.

[0079] The composition of the present invention may be, for example, a pharmaceutical composition or a diagnostic composition. The composition of the present invention may be formulated as a pharmaceutical composition. Examples of the dosage form of a pharmaceutical composition include injections. The composition of the present invention may also be used as a diagnostic composition. The term "diagnostic composition" refers to a composition administered to a subject for diagnosis (in vivo diagnosis or in vitro diagnosis, particularly in vivo diagnosis). The dosage form of a diagnostic composition is not particularly limited, and examples thereof include injections. The pharmaceutical or diagnostic composition of one embodiment of the present invention is a liquid composition containing the lipid microparticles of the above-described embodiment, a pharmaceutically acceptable vehicle, and other additives. When the composition is in liquid form, it can be used as an injection, either directly or after being suspended or dissolved in a pharmaceutically acceptable vehicle. The pharmaceutical or diagnostic composition of one embodiment of the present invention is a powder composition containing the lipid microparticles of the above-described embodiment and other additives. When the pharmaceutical composition is in powder form, it can be used as an injection by suspending or dissolving it in a pharmaceutically acceptable vehicle before use.

[0080] The composition, pharmaceutical composition, or diagnostic composition can be administered to a subject parenterally, for example, by intraarterial injection, intravenous injection, or subcutaneous injection. The dosage varies depending on the subject, target organ, symptoms, and administration method. The subject of administration is not limited, and the composition can be applied to various animals, but is particularly applicable to mammals, preferably humans, and experimental animals used in clinical trials, screening, and experiments.

[0081] 4. Uses The ICG lipid derivatives, lipid microparticles, and compositions comprising lipid microparticles of the above embodiments can be used for a variety of applications. For example, the compositions (pharmaceutical compositions, diagnostic compositions) comprising the ICG lipid derivatives, lipid microparticles, and lipid microparticles of the above embodiments can be used for at least one selected from photothermal therapy, photodynamic therapy, and fluorescence imaging. In some embodiments, a photothermal therapy, photodynamic therapy, or fluorescence imaging method is provided, which includes administering to a subject a composition (pharmaceutical composition or diagnostic composition) comprising the lipid complex or lipid microparticles of the above embodiments. In some embodiments, use of a composition (pharmaceutical composition or diagnostic composition) comprising the lipid complex or lipid microparticles of the above embodiments for at least one selected from photothermal therapy, photodynamic therapy, and fluorescence imaging is provided.

[0082] (Fluorescence Imaging) In some embodiments, a composition containing the lipid microparticles of the above embodiment is used for fluorescence imaging. The lipid microparticles of the above embodiment encapsulate an ICG lipid derivative, and thus can absorb near-infrared light and emit fluorescence, making them useful as probes (contrast agents) for fluorescence imaging. The lipid microparticles of one embodiment are probes for fluorescence imaging. A fluorescence imaging method of the embodiment is characterized by the use of the lipid microparticles of the above embodiment. The imaging method of the embodiment is useful as a non-invasive identification method that can visualize biological tissue while preventing or reducing damage. The fluorescence imaging method of the embodiment typically includes the steps of administering a composition (probe) containing the lipid microparticles (ICG lipid derivatives) of the above embodiment to a subject, irradiating the subject with excitation light (e.g., near-infrared light) from an excitation light source, and then detecting fluorescence generated from the lipid microparticles (ICG lipid derivatives) by the excitation light. The excitation wavelength varies depending on the ICG lipid derivative contained in the lipid microparticles, and is not limited as long as the lipid microparticles (ICG lipid derivatives) effectively emit fluorescence in the near-infrared region. The excitation light is, for example, near-infrared light. Fluorescence imaging is performed according to known methods, with parameters such as excitation wavelength and fluorescence wavelength to be detected being appropriately determined depending on the type of ICG lipid derivative contained in the lipid microparticles and the recipient. The subject of the above-mentioned fluorescence imaging method is not limited and can be applied to various animals, but it is particularly applicable to mammals, preferably humans, and experimental animals in clinical trials, screening, and experiments. It may be performed in vivo or in vitro. One embodiment is a fluorescence imaging method for identifying sentinel lymph nodes. In some embodiments, a composition containing the lipid microparticles of the above-mentioned embodiment is used as a diagnostic composition in the above-mentioned fluorescence imaging method. The diagnostic composition can be used for diagnosis or diagnostic assistance in animals, or for detecting specific cells or tissues.

[0083] (Photothermotherapy / Photodynamic Therapy) In some embodiments, the pharmaceutical composition comprising the lipid microparticles of the above embodiments is used for photothermotherapy and / or photodynamic therapy. The photothermotherapy and / or photodynamic therapy of the embodiments is characterized by the use of the lipid microparticles of the above embodiments. Photothermotherapy and / or photodynamic therapy typically involves administering (e.g., intravenously or intratumorally) a pharmaceutical composition comprising the lipid microparticles of the above embodiments to a subject, and irradiating the subject (the affected area of ​​the subject) with near-infrared light after a certain period of time. Photothermotherapy and / or photodynamic therapy is performed in accordance with known methods. Parameters such as the wavelength range of near-infrared light, light irradiation time, and lipid microparticle concentration are determined appropriately depending on the type of ICG lipid derivative contained in the lipid microparticles, the subject to be administered, and the desired therapeutic effect. Photothermotherapy and / or photodynamic therapy according to the embodiments exhibit therapeutic effects on various tumors, such as brain tumors, insulinomas, nasal cancer, oral cancer, kidney cancer, lung cancer, colon cancer, soft tissue sarcoma, metastatic cancer (pleural metastasis, peritoneal metastasis), etc. In some embodiments, a pharmaceutical composition comprising the lipid microparticles according to the above embodiments is used for the treatment of cancer.

[0084] (Drug Delivery and Related Uses) The lipid microparticles of the above embodiments can be used as a means for selectively and efficiently introducing a desired drug encapsulated therein to a target site. For example, the composition of the present invention can be used in treatments that deliver a desired drug to a target site. Thus, one embodiment of the present invention provides a method for treating various diseases using a pharmaceutical composition containing lipid microparticles containing the above-mentioned drug. Another embodiment provides the use of lipid microparticles of the above embodiments in the manufacture of a medicament for treating various diseases. Some embodiments provide a method for delivering a drug to a subject, the method comprising administering to the subject a pharmaceutical composition containing lipid microparticles containing the drug of the above embodiment. Another embodiment provides a method for releasing or controlling the release of a desired drug encapsulated in lipid microparticles at a target site. When irradiated with near-infrared light, the ICG lipid derivative contained in the lipid microparticles generates heat, and as the temperature of the lipid microparticles increases, the encapsulated drug is released outside the lipid microparticles (e.g., liposomes). Therefore, irradiating a target site with near-infrared light enables selective drug release at the target site. In one embodiment, the liposomes are temperature-sensitive liposomes. Thermosensitive liposomes can efficiently and rapidly release drugs at target sites.

[0085] (Cancer Treatment) In some embodiments, a pharmaceutical composition comprising the lipid microparticles of the above embodiments is used for the treatment of cancer. In some embodiments, a method for treating cancer is provided, comprising administering to a subject a pharmaceutical composition comprising the lipid complex or lipid microparticles of the above embodiments. In some embodiments, use of a pharmaceutical composition comprising the lipid complex or lipid microparticles of the above embodiments for the treatment of cancer is provided. In certain embodiments, the lipid microparticles contain an anticancer drug as a drug. Liposomes (particularly liposomes with a particle size of 200 nm or less) have already been confirmed in cancer-bearing animal experiments and human patients to be prone to accumulate in solid cancer tissue due to the so-called Enhanced Permeability and Retention (EPR) effect. Liposomes containing the ICG lipid derivatives of the above embodiments combine the functions of in vivo imaging of cancer tissue based on the infrared fluorescence properties of the ICG lipid derivative, cancer hyperthermia based on the heat-generating effect of the ICG lipid derivative, and anticancer drug-encapsulated thermosensitive liposomes (liposomes that specifically release anticancer drugs with increasing temperature) by encapsulating an anticancer drug, making them suitable for use in cancer treatment.

[0086] 5. Kit One embodiment of the present invention is a pharmaceutical delivery or imaging kit comprising the lipid microparticles. This kit can be used for various treatments or diagnoses. In the kit of this embodiment, the storage state of the lipid microparticles is not particularly limited, and they can be set in any state, such as a solution or powder, taking into consideration their respective stability (storage) and ease of use. In addition to the lipid microparticles, the kit of this embodiment may also include, for example, various media (pharmaceutically acceptable media, buffers), various drugs, and instructions for use (usage manuals). The kit of this embodiment is used to prepare a composition containing desired lipid microparticles to be introduced into a target site. For example, the lipid microparticles according to this embodiment and various media may be prepared as a kit, and the particles may be dispersed in the various media before administration to a living body.

[0087] The present invention will be described in further detail below with reference to examples, production examples, and test examples, but the present invention is not limited to these examples. In this specification, "room temperature" generally refers to about 10°C to about 35°C. "%" refers to weight percent unless otherwise specified. The starting materials, reagents, acids, bases, dehydrating agents, solvents, and catalysts used to synthesize the compounds of the present invention are all commercially available or can be produced by organic synthesis methods known to those skilled in the art. Furthermore, the compounds of the present invention can be produced by organic synthesis methods known to those skilled in the art, as shown in the following examples. Mass spectrometry was performed using a Waters Xevo™ G2-XS.

[0088] The names of the compounds shown below, except for commonly used reagents, were generated using "ChemDraw Professional" version 15.1.0.144 (PerkinElmer). The abbreviations used in the examples are conventional abbreviations well known to those skilled in the art. Some abbreviations are listed below. Ac ２ O Acetic anhydride AcONa Sodium acetate Br Bromo DCM Dichloromethane DIPEA N,N-Diisopropylethylamine DMF Dimethylformamide DOPE Dioleoylphosphatidylethanolamine DPPC Dipalmitoylphosphatidyl DSPC Distearoylphosphatidylcholine DSPE 1,2-distearoyl-sn-glycero-3-phosphoethanolamine HBTU 1-[bis(dimethylamino)methylene]-1H-benzotriazolium 3-oxide hexafluorophosphate MeOH Methanol MPEG Methoxypolyethylene glycol MPEG2000-DSPE (Methoxypoly(ethylene glycol)2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine SA Stearylamine o-DCB 1,2-Dichlorobenzene PEG polyethylene glycol reflux

[0089] A. Synthesis of ICG lipid derivatives [Production Example 1] Synthesis of ICG dicarboxylic acid (compound 5) Compound 5: 3-(2-((1E,3E,5E,7E)-7-(3-(2-carboxyethyl)-1,1-dimethyl-1,3-dihydro-2H-benzo[e]indol-2-ylidene)hepta-1,3,5-trien-1-yl)-1,1-dimethyl-1H-benzo[e]indol-3-ium)propanoate Compound 5 was synthesized according to the following scheme.

