JP7713734B2 - Amyloid fibril detection probe - Google Patents

Description

The present invention relates to a probe compound for detecting and observing amyloid fibrils in the body from outside the body.

More than 30 diseases, including Alzheimer's disease and Parkinson's disease, have been reported to be caused by insoluble amyloid fibrils derived from various types of proteins. However, the detailed pathological mechanisms of these diseases remain unknown.

One method of treatment that has been considered is the use of antibodies. For example, aducanumab (BIIB037) is known as a human monoclonal antibody that selectively binds to the aggregated form of beta-amyloid (Aβ) peptide, and it has been reported that amyloid plaques (amyloid fibrils) can be reduced by identifying the method of administering such a monoclonal antibody (Patent Document 1).

Meanwhile, methods for detecting amyloid fibrils include thioflavin fluorescence, staining with Congo Red, staining with FSB, and ELISA using antibodies that recognize amyloid fibrils (Patent Document 2).

In addition, a method for detecting amyloid fibril deposition in vivo has been proposed in which a compound represented by formula (99) radiolabeled at a carbon atom is detected by gamma-ray imaging, magnetic resonance imaging, or magnetic resonance spectroscopy (Patent Document 3).

[In the formula,
R ¹ is hydrogen, —OH, —NO 2 , —CN, —COOR, —OCH 2 OR, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy, or halo, wherein one or more of the atoms of R ¹ can be a radiolabeled atom;
R is a C ₁ -C ₆ alkyl, where one or more of the carbon atoms may be a radiolabeled atom;
^R2 is hydrogen, non-radioactive halo or radioactive halo;
^R3 is hydrogen, _C1 - _C6 alkyl, _C2 - _C6 alkenyl or _C2 - _C6 alkynyl;
R ⁴ is hydrogen, C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, or C ₂ -C ₆ alkynyl, where if R ² is hydrogen or non-radioactive halo, the alkyl, alkenyl, or alkynyl contains a radioactive carbon or is substituted with a radioactive halo;
With the proviso that when R ¹ is hydrogen or —OH, R ² is hydrogen, and R ⁴ is — ¹¹ CH ₃ , then R ³ is C ₂ -C ₆ alkyl, C ₂ -C ₆ alkenyl, or C ₂ -C ₆ alkynyl;
Furthermore, when R ¹ is hydrogen, R ² is hydrogen, and R ⁴ is —CH ₂ CH ₂ CH ₂ ¹⁸ F, then R ₃ is C ₂ -C ₆ alkyl, C ₂ -C ₆ alkenyl, or C ₂ -C ₆ alkynyl; or a pharmaceutically acceptable salt, hydrate, solvate, or prodrug of the compound.

Japanese Patent Application Laid-Open No. 2023-11002 Japanese Patent Application Laid-Open No. 2020-188700 Special Publication No. 2006-522104

Observing amyloid fibers formed in the body in vivo is a necessary technique for treating diseases related to amyloid fibers. In this regard, the method of Patent Document 2 shows a certain degree of effectiveness. However, gamma ray imaging and the like require huge equipment. If there was a simpler device capable of observing amyloid fibers in vivo, it would be of great help in diagnosis. Furthermore, it would be even more effective if it could play a role in treatment at the same time as observation.

The present invention was conceived in light of the above-mentioned problems, and provides a novel curcumin derivative. It also provides a probe that selectively binds to amyloid fibrils containing the curcumin derivative and emits light when exposed to infrared light, making it possible to observe amyloid fibrils.

Specifically, the compounds according to the present invention are characterized by having structures represented by formulas (2) to (8) and formulas (10) to (14).

Furthermore, the amyloid fiber detection probe according to the present invention that binds to amyloid-β amyloid fibers contains at least one curcumin derivative represented by formula (4), formula (5), formula (6), formula (10), or formula (14).

Furthermore, the amyloid fiber detection probe according to the present invention that binds to α-synuclein amyloid fibers contains at least one curcumin derivative represented by formula (3), formula (4), formula (7), or formula (8).

