A composition of albumin and mercapto dodecaborane and its application in preparing tumor therapeutic drugs

Abstract

The present invention relates to the technical field of tumor therapeutic drugs, and in particular to a composition of albumin and mercaptododecaborane and its application in the preparation of tumor therapeutic drugs. Its technical solution includes albumin, which is covalently coupled with mercaptododecaborane through the cysteine thiol group at position 34 to construct a nucleation site, and further point-loaded with mercaptododecaborane on the protein through the nucleation effect; the thiol group of the mercaptododecaborane is bound to the thiol group at position 34 of albumin cysteine through a disulfide bond. The present invention forms a nucleus by covalently coupling the free thiol group of albumin with mercaptododecaborane, and utilizes the nucleation effect to point-coat mercaptododecaborane, thereby avoiding the toxicity risks brought by organic solvents or chemical modifications in traditional solubilization methods, and effectively preventing oxidative degradation and free boron ion release, achieving pH-responsive targeted release, and significantly increasing the boron accumulation in tumor tissues, with both high stability, precise drug release, and low systemic toxicity.

Description

Technical Field

[0001] The present invention relates to the technical field of tumor therapeutic drugs, and in particular to a composition of albumin and mercaptododecaborane and application thereof in preparing tumor therapeutic drugs. Background Art

[0002] Boron Neutron Capture Therapy (BNCT) uses thermal neutrons to bombard tumor cells with 10 element B, produces alpha particles with high energy transfer linear density and ions, thereby achieving precise killing of cancer cells. 10 After capturing a neutron, the B element undergoes a nuclear reaction, generating alpha particles and Ions have the characteristics of short range (only about 5-10 μm, covering about 1-2 cell diameters) but high energy, which can achieve precise attack on tumors at the cellular level, and theoretically minimize damage to surrounding normal tissues. Among the many boron carrier molecules, mercapto dodecaborane (such as thiol-modified dodecaborane derivatives) has a high abundance of 10 B atoms (each molecule contains 12 boron atoms) show strong potential as boron carriers and are expected to provide efficient boron delivery for BNCT.

[0003] However, the inherently poor hydrophobicity and stability of mercaptododecaborane limit its application in BNCT. The inherently strong hydrophobicity of the dodecaborane backbone results in low water solubility. While the thiol group introduces a degree of polarity, it has limited impact on overall water solubility. Low water solubility not only complicates drug preparation and administration but can also lead to uneven drug distribution in the body. Currently, commonly used solubilization methods include chemical modification (such as the introduction of hydrophilic groups) and the addition of solubilizers (such as organic solvents). However, the introduction of polar groups or other chemical modifications may alter the bioactivity and safety of mercaptododecaborane, leading to increased toxicity or reduced therapeutic efficacy. Some solubilizers, such as cyclodextrin, PEG, and Tween, are toxic and may induce adverse reactions in vivo, including allergic reactions and inflammation. Furthermore, the thiol groups in mercaptododecaborane are easily oxidized in body fluids, especially under neutral or alkaline conditions (such as blood pH 7.4), forming disulfide bonds (-SS-). As the oxidation process proceeds, the structural stability of the borane molecule is destroyed, and degradation occurs: the free boron ions (B 3+) will be released. This will destroy the specific targeting properties of the borane compound, preventing it from precisely acting on the target tissue or cells. Normal tissues are also exposed to an increased risk of boron ion release, potentially leading to toxic effects or adverse reactions. Forming a protective layer on the surface of mercaptododecaborane (such as polydopamine modification) can isolate oxygen and water to enhance its stability, but this will increase the molecular size and surface charge of mercaptododecaborane, altering its physicochemical properties and potentially affecting its pharmacokinetic behavior in vivo.

[0004] Serum albumin, with its excellent biocompatibility and water solubility, plays an important role in the solubilization of hydrophobic drugs. For example, the clinically used albumin paclitaxel uses its amphiphilic structure to encapsulate hydrophobic drugs, thus solving the allergic risk of traditional solvent-based drugs. However, traditional methods mainly rely on hydrophobic interactions to achieve albumin coating of drugs. This binding method cannot precisely control the interaction sites between drugs and proteins. The drug is randomly adsorbed into the hydrophobic cavity inside the protein, which may cause changes in the spatial conformation of albumin, resulting in recognition barriers to key target functional areas such as surface glycosylation sites and receptor binding domains, resulting in a lack of biological targeting.