[0090] (Step i) 1,1,2-trimethyl[1H]-benz[e]indole (10.0 g, Compound 3) and 3-bromopropionic acid (7.3 g) were added and dissolved in 50 ml of 1,2-dichlorobenzene, followed by stirring at 110°C for 18 hours. After allowing to cool to room temperature, the precipitated solid was filtered off and washed with dichloromethane. After filtering, the solid was dried under reduced pressure to obtain 13.9 g of 3-(2-carboxyethyl)-1,1,2-trimethyl-1H-benzo[e]indol-3-ium bromide (Compound 4).

[0091] (Step ii) Glutaconaldehyde dianilide monohydrochloride (2.84 g; Compound 1), N,N-diisopropylethylamine (2.60 g), and 20 ml of dichloromethane were added to the mixture, and a solution of acetic anhydride (1.20 g) in dichloromethane (5 ml) was added dropwise under ice cooling. The mixture was stirred for 3 hours, and the solvent was distilled off. (Step iii) The residue from Step ii was dissolved in 5 ml of methanol, and the solution was added dropwise to a solution of Compound 4 (10.0 g) and sodium acetate (3.9 g, 47.54 mmol) in methanol (50 ml) while refluxing. After the addition, the mixture was reacted under reflux for 16 hours, and the solvent was distilled off. The residue was washed with ethyl acetate, 5% hydrochloric acid, and ethyl acetate, in that order, and the resulting mixture was recrystallized from acetonitrile / water (3 / 7) to obtain 4.3 g of Compound 5.

[0092] Example A1 ICG-diSA: 2-((1E,3E,5E,7E)-7-(1,1-dimethyl-3-(3-(octadecylamino)-3-oxopropyl)-1,3-dihydro-2H-benzo[e]indol-2-ylidene)hepta-1,3,5-trien-1-yl)-1,1-dimethyl-3-(3-(octadecylamino)-3-oxopropyl)-1H-benzo[e]indol-3-ium According to the following scheme, an ICG lipid derivative (ICG-diSA) was synthesized from compound 5 obtained in Production Example 1. A mixture of compound 5 (502 mg), dimethylformamide (85 ml), stearylamine (1.01 g, compound 6), 1-[bis(dimethylamino)methylene]-1H-benzotriazolium 3-oxide hexafluorophosphate (2.71 g), and N,N-diisopropylethylamine (413 mg) was stirred at room temperature for 3 hours. After the solvent was distilled off, the residue was dissolved in ethyl acetate (130 ml) and washed twice with 50 ml of saturated aqueous sodium hydrogen carbonate solution and twice with 50 ml of saturated saline, and the organic layer was dehydrated over anhydrous magnesium sulfate. After the solvent was distilled off, the residue was passed through a silica gel column (Cosmosil 75C １８ The resulting mixture was purified with 0.1% trifluoroacetic acid / methanol (100 / 0 to 0 / 100) using OPN (30 g), and then further purified with silica gel (Cosmosil 75C １８ The mixture was purified with OPN (40 g) and methanol / water (75 / 25 to 92 / 8) to obtain ICG-diSA (222 mg). １ The H NMR chart is shown in Figure 1.

[0093] [Examples A2 and A3] ICG-diDOPE: 2-(3-(2-((1E,3E,5E,7E)-7-(3-(3-((2-(((2,3-bis(oleoyloxy)propoxy)(hydroxy)phosphoryl)oxy)ethyl)amino)-3-oxopropyl)-1,1-dimethyl-1,3-dihydro-2H-benzo[e]indol-2-ylidene)hepta-1,3,5-trien-1-yl)-1,1-dimethyl-1H-benzo[e]indol-3-ium-3-yl)propanamido)ethyl (2,3-bis(oleoyloxy)propyl)phosphate ICG-diDSPE: 2-(3-(2-((1E,3E,5E,7E)-7-(3-(3-((2-(((2,3-bis(stearoyloxy)propoxy)(hydroxy)phosphoryl)oxy)ethyl)amino)-3-oxopropyl)-1,1-dimethyl-1,3-dihydro-2H-benzo[e]indol-2-ylidene)hepta-1,3,5-trien-1-yl)-1,1-dimethyl-1H-benzo[e]indol-3-ium-3-yl)propanamido)ethyl (2,3-bis(stearoyloxy)propyl)phosphate ICG lipid derivatives ICG-diDOPE and ICG-diDSPE were synthesized from compound 5 obtained in Production Example 1 according to the following scheme.

[0094] (1) ICG-diDOPE To a mixture of compound 5 (501 mg), dichloromethane (15 ml), N-hydroxysuccinimide (278 mg), and acetonitrile (3 ml) was added dropwise a solution of N,N'-dicyclohexylcarbodiimide (511 mg) in dichloromethane (3 ml) at room temperature while stirring. After filtration, the solvent was evaporated and the mixture was recrystallized from ethyl acetate (20 ml). The resulting solid was dissolved in dichloromethane (20 ml), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE, 738 mg, compound 8) and triethylamine (0.22 g) were added, followed by stirring overnight at room temperature. After evaporation of the solvent, the mixture was filtered and recrystallized with silica gel (Cosmosil 75C １８The mixture was purified with OPN (10 g), methanol / water (50 / 50 to 100 / 0), and methanol / chloroform (90 / 10 to 80 / 20), and then further purified with silica gel (Cosmosil 75C １８ The resulting mixture was purified with OPN (40 g) and chloroform / methanol (100 / 0 to 75 / 25) to obtain ICG-diDOPE (97 mg). １ The H NMR chart is shown in FIG.

[0095] (2) ICG-diDSPE Compound 5 (1.01 g, 1.60 mmol), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE, 2.50 g, Compound 9), chloroform (170 ml), 1-[bis(dimethylamino)methylene]-1H-benzotriazolium 3-oxide hexafluorophosphate (2.43 g), and N,N-diisopropylethylamine (435 mg) were added and stirred at room temperature overnight. After filtration, the solvent was distilled off, and the resulting mixture was passed through silica gel (Cosmosil 75C １８ The mixture was purified with OPN (120 g) and chloroform / methanol (100 / 0 to 90 / 10) to give ICG-diDSPE (299 mg). MS (ESI) m / z: 2082.4 [M−H] － ICG-diDSPE １ The H NMR chart is shown in FIG.

[0096] [Comparative Example A1] ICG-DOPE: 4-(2-((1E,3E,5E,7E)-7-(3-(3-((2-(((2,3-bis(oleoyloxy)propoxy)oxidophosphoryl)oxy)ethyl)amino)-3-oxopropyl)-1,1-dimethyl-1,3-dihydro-2H-benzo[e]indol-2-ylidene)hepta-1,3,5-trien-1-yl)-1,1-dimethyl-1H-benzo[e]indol-3-ium-3-yl)butane-1-sulfonate ICG-DOPE was synthesized based on the following scheme with reference to the methods described in paragraphs

[0050] to

[0053] and

[0068] of WO 2011 / 152046. １ The H NMR chart is shown in FIG.

[0097] <Calculation of Molar Absorption Coefficient> The molar absorption coefficients of the ICG lipid derivatives and ICG synthesized in Examples A1 to A3 and Comparative Example A1 were calculated using the following method. Each ICG lipid derivative or ICG was dissolved in ethanol at 5 μM in an ICG lipid solution, which was placed in a cuvette with a path length of 1 cm, and the absorption spectrum was measured from 230 to 1000 nm using an Infinite M200 (Tecan). Figure 5 shows the absorption spectrum of each ICG lipid derivative. The absorption spectrum in Figure 5 shows the absorbance at each wavelength minus the absorption spectrum of ethanol. The maximum absorption wavelength in the absorption spectrum for each ICG lipid was 790 nm. The molar absorption coefficient ε (L / mol / cm) was calculated from the absorbance at the maximum absorption wavelength of 790 nm (minus the absorbance of ethanol). In calculating the molar absorption coefficient, the molar concentration calculated assuming that the ICG lipid derivative was 100% pure and had not formed a salt was used. The molar extinction coefficient of each ICG lipid derivative is shown in Table A. As shown in Figure 5, all ICG lipid derivatives exhibited absorption in the near-infrared region of 700 to 900 nm. In addition, all ICG lipid derivatives had a molar absorption coefficient of 1.6 x 10 ５ [L / mol / cm] or more. The ICG lipid derivative of the present invention was confirmed to have excellent near-infrared light absorption properties. As described above, the ICG lipid derivative of the present invention exhibits near-infrared light absorption properties similar to ICG, and therefore lipid microparticles comprising the ICG lipid derivative are applicable to fluorescence imaging and photoacoustic imaging, and are considered to be useful as diagnostic agents and / or contrast agents. In other words, the ICG lipid derivative of the present invention and lipid microparticles comprising the same can also be used in theranostics (the fusion of diagnosis and treatment).

[0098] B. Preparation of ICG lipid derivative-modified liposomes and doxorubicin-encapsulating ICG lipid derivative-modified liposomes 1. Preparation of ICG lipid derivative-modified liposomes (iLip-1) and doxorubicin-encapsulated ICG lipid derivative-modified liposomes (DOX-iLip-1) Using the ICG lipid derivatives synthesized in Examples A1 to A3 above and doxorubicin as a drug, ICG lipid derivative-modified liposomes (iLip-1(ICG-diSA), iLip-1(ICG-diDOPE), iLip-1(ICG-diDSPE)) and doxorubicin-encapsulated ICG lipid derivative-modified liposomes (DOX-iLip-1(ICG-diSA), DOX-iLip-1(ICG-diDOPE), DOX-iLip-1(ICG-diDSPE)) were prepared using an ammonium sulfate gradient and a pH gradient according to the following procedure. In addition, using the ICG lipid derivative synthesized in Comparative Example A1 and doxorubicin as a drug, ICG lipid derivative-modified liposomes (iLip-1(ICG-DOPE)) and doxorubicin-encapsulated ICG lipid derivative-modified liposomes (DOX-iLip-1(ICG-DOPE)) were prepared using an ammonium sulfate gradient and a pH gradient according to the following procedures.