The amyloid fiber detection probe of the present invention selectively binds to amyloid fibers and emits light when exposed to infrared light, so the light emission can be detected even through the skin. Therefore, the distribution and amount of amyloid fibers can be easily estimated using an infrared light emitting device and an infrared camera. In addition, it is possible that the amyloid fibers themselves can be destroyed by continuing to emit near-infrared light.

Furthermore, since the amyloid fibril detection probe according to the present invention can relatively easily incorporate ¹³ C or F into its structure, it is also possible to obtain high-resolution cross-sectional images of amyloid fibrils using a nuclear magnetic resonance (MRI) apparatus.

In addition, because the amyloid fiber detection probe of the present invention contains boron in its structure, it may be possible to perform neutron capture therapy on the bound amyloid fibers, potentially breaking down the amyloid fibers.

FIG. 1 shows a concept of biosynthesis of curcumin analogues according to the present invention. FIG. 1 shows precursors in the biosynthesis of curcumin analogues. FIG. 1 shows the structure of a BF2-modified acurcumin analogue according to the present invention. FIG. 1 shows the structure of a BF2-modified acurcumin analogue according to the present invention. FIG. 1 shows the structure of a BF2-modified acurcumin analogue according to the present invention. FIG. 1 shows the fluorescence emission characteristics of an amyloid fibril detection probe. FIG. 1 shows amyloid β binding to amyloid fibrils. 1 is a photograph of SDS-PAGE showing the selection of BF2-modified acurcumin analogs that bind to amyloid β amyloid fibrils. 1 is a Native-PAGE photograph showing the selection of BF2-modified acurcumin analogs that bind to synuclein amyloid fibrils.

The amyloid fiber detection probe according to the present invention will be described below with reference to drawings and examples. Note that the following description illustrates one embodiment of the present invention and one example, 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.

The amyloid fibril detection probe of the present invention is a derivative obtained from curcumin and generally has the structure of formula (100).

The coordination numbers indicate the numbers of the basic skeleton of curcumin. The functional groups that bond to the left and right benzene rings with the 1-position at the center are designated Rp1 and Rp2. There are no particular limitations on each functional group, and the functional group Rp1 and the functional group Rp2 may each be multiple functional groups. In addition, the functional group Rp1 and the functional group Rp2 may be the same. Hereinafter, for the sake of explanation, the right side of the 1-position at the center will be called the right site, and the left side will be called the left site.

The amyloid fiber detection probe of the present invention has this structure, emits fluorescence in the infrared range, and has the ability to bind to amyloid fibers. These amyloid fiber detection probes were produced by biosynthesis. Biosynthesis is carried out by introducing an enzyme gene involved in the curcumin synthesis pathway into Escherichia coli, and culturing it in a medium containing a precursor substance that serves as the material for the derivative. In this way, derivatives with different functional groups can be easily obtained. Also, specific carbons are replaced with stable isotopes.

Figure 1 shows an overview of the biosynthesis. A plasmid carrying the genes for the enzymes that synthesize curcumin (4CL, DCS, CURS1) is introduced into E. coli. The introduced E. coli is designated "mut-ecoil." This E. coli is cultured in a medium containing two compounds that are precursors of curcumin (referred to as precursor P1 and precursor P2, respectively). These two precursors are phenylpropionic acid to which functional groups have been bound, and are designated Rp1 and Rp2 in Figure 1. There are no particular limitations on these functional groups.

E. coli mut-ecoli synthesizes curcumin analogues from these precursors. In this process, there is no distinction between the precursors, so three types of analogues can be obtained: precursor P1 used at the left and right sites (homo-P1), precursors P1 and P2 used at the left and right sites (hetero-P1_P2), and precursor P2 used at the left and right sites (homo-P2).

For example, if precursor P1 and precursor P2 are both hydroxymethoxyphenylpropionic acid of formula (100), all three synthesized compounds will be curcumin.

The curcumin derivative obtained in this way undergoes keto-enol tautomerism, converting between the keto and enol forms as shown in formula (101).