[0005] In summary, the present application proposes a composition of albumin and mercaptododecaborane and its application in the preparation of tumor therapeutic drugs. Summary of the Invention

[0006] The purpose of the present invention is to address the problems of poor hydrophobicity and stability of the boron carrier molecule mercaptododecaborane in the background art, and to propose a composition of albumin and mercaptododecaborane and its use in the preparation of tumor therapeutic drugs.

[0007] In a first aspect, the present invention provides a composition of albumin and mercapto dodecaborane, comprising:

[0008] Albumin, which is covalently coupled to mercapto-dodecaborane via the thiol group of cysteine at position 34, constructs a nucleation site, and further loads mercapto-dodecaborane on the protein through the nucleation effect;

[0009] The thiol group of the mercaptododecaborane is bound to the thiol group at position 34 of albumin cysteine via a disulfide bond, wherein the disulfide bond is stably present in a physiological environment and reversibly broken in the acidic microenvironment of the tumor for targeted release of the mercaptododecaborane.

[0010] Optionally, the pH of the physiological environment is 7.3-7.5, and the pH of the tumor acidic microenvironment is 6.5-7.0.

[0011] Optionally, the albumin is selected from any one or more of the following: human serum albumin, ovalbumin, lactalbumin, myoalbumin, wheat albumin, and soy albumin.

[0012] Optionally, it is characterized in that the mass ratio of albumin to mercaptododecaborane is 1:1 to 1000:1.

[0013] Optionally, the composition further comprises a pharmaceutically acceptable excipient, wherein the excipient is selected from one or more of a stabilizer, a buffer, a preservative, and an osmotic pressure regulator.

[0014] Optionally, the composition is in the form of an intravenous injection or a lyophilized powder injection.

[0015] In a second aspect, the present application provides a drug comprising the albumin-mercaptododecaborane composition described in the first aspect, and a pharmaceutically acceptable carrier.

[0016] In a third aspect, the present application provides the use of the drug described in the second aspect in the preparation of a tumor treatment drug, characterized in that the tumor treatment is boron neutron capture therapy, and the tumor is any one of colon cancer, triple-negative breast cancer, malignant melanoma, non-small cell lung cancer, liver cancer, renal cell carcinoma, prostate cancer, ovarian cancer, and gastric cancer.

[0017] Optionally, after intravenous injection of the drug, the ratio of boron concentration in tumor tissue to boron concentration in normal tissue is T / N≥3:1.

[0018] Optionally, the drug is released in the acidic microenvironment of the tumor via a pH-responsive release mechanism. 10 B, used to kill tumor cells.

[0019] Compared with the prior art, this application has at least one of the following beneficial technical effects:

[0020] This application utilizes the only free thiol group (Cys34) in the albumin molecule to bind to thiol dodecaborane through covalent coupling. This process is highly site-specific and avoids the random binding defects of traditional methods. The rigid cage-like structure of the dodecaborane molecule can produce a "nucleation effect" on the protein surface, inducing the dodecaborane molecules to form stable aggregates on the albumin molecule. This structural modification brings multiple advantages: on the one hand, the complex formed by covalent coupling can enhance the stability of the protein preparation; on the other hand, because the covalent modification is located in the non-functional region of albumin, away from the receptor binding domain, it effectively retains the natural targeting of albumin.

[0021] Leveraging the acidic cleavage properties of thiol groups, a pH-sensitive drug release mechanism has been developed, enabling controlled drug release under specific conditions. This technology overcomes the limitations of traditional albumin carriers, which rely on physical interactions. By leveraging site-specific covalent modification and nucleation effects, it achieves synergistic optimization of carrier stability, targeting, and responsiveness. This technology has broad potential for application in drug delivery and provides new insights into addressing common technical challenges faced by protein-based drug carriers. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Figure 1 is the result of cell uptake detection;

[0023] Figure 2 This is a diagram of tissue distribution detection results. DETAILED DESCRIPTION

[0024] The technical solution of the present invention is further described below with reference to the accompanying drawings and specific embodiments.

