Amyloid fiber detection probe

Abstract

To provide an effective technique to enable amyloid fibrils formed within the body to be observed in vivo from outside the body.SOLUTION: The present invention provides an amyloid fibril detection probe that binds to amyloid β amyloid fibrils, containing a specific curcumin derivative, for example, a derivative represented by the following formula.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to a probe compound for detecting and observing amyloid fibers in the body from outside the body.

Background Art

[0002] More than 30 diseases caused by insoluble amyloid fibers derived from various types of proteins have been reported, such as Alzheimer's disease and Parkinson's disease. However, the detailed pathological mechanisms of these diseases are not yet understood.

[0003] As one method of treatment, a method using an antibody has been considered. For example, aducanumab (BIIB037) is known as a human monoclonal antibody that selectively binds to the aggregated form of beta-amyloid (Aβ) peptide, and there is also a report that amyloid plaques (amyloid fibers) can be reduced by specifying the administration method of such a monoclonal antibody (Patent Document 1).

[0004] On the other hand, as methods for detecting amyloid fibers, methods such as the thioflavin fluorescence method, staining using Congo Red, staining using FSB, and ELISA using an antibody that recognizes amyloid fibers have been shown (Patent Document 2).

[0005]

[0006]

Chemical Formula

[0007] [In the formula, R​is hydrogen, -OH, -NO2, -CN, -COOR, -OCH2OR, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy or halo, where R 1 one or more of the atoms of; may be a radiolabeled atom; R is C1-C6 alkyl, where one or more of the carbon atoms may be a radiolabeled atom; R 2 is hydrogen, non-radioactive halo or radioactive halo; R 3 is hydrogen, C1-C6 alkyl, C2-C6 alkenyl or C2-C6 alkynyl; R 4 is hydrogen, C1-C6 alkyl, C2-C6 alkenyl or C2-C6 alkynyl, where when R 2 is hydrogen or non-radioactive halo, the alkyl, alkenyl or alkynyl contains radioactive carbon or is substituted with radioactive halo; provided that when R 1 is hydrogen or -OH, R 2 is hydrogen, R 4 is - 11 CH3, R 3 is C2-C6 alkyl, C2-C6 alkenyl or C2-C6 alkynyl; Furthermore, when R 1 is hydrogen, R 2 is hydrogen, R 4 is -CH2CH2CH2 18 F, R3 is C2-C6 alkyl, C2-C6 alkenyl or C2-C6 alkynyl], or a pharmaceutically acceptable salt, hydrate, solvate or prodrug of the compound.

Prior Art Documents

Patent Documents

[0008]

Patent Document 1

Patent Document 2

[0009] Observation of amyloid fibers generated in vivo in vivo is an essential technique for diseases related to amyloid fibers. In that regard, the method of Patent Document 2 shows a certain effect. However, methods such as gamma-ray imaging require a large-sized apparatus itself. If there is a simpler apparatus that can observe amyloid fibers in vivo, it is considered to be very contributive in diagnosis. Moreover, if it can play a role in treatment simultaneously with observation, it can be said to be even more effective. [Means for Solving the Problems]

[0010] The present invention was conceived in view of the above problems and provides a novel curcumin derivative, and a probe that can selectively bind to amyloid fibers having the curcumin derivative and emit light by infrared rays to observe amyloid fibers.

[0011] Specifically, the present invention relates to Compound It is characterized by having a structure represented by formulas (2) to (8) and (10) to (14).

[0012] [Chemical Formula]

[0013] [Chemical Formula]

[0014] [Chemical Formula]

[0015] [Chemical formula]

[0016] [Chemical formula]

[0017] [Chemical formula]

[0018] [Chemical formula]

[0019] [Chemical formula]

[0020] [Chemical formula]

[0021] [Chemical formula]

[0022] [Chemical formula]

[0023] [Chemical formula]

[0024] Further, the amyloid fiber detection probe that binds to the amyloid β amyloid fiber according to the present invention contains at least one curcumin derivative represented by formula (4), formula (5), formula (6), formula (10), and formula (14).

[0025] In addition, the amyloid fiber detection probe according to the present invention contains at least one curcumin derivative represented by formula (3), formula (4), formula (7), and formula (8).

Advantages of the Invention

[0026] The amyloid fiber detection probe according to the present invention selectively binds to amyloid fibers and emits light by infrared rays, so that the light emission can be detected even through the skin. Therefore, the distribution and amount of amyloid fibers can be easily estimated by an infrared light-emitting device and an infrared camera. In addition, there is also a possibility of disintegrating the amyloid fibers themselves by continuing to emit light in the near-infrared range.