[0099] [Example B1] (1) Preparation of iLip-1 (ICG-diSA) ICG-diSA synthesized in Example A1 above was used as the ICG lipid derivative. 200 μL of a chloroform solution of 0.1 mol / L dipalmitoylphosphatidylcholine (DPPC, manufactured by Nippon Fine Chemicals), 100 μL of a chloroform solution of 0.1 mol / L cholesterol (manufactured by Nippon Fine Chemicals), 100 μL of a chloroform solution of 0.01 mol / L MPEG2000-DSPE (manufactured by Nippon Fine Chemicals, PEG average molecular weight: 2,000), and 200 μL of a chloroform solution of 0.001 mol / L ICG lipid derivative (ICG-diSA) were added and mixed in chloroform. The mixture was then evaporated under reduced pressure and dried under vacuum for at least 1 hour. To the lipid thin film formed on the wall of the recovery flask, 2 ml of 0.3 mol / L ammonium sulfate aqueous solution (pH 5.5) was added, heated to 50°C, and stirred using a vortex mixer. The resulting crude liposome dispersion was frozen in liquid nitrogen, thawed at 50°C, and gently stirred using a vortex mixer. This freezing and thawing procedure was repeated three times, followed by treatment for 5 minutes in a bath-type ultrasonicator (Branson). The resulting liposome dispersion was further passed through a 100 nm pore size polycarbonate Nuclepore membrane filter (GE Healthcare) five times using an extruder (Transfera Nanosciences) heated to 50°C to adjust the liposome particle size. The resulting liposome dispersion was then subjected to separation for 15 minutes using an ultracentrifuge (CS120GXL, Hitachi Koki Co., Ltd.) set at 453,000 x g and 4°C. The supernatant was then removed and the mixture was resuspended in 1 ml of 0.02 mol / l Hepes buffer (pH 7.4) to obtain a dispersion of liposomes modified with an ICG lipid derivative (iLip-1 (ICG-diSA)).

[0100] (2) DOX-iLip-1 (ICG-diSA) To the dispersion of liposomes (iLip-1 (ICG-diSA)) obtained above, 1 ml of a solution of doxorubicin (Kyowa Hakko Kirin Co., Ltd.) dissolved in 0.02 mol / L Hepes buffer (pH 7.4) to a concentration of 2 mg / ml was added, and the mixture was stirred at 50°C for 30 minutes to encapsulate doxorubicin within the liposomes. The liposome dispersion was subjected to a separation procedure for 15 minutes using the ultracentrifuge described above set at 453,000 x g and 4°C. After removing the supernatant, 2 ml of ultrapure water was added to obtain a dispersion of doxorubicin-encapsulated ICG lipid derivative-modified liposomes (DOX-iLip-1 (ICG-diSA)).

[0101] [Example B2] A dispersion of ICG lipid derivative-modified liposomes (iLip-1(ICG-diDOPE)) and a dispersion of doxorubicin-encapsulated ICG lipid derivative-modified liposomes (DOX-iLip-1(ICG-diDOPE)) were obtained in the same manner as in Example B1, except that ICG-diDOPE obtained in Example A2 was used instead of ICG-diSA as the ICG lipid derivative.

[0102] [Example B3] A dispersion of ICG lipid derivative-modified liposomes (iLip-1(ICG-diDSPE)) and a dispersion of doxorubicin-encapsulated ICG lipid derivative-modified liposomes (DOX-iLip-1(ICG-diDSPE)) were obtained in the same manner as in Example B1, except that ICG-diDSPE obtained in Example A3 was used instead of ICG-diSA as the ICG lipid derivative.

[0103] Comparative Example B1 A dispersion of ICG lipid derivative-modified liposomes (iLip-1(ICG-DOPE)) and a dispersion of doxorubicin-encapsulated ICG lipid derivative-modified liposomes (DOX-iLip-1(ICG-DOPE)) were obtained in the same manner as in Example B1, except that ICG-DOPE obtained in Comparative Example A1 was used instead of ICG-diSA as the ICG lipid derivative.

[0104] <Liposome Analysis 1> (1) Measurement of Liposome Particle Size Distribution and Zeta Potential The liposome particle size distribution (mean particle size (Z-average) (d) and polydispersity index) and zeta potential of the liposome (iLip-1) samples before doxorubicin encapsulation and the liposome (DOX-iLip-1) samples after doxorubicin encapsulation obtained in Examples B1 to B3 and Comparative Example B1 above were measured using a Zetasizer Nano ZS (manufactured by Malvern Instruments, Spectris; solvent: Water) in a standard manner. Table 1 shows the particle size distribution and zeta potential of each liposome (iLip-1) before doxorubicin encapsulation. Table 2 shows the particle size distribution and zeta potential of each liposome (DOX-iLip-1) after doxorubicin encapsulation.

[0105] (2) Quantification of doxorubicin encapsulated in liposomes and calculation of encapsulation rate 35 μl of each liposome (DOX-iLip-1) dispersion after doxorubicin encapsulation obtained in Examples B1 to B3 and Comparative Example B1 above was mixed with 35 μl of 10% reduced Triton X-100 (Sigma-Aldrich) solution and 280 μl of ultrapure water to obtain a solubilized solution of each liposome. To prepare a calibration curve for doxorubicin, solutions containing doxorubicin aqueous solutions (doxorubicin diluted with ultrapure water to final concentrations of 0, 3.125, 6.25, 12.5, 25, 50, 100, 200, 400, and 800 μg / ml) were also prepared in place of the liposome dispersion. 100 μl of each of these mixtures was dispensed into wells of a 96-well microplate (Corning), and the absorbance at a wavelength of 495 nm was measured using a multiplate reader (Infinite® M200; manufactured by Tecan). The amount of doxorubicin encapsulated in DOX-iLip-1 was determined from the obtained absorbance using a calibration curve method. The ratio of the amount of doxorubicin encapsulated in DOX-iLip-1 (doxorubicin concentration; DOX concentration) to the amount of doxorubicin added to the dispersion of liposomes (iLip-1) for encapsulation in the liposomes (concentration of the doxorubicin solution) was calculated, and this was taken as the encapsulation rate of doxorubicin in each liposome (DOX encapsulation rate, %). The results are shown in Table 3.

[0106] As shown in Table 1 and Figure 6, regardless of which ICG lipid derivative was used, uniform ICG lipid derivative-modified liposomes (iLip-1) with an average particle size of approximately 100 to 140 nm and a polydispersity index of 0.1 or less were prepared before doxorubicin encapsulation. The results in Table 1 demonstrate that the ICG lipid derivatives of the present invention can be used as components of lipid microparticles. As shown in Table 2 and Figure 7, for iLip-1 prepared using each of the ICG lipid derivatives of the present invention (ICG-diSA, ICG-diDOPE, ICG-diDSPE), the particle size remained almost unchanged, at 130 to 140 nm, even after doxorubicin encapsulation, compared with DOX-iLip-1. On the other hand, for DOX-iLip-1 prepared using the known substance ICG-DOPE, a large increase in average particle size and polydispersity index, likely due to aggregation, was observed after doxorubicin encapsulation into the liposomes. These results confirmed that the ICG lipid derivative of the present invention is useful as a lipid microparticle for encapsulating drugs such as doxorubicin, and is capable of forming drug-encapsulating liposomes with excellent stability. The reason why stable drug-encapsulating liposomes could not be obtained when using the known substance ICG-DOPE is not limited to the following theory, but it is thought that the known substance ICG-DOPE has a sulfo group (-SO ３H) is exposed on the surface of the liposome membrane, and it is presumed that the interaction between this sulfo group and the drug (doxorubicin) prevents encapsulation inside the liposome. As shown in Table 3, the doxorubicin encapsulation rate of each DOX-iLip-1 was about 50% for DOX-iLip-1 (ICG-DOPE) of Comparative Example B1, which was prepared using the known substance ICG-DOPE, whereas the liposomes DOX-iLip-1 (ICG-diSA), DOX-iLip-1 (ICG-diDOPE), and DOX-iLip-1 (ICG-diDSPE) prepared using the ICG lipid derivatives of the present invention (ICG-diSA, ICG-diDOPE, ICG-diDSPE) showed high doxorubicin encapsulation rates of 70 to 90%. From the above, it was revealed that liposomes prepared with the ICG lipid derivatives of the present invention (ICG-diSA, ICG-diDOPE, ICG-diDSPE) have superior stability of drug-encapsulated liposomes formed by preparing empty liposomes and then encapsulating a drug from the outside of the liposomes into the interior of the liposomes, compared to liposomes prepared with a known ICG lipid derivative (ICG-DOPE), and 2) have a higher efficiency of encapsulating the drug into the interior of the liposomes.

[0107] 2. Preparation of ICG lipid derivative-modified liposomes (iLip-2) and doxorubicin-encapsulated ICG lipid derivative-modified liposomes (DOX-iLip-2) ICG lipid derivative-modified liposomes (iLip-2(ICG-diSA), iLip-2(ICG-diDOPE), iLip-2(ICG-diDSPE)) and doxorubicin-encapsulated ICG lipid derivative-modified liposomes (DOX-iLip-2(ICG-diSA), DOX-iLip-2(ICG-diDOPE), DOX-iLip-2(ICG-diDSPE)) were prepared using the ICG lipid derivatives synthesized in Examples A1 to A3 above and doxorubicin as the drug by the pH gradient method using citric acid, which is another representative encapsulation method. In addition, using the ICG lipid derivative synthesized in Comparative Example A1 and doxorubicin as a drug, ICG lipid derivative-modified liposomes (iLip-2(ICG-DOPE)) and doxorubicin-encapsulated ICG lipid derivative-modified liposomes (DOX-iLip-2(ICG-DOPE)) were prepared using an ammonium sulfate gradient and a pH gradient according to the following procedures.