[0025] Example 1: Preparation of pharmaceutical composition (AJ)

[0026] Prepare 2.5 mL of a 20 mg / mL human serum albumin solution (dissolved in PBS) and 0.5 mL of a 100 mg / mL mercaptododecaborane solution (dissolved in chloroform). Combine the two solutions in a vial and homogenize using an ultrasonic probe deep into the vial, near the bottom of the liquid. Ultrasonication was performed at 500 W, with a 2-second interval followed by a 3-second pause. Ultrasonication was repeated three times, each lasting 2 minutes. Chloroform was then removed using a rotary evaporator at 30°C under reduced pressure to obtain the albumin-mercaptododecaborane composition A. The mass ratio of albumin to mercaptododecaborane was 1:1.

[0027] Example 2: Preparation of pharmaceutical composition (AJ)

[0028] Prepare 2.5 mL of a 40 mg / mL human serum albumin solution (dissolved in PBS) and 0.5 mL of a 100 mg / mL mercaptododecaborane solution (dissolved in chloroform). Combine the two solutions in a vial and homogenize using an ultrasonic probe deep into the vial, near the bottom of the liquid. Ultrasonication was performed at 500 W, with a 2-second interval followed by a 3-second pause. The sonication was repeated three times, each lasting 2 minutes. Chloroform was then removed using a rotary evaporator at 30°C under reduced pressure to obtain the albumin-mercaptododecaborane composition B. The mass ratio of albumin to mercaptododecaborane was 2:1.

[0029] Example 3: Preparation of pharmaceutical composition (AJ)

[0030] Prepare 2.5 mL of a 100 mg / mL human serum albumin solution (dissolved in PBS) and 0.5 mL of a 100 mg / mL mercaptododecaborane solution (dissolved in chloroform). Combine the two solutions in a vial and homogenize using an ultrasonic probe deep into the vial, near the bottom of the liquid. Ultrasonication was performed at 500 W, with a 2-second interval followed by a 3-second pause. The sonication was repeated three times, each lasting 2 minutes. Chloroform was then removed using a rotary evaporator at 30°C under reduced pressure to obtain the albumin-mercaptododecaborane composition C. The mass ratio of albumin to mercaptododecaborane was 5:1.

[0031] Example 4: Preparation of pharmaceutical composition (AJ)

[0032] Prepare 2.5 mL of a 100 mg / mL human serum albumin solution (dissolved in PBS) and 0.5 mL of a 50 mg / mL mercaptododecaborane solution (dissolved in chloroform). Combine the two solutions in a vial and homogenize using an ultrasonic probe deep into the vial, near the bottom. Ultrasonication was performed at 500 W, with a 2-second interval followed by a 3-second pause. The sonication was repeated three times, each lasting 2 minutes. Chloroform was then removed using a rotary evaporator at 30°C under reduced pressure to obtain the albumin-mercaptododecaborane composition D. The mass ratio of albumin to mercaptododecaborane was 10:1.

[0033] Example 5: Preparation of pharmaceutical composition (AJ)

[0034] Prepare 2.5 mL of a 100 mg / mL human serum albumin solution (dissolved in PBS) and 0.5 mL of a 25 mg / mL mercaptododecaborane solution (dissolved in chloroform). Combine the two solutions in a vial and homogenize using an ultrasonic probe deep into the vial, near the bottom of the liquid. Ultrasonication was performed at 500 W, with a 2-second interval followed by a 3-second pause. The sonication was repeated three times, each lasting 2 minutes. Chloroform was then removed using a rotary evaporator at 30°C under reduced pressure to obtain the albumin-mercaptododecaborane composition E. The mass ratio of albumin to mercaptododecaborane was 20:1.

[0035] Example 6 Preparation of Pharmaceutical Composition (AJ)

[0036] Prepare 2.5 mL of a 200 mg / mL human serum albumin solution (dissolved in PBS) and 0.5 mL of a 20 mg / mL mercaptododecaborane solution (dissolved in chloroform). Combine the two solutions in a vial and homogenize using an ultrasonic probe deep into the vial, near the bottom of the liquid. Ultrasonication was performed at 500 W, with a 2-second interval followed by a 3-second pause. Ultrasonication was repeated three times, each lasting 2 minutes. Chloroform was then removed using a rotary evaporator at 30°C under reduced pressure to obtain the albumin-mercaptododecaborane composition F. The mass ratio of albumin to mercaptododecaborane was 50:1.

