Boron nanocarrier for encapsulating water-soluble protein and boron nanocarrier complex
Boron nanocarriers that modify polysaccharides with boronic acid compounds address the challenges of protein retention and release, enhancing both boron neutron capture therapy and immunostimulation for effective cancer treatment.
Patent Information
- Application Number
- JP2024150066
- Authority / Receiving Office
- JP · JP
- Patent Type
- Applications
- Current Assignee / Owner
- Priority Date
- 2023-09-19
- Filing Date
- 2024-08-30
- Publication Date
- 2025-04-01
AI Technical Summary
Existing boron nanocarriers face challenges in stably retaining proteins and controlling protein release, as well as conjugating proteins with polymers to resist degradation enzymes effectively.
Development of boron nanocarriers that modify polysaccharides with boronic acid compounds, allowing for stable conjugation of water-soluble proteins and external stimulus-induced release, enhancing delivery to target tissues and protecting proteins from degradation.
The boron nanocarriers improve the response rate of primary foci by boron neutron capture therapy and enable treatment of metastatic foci through immunostimulation, with efficient protein delivery and minimal side effects.
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Abstract
Description
[Technical field]
[0001] The present disclosure relates to boron nanocarriers and boron nanocarrier complexes for encapsulating water-soluble proteins. [Background technology]
[0002] With the advancement of molecular biology, it has been a long time since we could design and synthesize any protein in a test tube, and the development of protein drugs using this technology is accelerating. Among them, antibody drugs have been shown to be effective in treating diseases including cancer, and the competition to develop them is fierce. In order to improve the efficacy of these protein drugs, it is important to develop a delivery system that improves the delivery to the target site.
[0003] Conventionally, antibody drugs have a longer blood retention time than general protein drugs, and therefore have superior delivery to tumor tissues, but it has been difficult to express their functions only at the target site. Some antibodies have been designed to induce conformational changes in the antibody by binding to ATP in the tumor environment, and to show affinity to the target, but the technology is still in development.
[0004] In recent years, boron compounds have been attracting attention as carriers for transporting proteins into cells. For example, Non-Patent Document 1 discloses a boron delivery system using a complex composed of a fructose-modified poly(ethylene glycol)-poly(L-lysine) block copolymer and p-boronophenylalanine. This complex is used in boron neutron capture therapy (BNCT). In addition, Non-Patent Document 2 describes a boron compound that mainly uses a dendrimer as a protein delivery system.
[0005] Boron neutron capture therapy is a treatment in which cancer cells are locally destroyed by irradiating them with neutrons after incorporating boron compounds into them. In this treatment, it is important to selectively accumulate the boron compounds within the tumor. [Prior art documents] [Non-patent literature]
[0006] [Non-Patent Document 1] T.Nomoto et al., J.Control.Release,332(2021)184-193 [Non-Patent Document 2] Q.Ren et al., Adv.Healthcare Mater.2023,12,2202049 Summary of the Invention [Problem to be solved by the invention]
[0007] The boron compound described in Non-Patent Document 1 forms micelle-type nanoparticles in which the polymer is highly accumulated. Therefore, it is difficult to stably hold a protein inside and control its release. In addition, although the boron compound described in Non-Patent Document 2 has high structural stability, it is difficult to complex a protein in a stable state and in a state in which the protein is covered with a polymer to impart resistance to decomposition enzymes.
[0008] The technology disclosed herein has been made in view of the above, and its purpose is to provide a boron nanocarrier that can treat not only primary lesions by boron neutron capture therapy, but also metastatic lesions by immunostimulation. It also aims to improve the delivery of water-soluble proteins to target tissues, reduce side effects as a drug, and release water-soluble proteins in response to external stimuli. [Means for solving the problem]
[0009] The inventors of the present application have developed a boron nanocarrier that can stably complex water-soluble proteins, such as antigens and antibodies, inside it and release them in response to an external stimulus.
[0010] The present disclosure relates to a boron nanocarrier for encapsulating a water-soluble protein, which can encapsulate a water-soluble protein used as an immune checkpoint inhibitor and can be used in boron neutron capture therapy (BNCT). This boron nanocarrier has a structure in which a polysaccharide having a cis-diol group is modified at the diol group with a boronic acid compound that can be used as an immune checkpoint inhibitor, and is represented by the following formula (I).
[0011] [ka]
[0012] In formula (I), R 1 indicates a structure derived from a boronic acid compound, and R 2 and R 3 indicates a polysaccharide-derived structure.