[0108] [Example B4] (1) Preparation of iLip-2 (ICG-diSA) ICG-diSA synthesized in Example A1 above was used as the ICG lipid derivative. 360 μl of a chloroform solution of 0.1 mol / L dipalmitoylphosphatidylcholine (DPPC, manufactured by Nippon Fine Chemicals), 200 μl of a chloroform solution of 0.01 mol / L distearoylphosphatidylcholine (DSPC, manufactured by Nippon Fine Chemicals), 200 μl of a chloroform solution of 0.01 mol / L MPEG2000-DSPE (manufactured by Nippon Fine Chemicals, average molecular weight of PEG: 2,000), and 400 μl of a chloroform solution of 0.001 mol / L ICG lipid derivative (ICG-diSA) were added and mixed in chloroform. The residue was then evaporated under reduced pressure and dried under vacuum for at least 1 hour, followed by the addition of 2 ml of 0.3 mol / L citrate buffer (pH 4.0), heating to 50°C, and stirring with a vortex mixer to obtain a crude liposome dispersion. Next, using an extruder (Transfera Nanoscience) heated to 50°C, the mixture was passed through polycarbonate Nuclepore membrane filters (GE Healthcare) with pore sizes of 200 nm, 100 nm, and 50 nm, five times each, to obtain a dispersion of liposomes (iLip-2 (ICG-diSA)).

[0109] (2) DOX-iLip-2 (ICG-diSA) To 2 ml of the resulting liposome dispersion, 1.56 ml of 0.5 mol / L aqueous sodium bicarbonate solution and 0.44 ml of 0.02 mol / L Hepes buffer (pH 7.4) were added. Then, 2 ml of a solution of doxorubicin (Kyowa Hakko Kirin Co., Ltd.) dissolved in 0.02 mol / L Hepes buffer (pH 7.4) to a concentration of 2 mg / ml was added, and the mixture was stirred at 50°C for 2 minutes to encapsulate doxorubicin within the liposomes. The liposome dispersion was subjected to a separation procedure for 30 minutes using an ultracentrifuge (CS120GXL, Hitachi Koki Co., Ltd.) set at 511,000 x g and 4°C. After removing the supernatant, 4 ml of a 0.9% aqueous sodium chloride solution was added to obtain a dispersion of various doxorubicin-encapsulating ICG lipid derivative-modified liposomes (DOX-iLip-2 (ICG-diSA)).

[0110] [Example B5] A dispersion of ICG lipid derivative-modified liposomes (iLip-2(ICG-diDOPE)) and a dispersion of doxorubicin-encapsulated ICG lipid derivative-modified liposomes (DOX-iLip-2(ICG-diDOPE)) were obtained in the same manner as in Example B4, except that ICG-diDOPE obtained in Example A2 was used instead of ICG-diSA as the ICG lipid derivative.

[0111] [Example B6] A dispersion of ICG lipid derivative-modified liposomes (iLip-2(ICG-diDSPE)) and a dispersion of doxorubicin-encapsulated ICG lipid derivative-modified liposomes (DOX-iLip-2(ICG-diDSPE)) were obtained in the same manner as in Example B4, except that ICG-diDSPE obtained in Example A3 was used instead of ICG-diSA as the ICG lipid derivative.

[0112] Comparative Example B2 A dispersion of ICG lipid derivative-modified liposomes (iLip-2(ICG-DOPE)) and a dispersion of doxorubicin-encapsulated ICG lipid derivative-modified liposomes (DOX-iLip-2(ICG-DOPE)) were obtained in the same manner as in Example B4, except that ICG-DOPE obtained in Comparative Example A1 was used instead of ICG-diSA as the ICG lipid derivative.

[0113] <Liposome Analysis 2> (1) Measurement of Liposome Particle Size Distribution and Zeta Potential For the liposome (DOX-iLip-2) samples after doxorubicin encapsulation obtained in Examples B4 to B6 and Comparative Example B2 above, the liposome particle size distribution (mean particle size (Z-average) and polydispersity index) and zeta potential were measured using a Zetasizer Nano ZS (manufactured by Malvern Instruments, Spectris) in the same manner as in (1) of <Liposome Analysis 1> above. Table 4 shows the particle size distribution and zeta potential of each liposome (DOX-iLip-2) after doxorubicin encapsulation.

[0114] (2) Quantification of doxorubicin encapsulated in liposomes and calculation of encapsulation rate For the liposome (DOX-iLip-2) dispersions after encapsulating doxorubicin obtained in Examples B4 to B6 and Comparative Example B2, the amount of doxorubicin encapsulated in the liposomes was quantified in the same manner as in (2) of <Liposome analysis 1> above, and the encapsulation rate of doxorubicin in each liposome (DOX encapsulation rate) was calculated. The results are shown in Table 5.

[0115] In Examples B4 to B6 and Comparative Example B2, uniform ICG lipid derivative-modified liposomes (ICG-iLip-2) with an average particle size of approximately 85 to 140 nm and a polydispersity index of 0.16 or less after doxorubicin encapsulation were successfully prepared using any of the ICG lipid derivatives (see Table 4 and Figure 8). On the other hand, there was a significant difference in the doxorubicin encapsulation rate of each DOX-iLip-2 obtained between Examples B4 to C3 and Comparative Example B2. That is, while the doxorubicin encapsulation rate was about 50% in DOX-iLip-2 (ICG-DOPE) of Comparative Example B2 prepared using the known substance ICG-DOPE, the ICG lipid derivatives of the present invention (ICG-diSA, ICG-diDOPE, ICG-diDSPE) showed a high doxorubicin encapsulation rate of 80 to 90% (see Table 5). This confirmed that the use of liposomes DOX-iLip-2 (ICG-diSA), DOX-iLip-2 (ICG-diDOPE), and DOX-iLip-2 (ICG-diDSPE) prepared using the ICG lipid derivatives of the present invention allowed for a higher doxorubicin encapsulation rate than DOX-iLip-2 (ICG-DOPE) prepared using the known substance ICG-DOPE. These results confirmed that the ICG lipid derivatives of the present invention (ICG-diSA, ICG-diDOPE, ICG-diDSPE) are useful as lipid microparticles for encapsulating drugs such as doxorubicin, and are capable of forming liposomes with excellent stability and encapsulating drugs at a higher encapsulation rate than the known substance ICG-DOPE. The reason for the lower drug encapsulation rate when the known substance ICG-DOPE was used is not limited to the following theory, but it is thought that the known substance ICG-DOPE has a sulfo group (-SO ) contained in its structure during liposome formation.３ H) is exposed on the surface of the liposome membrane, and it is presumed that the interaction between this sulfo group and the drug (doxorubicin) prevents the drug from being encapsulated inside the liposome.

[0116] (3) Investigation of DOX-iLip-2 Temperature Increase and Doxorubicin Release by Irradiation with Near-Infrared LED Light The temperature increase of the dispersion medium and the accompanying release behavior of doxorubicin from each liposome when the doxorubicin-encapsulated liposome (DOX-Lip) dispersion obtained in Examples B4 to B6 and Comparative Example B2 above were irradiated with an LED lamp were observed. Each DOX-iLip-2 dispersion was diluted with ultrapure water to a total lipid concentration (concentration of DPPC, cholesterol, MPEG2000-DSPE, and ICG lipid derivative in the dispersion) of 0.01 mol / L, and 500 μl was placed in a well of a 24-well Falcon microplate (Corning). Near-infrared light was irradiated from the bottom of the plate using an LED light source (maximum wavelength 810 nm, half-width 40 nm) in a thermostatic chamber (Tytec Co., Ltd.) set at 37°C. The temperature of the DOX-iLip-2 dispersion irradiated with near-infrared light was simultaneously measured using a temperature logger (SK-L400T, Sato Keiryoki Seisakusho Co., Ltd.) (data acquisition every 10 seconds). After 5, 10, and 60 minutes of near-infrared light irradiation, the DOX-iLip-2 dispersion was collected and mixed with 0.9% aqueous sodium chloride solution. The DOX-iLip-2 was then precipitated using an ultracentrifuge (CS120GXL, Hitachi Koki Co., Ltd.) set at 511,000 x g and 4°C. After removing the supernatant, 500 μl of 0.9% aqueous sodium chloride solution was added. 35 μl of the resulting liposome dispersion was mixed with 35 μl of 10% Triton X-100 solution and 280 μl of ultrapure water to obtain a solubilized solution of each liposome. Each mixture was treated in the same manner as in (2) of <Liposome Analysis 1> above to determine the amount of doxorubicin remaining in the liposomes, and the amount of doxorubicin (%) released from the liposomes over time was calculated. Note that each liposome dispersion was placed in a microplate well in a thermostatic bath (Titec Co., Ltd.) set at 37°C, and the amount of doxorubicin released over time (%) was also determined for a sample that was left without being irradiated with the LED lamp, and this was used as a control experiment for each formulation.

[0117] Tables 6 to 9 show the time course of the doxorubicin release rate (%) from each DOX-iLip-2 upon irradiation with near-infrared light.