[0037] Example 7 Preparation of Pharmaceutical Composition (AJ)

[0038] Prepare 2.5 mL of a 200 mg / mL human serum albumin solution (dissolved in PBS) and 0.5 mL of a 10 mg / mL mercaptododecaborane solution (dissolved in chloroform). Combine the two solutions in a vial and homogenize using an ultrasonic probe deep into the vial, near the bottom. Ultrasonication was performed at 500 W, with a 2-second interval followed by a 3-second pause. Repeat three times for 2 minutes each. Chloroform was then removed using a rotary evaporator at 30°C under reduced pressure to obtain the albumin-mercaptododecaborane composition G. The mass ratio of albumin to mercaptododecaborane was 100:1.

[0039] Example 8 Preparation of Pharmaceutical Composition (AJ)

[0040] Prepare 2.5 mL of a 200 mg / mL human serum albumin solution (dissolved in PBS) and 0.5 mL of a 5 mg / mL mercaptododecaborane solution (dissolved in chloroform). Combine the two solutions in a vial and homogenize using an ultrasonic probe deep into the vial, near the bottom. Ultrasonication was performed at 500 W, with a 2-second interval followed by a 3-second pause. Repeat three times for 2 minutes each. Chloroform was then removed using a rotary evaporator at 30°C under reduced pressure to obtain the albumin-mercaptododecaborane composition H. The mass ratio of albumin to mercaptododecaborane was 200:1.

[0041] Example 9 Preparation of Pharmaceutical Composition (AJ)

[0042] Prepare 2.5 mL of a 500 mg / mL human serum albumin solution (dissolved in PBS) and 0.5 mL of a 5 mg / mL mercaptododecaborane solution (dissolved in chloroform). Combine the two solutions in a vial and homogenize using an ultrasonic probe deep into the vial, near the bottom of the liquid. Ultrasonication was performed at 500 W, with a 2-second interval followed by a 3-second pause. Repeat three times for 2 minutes each. Chloroform was then removed using a rotary evaporator at 30°C under reduced pressure to obtain the albumin-mercaptododecaborane composition I. The mass ratio of albumin to mercaptododecaborane was 500:1.

[0043] Example 10: Preparation of pharmaceutical composition (AJ)

[0044] Prepare 2.5 mL of a 500 mg / mL human serum albumin solution (dissolved in PBS) and 0.5 mL of a 2.5 mg / mL mercaptododecaborane solution (dissolved in chloroform). Combine the two solutions in a vial and homogenize using an ultrasonic probe deep into the vial, near the bottom of the liquid. Ultrasonication was performed at 500 W, with a 2-second interval followed by a 3-second pause. Ultrasonication was repeated three times, each lasting 2 minutes. Chloroform was then removed using a rotary evaporator at 30°C under reduced pressure to obtain the albumin-mercaptododecaborane composition J. The mass ratio of albumin to mercaptododecaborane was 1000:1.

[0045] Example 11: Cellular uptake experiment

[0046] (1) Experimental groups

[0047] Control group 1: blank culture medium group (negative control group);

[0048] Control group 2: free mercapto dodecaborane group (positive control group);

[0049] Experimental group 1: albumin-mercaptododecaborane composition A (mass ratio 1:1);

[0050] Experimental group 2: albumin-mercaptododecaborane composition B (mass ratio 2:1);

[0051] Experimental group 3: albumin-mercaptododecaborane composition C (mass ratio 5:1);

[0052] Experimental group 4: albumin-mercaptododecaborane composition D (mass ratio 10:1);

[0053] Experimental group 5: albumin-mercaptododecaborane composition E (mass ratio 20:1);

[0054] Experimental group 6: albumin-mercaptododecaborane composition F (mass ratio 50:1);

[0055] Experimental group 7: albumin-mercaptododecaborane composition G (mass ratio 100:1);

