Fluorescent probe for imaging lymph nodes
09957392 ยท 2018-05-01
Assignee
Inventors
- Hideki Hayashi (Chiba, JP)
- Masanori Fujinami (Chiba, JP)
- Taro Toyota (Chiba, JP)
- Yutaka Tamura (Chiba, JP)
- Akira AOKI (Kyoto, JP)
- Yutaka Muraki (Kyoto, JP)
- Kazuki Onoue (Kyoto, JP)
Cpc classification
C07D209/60
CHEMISTRY; METALLURGY
A61K9/127
HUMAN NECESSITIES
International classification
C09B23/08
CHEMISTRY; METALLURGY
A61K9/127
HUMAN NECESSITIES
C07D209/60
CHEMISTRY; METALLURGY
Abstract
Disclosed is a near-infrared fluorescent imaging agent comprising an indocyanine-based fluorescent dye and a liposome. The near-infrared fluorescent imaging agent of the present invention demonstrates high fluorescence intensity and a long anchoring time in sentinel lymph nodes, thereby making it useful for detecting sentinel lymph nodes in sentinel lymph node navigation surgery. Also disclosed is an indocyanine green derivative that is particularly suitable for use in the near-infrared fluorescent imaging agent of the present invention.
Claims
1. A fluorescent probe for near-infrared fluorescent imaging in sentinel lymphadenography comprising a liposome containing a fluorescent dye having a hexatriene skeletal structure, wherein the fluorescent dye having a hexatriene skeletal structure is a compound represented by chemical formula (V): ##STR00042## wherein, n represents an integer of 4 to 18.
2. The fluorescent probe for near-infrared fluorescent imaging in sentinel lymphadenography according to claim 1, wherein the particle diameter of the liposome is 100 nm to 300 nm.
3. The fluorescent probe for near-infrared fluorescent imaging in sentinel lymphadenography according to claim 1, wherein the particle diameter of the liposome is 150 nm to 250 nm.
4. A near-infrared fluorescent imaging agent comprising a compound represented by chemical formula (V): ##STR00043## wherein, n represents an integer of 4 to 18.
5. A method for identifying a sentinel lymph node in a subject, comprising administering to the subject the fluorescent probe according to claim 1 and detecting fluorescence generated from the fluorescent probe.
6. A compound represented by chemical formula (V): ##STR00044## wherein, n represents an integer of 4 to 18.
7. A liposome comprising the compound according to claim 6.
8. The compound according to claim 6, wherein n is 4.
9. The compound according to claim 6, wherein n is 6.
10. The compound according to claim 6, wherein n is 8.
11. The compound according to claim 6, wherein n is 10.
12. The compound according to claim 6, wherein n is 18.
13. A liposome comprising the compound according to claim 8.
14. A liposome comprising the compound according to claim 9.
15. A liposome comprising the compound according to claim 10.
16. A liposome comprising the compound according to claim 11.
17. A liposome comprising the compound according to claim 12.
18. A method for identifying a sentinel lymph node in a subject, comprising administering to the subject the compound according to claim 8 and detecting fluorescence generated from the compound.
19. A method for identifying a sentinel lymph node in a subject, comprising administering to the subject the compound according to claim 9 and detecting fluorescence generated from the compound.
20. A method for identifying a sentinel lymph node in a subject, comprising administering to the subject the compound according to claim 10 and detecting fluorescence generated from the compound.
21. A method for identifying a sentinel lymph node in a subject, comprising administering to the subject the compound according to claim 11 and detecting fluorescence generated from the compound.
22. A method for identifying a sentinel lymph node in a subject, comprising administering to the subject the compound according to claim 12 and detecting fluorescence generated from the compound.
23. A method for identifying a sentinel lymph node in a subject, comprising administering to the subject the liposome according to claim 13 and detecting fluorescence generated from the liposome.
24. A method for identifying a sentinel lymph node in a subject, comprising administering to the subject the liposome according to claim 14 and detecting fluorescence generated from the liposome.
25. A method for identifying a sentinel lymph node in a subject, comprising administering to the subject the liposome according to claim 15 and detecting fluorescence generated from the liposome.
26. A method for identifying a sentinel lymph node in a subject, comprising administering to the subject the liposome according to claim 16 and detecting fluorescence generated from the liposome.
27. A method for identifying a sentinel lymph node in a subject, comprising administering to the subject the liposome according to claim 17 and detecting fluorescence generated from the liposome.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
(1)
(2)
(3)
(4)
(5)
(6)
(7)
(8)
(9)
PREFERRED EMBODIMENTS OF THE INVENTION