[0027] In addition, the amyloid fiber detection probe according to the present invention can relatively easily incorporate 13 C or F into its structure, so that amyloid fibers can also be tomographed with high resolution by a nuclear magnetic resonance apparatus (MRI apparatus).

[0028] In addition, since the amyloid fiber detection probe according to the present invention contains boron in its structure, neutron capture therapy can be performed on the bound amyloid fibers, and there is a possibility of decomposing the amyloid fibers.

Brief Description of the Drawings

[0029]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Mode for Carrying Out the Invention

[0030] The amyloid fiber detection probe according to the present invention will be described below with reference to the drawings and examples. The following description illustrates one embodiment and one example of the present invention, and the present invention is not limited to the following description. The following description can be modified without departing from the spirit of the present invention.

[0031] The amyloid fiber detection probe according to the present invention is a derivative obtained from curcumin and generally has a structure of formula (100).

[0032]

Chemical formula

[0033] The coordination numbers indicate the numbers of the basic skeleton of curcumin. The functional groups bonded to the left and right benzene rings centered on the 1-position are defined as functional group Rp1 and functional group Rp2. There are no particular restrictions on each functional group, and the functional groups Rp1 and Rp2 may each be a plurality of functional groups. Also, it does not prevent the functional groups Rp1 and Rp2 from being the same. Hereinafter, for the sake of explanation, the right side centered on the position of the 1-position will be referred to as the right site, and the left side will be referred to as the left site.

[0034] The amyloid fiber detection probe according to the present invention has fluorescence emission in the infrared region and a binding force to amyloid fibers among those having this structure. These amyloid fiber detection probes are produced by biosynthesis. Biosynthesis is carried out by introducing an enzyme gene related to the curcumin synthesis pathway into Escherichia coli and culturing it in a medium containing a precursor serving as a material for derivatives. By doing so, derivatives with different functional groups can be easily obtained. Also, specific carbon is replaced with a stable isotope.

[0035] Fig. 1 shows an outline of biosynthesis. A plasmid into which an enzyme gene (4CL, DCS, CURS1) for synthesizing curcumin is introduced is introduced into Escherichia coli. The introduced Escherichia coli is designated as "mut-ecoil". This Escherichia coli is cultured in a medium containing two compounds (hereinafter referred to as precursor P1 and precursor P2, respectively) that serve as precursors of curcumin. These two precursors are those in which a functional group is bonded to phenylpropionic acid, and are designated as Rp1 and Rp2 in Fig. 1. There is no particular limitation on this functional group.

[0036] Escherichia coli mut-ecoli synthesizes curcumin analogs from these precursors. At this time, since there is no distinction between the precursors, three types of analogs can be obtained: a substance in which precursor P1 is used at both the left and right sites (homo-P1), a substance in which precursors P1 and P2 are used at both the left and right sites (hetero-P1_P2), and a substance in which precursor P2 is used at both the left and right sites (homo-P2).

[0037] For example, if both precursor P1 and precursor P2 are made into hydroxymethoxyphenylpropionic acid of formula (100), all three synthesized substances will be curcumin.

[0038]

Chemical formula

[0039] The curcumin derivative thus obtained tautomerizes between the keto form and the enol form as shown in formula (101) by keto-enol tautomerism.

[0040] [Chemistry]

[0041] Therefore, the oxygen atoms at the 2-position and the 2'-position are bonded to each other with difluoroboron. This is called "BF2 conversion". As a result, curcumin BF2-converted as shown in formula (102) is produced.

[0042] [Chemistry]

[0043] The curcumin BF2-converted has no tautomerism shown in formula (102) and exists in a stable structure. The curcumin BF2-converted has the property of fluorescence emission. And by changing the functional groups bonded to the benzene rings on the left and right sites, the fluorescence emission in the infrared band can be enhanced to a sufficient intensity for observation.

[0044] Furthermore, by adjusting the functional groups bonded to the benzene rings on the left and right sites, a specific binding force to amyloid fibers can be imparted.

[0045] Also, by mixing 13 C glucose into the medium during culturing, the carbon at the 1-position (refer to formula (100)) can also be modified with a stable isotope. 13 If there is C, it can be imaged by MRI, and detailed imaging of amyloid fibers in the brain is possible. Also, since it is BF2-converted, it has F in the skeleton. Therefore, nuclear magnetic resonance imaging using F is also possible.

[0046] Thus, the amyloid fiber detection probe according to the present invention is composed of a curcumin derivative and has the ability of fluorescence emission in the infrared region, so the presence several centimeters under the skin can be observed with an infrared camera or the like.