[0056] Experimental group 8: albumin-mercaptododecaborane composition H (mass ratio 200:1);

[0057] Experimental group 9: albumin-mercaptododecaborane composition I (mass ratio 500:1);

[0058] Experimental group 10: albumin-mercaptododecaborane composition J (mass ratio 1000:1);

[0059] (2) Experimental steps

[0060] B16 tumor cells were seeded into 6-well plates at a cell seeding density of , cultured for 24 hours until cells adhered;

[0061] The experimental group was added with a culture medium containing an albumin-mercaptododecaborane composition containing 200 μg / mL mercaptododecaborane, the positive control group was added with an equal concentration of free mercaptododecaborane, and the negative control group was added with an equal volume of blank culture medium. The cells were incubated for 1, 2, 4, and 8 hours.

[0062] After incubation, the cells were washed three times with PBS and collected by trypsin digestion; the cells were collected by centrifugation (1000 rpm, 5 minutes) and counted, and 1×10 6 The cells were digested with nitric acid, and the intracellular boron content was quantified by ICP-MS.

[0063] (3) Experimental results

[0064] Cellular uptake assay results Figure 1 As shown, the cellular uptake in the experimental group was significantly higher than that in the control group. The experimental group exhibited high cellular uptake efficiency at various mass ratios, with peak cellular uptake occurring within the mass ratio range of 10:1 to 500:1. This demonstrates that the albumin-mercaptododecaborane composition significantly enhances boron uptake by tumor cells.

[0065] Example 12: Mouse tumor-targeted accumulation experiment

[0066] (1) Experimental groups

[0067] Control group 1: normal saline (negative control group);

[0068] Control group 2: free mercapto dodecaborane group (positive control group);

[0069] Experimental group 1: albumin-mercaptododecaborane composition A (mass ratio 1:1);

[0070] Experimental group 2: albumin-mercaptododecaborane composition B (mass ratio 2:1);

[0071] Experimental group 3: albumin-mercaptododecaborane composition C (mass ratio 5:1);

[0072] Experimental group 4: albumin-mercaptododecaborane composition D (mass ratio 10:1);

[0073] Experimental group 5: albumin-mercaptododecaborane composition E (mass ratio 20:1);

[0074] Experimental group 6: albumin-mercaptododecaborane composition F (mass ratio 50:1);

[0075] Experimental group 7: albumin-mercaptododecaborane composition G (mass ratio 100:1);

[0076] Experimental group 8: albumin-mercaptododecaborane composition H (mass ratio 200:1);

[0077] Experimental group 9: albumin-mercaptododecaborane composition I (mass ratio 500:1);

[0078] Experimental group 10: albumin-mercaptododecaborane composition J (mass ratio 1000:1);

[0079] (2) Experimental steps

[0080] Twenty-four female BALB / c nude mice aged 6-8 weeks were randomly divided into two groups (12 mice in each group). U87MG cells were injected subcutaneously Establish a human glioma xenograft model. Start the experiment when the tumor volume reaches 100-150 mm³;

[0081] The experimental group received an albumin-mercaptododecaborane complex (same boron, 10 mg / kg) via tail vein injection, while the control group received an equal dose of free mercaptododecaborane. A blank group received an equal volume of saline. Animals were sacrificed 0.5, 1, 2, 4, 8, and 24 hours after administration (3 animals per time point).

[0082] Tumor tissue, blood, salivary gland, kidney, liver, heart, spleen, and lung tissue were collected, washed with ultrapure water for residual blood, weighed, and homogenized in a Tissue-Tearor. The boron content of each sample was determined using ICP-MS.

[0083] (3) Experimental results

[0084] Tissue distribution test results Figure 2As shown: 6 hours after administration, the boron content in the tumor tissue of the experimental group was significantly higher than that in the control group, both exceeding 10 μg / g, meeting the effective therapeutic threshold of boron neutron capture therapy (BNCT).