(10) Near-Infrared Fluorescent Dye
(11) The near-infrared fluorescent imaging agent of the present invention comprises an indocyanine-based fluorescent dye and a liposome. Examples of the indocyanine-based fluorescent dyes that can be used in the present invention include a compound represented by chemical formula (I):
(12) ##STR00023##
wherein, Y represents C(CH.sub.3).sub.2, O or S, Z.sub.1 and Z.sub.3 represent hydrogen or OCH.sub.3, Z.sub.2 and Z.sub.4 represent hydrogen, or Z1 and Z2, and Z3 and Z4 may respectively together form a benzene ring fused with a ring to which they are bound, R.sub.1 represents (CH.sub.2).sub.nSO.sub.3, (CH.sub.2).sub.nX.sub.1(CH.sub.2).sub.mSO.sub.3, (CH.sub.2).sub.nCOO, (CH.sub.2).sub.nX.sub.1(CH.sub.2).sub.mCOO, (CH.sub.2).sub.nPO.sub.4, (CH.sub.2).sub.nX.sub.1(CH.sub.2).sub.mPO.sub.4, (CH.sub.2).sub.nOH(A), (CH.sub.2).sub.nX.sub.1(CH.sub.2).sub.mOH(A), (CH.sub.2).sub.nSH(A), (CH.sub.2).sub.nX.sub.1(CH.sub.2).sub.mSH(A), (CH.sub.2).sub.nNH.sub.2(A), (CH.sub.2).sub.nX.sub.1(CH.sub.2).sub.mNH.sub.2(A), (CH.sub.2).sub.n(CH.sub.2CH.sub.2O).sub.xH(A), (CH.sub.2).sub.nX.sub.1(CH.sub.2).sub.m(CH.sub.2CH.sub.2O).sub.xH(A), (CH.sub.2).sub.nCH.sub.3(A), (CH.sub.2).sub.nX.sub.1(CH.sub.2).sub.mCH.sub.3(A), (CH.sub.2).sub.nPO.sub.4(CH.sub.2).sub.mCH.sub.3, (CH.sub.2).sub.nPO.sub.3(OC.sub.lH.sub.2l+1)(CH.sub.2).sub.mCH.sub.3, (CH.sub.2).sub.nCON(C.sub.lH.sub.2l+1)(CH.sub.2).sub.mCH.sub.3(A), (CH.sub.2).sub.nN(C.sub.lH.sub.2l+1)(CH.sub.2).sub.mCH.sub.3(A), (CH.sub.2).sub.nX.sub.1(CH.sub.2).sub.pCON(C.sub.lH.sub.2l+1)(CH.sub.2).sub.mCH.sub.3(A) or (CH.sub.2).sub.nX.sub.1(CH.sub.2).sub.pN(C.sub.lH.sub.2l+1)(CH.sub.2).sub.mCH.sub.3(A), where X.sub.1 represents O, NH, S, SO.sub.2, CHCH, SO.sub.2NH, NHSO.sub.2, CONH, NHCO, CO, COO, OCO or C.sub.6H.sub.4, A represents a monovalent anion in the form of a chloride ion, bromide ion or iodide ion, n and m represent an integer of 0 to 22, l represents an integer of 1 to 22, p represents an integer of 0 to 17, and x represents an integer of 2 to 2000, R.sub.2 represents a group selected from the group consisting of the following groups:
(13) ##STR00024##
where, X.sub.1 or X.sub.2 represents O, NH, S, SO.sub.2, CHCH, SO.sub.2NH, NHSO.sub.2, CONH, NHCO, CO, COO, OCO or C.sub.6H.sub.4, and B represents a monovalent cation in the form of a hydrogen ion, lithium ion, sodium ion or potassium ion, and R.sub.3 represents an alkane group, alkene group or alkyne group having 1 to 18 carbon atoms, n and m represent an integer of 0 to 22, l represents an integer of 1 to 22 and p represents an integer of 0 to 17; or a compound represented by chemical formula (II):
(14) ##STR00025##
wherein, Y represents C(CH.sub.3).sub.2, O or S, Z.sub.1 and Z.sub.3 represent hydrogen or OCH.sub.3, Z.sub.2 and Z.sub.4 represent hydrogen, or Z1 and Z2, and Z3 and Z4 may respectively together form a benzene ring fused with a ring to which they are bound, R.sub.1 represents (CH.sub.2).sub.nSO.sub.3, (CH.sub.2).sub.nX.sub.1(CH.sub.2).sub.mSO.sub.3, (CH.sub.2).sub.nCOO, (CH.sub.2).sub.nX.sub.1(CH.sub.2).sub.mCOO, (CH.sub.2).sub.nPO.sub.4, (CH.sub.2).sub.nX.sub.1(CH.sub.2).sub.mPO.sub.4, (CH.sub.2).sub.nOH(A), (CH.sub.2).sub.nX.sub.1(CH.sub.2).sub.mOH(A), (CH.sub.2).sub.nSH(A), (CH.sub.2).sub.nX.sub.1(CH.sub.2).sub.mSH(A), (CH.sub.2).sub.nNH.sub.2(A), (CH.sub.2).sub.nX.sub.1(CH.sub.2).sub.mNH.sub.2(A), (CH.sub.2).sub.n(CH.sub.2CH.sub.2O).sub.xH(A), (CH.sub.2).sub.nX.sub.1(CH.sub.2).sub.m(CH.sub.2CH.sub.2O).sub.xH(A), (CH.sub.2).sub.nCH.sub.3(A), (CH.sub.2).sub.nX.sub.1(CH.sub.2).sub.mCH.sub.3(A), (CH.sub.2).sub.nPO.sub.4(CH.sub.2).sub.mCH.sub.3, (CH.sub.2).sub.nPO.sub.3(OC.sub.lH.sub.2l+1)(CH.sub.2).sub.mCH.sub.3, (CH.sub.2).sub.nCON(C.sub.lH.sub.2l+1)(CH.sub.2).sub.mCH.sub.3(A), (CH.sub.2).sub.nN(C.sub.lH.sub.2l+1)(CH.sub.2).sub.mCH.sub.3(A), (CH.sub.2).sub.nX.sub.1(CH.sub.2).sub.pCON(C.sub.lH.sub.2l+1)(CH.sub.2).sub.mCH.sub.3(A) or (CH.sub.2).sub.nX.sub.1(CH.sub.2).sub.pN(C.sub.lH.sub.2l+1)(CH.sub.2).sub.mCH.sub.3(A), where X.sub.1 represents O, NH, S, SO.sub.2, CHCH, SO.sub.2NH, NHSO.sub.2, CONH, NHCO, CO, COO, OCO or C.sub.6H.sub.4, A represents a monovalent anion in the form of a chloride ion, bromide ion or iodide ion, n and m represent an integer of 0 to 22, l represents an integer of 1 to 22, p represents an integer of 0 to 17, and x represents an integer of 2 to 2000, R.sub.2 represents (CH.sub.2).sub.nSO.sub.3(M), (CH.sub.2).sub.nX.sub.2(CH.sub.2).sub.mSO.sub.3(M), (CH.sub.2).sub.nCOO(M), (CH.sub.2).sub.nX.sub.2(CH.sub.2).sub.mCOO(M), (CH.sub.2).sub.nPO.sub.4(M), (CH.sub.2).sub.nX.sub.2(CH.sub.2).sub.mPO.sub.4(M), (CH.sub.2).sub.nOH, (CH.sub.2).sub.nX.sub.2(CH.sub.2).sub.mOH, (CH.sub.2).sub.nSH, (CH.sub.2).sub.nX.sub.2(CH.sub.2).sub.mSH, (CH.sub.2).sub.nNH.sub.2, (CH.sub.2).sub.nX.sub.2(CH.sub.2).sub.mNH.sub.2, (CH.sub.2).sub.n(CH.sub.2CH.sub.2O).sub.xH, (CH.sub.2).sub.nX.sub.2(CH.sub.2).sub.m(CH.sub.2CH.sub.2O).sub.xH, (CH.sub.2).sub.nCH.sub.3, (CH.sub.2).sub.nX.sub.2(CH.sub.2).sub.mCH.sub.3, (CH.sub.2).sub.nPO.sub.4(CH.sub.2).sub.mCH.sub.3(M), (CH.sub.2).sub.nPO.sub.3(OC.sub.lH.sub.2l+1)(CH.sub.2).sub.mCH.sub.3(M), (CH.sub.2).sub.nCON(C.sub.lH.sub.2l+1)(CH.sub.2).sub.mCH.sub.3, (CH.sub.2).sub.nN(C.sub.lH.sub.2l+1)(CH.sub.2).sub.mCH.sub.3, (CH.sub.2).sub.nX.sub.2(CH.sub.2).sub.pCON(C.sub.lH.sub.2l+1)(CH.sub.2).sub.mCH.sub.3, or (CH.sub.2).sub.nX.sub.2(CH.sub.2).sub.pN(C.sub.lH.sub.2l+1)(CH.sub.2).sub.mCH.sub.3, where X.sub.2 represents O, NH, S, SO.sub.2, CHCH, SO.sub.2NH, NHSO.sub.2, CONH, NHCO, CO, COO, OCO or C.sub.6H.sub.4, where M represents a monovalent cation in the form of a hydrogen ion, lithium ion, sodium ion or potassium ion, n and m represent an integer of 0 to 22, l represents an integer of 1 to 22, p represents an integer of 0 to 17, and x represents an integer of 2 to 2000, and R.sub.3 represents a group selected from the group consisting of the following groups:
(15) ##STR00026##
where, B represents a monovalent cation in the form of a hydrogen ion, lithium ion, sodium ion or potassium ion, X.sub.1 or X.sub.2 represents O, NH, S, SO.sub.2, CHCH, SO.sub.2NH, NHSO.sub.2, CONH, NHCO, CO, COO, OCO or C.sub.6H.sub.4, R.sub.4 represents an alkane group, alkene group or alkyne group having 1 to 24 carbon atoms, n and m represent an integer of 0 to 22, l represents an integer of 1 to 22, and p represents an integer of 0 to 17.
(16) These compounds can be easily synthesized using methods commonly known in the art, examples include those disclosed in Japanese Patent Application Laid-open No. H09-124599, WO 1995/007888, Japanese Patent Application Laid-open No. H03-171136 and J. Org. Chem., 60, 2391 (1995). Among these compounds, indocyanine green (ICG):
(17) ##STR00027##
is already commercially available as a contrast agent, and can be preferably used in the present invention.
(18) The inventors have synthesized a large number of derivatives having a hexatriene skeletal structure similar to that of indocyanine green and investigated their fluorescence properties. It was found that, in particular, the following compounds have high fluorescence intensity, and have high stability on a liposome lipid bilayer membrane: [4-(2-((1E,3E,5E,7E)-7-(3-(2-carboxyethyl)-1,1-dimethyl-1H-benzo[e]indol-2-(3H)-ylidene) hepta-1,3,5-trienyl)-1,1-dimethyl-1H-benzo[e]indolium-3-yl) butane-1-sulfonic acid] (referred to as ICG-6 herein):
(19) ##STR00028##
the compound [4(2-((1E,3E,5E,7E)-7-(1,1-dimethyl-3-octadecyl-1H-benzo[e]indol-2(3H)-ylidene)hepta-1,3,5-trienyl)-1,1-dimethyl-1H-benzo[e]indolium-3-yl)butane-1-sulfonic acid] (referred to as ICG-8 herein):
(20) ##STR00029##
the compound sodium [4-(2-((E)-2-((E)-2-(4-(dihexyamino)phenoxy)-3-((E)-2-(1,1-dimethyl-3-(4-sulfonatebutyl)-1H-benzo[e]indol-2(3H)-ylidene)ethylidene) cyclohexenyl)vinyl)-1,1-dimethyl-1H-benzo[e]indolium-3-yl) butane-1-sulfonate] (referred to as ICG-9 herein):
(21) ##STR00030##
a compound represented by the chemical formula:
(22) ##STR00031##
(referred to as ICG-8-C4 herein), or a compound represented by the chemical formula:
(23) ##STR00032##
(referred to as ICG-8-C6 herein), or a compound represented by the chemical formula:
(24) ##STR00033##
(referred to as ICG-8-C8 herein), or a compound represented by the chemical formula:
(25) ##STR00034##
(referred to as ICG-8-C10 herein), or a compound represented by the chemical formula:
(26) ##STR00035##
(referred to as ICG-11-DOPE herein). These compounds are particularly suitable for use in the near-infrared fluorescent imaging agent of the present invention.
(27) The novel fluorescent dyes ICG-6 and ICG-8 used in the near-infrared fluorescent imaging agent of the present invention can be synthesized according to the following scheme in accordance with the descriptions of WO 1995/007888 and Japanese Patent Application Laid-open No. H09-124599.
(28) ##STR00036##
(29) Commercially available 2,3,3-trimethyl-4,5-benzo-3H-indole is reacted with 1,4-butanesultone to form one of the units indicated in the chemical formula in the form of 2,3,3-trimethyl-1-(sulfobutyl)-4,5-benzoindole (1), and then coupled to a hexatriene chain by reacting with glutaconaldehyde dianil hydrochloride to obtain 2-(6-acetoanilido-1,3,5-hexatrienyl)-3,3-dimethyl-1-(sulfobutyl)-4,5-benzo[e]indole (3). The other unit indicated in the chemical formula of ICG-6 in the form of 2,3,3-trimethyl-1-(2-carboxyethyl)-4,5-benzoindole (2) can be obtained by reacting 2,3,3-trimethyl-4,5-benzo-3H-indole with a halogenated propionic acid. ICG-6 can be obtained by coupling these two units in the presence of pyridine.
(30) The other unit indicated in the chemical formula of ICG-8 in the form of 1-octadecyl-2,3,3-trimethyl-4,5-benzo[e]indole (4) can be obtained by alkylating 2,3,3-trimethyl-4,5-benzo-3H-indole with a halogenated octadecane, which is then coupled to 2-(6-acetoanilido-1,3,5-hexatrienyl)-3,3-dimethyl-1-(sulfobutyl)-4,5-benzo[e]indole (3) to obtain ICG-8.