[0047] In addition, the amyloid fiber detection probe according to the present invention contains boron in its backbone because it is BF2-modified. Boron clusters are used in neutron capture therapy, but the problem is how to deliver the boron clusters to the target. However, the amyloid fiber detection probe according to the present invention binds to amyloid fibers and has boron in its backbone. Therefore, the position of amyloid fibers can be identified by fluorescence emission in the infrared region, and at the same time, it can be used as a target for neutron irradiation and can be used as a composition for neutron capture therapy.

Example

[0048] Curcumin analogs were produced and purified by the following procedure. Three enzyme genes 4CL, DCS, and CURS1 of the curcumin synthesis pathway were introduced into Escherichia coli BL21(DE3) using a plasmid.

[0049] After shaking culture at 37 °C overnight in TB medium, the culture temperature was lowered to 27 °C, and isopropyl-β-thiogalactopyranoside (IPTG, final concentration 1 mM) was added, followed by further shaking culture overnight. A precursor dissolved in DMSO to a concentration of 10 mg / mL was added in an amount of 1 / 1000 of the medium volume, and the culture was continued for another 24 hours. The cells were collected by centrifugation, dissolved in PBS(-), and stored at -30 °C. The culture supernatant was stored at 4 °C.

[0050] Lipophilic metabolites were extracted from the cells by the Bligh & Dyer method and then spray-dried. After dissolving in methanol and confirming the presence of curcumin analogs by TLC, purification was performed using a silica column (10 mL). The elution fractions were collected 1 mL at a time and immediately dried. After suspending in methanol, the fractions containing curcumin analogs were identified and collected by TLC. At this point, the absorption spectrum and fluorescence spectrum were measured.

[0051] The lipophilic substances contained in the culture supernatant were eluted by solid-phase extraction using a C18 sep-pak column, then spray-dried and suspended in methanol. Thereafter, purification was performed using a silica column in the same manner as for the curcumin analogs derived from the cells.

[0052] The precursors used are shown in Figure 2. There are eight precursor substances used, namely precursor substance - 8, precursor substance - 13, precursor substance - 14, precursor substance - 15, precursor substance - 18, precursor substance - 22, precursor substance ferulate, and precursor substance coumalate. Precursor substance ferulate and precursor substance coumalate are hydroxymethoxyphenylpropionic acid and methoxyphenylpropionic acid respectively. Curcumin analogs were produced using these. Table 1 shows the precursor substances used (designated as the first precursor substance and the second precursor substance respectively), the sample names and sample numbers of the curcumin analogs.

[0053]

Table 1

[0054] Note that curu8 and curu9 are the same substance but with different production dates. Also, curu14 and curu15 were obtained from inside (ppt) and outside (supernatant: sup) of Escherichia coli and were the same substance.

[0055] <BF2 Modification of Curcumin Analogs> The curcumin analogs in Table 1 were transferred to a glass vial, a stir bar was added, and after complete drying, it was sealed. A syringe filled with N2 was inserted into the vial to replace the oxygen and water vapor in the vial with N2. 250 μL of dichloromethane (DCM) dehydrated by molecular sieve since the previous day was injected into the vial while stirring with a stirrer to completely dissolve the curcumin analogs. 250 μL of boron trifluoride diethyl ether complex mixed with DCM in a 1:1 ratio was slowly added while stirring with a stirrer and left at room temperature for more than 2 hours.

[0056] After the reaction, drying and washing with diethyl ether were repeated to remove the unreacted boron trifluoride diethyl ether complex. After complete drying, it was suspended in methanol, filtered through a PFTE filter, and stored at 4 °C. At this point, the absorption spectrum and fluorescence spectrum were measured. The BF2 - modified curcumin analogs are shown in Table 2. Also, the structures of each compound are shown in Figures 3 to 5. The structures of Compounds 1 to 15 are represented by Formula (1) to Formula (15), respectively. Note that Compound 1 is curcumin itself, which is the same as Formula (103).

[0057]

Table 2

[0058] <Luminescence properties> The luminescence properties of the BF2 - modified curcumin analogs change compared to before BF2 modification. In particular, the luminescence in the infrared region increases. Figure 6 shows the luminescence properties of Compound 1 (curcumin) and curu1 (curcumin itself before BF2 modification). Figure 6(a) is curu1, and Figure 6(b) is Compound 1.

[0059] Referring to each graph, the horizontal axis is the excitation light wavelength (nm), and the vertical axis is the luminescence intensity (unitless). Also, the wavelength of the fluorescence emitted for each excitation light wavelength is shown by vertical numbers above the bar graph.