[0118] From the results of measuring the temperature of the liposome dispersion over time shown in Figures 9 to 12, it was confirmed that the temperature of the liposome dispersion rose with irradiation of near-infrared light for all four types of liposomes (DOX-iLip-2) in Examples B4 to B6 and Comparative Example B2, reaching a temperature of approximately 50°C. This indicates that the ICG lipid derivative (ICG-modified liposome), which is a liposome-modifying substance, generates heat upon irradiation with near-infrared light, resulting in an increase in the temperature of the entire dispersion medium. This suggests that the ICG lipid derivative of the present invention is applicable to hyperthermia using near-infrared light irradiation. It is generally believed that cancer cells die when the temperature reaches 42°C or higher. Under actual in vivo conditions, compared to the experimental system used this time, liposome particles are distributed in a more diluted state within cancer tissue, and the heat generation temperature of the entire solvent is expected to be lower. However, considering the amount of ICG lipid derivative added to the liposome particles, it is expected that the liposome particles themselves approaching the vicinity of cancer cells will generate heat upon near-infrared light irradiation, killing the cancer cells in that vicinity. When the release of doxorubicin from liposomes due to near-infrared light irradiation was measured, the release of the drug (doxorubicin) over time was confirmed after 5, 10, and 60 minutes of near-infrared light irradiation for all four liposomes (DOX-iLip-2) in Examples B4 to B6 and Comparative Example B2 (see Tables 6 to 9 and Figures 9 to 12 with near-infrared light irradiation (NIRL+)). This indicates that near-infrared light irradiation causes heat generation in the ICG lipid derivative and the liposome particles themselves, resulting in increased fluidity of the liposome membrane and the release of the encapsulated drug from the liposome particles. This suggests that the lipid microparticles composed of the ICG lipid derivative of the present invention may have the function of so-called temperature-sensitive liposomes. In samples left at 37°C without near-infrared light irradiation, a significant difference occurred between Examples B4 to B6 and Comparative Example B2.That is, in DOX-iLip-2 prepared in Examples B4 to B6 using three types of ICG lipid derivatives, ICG-diSA, ICG-diDOPE, and ICG-diDSPE, almost no leakage of doxorubicin was observed (see Tables 6 to 8 and Figures 9 to 11, without near-infrared light irradiation (NIRL-)), whereas in DOX-iLip-2 prepared in Comparative Example B2 using the known ICG lipid derivative ICG-DOPE, leakage of doxorubicin was observed immediately after standing, and almost no doxorubicin remained in the liposomes after 60 minutes of standing (see Table 9 and Figure 12, without near-infrared light irradiation (NIRL-)).

[0119] 3. Preparation of ICG lipid derivative-modified liposomes (ICGLip73, ICG Lip82) and doxorubicin-encapsulated ICG lipid derivative-modified liposomes (ICGLip73-DOX, ICG Lip82-DOX) Using the ICG lipid derivative synthesized in Example A3 above and doxorubicin as the drug, ICG lipid derivative-modified liposomes (ICGLip73, ICG Lip82) and doxorubicin-encapsulated ICG lipid derivative-modified liposomes (ICGLip73-DOX, ICG Lip82-DOX) were prepared using an ammonium sulfate gradient and a pH gradient according to the following procedure. Furthermore, liposomes containing no ICG lipid derivative (Lip73, Lip82) and doxorubicin-encapsulated liposomes (Lip73-DOX, Lip82-DOX) were prepared using an ammonium sulfate gradient and a pH gradient according to the following procedures.

[0120] Example B7 Preparation of ICG Lip73-DOX (1) ICG Lip73 (i) 280 μl of a chloroform solution of 0.1 mol / L dipalmitoylphosphatidylcholine (DPPC, manufactured by Nippon Fine Chemicals), 600 μl of a chloroform solution of 0.01 mol / L distearoylphosphatidylcholine (DSPC, manufactured by Nippon Fine Chemicals), 200 μl of a chloroform solution of 0.01 mol / L MPEG2000-DSPE (manufactured by Nippon Fine Chemicals, average molecular weight of PEG: 2,000), and 200 μl of a chloroform solution of 0.01 mol / L ICG-diDSPE (manufactured by Nippon Fine Chemicals) were mixed in chloroform. (ii) Thereafter, chloroform was distilled off under reduced pressure, and the mixture was dried under vacuum for at least 1 hour. 2 ml of a 0.3 mol / L aqueous ammonium sulfate solution (pH 5.5) was added, and the mixture was heated to 55°C and stirred with a vortex mixer. The obtained crude liposome dispersion was passed through polycarbonate Nuclepore membrane filters (GE Healthcare) with pore sizes of 400, 200, and 100 nm, five times each, in descending order of pore size, using an extruder (Northern Lipids) heated to 55°C. The obtained liposome dispersion was dialyzed in ultrapure water for at least 4 hours using a dialysis membrane (Spectra / Por (registered trademark) 4 Dialysis Membrane, Spectra) with a molecular weight cutoff of 12-14 kDa, and then centrifuged in an ultracentrifuge (Optima) set at 543,200 x g and 4°C. TMA separation procedure was performed for 15 minutes using a centrifuge (TLX; manufactured by Beckman Coulter). The supernatant was then removed and the mixture was suspended in 2 ml of 0.02 mol / L Hepes buffer (pH 7.4) to obtain a liposome dispersion (ICGLip73). (2) ICG Lip73-DOX: 1 ml of a solution of doxorubicin (manufactured by Kyowa Hakko Kirin Co., Ltd.) dissolved in 0.02 mol / L Hepes buffer (pH 7.4) to a concentration of 2 mg / ml was then added, heated to 55°C, and stirred (750 rpm) for 5 minutes to encapsulate doxorubicin within the liposomes. After cooling on ice, the resulting liposome dispersion was subjected to three 30-minute separation procedures using the ultracentrifuge described above set at 543,200 x g and 4°C. After removing the supernatant, 2 ml of 0.066 mol / L phosphate buffer (pH 7.4) was added to obtain a dispersion of doxorubicin-encapsulating ICG lipid derivative-modified liposomes (ICGLip73-DOX).

[0121] Comparative Example B3 Preparation of Lip73-DOX 280 μL of a chloroform solution of 0.1 mol / L dipalmitoylphosphatidylcholine (DPPC, manufactured by Nippon Fine Chemicals Co., Ltd.), 1000 μL of a chloroform solution of 0.01 mol / L distearoylphosphatidylcholine (DSPC, manufactured by Nippon Fine Chemicals Co., Ltd.), and 200 μL of a chloroform solution of 0.01 mol / L MPEG2000-DSPE (manufactured by Nippon Fine Chemicals Co., Ltd., PEG average molecular weight: 2,000) were mixed in chloroform. The subsequent operations were the same as those for the preparation of ICG Lip73-DOX in Example B7 (1), (ii), and (2), to obtain a liposome (Lip73) dispersion and a doxorubicin-encapsulated liposome (Lip73-DOX) dispersion.

[0122] [Example B8] 320 μl of a chloroform solution of 0.1 mol / L dipalmitoylphosphatidylcholine (DPPC, manufactured by Nippon Fine Chemicals), 200 μl of a chloroform solution of 0.01 mol / L distearoylphosphatidylcholine (DSPC, manufactured by Nippon Fine Chemicals), 200 μl of a chloroform solution of 0.01 mol / L MPEG2000-DSPE (manufactured by Nippon Fine Chemicals, average molecular weight of PEG: 2,000), and 200 μl of a chloroform solution of 0.01 mol / L ICG-diDSPE (manufactured by Nippon Fine Chemicals) were mixed in chloroform. The subsequent operations were the same as in the preparation of ICG Lip73-DOX in Example B7 (1) (ii) and (2), to obtain an ICG lipid derivative-modified liposome (ICGLip82) dispersion and a doxorubicin-encapsulated ICG lipid derivative-modified liposome (ICGLip82-DOX) dispersion.

[0123] Comparative Example B4 Preparation of Lip82-DOX 320 μL of a chloroform solution of 0.1 mol / L dipalmitoylphosphatidylcholine (DPPC, manufactured by Nippon Fine Chemicals Co., Ltd.), 600 μL of a chloroform solution of 0.01 mol / L distearoylphosphatidylcholine (DSPC, manufactured by Nippon Fine Chemicals Co., Ltd.), and 200 μL of a chloroform solution of 0.01 mol / L MPEG2000-DSPE (manufactured by Nippon Fine Chemicals Co., Ltd., PEG average molecular weight: 2,000) were mixed in chloroform. The subsequent operations were the same as those for the preparation of ICGLip73-DOX in Example B7 (1), (ii), and (2), to obtain a liposome (Lip82) dispersion and a doxorubicin-encapsulated liposome (Lip82-DOX) dispersion.

[0124] <Liposome Analysis 3> (1) Measurement of liposome particle size and zeta potential The particle size (average particle size (Z-average) and polydispersity index) and zeta potential of each liposome after encapsulating doxorubicin obtained in Examples B7 and B8 and Comparative Examples B3 and B4 were measured by standard methods using a zeta potential / particle size measurement system ELSZ-1000 (manufactured by Otsuka Electronics Co., Ltd.). The results are shown in Table 10.

[0125] (2) Quantification of doxorubicin encapsulated in liposomes: 10 μl of each liposome dispersion after doxorubicin encapsulation obtained in Examples B7-B8 and Comparative Examples B3-B4 above was mixed with 10 μl of 10% reduced Triton X-100 (Sigma-Aldrich) solution and 80 μl of ultrapure water in a 96-well microplate (Corning) to obtain a liposome solubilized solution. To prepare a doxorubicin calibration curve, doxorubicin aqueous solutions (diluted with ultrapure water to final doxorubicin concentrations of 0, 12.5, 25, 50, 100, 200, 400, and 800 μg / ml) were also prepared in place of the liposome dispersion. Absorbance at 495 nm was measured using a multiplate reader (Synergy H1; Biotech). The amount of doxorubicin encapsulated in the liposomes (doxorubicin (DOX) concentration) was calculated from the absorbance obtained using a calibration curve. The results are shown in Table 10.

[0126] (3) Quantification of Indocyanine Green (ICG) in Liposomes: 10 μl of a 10-fold dilution of each liposome dispersion after doxorubicin encapsulation obtained in Examples B7-B8 and Comparative Examples B3-B4 above was added to a 96-well microplate (Corning) with 10 μl of 10% reduced Triton X-100 (Sigma-Aldrich) solution and 80 μl of ultrapure water, and mixed to obtain a liposome solubilized solution. To prepare a calibration curve for ICG, an ICG aqueous solution (Diagnogreen (Daiichi Sankyo Co., Ltd.) diluted with saline to final ICG concentrations of 0, 5, 10, 25, 50, or 100 μg / ml) was also prepared in place of the liposome dispersion. The absorbance at a wavelength of 800 nm was measured using a multiplate reader (Synergy H1; Biotec). The amount of ICG in the liposomes was calculated from the absorbance using a calibration curve. The results are shown in Table 10.