(31) The novel fluorescent dye ICG-9 used in the near-infrared fluorescent imaging agent of the present invention can be synthesized according to the following scheme in accordance with the descriptions of Japanese Patent Application Laid-open No. H03-171136 and J. Org. Chem., 60, 2391 (1995).
(32) ##STR00037##
(33) 3-chloro-2,4-trimethylene glutaconaldehyde dianil hydrochloride (5), which forms the triene skeleton, can be obtained by converting cyclohexanone to an aldehyde using phosphorous oxychloride and coupling the aldenyde with aniline. A cyanine dye can be obtained by coupling the compound (5) with 2,3,3-trimethyl-1-(sulfobutyl)-4,5-benzoindole (1) unit formed as described above in the presence of triethylamine, methanol and acetic anhydride. The pendant group on the triene skeleton in the form of p-(di-n-hexylamino)phenol (4) is obtained by alkylating p-aminophenol with an alkyl halide, which is then reacted with sodium hydride to form a sodium phenoxide and coupled with the cyanine dye to obtain ICG-9.
(34) The phospholipid-modified fluorescent dye ICG-11-DOPE used in the near-infrared fluorescent imaging agent of the present invention, can be synthesized according to the following scheme from a commercially available dioleoylphosphatidylethanolamine (Coatsome ME-8181).
(35) ##STR00038##
(36) A fluorescent dye of the present invention that contains another phospholipid moiety can be prepared from ICG-11-DOPE by hydrolyzing a fatty acid phosphate in the presence of a base or acid catalyst or with lipase, and converting the fatty acid moiety by dehydration reaction using a carbodiimide or by an actylation reaction. Alternatively, the fatty acid moiety may be reacted with acyltransferase. These conversion reactions can be easily carried out using methods commonly known in the art, and examples may include those schemes disclosed in Organic Chemistry, Maitland Jones Jr., Tokyo Kagaku Dojin, 2000, and Biochemistry, Lubert Stryer, Tokyo Kagaku Dojin, 2004.
(37) As is demonstrated in the following examples, the fluorescent dyes used in the near-infrared fluorescent imaging agent of the present invention are able to generate fluorescence at wavelengths in the near-infrared region, and have high molar extinction coefficients similar to that of ICG. The molar extinction coefficients of the fluorescent dyes are shown in Table 1.
(38) TABLE-US-00001 TABLE 1 Molar Extinction Name max (EtOH) Coefficient () ICG (commercially 787 nm 1.47 10.sup.5 available*) ICG-6 786 nm 1.86 10.sup.5 ICG-8 787 nm 2.30 10.sup.5 ICG-9 810 nm 1.24 10.sup.5 ICG-8-C4 787 nm 2.52 10.sup.5 ICG-8-C6 787 nm 2.34 10.sup.5 ICG-8-C8 788 nm 2.63 10.sup.5 ICG-8-C10 788 nm 2.50 10.sup.5 ICG-11-DOPE 790 nm 1.61 10.sup.5 *Manufactured by Tokyo Chemical Industry Co., Ltd. (purity: 80% or more)
(39) Preparation of Fluorescent Probe
(40) The near-infrared fluorescent dye used in the present invention has high affinity for lipids, and is able to stably generate intense fluorescence when conjugated with a liposome. In this description, a conjugate of a near-infrared fluorescent dye and a liposome is referred to as a fluorescent probe.
(41) The fluorescent probe of the present invention can be prepared by forming a liposome having a near-infrared fluorescent dye using any method known in the art. For example, the fluorescent probe of the present invention can be prepared by dissolving an indocyanine-based near-infrared fluorescent dye and phospholipid in a suitable organic solvent, drying, dispersing in an aqueous solution, and repeatedly passing through a filter. Alternatively, the fluorescent probe of the present invention may be prepared by ultrasound treatment or reverse phase evaporation as is known in the related art.
(42) Examples of phospholipids for preparing the probe may include phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, phosphatidylglycerol, sphingomyelin and phosphatidic acid. Moreover, a lipid component such as cholesterol is preferably added to stabilize the lipid membrane.
(43) The outer surface of the liposome can be modified with a hydrophilic polymer to improve the stability of the liposome in vivo. Examples of such hydrophilic polymers include polyethylene glycol, polymethylethylene glycol, polyhydroxypropylene glycol, polypropylene glycol, polymethylpropylene glycol and polyhydroxypropylene oxide. Polyethylene glycol is particularly preferable.
(44) An example of preparation process of the fluorescent probe of the present invention comprises first dissolving an indocyanine-based near-infrared fluorescent dye, a phospholipid (such as egg yolk lecithin containing phosphatidylcholine), and a lipid (such as cholesterol) or a phospholipid derivative of a hydrophilic polymer (such as a polyethyleneglycol-modified phospholipid) in a suitable organic solvent. The mixing ratio of the near-infrared fluorescent dye to the phospholipid is about 1/1000 to 5/100. Next, the solution is dispersed in an aqueous solution. Alternatively, the organic solvent is removed from the mixture to form a thin film, and an aqueous solution is added. Any physiologically acceptable buffer may be used as an aqueous solution. Preferably, a buffer containing albumin may be used for the purpose of pretreatment that allows the fluorescent probe to form a stable structure in vivo.
(45) Next, liposomes can be formed by passing a suspension containing a phospholipid is dispersed in a buffer through a filter with a pore size of 0.1 m to 0.2 m about 10 times to about 30 times. The particle diameter of the liposome can be suitably adjusted by selecting the phospholipid employed, the type and concentration of the lipid and/or modified phospholipid, the pore size of the filter, materials of the filter or the number of times the suspension is passed through the filter. A liposome having a desired diameter can be prepared by sizing the liposome thus formed as necessary. The particle diameter of the liposome is preferably 100 nm to 300 nm and more preferably 150 nm to 250 nm in order to ensure a long anchoring time in lymph nodes. The optimum particle diameter suitable for the target lymph node varies dependent on the tissue being imaged and the type of lymph node, but can be easily selected by those ordinary skill in the art by conducting simple preliminary experiments.