[0013] As a result of intensive research by the present inventors, the findings obtained by the present disclosure show that the boron nanocarrier for encapsulating water-soluble proteins can improve the response rate of primary lesions by boron neutron capture therapy (BNCT), which is a radiotherapy, and can also treat metastatic lesions by immunoactivation, by combining the boron neutron capture therapy (BNCT) and immune checkpoint inhibitors. In addition, the boron nanocarrier for encapsulating water-soluble proteins of the present disclosure can easily and stably complex proteins by weak supramolecular chemical interactions, efficiently deliver proteins to the target site, and release the complexed proteins by external stimuli. In addition, since the polymer density is relatively coarse, it is possible to exhibit complexation behavior sufficient to protect proteins from decomposition enzymes.
[0014] Furthermore, according to the present disclosure, it is possible to provide a boron nanocarrier complex in which a water-soluble protein that acts as an immune checkpoint inhibitor is encapsulated in the boron nanocarrier.
[0015] The polysaccharide is preferably at least one selected from the group consisting of dextran, dextrin, glycogen, cellulose, oxidized cellulose, tamarind gum, mannan, glucan, and β-glucan, and is more preferably dextran, dextrin, or a derivative thereof, and has a linear or branched structure.
[0016] The boronic acid compound is preferably at least one selected from the group consisting of boronophenylalanine, bortezomib, ixazomib, tavaborole, crisaborole, vaborbactam, and borofarane, and more preferably boronophenylalanine or bortezomib. Effect of the Invention
[0017] As described above, according to the present disclosure, it is possible to provide a boron nanocarrier that can not only treat primary lesions through boron neutron capture therapy, but also treat metastatic lesions through immune activation. [Brief description of the drawings]
[0018] [Figure 1] This is a 1H-NMR spectrum of the boron nanocarrier of Example 1. [Diagram 2] TEM image of an aggregate of boron nanocarriers from Example 1. [Diagram 3] 1 is a graph showing the interaction between a boron nanocarrier of Example 1 and an antibody. [Figure 4] 1 is a graph showing the BNCT activity of a boron nanocarrier according to Example 1. [Diagram 5] 1 is a graph showing the BNCT activity of a clinical drug as a comparative control. [Figure 6] 1 shows photographs showing the tumor accumulation of a fluorescently labeled antibody. [Figure 7] 1 is a graph showing changes in tumor volume in the primary focus after BNCT treatment. [Figure 8] 1 is a graph showing changes in tumor volume in metastatic lesions after BNCT treatment. [Figure 9] 1 is a graph showing changes in mouse body weight after BNCT treatment. [Figure 10] 1 is a graph showing the amount of HMGB-1 released in a cell experiment. [Figure 11] 1 is a graph showing the amount of HMGB-1 released in an animal experiment. [Figure 12] 1 is a graph showing the amount of immune activator released. [Figure 13] This is a 1H-NMR spectrum of the boron nanocarrier of Example 2. [Figure 14] This is a 1H-NMR spectrum of the boron nanocarrier of Example 3. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0019] The boron nanocarriers of the present disclosure are described in detail below.
[0020] The boron nanocarrier disclosed herein is designed to stably encapsulate water-soluble proteins such as antigens and antibodies and deliver the water-soluble proteins to target tissues. It can be used as an immune checkpoint inhibitor and can be used in boron neutron capture therapy (BNCT).
[0021] The boron nanocarrier of the present disclosure is an amphiphilic polysaccharide in which a boronic acid compound is introduced into a polysaccharide having a cis-diol group. The boron nanocarrier of the present disclosure is represented by the following formula (I) in which a polysaccharide having a cis-diol group is modified at its diol group with a boronic acid compound used as an immune checkpoint inhibitor.
[0022] [ka]
[0023] In formula (I), R 1 is a structure derived from a boronic acid compound, and R 2 and R 3 indicates a polysaccharide-derived structure.
[0024] The polysaccharides applied to the boron nanocarrier of the present disclosure are not limited as long as they have a cis-diol group, and examples thereof include dextran, dextrin, glycogen, cellulose, oxidized cellulose, tamarind gum, mannan, glucan, and β-glucan. Any of these polysaccharides can be bound to boronic acid at the cis-diol group, and when the boron nanocarrier is formed, it can encapsulate water-soluble proteins in a stable state. More preferred polysaccharides are dextran, dextrin, or derivatives thereof. Dextran, dextrin, or derivatives thereof may have either a linear structure or a branched structure.
