Boron-containing drug and its manufacturing method

Abstract

The present invention relates to a boron-containing drug and a method for preparing the same, which can be obtained by polymerization of dopamine or its derivatives with a boron-containing compound. The boron-containing drug has a high tumor concentration of 70 μg / g, significantly improves the tumor / blood concentration ratio of boron, reaching 70:1, and is rapidly removed from the blood and normal organs, significantly reducing the drug dosage and side effects, and significantly improving the tumor treatment effect.

Description

[Technical Field]

[0001] The present invention belongs to the technical field of tumor treatment drugs, and specifically relates to boron-containing drugs and methods for preparing the same. [Background technology]

[0002] Currently, two boron-containing drugs have been clinically used to some extent: 4-boron-10 acid-L-phenylalanine (BPA) and mercaptododecylborane disodium salt (sodium mercaptoundecahydrocloso-dodecaborate (BSH)). Currently, BSH-based drugs have undergone limited clinical trials, but have not yet received full-scale marketing approval. BSH is favored by researchers due to its high boron-10 content; however, the synthesis and purification process of the active pharmaceutical ingredient is difficult, making it expensive and limited in source. Furthermore, the clinical safety and efficacy of BSH have not yet been fully validated, and research based on BSH is currently declining. BPA has attracted more attention in recent years due to its relatively simple synthesis and purification process, good compound stability, and more thorough research and validation of its clinical safety and efficacy.

[0003] Currently, BPA-based drug formulations are mainly produced as solution formulations or lyophilized powders using BPA as an active ingredient. Due to their low tumor uptake rate and rapid blood clearance, the clinical use of such drug formulations is extremely large. The main challenge currently facing boron-containing drugs is how to improve the tumor targeting and utilization rate of boron-containing drugs.

[0004] When tumors are treated clinically using boron neutron capture therapy, 10The B concentration must be maintained at about 30 μg / g during the neutron irradiation process to achieve a good therapeutic effect. The tumor / normal tissue concentration ratio (T / N ratio) of BPA can only reach 3:1, and when the BPA level in cancer cells increases, it is excreted by the "reverse transport" mechanism. Steboronine®, a BPA-based solution formulation that has already been clinically available, has been shown to effectively suppress tumor localization during the treatment process. 10 To maintain B levels, adult patients before treatment should receive borophalan ( 10 B) should be infused for 2 hours, and adult patients should receive borophalan ( 10 B) requires a 30-60 minute infusion. Due to its poor tumor targeting, low T / N ratio, and rapid metabolism, 36 g of BPA is required for a single treatment of a 60 kg patient, resulting in a high clinical dose, which not only increases treatment costs but also safety risks. Furthermore, the low tumor targeting and T / N ratio require greater precision in neutron source irradiation in clinical applications and increase the risk of over-standardization of normal tissue exposure doses (irradiating normal tissues is prone to damage due to the high boron content of normal tissues caused by the low T / N ratio). This further limits the clinical application and broadening of indications for this drug.

[0005] The present invention has been made in view of such circumstances. Summary of the Invention

[0006] In order to solve the technical problems in the background art, one object of the present invention is to develop a safe and effective boron-containing drug formulation with better tumor targeting and higher utilization rate based on dopamine and its derivatives and boron-containing compounds, to develop a method for producing such a boron-containing drug, and to study its potential application in the treatment of diseases such as tumors. Such a boron-containing drug formulation is safe and effective, and can significantly reduce the amount of boron-containing drug used. Furthermore, due to its good tumor targeting distribution, it can expand the range of indications.

[0007] To achieve the above object, the present invention adopts the following first technical solution.

[0008] A boron-containing drug obtained by polymerization of dopamine or its derivatives with a boron-containing compound.

[0009] Preferably, the boron-containing compound includes, but is not limited to, any one or more of boric acid, phenylalanine borate and its derivatives, proline borate, aspartic acid borate, tyrosine borate, cysteine ​​borate, methionine borate, serine borate, 5-amino-2,3-difluorophenylboronic acid, 3-amino-5-cyanophenylboronic acid, 3-amino-4-methylphenylboronic acid, 3-aminophenylborate hydrochloride, and 4-carbamoylphenylboronic acid.

[0010] Preferably, the boron-containing compound includes phenylalanine borate and any of its derivatives.

[0011] Preferably, the borate phenylalanine and its derivatives is 4-boron-10-acid-L-borate phenylalanine (BPA).

[0012] Preferably, the dopamine and derivatives thereof include one or more of dopamine, 4-(2-aminopropyl)benzene-1,2-diol, noradrenaline (1-(3,4-dihydroxyphenyl)-2-aminoethanol), L-methyldopa (2-methyl-3-(3,4-dihydroxyphenyl)-L-alanine), droxidopa ((2S,3R)-2-amino-3-(3,4-dihydroxyphenyl)-3-hydroxypropionic acid), and 5-hydroxydopamine, and preferably dopamine, L-methyldopa (2-methyl-3-(3,4-dihydroxyphenyl)-L-alanine), or 5-hydroxydopamine.

[0013] Preferably, the mass ratio of the dopamine and derivatives thereof to the boron-containing compound is (1-10):1.

[0014] Preferably, the mass ratio of the dopamine and derivatives thereof to the boron-containing compound is (3-4):1.

[0015] Preferably, the boron content in the boron-containing drug is 0.1% to 1.0%.