(46) Identification of Sentinel Lymph Node Using Fluorescent Probe for Lymph Node Imaging
(47) In order to identify a sentinel lymph node using the fluorescent probe of the present invention, about 200 L to about 500 L of the fluorescent probe are directly injected into the submucosal layer, muscle layer or subserous layer around the primary lesion of a tumor. The dispersion medium for dispersion of the fluorescent probe is water. A salt is added to stabilize the fluorescent probe that has the buffering action from pH 6.5 to pH 8.0 at a concentration of 10 mM to 200 mM, such as a phosphate or carbonate, or a sodium salt or potassium salt at a concentration of 10 mM to 500 mM. Serum containing albumin is also effective as a stabilizer. In the case of injecting the dispersion using an endoscope, the fluorescent probe is preferably injected directly using a centesis needle or injection needle for endoscopic treatment. Immediately after direct injection of the fluorescent probe, lymph nodes are observed using a monitoring device and a bioscope such as an endoscope or laparoscope equipped with a filter that only transmits near-infrared light. In this way, lymph nodes where the fluorescent probe of the present invention has accumulated are detected and identified.
(48) Since the fluorescent probe of the present invention has a long anchoring time in sentinel lymph nodes, the sentinel lymph node can be identified with high precision, thereby making is extremely useful for sentinel lymph node navigation surgery.
(49) As used herein, embodiments represented by the expression comprising include embodiments represented by the expression essentially consisting of as well as aspects represented by the expression consisting of.
(50) The contents of all patent and reference documents explicitly cited in the description are incorporated herein by reference.
(51) The following provides a more detailed explanation of the present invention through examples thereof, but the present invention is not limited to these examples.
EXAMPLES
Example 1Synthesis of Fluorescent Dyes ICG-6 and ICG-8
(52) Fluorescent dyes ICG-6 and ICG-8 of the present invention were synthesized according to the scheme indicated below.
(53) ##STR00039##
2,3,3-trimethyl-1-(sulfobutyl)-4,5-benzoindolium inner salt (1)
(54) 2,3,3-trimethyl-4,5-benzo-3H-indole (3.1 g, 15 mmol) and 1,4-butanesultone (2.1 g, 15 mmol) were mixed in a 25 ml four-mouth flask and filled with nitrogen. After stirring for 4 hours at 80 C., the mixture was allowed to stand to cool to room temperature. Acetone was added and stirred briefly, then the resulting crystals were filtered, washed with 10 ml of acetone, and dried to obtain gray crystals (1.17 g, 23%).
2,3,3-trimethyl-1-(2-carboxyethyl)-4,5-benzoindolium bromide (2)
(55) A mixture of 2,3,3-trimethyl-4,5-benzo-3H-indole (2.3 g, 11 mmol), 3-bromo-1-propionic acid (1.5 g, 9.8 mmol) and acetonitrile (10 ml) was stirred for 16 hours at 65 C., allowed to stand to cool to room temperature, and poured into ethyl acetate (50 ml). The crystals were filtered, washed with acetone, and filtered again, and dried to obtain the titled compound as gray crystals (1.68 g, 47%).
2-(6-acetoanilido-1,3,5-hexatrienyl)-3,3-dimethyl-1-(sulfobutyl)-4,5-benzo[e]indolium inner salt (3)
(56) The indolium salt (1) (1.04 g, 3.0 mmol) and glutaconaldehyde dianil hydrochloride (0.94 g, 3.3 mmol) were mixed in a 25 ml four-mouth flask and stirred for 1 hour at 120 C. The mixture was allowed to stand to cool to room temperature and stirred for 1 hour. The resulting crystals were filtered, washed with acetone, filtered and dried to obtain the titled compound as dark violet crystals (0.91 g, 58%).
4-(2-((1E,3E,5E,7E)-7-(3-(2-carboxyethyl)-1,1-dimethyl-1H-benzo[e]indol-2-(3H)-ylidene) hepta-1,3,5-trienyl)-1,1-dimethyl-1H-benzo[e]indolium-3-yl) butane-1-sulfonic acid (ICG-6)
(57) Compound (2) (0.55 g, 1.5 mmol), Compound (3) (0.80 g, 1.5 mmol) and pyridine (8 ml) were mixed in a 25 ml four-mouth flask, stirred for 45 minutes at 50 C. and allowed to stand to cool to room temperature. The reaction liquid was then concentrated under reduced pressure and water (20 ml) was added to the residue and stirred. 10% hydrochloric acid was added slowly and stirred briefly (pH 3 to pH 4). The resulting crystals were filtered and dried to obtain a crude product (1.12 g). 20 ml of a mixture of methanol and chloroform (5/1) were added to the resulting crystals, stirred with heating, and allowed to stand to cool. The crystals were filtered and recrystallized from a mixture of methanol and chloroform, filtered and allowed to air-dry to obtain ICG-6 as deep green crystals (0.38 g, 37%).
(58) The primary molecular ion peak as determined by LC-MS was m/z=687 (negative). The maximum absorbance wavelength and molar extinction coefficient in ethanol were max=786 nm and =1.8610.sup.5, respectively.
1-octadecyl-2,3,3-trimethyl-4,5-benzo[e]indolium iodide (4)
(59) 2,3,3-trimethyl-4,5-benzo-3H-indole (8.4 g, 40 mmol), 1-iodooctadecane (16.8 g, 44 mmol) and 2-butanone (30 ml) were mixed in a 100 ml four-mouth flask, stirred for 18 hours at 70 C., allowed to stand to cool to room temperature and ethyl acetate (40 ml) was added. The resulting crystals were filtered, washed (twice) with ethyl acetate, filtered again, and air-dried to obtain the titled compound as gray crystals (4.4 g, 19%).
4(2-((1E,3E,5E,7E)-7-(1,1-dimethyl-3-octadecyl-1H-benzo[e]indol-2 (3H)-ylidene) hepta-1,3,5-trienyl)-1,1-dimethyl-1H-benzo[e]indolium-3-yl) butane-1-sulfonic acid (ICG-8)
(60) Compound (3) (1.58 g, 3.0 mmol), the indolium salt (4) (1.77 g, 3.0 mmol) and pyridine (16 ml) were mixed and dissolved in a 50 ml three-mouth flask, filled with nitrogen and allowed to react for 1 hour at 50 C. The reaction liquid was poured into water (40 ml) and the precipitated crystals were filtered. The resulting crude crystals were dissolved in ethyl acetate, filtered and recrystallized from 40 ml of a mixture of chloroform and ethyl acetate (1/1) to obtain the titled compound as dark green crystals (1.39 g, 53%).