[0025] The boronic acid compound applied to the boron nanocarrier of the present disclosure is not limited as long as it is a compound that can be introduced into the diol group of a polysaccharide and can be used for boron neutron capture therapy. For example, boronophenylalanine (BPA), which is known to be selectively taken up by malignant melanoma cells and various types of cancer cells, and bortezomib, ixazomib, tavaborole, crisaborole, vaborbactam, borofarane, and the like, which are known as anticancer drugs, can be used. Due to the electronic interaction between the boron atom and the water-soluble protein, the boron nanocarrier can carry the water-soluble protein in a stable state. By accumulating such a structure derived from a boronic acid compound in the primary lesion, not only can boron neutron capture therapy be performed effectively, but also the action of the water-soluble protein loaded as a complex can be used to treat metastatic lesions by immune activation.
[0026] The boron nanocarrier of the present disclosure can easily form a boron nanocarrier complex encapsulating a water-soluble protein by simply mixing it with a water-soluble protein. The water-soluble protein that can be encapsulated in the boron nanocarrier acts as an immune checkpoint inhibitor, and various substances, including antigens and antibodies, can be used. EXAMPLES
[0027] The present invention will be described in detail below with reference to examples. These examples are merely illustrative for suitably explaining the present invention, and are not intended to limit the present invention.
[0028] Example 1: L-BPA modified dextran (Dex-BPA) [Synthesis of boron nanocarriers] The L-BPA-modified dextran (Dex-BPA) as the boron nanocarrier in Example 1 was synthesized by modifying dextran with L-BPA, as shown in the following formula (II).
[0029] [ka]
[0030] Specifically, dextran (100 mg, 0.62 mmol) and L-BPA (64.8 mg, 0.31 mmol) were dissolved in dehydrated DMSO (10 mL), DBU (0.65 mL, 2.2 mmol) was added as a base, and the mixture was stirred at 45°C for 24 hours. The resulting polymer (Dex-BPA) was recovered by reprecipitation, redissolved in DMSO, and purified by dialysis. As shown in Figure 1, the polymer was analyzed by nuclear magnetic resonance spectroscopy ( 1 The synthesis of Dex-BPA was confirmed by H-NMR (Figure 1).
[0031] Dex-BPA was dissolved in water to a concentration of 1 mg / mL, stirred for 24 hours, and then irradiated with ultrasound (10 mW, 10 minutes). The particle size and boron concentration were measured by dynamic light scattering (DLS) and ICP emission spectrometry, and were found to be approximately 120 nm and 12 ppm, respectively. When observed with a transmission electron microscope (TEM), spherical aggregates were observed, as shown in Figure 2.
[0032] [Combination of boron nanocarriers with antibodies and release of antibodies] Dex-BPA and the antibody were mixed in phosphate buffer solution (PBS) so that the substance ratios were equal. The antibody used here was fluorescently labeled with FITC. The complexation was then measured by fluorescence correlation spectroscopy (FCS). Furthermore, the release behavior was measured by adding 1 mM ATP to the complex. As a result, it was confirmed that the antibody complexation proceeded simply by mixing, and that the antibody was completely released by adding 1 mM ATP, as shown in Figure 3. The particle size and morphology of the obtained complex were measured and observed by DLS, and it was found that there was no significant change before and after complexation, at about 120 nm.
[0033] It was also confirmed that Dex-BPA could form a complex when mixed with insulin and bovine serum albumin as model proteins in a molecular weight ratio of 1:1.
[0034] The above results confirm that the boron nanocarrier disclosed herein is capable of stably complexing water-soluble proteins, transporting them to target tissues (tumors), and releasing the water-soluble proteins in response to ATP, which is abundant in tumors.
[0035] [BNCT treatment effect of boron nanocarriers on cancer cells] The therapeutic performance of Dex-BPA against cancer cells (mouse colon cancer cells, Colon26) was evaluated. As a control, a clinical drug, L-BPA / fructose complex, was used. Colon26 cells were exposed to a given boron concentration (0, 0.001, 0.05, 0.1, 1, 5, 10 ppm) for 24 hours, and neutron beams were irradiated for 70 minutes at 1 MW using a heavy water dolly at the Kyoto University Institute for Integrated Radiation Science. Then, after 24 hours, the cell survival rate was calculated using Cell Counting Kit-8. FIG. 4 is a graph showing the BNCT activity of the boron nanocarrier (Dex-BPA) according to Example 1, and FIG. 5 is a graph showing the BNCT activity of the clinical drug (L-BPA / fructose complex). The 50% inhibitory concentration indicating BNCT activity was determined, and Dex-BPA (0.065 ppm) demonstrated therapeutic performance superior to that of the L-BPA / fructose complex (0.080 ppm).