[0127] As shown in Table 10, uniform doxorubicin-encapsulated ICG lipid derivative-modified liposomes (ICGLip73-DOX, ICG Lip82-DOX) and doxorubicin-encapsulated liposomes (Lip73-DOX, Lip82-DOX) with particle sizes of approximately 100 to 120 nm and polydispersity indices of 0.12 or less were obtained. Furthermore, the results of quantification of doxorubicin (DOX) and ICG indicated that these liposomes encapsulated high concentrations of doxorubicin. In particular, ICG Lip73-DOX and ICG Lip82-DOX prepared using the ICG lipid derivative of the present invention (ICG-diDSPE) encapsulated higher concentrations of doxorubicin compared to Lip73-DOX and Lip82-DOX that did not contain the ICG lipid derivative. HPLC analysis also confirmed that ICG Lip73-DOX and ICG Lip82-DOX contained ICG-diDSPE.

[0128] (4) Investigation of doxorubicin release from ICG Lip-DOX by near-infrared LED light irradiation Next, the liposome dispersions after encapsulating doxorubicin in Examples B7 to B8 and Comparative Examples B3 to B4 (ICGLip73-DOX, Lip73-DOX, ICG Lip82-DOX, Lip82-DOX) were irradiated with LED light, and the temperature rise of the dispersion medium and the accompanying release behavior of doxorubicin from each liposome were observed. Specifically, 200 μl of each liposome dispersion diluted with 0.066 mol / L phosphate buffer (pH 7.4) to an ICG concentration of 0.1 mg / mL was placed in each well of a 96-well microplate (manufactured by Corning Incorporated). (Note: For Lip73-DOX and Lip82-DOX liposome dispersions, the liposome dispersions were diluted to the same doxorubicin concentration before addition.) The plate was then irradiated from below with an LED irradiator (a set including an L800 irradiator (EL-SPI-800-01, LED: USHIO L800-66-60) and an L800 irradiator power supply (EL-DGI-10008-01) (manufactured by Ebisu Electronics Co., Ltd.); maximum wavelength: 808 nm, half-width: 32 nm, average output: 767 mW / cm). ２After irradiation with near-infrared light, each liposome dispersion was collected, mixed with 2.5 ml of 0.066 mol / L phosphate buffer (pH 7.4), and then centrifuged at 543,200 x g and 4°C in an ultracentrifuge (Optima TM The liposomes were sedimented for 30 minutes using a TLX (Beckman Coulter) and the supernatant was removed, followed by the addition of 200 μl of 0.066 mol / L phosphate buffer (pH 7.4). Each suspension was treated in the same manner as in (2) of <Liposome Analysis 3> above to determine the amount of doxorubicin remaining in the liposomes, and the amount of doxorubicin (%) released from the liposomes was calculated. Each liposome dispersion was placed in a microplate well in a thermostatic bath (Tytec) set at 37°C and allowed to stand for 10 minutes without LED light irradiation, and the amount of doxorubicin released (%) was also determined for each formulation as a control experiment.

[0129] The results for ICG Lip73-DOX, Lip73-DOX, ICG Lip82-DOX, and Lip82-DOX are shown in Figure 6. Figure 6 confirms that 10 minutes of LED light irradiation of ICG Lip73-DOX resulted in the release of approximately 50% of the doxorubicin encapsulated in the liposomes, while no doxorubicin was released from ICG Lip73-DOX that was not exposed to LED light. On the other hand, LED light irradiation of Lip73-DOX resulted in the same level of release (approximately 10%) as that observed without LED light irradiation. Similarly, it was confirmed that LED light irradiation of ICG Lip82-DOX resulted in the release of approximately 50% of doxorubicin from the liposomes, while no doxorubicin was released from ICG Lip82-DOX that was not exposed to LED light. No release of doxorubicin was confirmed for Lip82-DOX, either with or without LED light irradiation. The liposome dispersions (ICGLip73-DOX, ICG Lip82-DOX) after doxorubicin encapsulation in Examples B7 and B8 showed superior doxorubicin encapsulation stability compared to DOX-iLip-2 (ICG-diDSPE) in Example B6 using ICG-diDSPE (DOX release rate at 10 minutes of near-infrared light irradiation: 40.3% in Figure 11A).

[0130] The results of the above examples demonstrate that liposomes prepared from the ICG lipid derivatives of the present invention (ICG-diSA, ICG-diDOPE, ICG-diDSPE) stably retain doxorubicin within the liposomes in vivo, and also have a heat-generating effect when irradiated with near-infrared light, and can simultaneously release the encapsulated doxorubicin to the outside of the liposomes.

[0131] C. Preparation of cisplatin-encapsulated ICG lipid derivative-modified liposomes Using the ICG lipid derivative synthesized in Example A3 above and cisplatin (Cis) as a drug, cisplatin-encapsulated ICG lipid derivative-modified liposomes (ICGLip73-Cis, ICG Lip82-Cis) were prepared according to the following procedure. In addition, ICG lipid derivative-free liposomes (Lip73, Lip82) and cisplatin-encapsulated liposomes (Lip73-DOX, Lip82-DOX) were prepared according to the following procedure.

[0132] [Examples C1 to C2, Comparative Examples C1 to C2] Preparation of Lip73-Cis, ICG Lip73-Cis, Lip82-Cis, and ICG Lip82-Cis According to the table below, DPPC (manufactured by NOF), DSPC (manufactured by Nippon Fine Chemicals), MPEG2000-DSPE (manufactured by Genzyme), and ICG-diDSPE were weighed into glass vials, and the molar ratios (DPPC:DSPC:MPEG2000-DSPE:ICG-diDSPE) were adjusted to 7:2.5:0.5:0 (Lip73-Cis), 7:1.5:0.5:0.5 (ICGLip73-Cis), 8:1.5:0.5:0 (Lip82-Cis), and 8:0.5:0.5:0.5 (ICGLip82-Cis), respectively. 1 mL of ethanol was added to each glass vial and heated to 80°C to prepare a lipid solution. 150 mg of cisplatin was weighed, 18 mL of saline was added, and the mixture was heated to 80°C to prepare an 8.3 mg / mL cisplatin aqueous solution. 4 mL of cisplatin aqueous solution was added to each lipid solution to prepare a liposome aqueous solution. Using an extruder (manufactured by LIPEX) and heated at 80°C, the liposome aqueous solution was passed through a 100 nm pore size filter (manufactured by Whatman) five times. The solution was cooled at 4°C for 30 minutes and centrifuged (approximately 1600 g, Kubota 7780II) for 10 minutes to separate the precipitate and liposome aqueous solution. The supernatant was dialyzed against saline for 12 hours at 4°C using a Float-A-Lyzer G2 Dialysis Device (100 kD). After dilution with saline, the mixture was ultracentrifuged (Beckman, Optima XPN-80) at approximately 370,000 g. The supernatant was removed and the mixture was suspended in saline. This was passed through a 0.45 μm pore size PES (polyethersulfone) filter (Whatman) to obtain aqueous solutions of cisplatin-encapsulated liposomes (ICGLip73-Cis, ICG Lip82-Cis) and aqueous solutions of cisplatin-encapsulated liposomes (Lip73-DOX, Lip82-DOX) that did not contain ICG lipid derivatives.

[0133] <Liposome Analysis 4> The aqueous liposome solutions after encapsulating cisplatin obtained in Examples C1 and C2 and Comparative Examples C1 and C2 were analyzed as follows.

[0134] (1) Measurement of liposome particle size and zeta potential The particle size (mean particle size (Z-average) and polydispersity index) and zeta potential of each liposome were measured by standard methods using a zeta potential / particle size measurement system ELSZ-1000 (manufactured by Otsuka Electronics Co., Ltd.). The results are shown in Table 12.

[0135] (2) Quantification of cisplatin encapsulated in liposomes The lipid concentrations and cisplatin concentrations of each liposome aqueous solution were measured by HPLC (Shimadzu Corporation). The results are shown in Table 12.

[0136] (3) Quantification of indocyanine green (ICG) in liposomes. 10 μl of a 50-fold dilution of each liposome dispersion obtained was added to a 96-well microplate (Corning) with 10 μl of 10% reduced Triton X-100 (Sigma-Aldrich) solution and 80 μl of ultrapure water, and mixed to obtain a liposome solubilized solution. The absorbance at a wavelength of 800 nm was measured using a multiplate reader (Synergy H1; Biotec). The amount of ICG in the liposomes was calculated from the obtained absorbance using a calibration curve. The results are shown in Table 12.

[0137] As shown in Table 12, uniform cisplatin-encapsulated ICG lipid derivative-modified liposomes (ICGLip73-Cis, ICGlip82-Cis) and cisplatin-encapsulated liposomes (Lip73-Cis, Lip82-Cis) with particle sizes of 100 nm or less and polydispersity indexes of 0.12 or less were obtained. Furthermore, quantification of cisplatin and ICG indicated that these liposomes encapsulated high concentrations of cisplatin. HPLC analysis of ICGlip73-Cis and ICGlip82-Cis also confirmed that they contained ICG-diDSPE.

[0138] (4) Investigation of cisplatin release from ICG Lip-Cis upon near-infrared LED light irradiation Next, the liposome dispersions of Examples C1 to C2 and Comparative Examples C1 to C2 (ICGLip73-Cis, Lip73-Cis, ICG Lip82-Cis, Lip82-Cis) were irradiated with LED light, and the temperature rise of the dispersion medium and the accompanying release behavior of cisplatin from each liposome were observed. 200 μl of each liposome dispersion diluted with 0.066 mol / L phosphate buffer (pH 7.4) to an ICG concentration of 0.1 mg / mL was placed in each well of a 96-well microplate (manufactured by Corning Incorporated). (Note: For Lip73-Cis and Lip82-Cis liposome dispersions, the liposome dispersions were diluted to the same cisplatin concentration before addition.) The plate was then irradiated from below in a thermostatic chamber (Taitec Co., Ltd.) set at 37°C with an LED irradiator (a set including an L800 irradiator (EL-SPI-800-01, LED: USHIO L800-66-60) and an L800 irradiator power supply (EL-DGI-10008-01) (manufactured by Ebisu Electronics Co., Ltd.); maximum wavelength: 808 nm, half-width: 32 nm, average output: 767 mW / cm). ２ The liposome dispersions were irradiated with near-infrared light for 10 minutes using a temperature logger (SK-L400T, manufactured by Sato Keiryoki Seisakusho Co., Ltd.) to measure the temperature of the liposome dispersions irradiated with near-infrared light every 10 seconds. After irradiation with near-infrared light, each liposome dispersion was collected, mixed with 2.5 ml of 0.066 mol / L phosphate buffer (pH 7.4), and then centrifuged in an ultracentrifuge (Optima) set at 543,200 x g and 4°C. TM The liposomes were sedimented for 30 minutes using a TLX (Beckman Coulter) and the supernatant was removed, followed by the addition of 200 μl of 0.066 mol / L phosphate buffer (pH 7.4). Each suspension was treated in the same manner as in (2) of <Liposome Analysis 4> above to determine the amount of cisplatin remaining in the liposomes, and the amount of cisplatin (%) released from the liposomes was calculated. Each liposome dispersion was placed in a microplate well in a thermostatic bath (Tytec) set at 37°C and allowed to stand for 10 minutes without LED light irradiation, and the amount of cisplatin released (%) was also determined for each formulation control.