(61) The primary molecular ion peak as determined by LC-MS was m/z=867 (negative). The maximum absorption wavelength and molar extinction coefficient in ethanol were max=787 nm and =2.3010.sup.5, respectively.
Example 2Synthesis of Fluorescent Dye ICG-9
(62) Fluorescent dye ICG-9 of the present invention was synthesized according to the scheme indicated below.
(63) ##STR00040##
p-(di-n-hexylamino)phenol (4)
(64) A mixture of p-aminophenol (0.55 g, 5.0 mmol), 1-iodohexane (2.5 g, 11.8 mmol), potassium carbonate (0.70 g, 5.1 mmol) and dimethylformamide (10 ml) in a 25 ml four-mouth flask was stirred for 3 hours at 75 C. Potassium carbonate (0.70 g, 5.1 mmol) was added and stirred for 6 hours. The mixture was allowed to stand to cool to room temperature, poured into water and extracted with ethyl acetate. The organic layer was washed with water (3 times) and saturated salt solution, dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain a crude product (1.3 g). The crude product was purified by silica gel column chromatography (toluene/ethyl acetate=25/1 to 15/1) to obtain the titled compound as an oil (0.81 g, 58%).
3-chloro-2,4-trimethylene glutaconaldehyde dianil hydrochloride (5)
(65) Dimethylformamide (55 ml) was placed in a 300 ml four-mouth flask and phosphorous oxychloride (50 ml, 0.54 mol) was added dropwise for 1 hour at 10 C. or lower. The mixture was slowly heated and cyclohexanone (15 g, 0.15 mol) and dimethylformamide (35 ml) were added dropwise for 1 hour at 45 C. to 50 C. After stirring for 3 hours at 50 C., the reaction liquid was allowed to stand to cool to room temperature, poured into water and stirred overnight. The crystals were filtered, washed with water, and filtered again, and dried overnight to obtain the aldehyde as yellow crystals (19.4 g, 74%). The aldehyde (19 g, 0.1 mol) and dimethylformamide (75 ml) were mixed in a 500 ml four-mouth flask, and concentrated hydrochloric acid (25 ml) was added dropwise at 8 C. or lower. After stirring for 15 minutes, a mixture of aniline (21.5 g, 0.23 mol) and ethanol (110 ml) was added dropwise at 7 C. or lower. The mixture was allowed to warm to room temperature for 1.5 hours, poured into water, and stirred for 1 hour. The resulting crystals were filtered and air-dried to obtain the titled compound (41.3 g, quantitative).
Sodium 4-(2-((E)-2-((E)-2-(4-(dihexyamino) phenoxy)-3-((E)-2-(1,1-dimethyl-3-(4-sulfonatebutyl)-1H-benzo[e]indol-2 (3H)-ylidene)ethylidene)cyclohexenyl)vinyl)-1,1-dimethyl-1H-benzo[e]indolium-3-yl)butane-1-sulfonate (ICG-9)
(66) Compound (1) (0.83 g, 2.4 mmol), 3-chloro-2,4-trimethylene glutaconaldehyde dianil hydrochloride (0.43 g, 1.2 mmol), triethylamine (1.1 ml, 7.9 mmol) and methanol (12 ml) were mixed in a 25 ml four-mouth flask and filled with nitrogen. Acetic anhydride was then added dropwise to the reaction liquid for 5 minutes, and stirred for 15 hours while protecting from light. Sodium acetate (0.49 g, 6.0 mmol) was added to the reaction liquid and evaporated the solvent under reduced pressure. The residue was then dissolved in acetone to remove insoluble materials, and the filtrate was concentrated under reduced pressure. The residue was washed with ethyl acetate and subsequently with acetone, filtered, and air-dried to obtain crude crystals of the cyanine dye (0.87 g).
(67) Sodium hydride (60%, 0.05 g, 1.3 mmol) and dimethylformamide (2 ml) were mixed in a 25 ml four-mouth flask and the phenol derivative (4) (0.30 g, 1.1 mmol) and dimethylformamide (3 ml) were added dropwise for 10 minutes in a nitrogen atmosphere at 65 C. or lower. After gradually warming to room temperature, the mixture was stirred for 1 hour to prepare a sodium phenoxide. Then the previously synthesized cyanine dye (0.80 g, 0.94 mmol) and dimethylformamide (9 ml) were mixed in a 50 ml four-mouth flask, phenoxide dimethylformamide solution was added dropwise for 10 minutes in a nitrogen atmosphere. After stirring for 3 hours, dry ice (1 g) was added and the reaction liquid was purified by silica gel column chromatography (chloroform/methanol=30/1 to 3/2). The resulting crude crystals were washed with a mixture of ethyl acetate and a small amount of acetone, filtered and air-dried to obtain ICG-9 as deep green crystals (0.49 g, 48%).
(68) The primary molecular ion peak as determined by LC-MS was m/z=1069 (negative). The maximum absorbance wavelength and molar extinction coefficient in ethanol were max=810 nm and =1.2410.sup.5, respectively.
Example 2-2
Synthesis of Additional Compounds
ICG-8-C4
4(2-((1E,3E,5E,7E)-7-(1,1-dimethyl-3-butyl-1H-benzo[e]indol-2(3H)-ylidene) hepta-1,3,5-trienyl)-1,1-dimethyl-1H-benzo[e]indolium-3-yl) butane-1-sulfonic acid (ICG-8-C4)
(69) 1-butyl-2,3,3-trimethyl-4,5-benzo[e]indolium iodide (10.7 g, 62%) was obtained in the same manner as Example 1 with replacing 1-iodooctadecane (16.8 g, 44 mmol) (used for the synthesis of 1-octadecyl-2,3,3-trimethyl-4,5-benzo[e]indolium iodide (4) in Example 1) with 1-iodobutane (12.1 g, 66 mmol). The titled compound (1.35 g, 44%) was obtained in the same manner as the synthesis of ICG-8. The primary molecular ion peak as determined by LC-MS was m/z=673 (positive). The maximum absorbance wavelength and molar extinction coefficient in ethanol were max=787 nm and =2.5210.sup.5, respectively.