[0036] [Intratumoral antibody delivery using boron nanocarriers] The tumor accumulation of the antibody when a complex of Dex-BPA and the antibody was used was evaluated using an in vivo imaging system. The antibody was fluorescently labeled by modifying it with a near-infrared responsive fluorophore. Colon26 cells (5.0 × 105 cells) were transplanted subcutaneously into the right thigh of Balb / c mice (male, 4 weeks old, 18 g). Ten days after transplantation, the tumor size was 80-100 mm. 3 After the mice had grown to about 100 mg / mL, an antibody or an antibody conjugated with Dex-BPA (Dex-BPA / antibody conjugate) was administered via the tail vein (Dex-BPA, 120 ppm; antibody, 0.1 mg / mL). Accumulation was visualized using an in vivo imaging system immediately after administration (0 hours), 3 hours, 6 hours, and 24 hours after administration. Accumulation in each organ (heart, lung, liver, kidney, spleen, tumor) after 24 hours was also visualized. As a result, as shown in FIG. 6, the boron nanocarrier conjugate of the present invention (IgG / Dex-BPA) showed a significantly increased accumulation compared to administration of the antibody alone (IgG).
[0037] [Treatment of metastatic cancer by combined therapy of immune checkpoint inhibitor delivery with boron nanocarriers and BNCT] The therapeutic performance of metastatic cancer by the combined therapy of immune checkpoint inhibitor (ICI) delivery by the boron nanocarrier (Dex-BPA) of Example 1 and BNCT was evaluated based on the tumor growth curves in the primary and metastatic lesions.
[0038] As a control, a mixture of L-BPA / fructose complex, a clinical drug, was used. Colon26 cells were subcutaneously transplanted into the right thigh (primary lesion 5.0 × 105 cells) of Balb / c mice (male, 4 weeks old, 18 g). Three days later, Colon26 was subcutaneously transplanted into the left thigh (metastatic lesion 2.5 × 105 cells). 3 After the mice had grown to about 100 mg / mL, drugs were administered via the tail vein of the mice (PBS, L-BPA / fructose complex, Dex-BPA, Dex-BPA+ICI, Dex-BPA / ICI complex: boron concentration 120 ppm, ICI concentration 0.1 mg / mL).
[0039] For the L-BPA / fructose complex system and the Dex-BPA system, neutron irradiation (1MW, 70 minutes) was performed 1 hour and 24 hours after administration. The tumor size of the primary and metastatic lesions was then measured. The tumor size was calculated as (long diameter) x (short diameter). 2 / 2 (Figures 7 and 8).
[0040] FIG. 7 is a graph showing the change in tumor volume in the primary lesion after BNCT treatment, and FIG. 8 is a graph showing the change in tumor volume in the metastatic lesion after BNCT treatment. The tumor volume in the mice not irradiated with neutron beams continued to grow over time. On the other hand, the tumor volume in the primary lesion of the mice irradiated with neutron beams after administration of boron drugs decreased. Here, the therapeutic performance of BNCT with Dex-BPA alone in the primary lesion exceeded that of the clinical drug L-BPA / fructose complex. In addition, co-administration of Dex-BPA and ICI (Dex-BPA+ICI in FIG. 7 and FIG. 8) improved the therapeutic performance of Dex-BPA, and the Dex-BPA / ICI complex (Dex-BPA / ICI in FIG. 7 and FIG. 8) further improved its therapeutic performance. Here, the tumor in the metastatic lesion in the group not containing ICI grew over time. Here, the therapeutic efficacy of metastatic lesions in mice co-administered with ICI and Dex-BPA and ICI was poor, whereas the group administered with the complex achieved very high therapeutic efficacy. The tumors in the treated mice, both primary and metastatic lesions, were observed to completely shrink. Moreover, as shown in Figure 9, there was no significant decrease in the body weight of the mice during this treatment, and the side effects were very low.
[0041] [Release of damage-associated pattern molecules by boron nanocarriers (cell experiments)] In activating intratumoral immunity, the amount of damage-associated molecular patterns (DAMPs) released by damaged cancer cells is important. Among them, the DAMP called HMGB-1 (High Mobility Group Box 1) is mainly released. Here, Dex-BPA or L-BPA was added to cultured cells (mouse colon cancer cells, Colon26), and the amount of HMGB-1 released in the culture supernatant immediately after and 24 hours after neutron irradiation (1MW, 70 minutes) was quantified by ELISA (enzyme-linked immunosorbent assay). As a result, as shown in Figure 10, Dex-BPA achieved a higher amount of HMGB-1 release than L-BPA. In Figure 10, the label "cold" indicates that neutron irradiation was not performed, and "hot" indicates that neutron irradiation was performed (similar to Figures 11 and 12).