[0139] The results for ICG Lip73-Cis, Lip73-Cis, ICG Lip82-Cis, and Lip82-Cis are shown in Figures 7A and 7B. Figure 7A shows that 10 minutes of LED light irradiation of ICG Lip73-Cis resulted in the release of more than 80% of the cisplatin encapsulated in the liposomes, while no cisplatin was released from ICG Lip73-Cis without LED light irradiation. On the other hand, no cisplatin release was observed with Lip73-Cis, regardless of whether or not it was irradiated with LED light. While some release was observed with ICG Lip82-Cis without LED light irradiation, it was confirmed that more than 90% of cisplatin was released from the liposomes with LED light irradiation. When Lip82-Cis was irradiated with LED light, release was confirmed to be similar to that observed without LED light irradiation. FIG. 7B confirmed that the temperature of the liposome dispersion containing the ICG lipid derivatives (ICGLip73-Cis, ICG Lip82-Cis) increased significantly upon irradiation with LED light.

[0140] The results of the above examples demonstrate that liposomes prepared from the ICG lipid derivative of the present invention (ICG-diDSPE) stably retain cisplatin within the liposomes and can release the encapsulated cisplatin to the outside of the liposomes upon irradiation with near-infrared light.

[0141] D. Therapeutic Effect on HT1080 Cancer-Bearing Mice 1. Preparation of Liposome Preparations (ICG-Lip, Lip-DOX, ICGLip-DOX) Using the ICG lipid derivative synthesized in Example A3 above and doxorubicin (DOX) as a drug, drug-encapsulated ICG lipid derivative-modified liposomes (ICGLip-DOX), drug-encapsulated liposomes not containing ICG lipid derivatives (Lip-DOX), and ICG lipid derivative-modified liposomes not containing a drug (ICG-Lip) were prepared according to the same procedures as in the above examples. (Preparation Procedure) DPPC (NOF), DSPC (Nippon Fine Chemicals), and MPEG2000-DSPE (Genzyme) were dissolved in ethanol to a concentration of 20 mg / mL, and ICG-diDSPE was dissolved in chloroform to a concentration of 5 mg / mL. The lipids were mixed to the weights shown in the table below. The molar ratios of the lipids (DPPC:DSPC:MPEG2000-DSPE:ICG-diDSPE) were 70:15:5:5 (Rp. 1) and 70:25:5:0 (Rp. 2), respectively. The organic solvent was removed under reduced pressure and dried in a vacuum for 12 hours to prepare a lipid mixture. Ammonium sulfate (Fujifilm Wako Pure Chemical Industries, Ltd.) was dissolved in water to a concentration of 300 mM to prepare an ammonium sulfate aqueous solution. Sucrose (Fujifilm Wako Pure Chemical Industries, Ltd.) and L-histidine (Fujifilm Wako Pure Chemical Industries, Ltd.) were dissolved in water to a concentration of 96 mg / mL and 10 mM, respectively, to prepare a 96 mg / mL sucrose / 10 mM / mL histidine aqueous solution. 10 mL of ammonium sulfate aqueous solution heated to 80°C was added to each lipid mixture (Rp. 1 and Rp. 2) and mixed by inversion to prepare a liposome aqueous solution. While heated at 80°C, the liposome aqueous solution was passed through a 50 nm pore size filter (Whatman) five times using an extruder (LIPEX). The resulting sized product was diluted with physiological saline and then subjected to ultracentrifugation (Beckman, Optima XPN-80) at approximately 370,000 g. The supernatant was removed, the product was suspended in physiological saline, and then ultracentrifuged at approximately 370,000 g. The supernatant was removed, and the product was suspended in a 96 mg / mL sucrose / 10 mM / mL histidine aqueous solution. The concentrations of each lipid were measured by HPLC (Shimadzu). Doxorubicin (Dox) (Sicor) was dissolved in a 5% glucose solution (Otsuka) to a concentration of 10 mg / mL to obtain a doxorubicin aqueous solution. Each liposome aqueous solution (Rp. 1 and Rp. 2) was mixed with a doxorubicin aqueous solution and heated at 50°C for 5 minutes to encapsulate doxorubicin. After cooling, the mixture was passed through a 0.45 μm pore size PES (polyethersulfone) filter (Whatman) to prepare doxorubicin-encapsulated liposome aqueous solutions (ICG-Lip-Dox and LipDOX). The lipid concentrations were measured by HPLC (Shimadzu). Particle size was measured using a Zetasizer NanoZS (Malvern). A portion of the obtained aqueous solution of doxorubicin-encapsulating liposomes was diluted 10-fold with physiological saline and subjected to ultracentrifugation (Hitachi, Himac CS120FX) at approximately 600,000 g. The doxorubicin concentrations in the supernatant and before ultracentrifugation were measured by HPLC to determine the encapsulation rates, which were 98.0% and 98.6%, respectively.Doxorubicin-free ICG-Lip was prepared by diluting the Rp.1 liposome solution before doxorubicin inclusion with a 96 mg / mL sucrose / 10 mM / mL histidine solution. The concentrations of doxorubicin (DOX) and each lipid (ICG, DPPC, DSPC) were as shown in the table below.

[0142] 2. Therapeutic experiment on HT1080 solid tumor-bearing mice (1) Preparation of HT1080 solid tumor-bearing mice A cell suspension of HT1080 cells, a human fibrosarcoma cell line, was prepared at 5x10 ７ The cells were suspended in D-MEM (high glucose) medium (manufactured by Fujifilm Wako Co., Ltd.) to a concentration of 5 × 10 cells / ml. Next, 0.1 ml of the HT1080 cell suspension was filled into a 1 ml syringe (manufactured by Terumo Co.) equipped with a 27 G needle (manufactured by Terumo Co., Ltd.), and injected into the right dorsal region of 6-week-old BALB / c nude male mice (purchased from Japan SLC Co., Ltd.). ６ The mice were then subcutaneously administered the drug to achieve a tumor density of 1000 cells / mouse (Day 0). Thereafter, the mice were kept under normal conditions until a solid tumor of an appropriate size was formed.

[0143] (2) Drug Administration and LED Light Irradiation On day 11 after cancer cell transplantation, mice were restrained in a mouse holder, and ICG Lip, Lip-DOX, and ICG Lip-DOX were administered intravenously via the tail vein at a doxorubicin dose of 10 mg / kg (body weight). However, the untreated group was administered saline, and the ICG Lip group was administered the same ICG-diDSPE dose as the ICG Lip-DOX group. 24 hours after administration, the mice were anesthetized by inhalation with isoflurane (Pfizer) using a small anesthesia machine for small animals (MK-A110, Muromachi Kikai Co., Ltd.) and placed on a heating pad (Heating Mat KN-475-35, Natsume Seisakusho Co., Ltd.) set to 35 ° C. LED irradiation device (a set including an L800 irradiator (EL-SPI-800-01, LED: USHIO L800-66-60) and a power supply for the L800 irradiator (EL-DGI-10008-01) (manufactured by Ebisu Electronics Co., Ltd.); maximum wavelength 808 nm, half-width 32 nm, average output 767 mW / cm ２The tip of the fiber was fixed as close as possible to the solid tumor, and the tumor was irradiated with LED light for 10 minutes under isoflurane inhalation anesthesia. A control group was also prepared that was not irradiated with LED light.

[0144] (3) Temperature Measurement of Solid Cancer Site Before and immediately after the LED light irradiation, the temperature of the solid cancer site was measured using a thermography camera (E5, manufactured by FLIR Systems). The results of the temperature measurement are shown in Figure 8.

[0145] (4) Evaluation of therapeutic effect and body weight measurement The short and long diameters of the solid tumors formed subcutaneously in the mice were measured using a vernier caliper. The volume of the solid tumor was calculated using the following formula: tumor volume (cm ３ ) = 0.4 x (minor axis (cm)) x (minor axis (cm)) x (major axis (cm)) Figure 9 shows a graph showing the percentage change in calculated tumor volume from the day of drug administration (relative tumor volume (RTV)). Figure 10 shows the results of measuring the body weight of the mice.

[0146] As shown in Figure 8, in HT1080 solid tumor-bearing mice administered ICG Lip and ICG Lip-DOX, liposomes containing an ICG lipid derivative (ICG-diDSPE), LED light irradiation of the solid tumor was confirmed to increase the temperature of the solid tumor to approximately 50°C. On the other hand, in mice administered saline or ICG Lip-DOX, LED light irradiation did not increase the temperature of the solid tumor. As shown in Figure 9, in HT1080 solid tumor-bearing mice administered ICG Lip-DOX and then irradiated with LED light (ICGLip-DOX + LED), a significant tumor regression effect was confirmed, and some mice even experienced tumor disappearance. In mice administered ICG Lip and irradiated with LED light (ICGLip + LED), temporary tumor shrinkage was observed, but the tumor subsequently grew larger. Furthermore, mice administered ICG Lip-DOX (without LED light irradiation) (ICGLip-DOX), mice administered Lip-DOX (without LED light irradiation) (Lip-DOX), and mice administered Lip-DOX and then irradiated with LED light (Lip-DOX+LED) showed a tumor growth inhibitory effect compared to mice administered saline (without LED light irradiation) (saline), mice administered saline and then irradiated with LED light (saline+LED), and mice administered ICG Lip (without LED light irradiation) (ICGLip). As shown in Figure 10, changes in mouse weight were not affected by drug administration or LED light irradiation, and mice administered ICG Lip-DOX and then irradiated with LED light to solid tumors (ICGLip-DOX+LED) showed a tendency to recover from weight loss due to tumor bearing.