ICG-8-C6
4(2-((1E,3E,5E,7E)-7-(1,1-dimethyl-3-hexyl-1H-benzo[e]indol-2 (3H)-ylidene) hepta-1,3,5-trienyl)-1,1-dimethyl-1H-benzo[e]indolium-3-yl) butane-1-sulfonic acid (ICG-8-C6)
(70) 1-hexyl-2,3,3-trimethyl-4,5-benzo[e]indolium iodide (5.63 g, 54%) was obtained in the same manner as Example 1 with replacing 1-iodooctadecane (16.8 g, 44 mmol) (used for the synthesis of 1-octadecyl-2,3,3-trimethyl-4,5-benzo[e]indolium iodide (4) in Example 1) with 1-iodohexane (6.4 g, 30 mmol). The titled compound (1.18 g, 57%) was obtained in the same manner as the synthesis of ICG-8. The primary molecular ion peak as determined by LC-MS was m/z=701 (positive). The maximum absorbance wavelength and molar extinction coefficient in ethanol were max=787 nm and =2.3410.sup.5, respectively.
ICG-8-C8
4(2-((1E,3E,5E,7E)-7-(1,1-dimethyl-3-octyl-1H-benzo[e]indol-2 (3H)-ylidene) hepta-1,3,5-trienyl)-1,1-dimethyl-1H-benzo[e]indolium-3-yl) butane-1-sulfonic acid (ICG-8-C8)
(71) 1-octyl-2,3,3-trimethyl-4,5-benzo[e]indolium iodide (11.7 g, 59%) was obtained in the same manner as Example 1 with replacing 1-iodooctadecane (16.8 g, 44 mmol) (used for the synthesis of 1-octadecyl-2,3,3-trimethyl-4,5-benzo[e]indolium iodide (4) in Example 1) with 1-iodooctane (15.8 g, 66 mmol). The titled compound (0.93 g, 59%) was obtained in the same manner as the synthesis of ICG-8. The primary molecular ion peak as determined by LC-MS was m/z=727 (positive). In addition, the maximum absorbance wavelength and molar extinction coefficient in ethanol were max=788 nm and =2.6310.sup.5, respectively.
ICG-8-C10
4(2-((1E,3E,5E,7E)-7-(1,1-dimethyl-3-decyl-1H-benzo[e]indol-2(3H)-ylidene)hepta-1,3,5-trienyl)-1,1-dimethyl-1H-benzo[e]indolium-3-yl)butane-1-sulfonic acid (ICG-8-C10)
(72) 1-decyl-2,3,3-trimethyl-4,5-benzo[e]indolium iodide (4.83 g, 23%) was obtained in the same manner as Example 1 with replacing 1-iodooctadecane (16.8 g, 44 mmol) (used for the synthesis of 1-octadecyl-2,3,3-trimethyl-4,5-benzo[e]indolium iodide (4) in Example 1) with 1-iododecane (17.7 g, 66 mmol). The titled compound (0.79 g, 46%) was obtained in the same manner as the synthesis of ICG-8.
(73) ICG-11-DOPE:
(74) Coatsome ME-8181 (manufactured by NOF Corp.) was used as a phospholipid to be modified.
(75) ##STR00041##
(76) ICG-6 (0.5 g, 0.73 mmol), N-hydroxysuccinimide (0.17 g, 1.5 mmol), acetonitrile (3 mL) and chloroform (10 mL) were placed in a 25 mL four-mouth flask and filled with nitrogen. A mixture of dicyclohexylcarbodiimide (0.30 g, 1.5 mmol) and chloroform (2 mL) was added dropwise for 10 minutes at 2 C. The reaction liquid was slowly warmed to room temperature, stirred for 30 minutes and filtered. The filtrate was concentrated under reduced pressure at 25 C. or lower, and the residue was triturated with ethyl acetate. The crystals were collected by filtration and washed with acetone to obtain crude crystals of ICG-11 (0.66 g). Then the resulting crystals (0.66 g, ca. 0.73 g) and chloroform (10 mL) were placed in a 25 mL four-mouth flask and a mixture of Coatsome ME-8181 (0.54 g, 0.73 mmol), triethylamine (0.10 g, 1.0 mmol) and chloroform (5 mL) was added dropwise for 20 minutes in a nitrogen atmosphere. The reaction liquid was stirred for 2.5 hours at room temperature, filtered and concentrated. The residue was purified with a flash column (SiO.sub.2, CHCl.sub.3/MeOH=4/1 to 3/1) to obtain ICG-11-DOPE (0.37 g, 36%).
Example 3Preparation of Fluorescent Probe
(77) Egg yolk phosphatidylcholine (7.3 mg), cholesterol (0.4 mg), phosphatidylethanolamine-N-methoxypolyethylene glycol 5000-dioleoyl-glyceroammonium salt (3.2 mg) and a fluorescent dye (ICG, ICG-6, ICG-8 or ICG-9) (3.2 mM to 3.210.sup.4 mM) were dissolved in a mixture of chloroform and methanol (volume percentage: 9:1). The organic solvent was evaporated under reduced pressure to form a thin film. 1 mL of an aqueous solution of 5% human albumin (CSL Behring GmbH) was then added and allowed to stand for several hours at room temperature. The suspension was then passed through a polycarbonate filter (pore size: 0.1 m to 0.2 m, Millipore Corp.) attached to a membrane holder (Millipore Corp.) about 10 times to form liposomes.
(78) Fluorescence properties of the ICG-8-containing fluorescent probe obtained in this manner were then examined. The fluorescence spectrum was measured by exciting at 738 nm. The excitation spectrum was measured by detecting fluorescence at 850 nm when excited at each wavelength from 600 nm to 800 nm. The results are shown in
(79) The particle diameter distribution of the resulting liposomes was measured using a dynamic light scattering particle size distribution analyzer (Nikkiso Co., Ltd.). The results are shown in
Example 4-1Fluorescence Detectability of Fluorescent Probe In Vitro
(80) Near-infrared fluorescence intensity was measured for the fluorescent dye-containing liposomes prepared in the manner described above with an aqueous solution of ICG serving as a control. 100 L of liposome suspension containing a dye of a predetermined concentration or the ICG aqueous solution were added to a 96-well plate and irradiated with excitation light (LED light source, <780 nm) to detect the generated fluorescence (>810 nm). Brightness was evaluated using Image J image processing software.