[0042] [Release of damage-associated pattern molecules by boron nanocarriers (animal experiments)] Regarding the amount of DAMPs actually released into tumor tissues by BNCT treatment, blood samples were taken from mice treated as described below, and the amount of HMGB-1 was quantified by ELISA. Blood samples were taken immediately after neutron irradiation (1 MW, 70 min), 1 day after irradiation, and 7 days after irradiation. As a result, as shown in FIG. 11, Dex-BPA was confirmed to release more DAMPs than L-BPA. In addition, Dex-BPA co-administered with ICI (Dex-BPA+ICI hot in FIG. 11) showed a high release level, and furthermore, in the complex of Dex-BPA and ICI (Dex-BPA / ICI hot in FIG. 11), the release level of DAMPs was maintained at a high level even 7 days after irradiation.
[0043] [Release of immune activating factors] The activation of CTLs (cytotoxic T cells), which directly attack cancer cells through tumor immunity, was evaluated by quantifying IFN-γ using ELISA. As a result, as shown in Figure 12, it was revealed that Dex-BPA achieved a higher degree of IFN-γ activation than L-BPA, and in particular, by combining it with ICI, a higher level of CTL activation was induced (Dex-BPA / ICI hot in Figure 12).
[0044] <Example 2: L-BPA modified dextrin (CDex-BPA)> The boron nanocarrier of Example 2 was synthesized in the same manner as in Example 1, using cluster dextrin (registered trademark), which is a branched polysaccharide, as the polysaccharide and L-BPA as the boronic acid compound. As shown in FIG. 13, the modification with the boron compound was confirmed, and the DLS measurement results confirmed that nanoparticles with a diameter of 80 nm were formed. Since nanoparticles of the same size as those of the boron nanocarrier of Example 1 were formed, it is possible to complex with water-soluble proteins like the boron nanocarrier of Example 1, and it is expected to show the same action.
[0045] <Example 3: Bortezomib-modified dextran (Dex-Bor)> The boron nanocarrier of Example 3 was synthesized in the same manner as in Example 1, using dextran as the polysaccharide and bortezomib, an anticancer drug, as the boronic acid compound. As shown in FIG. 14, the boron compound modification was confirmed, and the DLS measurement results confirmed that nanoparticles with a diameter of 230 nm were formed. Since nanoparticles of the same size as those of the boron nanocarrier of Example 1 were formed, it is possible to complex with water-soluble proteins like the boron nanocarrier of Example 1, and it is expected to show the same action.
Claims
1. A boron nanocarrier for encapsulating a water-soluble protein, which can encapsulate a water-soluble protein used as an immune checkpoint inhibitor and can be used in boron neutron capture therapy (BNCT), A boron nanocarrier for encapsulating water-soluble proteins, represented by the following formula (I), in which a polysaccharide having a cis-diol group is modified at the diol group with a boronic acid compound used as an immune checkpoint inhibitor. 【Chemistry 1】 [In formula (I), R １ represents a structure derived from a boronic acid compound, and R ２ and R ３ indicates a polysaccharide-derived structure.
2. The boron nanocarrier for encapsulating water-soluble proteins according to claim 1, wherein the polysaccharide is at least one selected from the group consisting of dextran, dextrin, glycogen, cellulose, oxidized cellulose, tamarind gum, mannan, glucan, and β-glucan.
3. The boron nanocarrier for encapsulating a water-soluble protein according to claim 1 , wherein the polysaccharide is dextran, dextrin or a derivative thereof and has a linear or branched structure.
4. The boron nanocarrier for encapsulating a water-soluble protein according to claim 2, wherein the boronic acid compound is at least one selected from the group consisting of boronophenylalanine, bortezomib, ixazomib, tavaborole, crisaborole, vaborbactam, and borofarane.
5. The boron nanocarrier for encapsulating a water-soluble protein according to claim 3 , wherein the boronic acid compound is boronophenylalanine or bortezomib.
6. A boron nanocarrier complex comprising a boron nanocarrier according to any one of claims 1 to 5 encapsulating a water-soluble protein as an immune checkpoint inhibitor.