[0147] E. Therapeutic Effect on BxPC3 Tumor-Bearing Mice 1. Preparation of Liposome Formulations (ICG-Lip, Lip-Cis, ICGLip-Cis) Using the ICG lipid derivative synthesized in Example A3 above and cisplatin (Cis) as the drug, drug-encapsulated ICG lipid derivative-modified liposomes (ICGLip-Cis), drug-encapsulated liposomes without ICG lipid derivatives (Lip-Cis), and ICG lipid derivative-modified liposomes without drug encapsulation (ICG-Lip) were prepared according to the same procedures as in the above examples. (Preparation Procedure) DPPC (manufactured by NOF), DSPC (manufactured by Nippon Fine Chemicals), MPEG2000-DSPE (manufactured by Genzyme), and ICG-diDSPE were weighed into glass vials according to the table below. The molar ratios of each lipid (DPPC:DSPC:MPEG2000-DSPE:ICG-diDSPE) were 70:15:5:5 (ICG-Lip), 70:25:5:0 (Lip-Cis), and 70:15:5:5 (ICG-Lip-Cis), respectively. 3 mL of ethanol was added to each glass vial and heated to 80 °C to prepare a lipid solution. 304 mg of cisplatin was weighed, 35 mL of saline was added, and the mixture was heated to 80 °C to prepare an 8.7 mg / mL cisplatin aqueous solution. 12 mL of cisplatin aqueous solution was added to each lipid solution to prepare a liposome aqueous solution. Using an extruder (manufactured by LIPEX) and heated at 80 °C, the liposome aqueous solution was passed through a 100 nm pore size filter (manufactured by Whatman) five times. The solution was cooled at 4 °C for 30 minutes and centrifuged (approximately 1600 g, Kubota 7780II) for 10 minutes to separate the precipitate and liposome aqueous solution. The supernatant was dialyzed against saline for 12 hours at 4 °C using a Float-A-Lyzer G2 Dialysis Device (100 kD). After dilution with saline, ultracentrifugation (Beckman, Optima XPN-80) was performed at approximately 370,000 g. The supernatant was removed and the suspension was suspended in saline. This was passed through a 0.45 μm pore size PES (polyethersulfone) filter (Whatman) to obtain aqueous solutions of cisplatin-encapsulated liposomes (ICGLip-Cis, Lip-Cis) and cisplatin-free liposomes (ICGLip). The lipid concentrations and cisplatin (Cis) concentrations were measured using HPLC (Shimadzu). Particle size was measured using a Zetasizer NanoZS (Malvern). The results are shown in the table below.

[0148] 2. Treatment experiment on mice bearing BxPC3 solid tumors (1) Preparation of mice bearing BxPC3 solid tumors A cell suspension of BxPC3 cells, a human pancreatic adenocarcinoma cell line, was prepared at 2.5 x 10 ７ The cells were suspended in RPMI 1640 medium (manufactured by Fujifilm Wako Co., Ltd.) to a concentration of 100 cells / ml. Next, 0.2 ml of the BxPC3 cell suspension was filled into a 1 ml syringe (manufactured by Terumo Co., Ltd.) equipped with a 27 G injection needle (manufactured by Terumo Co., Ltd.), and 5 × 10 cells were injected into the right dorsal region of 5-week-old BALB / c nude male mice (purchased from Japan SLC Co., Ltd.). ６ The mice were then subcutaneously administered the drug to achieve a tumor density of 1000 cells / mouse (Day 0). Thereafter, the mice were kept under normal conditions until a solid tumor of an appropriate size was formed.

[0149] (2) Drug administration and LED light irradiation On day 18 after cancer cell transplantation (Day 18), mice were restrained in a mouse holder, and ICG Lip, Lip-Cis, and ICG Lip-Cis were administered intravenously via the tail vein at a cisplatin dose of 10 mg / kg (body weight). However, the untreated group was administered saline, and the ICG Lip group was administered the same ICG-diDSPE dose as the ICG Lip-Cis group. 24 hours after administration, the mice were anesthetized by inhalation with isoflurane (Pfizer) using a small anesthesia machine for small animals (MK-A110, Muromachi Kikai Co., Ltd.) and placed on a heating pad (Heating Mat KN-475-35, Natsume Seisakusho Co., Ltd.) set to 35 ° C. LED irradiation device (a set including an L800 irradiator (EL-SPI-800-01, LED: USHIO L800-66-60) and a power supply for the L800 irradiator (EL-DGI-10008-01) (manufactured by Ebisu Electronics Co., Ltd.); maximum wavelength 808 nm, half-width 32 nm, average output 767 mW / cm ２ The tip of the fiber was fixed as close as possible to the solid tumor, and the tumor was irradiated with LED light for 10 minutes under isoflurane inhalation anesthesia. A control group was also prepared that was not irradiated with LED light.

[0150] (3) Temperature Measurement of Solid Cancer Site Before and immediately after the LED light irradiation, the temperature of the solid cancer site was measured using a thermography camera (E5, manufactured by FLIR Systems). The temperature measurement results are shown in Figure 11.

[0151] (4) Evaluation of therapeutic effect and body weight measurement The short and long diameters of the solid tumors formed subcutaneously in the mice were measured using a vernier caliper. The volume of the solid tumor was calculated using the following formula: tumor volume (cm ３ ) = 0.4 x (minor axis (cm)) x (minor axis (cm)) x (major axis (cm)) Figure 12 shows a graph showing the percentage change in the calculated tumor volume from the day of drug administration (relative tumor volume (RTV)). Figure 13 shows the results of measuring the body weight of the mice.

[0152] As shown in Figure 11, in BxPC3 solid tumor-bearing mice administered ICG Lip and ICG Lip-Cis, liposomes containing ICG-diDSPE, it was confirmed that LED light irradiation of the solid tumor increased the temperature of the solid tumor to approximately 43°C. On the other hand, in mice administered saline or Lip-Cis, LED light irradiation did not increase the temperature of the solid tumor. As shown in Figure 12, a tumor regression effect was confirmed in BxPC3 solid tumor-bearing mice (ICGLip-Cis + LED) that were administered ICG Lip-Cis and then irradiated with LED light. In mice administered ICG Lip and irradiated with LED light (ICGLip + LED), temporary tumor regression was observed, but the tumor subsequently grew. Furthermore, in mice administered ICG Lip-Cis (but not irradiated with LED light) (ICGLip-Cis), mice administered Lip-Cis (but not irradiated with LED light) (Lip-Cis), and mice administered Lip-Cis and then irradiated with LED light (Lip-Cis+LED), a slight growth inhibitory effect was observed, but the effect was low. As shown in Figure 13, no effect of drug administration or LED light irradiation was observed on changes in mouse weight.

[0153] The ICG lipid derivative of the present invention can be suitably applied in the fields of fluorescence imaging, photothermal therapy and / or photodynamic therapy, or DDS.

Claims

1. A compound represented by the following formula (A) or a pharmaceutically acceptable salt thereof. 【Chemistry 21】 [In the formula, R １ and R ２ Each is independently - (CH ２ ) ｋ -CONH-R ３ This represents, R ３ is - (CH ２ ) ｍ -OPO ３ - -CH ２ -CH(CH ２ O COR ４ )(O COR ５ ), a branched-chain C １４ ~C ４０ alkyl, and a branched-chain C １４ ~C ４０ alkenyl, and represents a group selected from the group consisting of R ４ and R ５ Each is independently a linear or branched C １３ ~C ２１ Alkyl or linear or branched C １３ ~C ２1 Representing Alkenil, k represents an integer between 2 and 4. m represents an integer between 2 and 4.

2. R １ and R ２ Each is independently - (CH ２ ) ２ -CONH-R ３ This represents, R ３ ha- (CH ２ ) ｍ - OPO ３ － -CH ２ -CH(CH ２ OCOR ４ ) (OCOR ５ ) represents, R ４ and R ５ Each is independently a linear C １３ ~C ２１ Alkyl or linear C １３ ~C ２１ Representing Alkenil, The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein m represents an integer from 2 to 4.

3. A compound or a pharmaceutically acceptable salt thereof, selected from the compounds represented by the following formula (I) or formula (II). 【Chemistry 22】

4. Lipid microparticles containing a compound represented by the following formula (A) or a pharmaceutically acceptable salt thereof. 【Chemistry 23】 [In the formula, R １ and R ２ Each is independently - (CH ２ ) ｋ -CONH-R ３ This represents, R ３ ha- (CH ２ ) ｍ - OPO ３ - -CH ２ -CH(CH ２ OCOR ４ ) (OCOR ５ ), straight chain C １４ ~C ２２ Alkyl, linear C １４ ~C ２２ Alkenyl, branched chain C １４ ~C ４０ Alkyl and branched chain C １４ ~C ４０ It represents a group selected from the group consisting of alkenyls. R ４ and R ５ Each is independently a linear or branched C １３ ~C ２１ Alkyl or linear or branched C １３ ~C ２1 Representing Alkenil, k represents an integer between 2 and 4. m represents an integer between 2 and 4.

5. Lipid microparticles according to claim 4, comprising a compound selected from the group consisting of compounds represented by the following formulas (I) to (III), or a pharmaceutically acceptable salt thereof. 【Chemistry 24】 【change】

6. Lipid microparticles according to claim 4, further immobilizing or supporting at least one drug.

7. Lipid microparticles according to claim 6, wherein the drug contains an anticancer agent.

8. The lipid microparticles according to claim 4, further comprising at least one lipid selected from the group consisting of neutral lipids, polyethylene glycol-modified lipids, and sterols.

9. Lipid microparticles according to claim 4, wherein the average particle diameter is 30 to 200 nm.

10. The lipid microparticles according to claim 4, wherein the lipid microparticles are polymer micelles or liposomes.

11. A pharmaceutical composition or diagnostic composition comprising a compound according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof, or lipid microparticles according to any one of claims 4 to 10.

12. A pharmaceutical composition or diagnostic composition according to claim 11, used in at least one selected from photothermal therapy, photodynamic therapy, and fluorescence imaging.

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