[0085] This invention achieves precise molecular anchoring by covalently coupling the albumin protein with mercaptododecaborane through a specific disulfide bond formed through the unique free thiol group at cysteine 34 (Cys34). This process strictly circumvents the uncontrollable binding site issues associated with traditional physical packaging or random chemical modification, providing a foundation for structural certainty and high stability of the complex. By leveraging the rigid cage-like structure of the dodecaborane molecule, covalent coupling at Cys34 triggers a "nucleation effect," inducing the directed aggregation of mercaptododecaborane molecules on the albumin surface to form a stable complex. This effect significantly enhances drug loading and complex stability, addressing the low drug loading rate and easy dissociation associated with traditional albumin carriers, which rely on weak interactions (such as hydrophobic interactions).

[0086] Among them, the covalent modification is strictly located in the non-functional domain of albumin (away from the receptor binding region), completely retaining the natural tumor targeting ability of albumin (such as SPARC receptor-mediated targeted delivery), ensuring that the complex is efficiently enriched in tumor tissue in vivo, and the tumor / normal tissue boron concentration ratio (T / N ratio ≥ 3:1) is experimentally verified. Based on the stability of disulfide bonds in physiological environments (pH 7.3-7.5) and the reversible cleavage characteristics in the acidic microenvironment of tumors (pH 6.5-7.0), an intelligent drug release system was constructed. This mechanism achieves "zero leakage" of drugs in the circulatory system while specifically releasing active substances at the tumor site. 10 B. Significantly improve the accuracy and efficacy of boron neutron capture therapy (BNCT).

[0087] It is worth noting that the synergistic effect of covalent coupling and nucleation completely inhibits the oxidative degradation of mercaptododecaborane, preventing systemic toxicity (such as liver and kidney damage) caused by the release of free boron ions. It also avoids the use of organic solvents or surfactants in traditional solubilization processes, reducing the risk of formulation toxicity at the source. This is the first time that "site-specific covalent coupling" and "nucleation effect" have been synergistically applied to albumin carrier design, breaking through the limitations of existing technologies that rely on physical interactions and providing a universal solution for the development of highly stable, highly targeted, and intelligently responsive protein drug delivery systems.

[0088] The above specific embodiments are merely several optional embodiments of the present invention. Based on the technical solutions of the present invention and the relevant inspirations of the above embodiments, those skilled in the art may make various alternative improvements and combinations to the above specific embodiments.

Claims

1. A composition of albumin and mercapto dodecaborane, characterized in that: include: Albumin, which is covalently coupled to mercapto-dodecaborane via the thiol group of cysteine at position 34, constructs a nucleation site, and further loads mercapto-dodecaborane on the protein through the nucleation effect; The thiol group of the mercaptododecaborane is bonded to the thiol group at position 34 of albumin cysteine via a disulfide bond, wherein the disulfide bond is stable in a physiological environment and reversibly cleaved in the acidic microenvironment of the tumor, thereby being used for the targeted release of the mercaptododecaborane; The albumin is human serum albumin; The mass ratio of the albumin to the mercapto dodecaborane is 10:1 to 500:

1.

2. The composition of albumin and mercapto dodecaborane according to claim 1, characterized in that: The pH of the physiological environment is 7.3-7.5, and the pH of the tumor acidic microenvironment is 6.5-7.

0.

3. The composition of albumin and mercaptododecaborane according to claim 1, further comprising a pharmaceutically acceptable excipient, wherein the excipient is selected from one or more of a stabilizer, a buffer, a preservative, and an osmotic pressure regulator.

4. The composition of albumin and mercapto dodecaborane according to claim 1, characterized in that: The composition is in the form of intravenous injection or lyophilized powder injection.

5. A drug, characterized in that The invention comprises the albumin-mercaptododecaborane composition according to any one of claims 1 to 4, and a pharmaceutically acceptable carrier.

6. Use of the drug according to claim 5 in the preparation of a drug for treating tumors, characterized in that: The tumor treatment is boron neutron capture therapy, and the tumor is malignant melanoma.

7. Use of the drug according to claim 6 in the preparation of a drug for treating tumors, characterized in that: After the drug is intravenously injected, the ratio of the boron concentration in the tumor tissue to the boron concentration in the normal tissue is T / N≥3:

1.

8. Use of the drug according to claim 6 in the preparation of a drug for treating tumors, characterized in that: The drug is released in the acidic microenvironment of the tumor through a pH-responsive release mechanism. 10 B, used to kill tumor cells.

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