Therefore, the oxygen atoms at the 2- and 2'-positions are bonded together with difluoroboron. This is called "BF2 conversion." As a result, BF2-converted curcumin is produced, as shown in formula (102).

BF2-modified curcumin does not exhibit the tautomerism shown in formula (102), and exists in a stable structure, giving it the property of fluorescing. By changing the functional groups bonded to the benzene rings at the left and right sites, the intensity of the fluorescence emission in the infrared band can be increased to a level sufficient for observation.

Furthermore, by adjusting the functional groups attached to the benzene rings on the left and right sites, it is possible to impart specific binding strength to amyloid fibrils.

Furthermore, by mixing ^13C glucose into the culture medium, the carbon at position 1 (see formula 100) can be modified with a stable isotope. ^13C allows for MRI imaging, enabling detailed imaging of amyloid fibrils in the brain. Furthermore, since it is converted to BF2, it contains F in the skeleton. Therefore, nuclear magnetic resonance imaging using F is also possible.

As such, the amyloid fiber detection probe of the present invention is made of a curcumin derivative and has the ability to emit fluorescence in the infrared range, making it possible to observe its presence several centimeters beneath the skin using an infrared camera or similar device.

The amyloid fiber detection probe according to the present invention contains boron in the skeleton due to its conversion to BF2. Boron clusters are used in neutron capture therapy, but the challenge is how to deliver the boron clusters to the target. However, the amyloid fiber detection probe according to the present invention not only binds to amyloid fibers, but also has boron in the skeleton. Therefore, it is possible to identify the location of amyloid fibers by fluorescence emission in the infrared range and simultaneously target them for neutron irradiation, and it can be used as a composition for neutron capture therapy.

Curcumin analogs were produced and purified using the following procedure. The genes encoding three enzymes in the curcumin synthesis pathway, 4CL, DCS, and CURS1, were introduced into Escherichia coli BL21(DE3) via plasmids.

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

Lipid-soluble metabolites were extracted from the fungal cells using the Bligh & Dyer method, then spray-dried. The metabolites were dissolved in methanol, and the presence of curcumin analogues was confirmed by TLC, after which they were purified using a silica column (10 mL). The eluted fractions were collected in 1 mL portions and promptly dried. After suspension in methanol, fractions containing curcumin analogues were identified and collected by TLC. At this point, the absorption and fluorescence spectra were measured.

Lipid-soluble 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. They were then purified using a silica column, just like the curcumin analogues derived from the fungus cells.

The precursors used are shown in Figure 2. Eight precursors were used: precursor-8, precursor-13, precursor-14, precursor-15, precursor-18, precursor-22, precursor ferulate, and precursor coumalate. The precursors ferulate and coumalate are hydroxymethoxyphenylpropionic acid and methoxyphenylpropionic acid, respectively. These were used to produce curcumin analogs. Table 1 shows the precursors used (referred to as the first and second precursors, respectively) and the sample names and sample numbers of the curcumin analogs.

Note that curu8 and curu9 are the same substance but were produced on different days. Furthermore, curu14 and curu15 were obtained from the inside (ppt) and outside (supernatant: sup) of E. coli, respectively, and are the same substance.

BF2 modification of curcumin analogues
The curcumin analogs listed in Table 1 were transferred to glass vials, and after inserting a stir bar, the vials were completely dried and then sealed. A syringe filled with N2 was inserted into the vials, and the oxygen and water vapor in the vials were replaced with N2. 250 μL of dichloromethane (DCM), which had been dehydrated using molecular sieves the previous day, was added to the vials while stirring with a stirrer to completely dissolve the curcumin analogs. 250 μL of a 1:1 mixture of boron trifluoride diethyl ether complex with DCM was slowly added while stirring with a stirrer, and the mixture was left at room temperature for at least 2 hours.

After the reaction, the mixture was repeatedly dried and washed with diethyl ether to remove unreacted boron trifluoride diethyl ether complex. After complete drying, the mixture was suspended in methanol, filtered through a PFTE filter, and stored at 4°C. The absorption and fluorescence spectra were measured at this stage. Table 2 shows the BF2-modified curcumin analogues. Figures 3 to 5 show the structures of each compound. The structures of compounds 1 to 15 are represented by formulas (1) to (15), respectively. Compound 1 is curcumin itself, and is the same as formula (103).