[0060] When excited at a wavelength of 635 nm, curu1 without BF2 modification hardly emits fluorescence, but for Compound 1, which is a BF2 - modified curcumin analog, fluorescence emission was confirmed in the infrared region at 700 nm. This was also the case for the other Compounds 2 to 15, where fluorescence emission in the infrared region was confirmed.

[0061] <Binding to amyloid fibrils> The production mechanism of amyloid-β is considered as follows. Amyloid-β is produced by being cleaved from amyloid precursor protein (APP) by proteases. When APP is cleaved by α-secretase, amyloid-β is not produced. On the other hand, when it is cleaved by β-secretase (from the left side of the figure →), it is then cleaved by γ-secretase, and amyloid-β is produced. The component of γ-secretase is presenilin (PS2), which regulates γ-secretase activity.

[0062] In this study, the variants of APP and PS2 found in patients with familial Alzheimer's disease were co-expressed in cells, and the production of amyloid-β was confirmed. It has also been confirmed that amyloid-β is not produced when APP is not expressed.

[0063] Figure 7 shows an example. Figure 7(a) is the result of electrophoresing the extract of cells co-expressing the variant of APP and the variant of PS2, and labeling it with an anti-amyloid-β antibody (labeled as "anti-Aβ" in the figure). Amyloid-β oligomers were confirmed at around 40 kDa.

[0064] The result of SDS-PAGE after contacting compound 10 (hetero-13-14-BF2) with the cell extract is shown in Figure 7(b). The vertical axis represents mass (kDa), and the horizontal axis represents sample types. When compound 10 was contacted with 10 μL of the APP / PS2 extract at concentrations of 0, 2, 4, 6, and 8 μM respectively, luminescence was confirmed in a concentration-dependent manner at the same mass position as in electrophoresis. From the above, it was confirmed that compound 10 binds to amyloid-β.

[0065] Figure 8 shows the results of performing the same experiment with other compounds. As a result of SDS-PAGE, it was found that compounds 4, 5, 6, 10, 14, and 15 (compounds 14 and 15 are the same substance) have the ability to bind to amyloid-β.

[0066] Figure 9 shows the results of Native-PAGE examining the binding of each compound to α-synuclein amyloid fibrils, which are considered to be the cause of Parkinson's disease. From this, it can be seen that Compound 3, Compound 4, Compound 7, Compound 8, and Compound 9 (note that Compound 8 and Compound 9 are the same substance) bind to α-synuclein amyloid fibrils.

Industrial Applicability

[0067] The amyloid fibril detection probe according to the present invention specifically binds to amyloid fibrils and emits infrared light in the body, so it can be non-invasively observed from outside the body, which is very effective for the detection of amyloid fibrils. Further, the amyloid fibril detection probe according to the present invention can be modified with C and F in the structure and can also be detected by nuclear magnetic resonance imaging or the like. Furthermore, since it has boron in the structure, it can also be used as a composition for neutron capture therapy, and there is a possibility of destroying amyloid fibrils with neutron beams. 13 It can be modified with C and F and can also be detected by nuclear magnetic resonance imaging or the like. Furthermore, since it has boron in the structure, it can also be used as a composition for neutron capture therapy, and there is a possibility of destroying amyloid fibrils with neutron beams.

Claims

**Claim 1** A compound represented by formulas (2) to (8) and formulas (10) to (14). 【Chemical 101】 【Chemical Formula 102】 【Chemical Formula 103】 【Chemical 104】 【Chemical 105】 【Chemical 106】 【Chemical 107】 【Chemical 108】 【Chemical 109】 【Chemical 110】 【Chemical 111】 【Chemical 112】 **Claim 2** An amyloid fiber detection probe that binds to amyloid β amyloid fibers containing at least one compound represented by formulas (4), (5), (6), (10), and (14). 【Chemical 114】 【Chemical 115】 【Chemical 116】 【Chemical 117】 【Chemical 118】 **Claim 3** The amyloid fiber detection probe according to claim 2, wherein at least one carbon in the amyloid fiber detection probe is isotope-modified. **Claim 4** An amyloid fiber detection probe that binds to α-synuclein amyloid fibers containing at least one compound represented by formulas (3), (4), (7), and (8). 【Chemical 119】 【Chemical 120】 【Chemical 121】 【Chemical 122】 **Claim 5** A composition for neutron beam therapy comprising the amyloid fiber detection probe according to any one of claims 2 to 4.

Images