(81) The results are shown in Table 2. In the table, values indicate the brightness score as determined with the Image J software (maximum value: 255), with the values shown in parentheses indicate the standard deviation.
(82) TABLE-US-00002 TABLE 2 ICG ICG-6 ICG-8 ICG-9 ICG aqueous Concentration fluorescent fluorescent fluorescent fluorescent solution (mM) probe probe probe probe (control) 3.2 100 55(5) 72(6) 36(5) 31(6) 27(5) 3.2 101 56(5) 81(5) 84(6) 52(4) 38(6) 3.2 102 173(7) 195(6) 217(6) 143(5) 98(5) 3.2 103 158(4) 181(5) 189(4) 149(4) 118(10) 3.2 104 84(4) 95(4) 105(5) 77(6) 61(7) |ICG-8-C4 ICG-8-C6 ICG-8-C8 ICG-8-C10 ICG-8-C18 ICG-11-DOPE Concentration fluorescent fluorescent fluorescent fluorescent fluorescent fluorescent (mM) probe probe probe probe probe probe 3.2 101 47(5) 66(7) 49(8) 39(7) 51(8) 3.2 102 155(10) 161(13) 163(9) 156(11) 164(9) 187(12) 3.2 103 141(8) 137(6) 138(6) 153(10) 138(6) 3.2 104 68(6) 68(7) 67(8) 77(9) 58(6)
(83) The fluorescent probes obtained by incorporating a near-infrared fluorescent dye in the liposomes according to the present invention demonstrated extremely high fluorescence intensity as compared to the ICG aqueous solution.
Example 4-2Fluorescence Lifetime of Fluorescence Probes
(84) The fluorescence lifetimes of the fluorescence probes of the present invention were measured. The TemPro fluorescence lifetime photometer (manufactured by Horiba, Ltd.) was used for measurement. Table 3 indicates the values for a chloroform solution containing each near-infrared fluorescent dye at a concentration of 3.210.sup.2 mM and an aqueous solution of ICG (control), and Table 4 indicates values obtained from each of the near-infrared fluorescent dyes incorporated in the liposomes at a concentration of 3.210.sup.2 mM. A 740 nm semiconductor laser was used for the excitation light, and the lifetime spectra obtained at a fluorescence wavelength of 816 nm was analyzed for two components. The results are shown in Table 3 and Table 4.
(85) TABLE-US-00003 TABLE 3 ICG aqueous solution Name ICG-8-C6 ICG-8-C10 ICG-8-C18 (control) First component 1.25 1.27 1.26 0.01~ (ns) 0.15 First component 69 67 68 (%) Second component 0.31 0.41 0.37 (ns) Second component 31 33 32 (%)
(86) TABLE-US-00004 TABLE 4 ICG-8-C6 ICG-8-C10 ICG-8-C18 fluorescent fluorescent fluorescent Name probe probe probe First component (ns) 0.41 0.39 0.38 First component (%) 90 93 82 Second component 0.77 0.83 0.65 (ns) Second component (%) 10 7 18
(87) All of the fluorescent probe dispersions showed the fluorescence lifetime longer than the ICG aqueous solution, and demonstrating superior sensitivity of near-infrared fluorescent imaging. Moreover, differences in the alkyl chains of the substituents have little effect on fluorescence lifetime of the fluorophore. These results indicate that the design of the skeletal structure of the near-infrared fluorescent dye of the invention is useful for preparing fluorescent probes.
Example 5Fluorescence Detectability of Fluorescent Probes In Vivo
(88) In order to investigate the fluorescence detectability of the fluorescent probes in vivo, an ICG-8-containing fluorescent probe dispersion (0.2 mL, 10 mM as phospholipid concentration) was injected into the foreleg of mice (indicated with a yellow arrow in
Example 6Fluorescent Probe Anchoring Property in Mouse Popliteal Lymph Node
(89) In order to investigate e lymph node anchoring property of the fluorescent probe, an ICG-8-containing fluorescent probe dispersion (liposome mean particle diameter: 240 nm, 0.2 mL, phospholipid concentration: 10 mM) or an ICG aqueous solution serving as a control (0.2 mL, 3.2 mM) was injected into the foreleg of mice (
(90) When the ICG aqueous solution was injected, fluorescent was emitted at the first popliteal lymph node from the foreleg as well as from the second external iliac lymph node from the foreleg (solid white circles in
(91) To investigate primary lymph node anchoring rate after the passage of a prescribed amount of time, an ICG-8-containing fluorescent probe or ICG aqueous solution was administered to mice in the same manner as described above. The results are indicated as (number of mice in which fluorescence was observed in primary lymph node only/total number of mice in the experiment). The p-values were calculated using Fisher's exact test.
(92) TABLE-US-00005 TABLE 5 No. of mice in which fluorescence was Elapsed time observed in primary lymph node from only/total no. of mice in experiment administration ICG-8-containing ICG aqueous (h) fluorescent probe solution P value 1 13/13 0/4 0.01 3 15/19 2/5 0.13 6 6/12 0/4 0.12 24 1/4 0/3 0.57
(93) These results indicate that the ICG-8-containing fluorescent probe demonstrates a significantly higher anchoring rate to the primary lymph node than ICG at 1 hour after administration. The ICG-8-containing fluorescent probe tended to demonstrate a higher anchoring rate than ICG at 3 hours or 6 hours after administration as well.
Example 7Anchoring Property of Fluorescent Probe in Porcine Sigmoid Lymph Node
(94) 0.2 mL of ICG-8-containing fluorescent probe (liposome mean particle diameter: 240 nm) were injected into the digestive tract of a pig at the sigmoid colon (indicated with a yellow arrow in
Example 8Anchoring Property of Fluorescent Probe in Porcine Subpyloric Lymph Node
(95) 0.2 mL of ICG-8-containing fluorescent probe (liposome mean particle diameter: 160 nm) dispersed in physiological saline were injected into the stomach wall in the pyloric region of a pig (
INDUSTRIAL APPLICABILITY
(96) The near-infrared fluorescent imaging agent of the present invention is useful for detecting sentinel lymph nodes during cancer and other surgeries.