<Light Emitting Characteristics>
The luminescence properties of the BF2-modified curcumin analogue change compared to before BF2 modification. The luminescence is particularly enhanced in the infrared region. Figure 6 shows the luminescence properties of compound 1 (curcumin) and curu1 (curcumin itself before BF2 modification). Figure 6(a) shows curu1, and Figure 6(b) shows compound 1.

Referring to each graph, the horizontal axis represents the excitation light wavelength (nm) and the vertical axis represents the emission intensity (unitless). The wavelength of the emitted fluorescence for each excitation light wavelength is shown as a vertical number above the bar graph.

When excited at a wavelength of 635 nm, curu1 that is not BF2 fluoresces very little, but compound 1, a BF2 fluoresces in the infrared region at 700 nm. Fluorescence in the infrared region was also observed for the other compounds 2 to 15.

<Binding to amyloid fibrils>
The mechanism of amyloid beta production is thought to be as follows: Amyloid beta is produced by cleavage of amyloid precursor protein (APP) by protease. When APP is cleaved by alpha-secretase, amyloid beta is not produced. On the other hand, when APP is cleaved by beta-secretase (→ on the left side of the figure), it is then cleaved by gamma-secretase, producing amyloid beta. A component of gamma-secretase is presenilin (PS2), which regulates gamma-secretase activity.

In this study, the APP mutant and PS2 mutant found in patients with familial Alzheimer's disease were co-expressed in cells, and the production of amyloid beta was confirmed. It was also confirmed that amyloid beta was not produced without APP expression.

Figure 7 shows an example. Figure 7(a) shows electrophoresis of extracts from cells co-expressing mutant APP and mutant PS2, labeled with an anti-amyloid beta antibody (labeled "anti-Aβ" in the figure). Amyloid beta oligomers were confirmed at approximately 40 kDa.

Figure 7(b) shows the results of SDS-PAGE after contacting cell extract with compound 10 (hetero-13-14-BF2). The vertical axis indicates mass (kDa) and the horizontal axis indicates sample type. When compound 10 was contacted with 10 μL of APP/PS2 extract at concentrations of 0, 2, 4, 6, and 8 μM, concentration-dependent luminescence was observed at the same mass location as in electrophoresis. From the above, it was confirmed that compound 10 binds to amyloid β.

Figure 8 shows the results of similar experiments using other compounds. SDS-PAGE results indicated that Compounds 4, 5, 6, 10, 14, and 15 (note that Compounds 14 and 15 are the same substance) have the ability to bind to amyloid beta.

Figure 9 shows the results of Native-PAGE, which looked at the binding of each compound to α-synuclein amyloid fibrils, which are thought to be the cause of Parkinson's disease. 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.

The amyloid fiber detection probe of the present invention specifically binds to amyloid fibers and emits infrared light inside the body, allowing non-invasive observation from outside the body, making it highly effective for detecting amyloid fibers. Furthermore, the amyloid fiber detection probe of the present invention can be modified with ¹³ C or F in its structure, enabling detection by nuclear magnetic resonance imaging, etc. Furthermore, since the probe contains boron in its structure, it can also be used as a composition for neutron capture therapy, potentially enabling destruction of amyloid fibers with neutron beams.

Claims (5)

Compounds represented by formulas (2) to (8) and formulas (10) to (14).
An amyloid fiber detection probe that binds to amyloid-β amyloid fibers, comprising at least one compound represented by formula (4), formula (5), formula (6), formula (10), or formula (14).
The amyloid fibril detection probe according to claim 2 , wherein at least one carbon in the amyloid fibril detection probe is isotope-modified. An amyloid fibril detection probe that binds to α-synuclein amyloid fibrils, comprising at least one compound represented by formula (3), formula (4), formula (7), or formula (8).
A composition for neutron radiotherapy, comprising the amyloid fiber detection probe according to any one of claims 2 to 4.