[0016] Preferably, the indications for the boron-containing drug include diseases such as head and neck tumors, lung cancer, peritoneal cancer, hepatocellular carcinoma, gastric cancer, melanoma, pancreatic cancer, brain glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, colorectal cancer, uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, B-cell lymphoma or leukemia, and preferably head and neck tumors, melanoma or brain glioma.

[0017] The second technical solution adopted by the present invention is as follows.

[0018] The method for producing a boron-containing drug includes the following steps:

[0019] An aqueous solution of dopamine and its derivatives and an aqueous solution of a boron-containing compound are mixed and reacted, and then subjected to solid-liquid separation to obtain a solid, which is then washed and freeze-dried.

[0020] Preferably, the pH of the mixed reaction is 6 to 14.

[0021] Preferably, the pH of the mixed reaction is 8 to 11.

[0022] Preferably, the mixing and reaction time is 0.5 to 48 hours, and the stirring speed during the reaction is 200 rpm or more.

[0023] The present invention has the following advantageous effects compared to the prior art.

[0024] 1. The boron-containing drug prepared in the present invention has a high tumor concentration of 70 μg / g, and the tumor / blood boron concentration ratio is significantly improved, reaching 70:1, which is much higher than the tumor / normal tissue boron concentration ratio of current BPA drug preparations. This greatly improves the tumor uptake rate, significantly improves the concentration of boron in tumors, improves the therapeutic effect, and can be rapidly removed from the blood and normal organs, significantly reducing the drug usage and reducing side effects, thereby achieving better tumor targeting and higher utilization rate and significantly improving the tumor therapeutic effect.

[0025] 2. The drug of the present invention has the characteristic of long retention in tumor tissue, resulting in a long therapeutic window, overcoming the drawback of the prior art requiring continuous infusion during boron neutron irradiation. Experiments with subcutaneous brain glioma in mice have shown that at a dose of 150 mg / kg, the boron concentration is within the range of 24-36 h, maintained at 30 μg / g or higher, and the therapeutic window exceeds 12 h, eliminating the need for continuous infusion of boron-containing drugs during treatment. This simplifies the implementation of the treatment process.

[0026] 3. Conventional nano-drugs cannot pass through the blood-brain barrier and therefore cannot be used to treat brain tumors. However, the boron-containing drug of the present invention can be concentrated in brain gliomas through the blood-brain barrier, and the drug can be maintained at a therapeutic concentration for a long time, allowing it to be used to treat brain tumors.

[0027] 4. The boron-containing drug of the present invention has low toxicity and good biocompatibility.

[0028] 5. The boron-containing drug of the present invention has the property of generating heat under near-infrared light irradiation, i.e., it has photothermal properties. The photothermal conversion efficiency of the boron-containing drug is 39.46%, which can realize combined boron neutron and photothermal therapy. [Brief explanation of the drawings]

[0029] [Figure 1] FIG. 1 is a Fourier transform infrared (FT-IR) spectrum of the boron-containing drug in Example 1. [Figure 2] FIG. 2 is a particle size distribution diagram of the boron-containing drug in Example 1. [Figure 3] FIG. 3 is an SEM image of the boron-containing drug in Example 2. [Figure 4] FIG. 4 shows the cell viability at 24 hours, 48 ​​hours, and 72 hours after the toxicity of boron-containing drugs was measured using tumor cell lines HepG2, SKOV3, and H22 overexpressing three types of sialic acid in Example 3. [Figure 5] FIG. 5 is an experiment on the biodistribution of a boron-containing drug in a mouse subcutaneous brain glioma model in Example 4, showing the distribution of boron in the mouse body 12 hours, 24 hours, and 36 hours after administration. [Figure 6] FIG. 6 is a graph showing the distribution of boron in the body of a mouse in situ brain glioma model in Example 5, 24 hours after administration. [Figure 7] FIG. 10 is an SEM image of a boron-containing drug in Example 8. [Figure 8] 10 is a graph showing the temperature rise of a boron-containing drug under irradiation conditions of an 808 nm near-infrared laser with an output density of 1.5 W / cm 2 in Example 13. DETAILED DESCRIPTION OF THE INVENTION

[0030] In order to better understand the technical solution of the present invention, the technical solution of the present invention will be further described below with reference to the drawings and examples. The manner in which the present invention is realized includes, but is not limited to, the following examples, which are for the purpose of illustrating the present invention and do not limit the scope of protection of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. Unless otherwise specified, the test methods in the following examples are all conventional methods.

[0031] A first embodiment of the present invention provides a boron-containing drug obtained by a polymerization reaction between dopamine or a derivative thereof and a boron-containing compound.

[0032] The boron-containing drug of this embodiment has a particle size of 50 nm to 1000 nm, and the boron-containing drug can enter cells and penetrate the blood-brain barrier.

[0033] This embodiment mainly aims to improve the targeting properties of boron-containing drugs in the body through the optimization of composition and structure, improve the T / N ratio between tumors and normal tissues, prolong the residence time of drugs in tumors, and enable them to be metabolized relatively quickly from blood and normal tissues and organs, thereby reducing the amount of boron-containing drugs used, reducing safety risks during treatment, achieving better tumor targeting and higher utilization rate, and significantly improving the therapeutic efficacy of tumors. Improve the therapeutic effect of boron-containing drugs, expand the indications, and significantly reduce the cost of drug treatment.

[0034] The boron-containing drug of this embodiment can be taken up into cells via cells, but cannot be excreted by tumor cells via the reverse transport mechanism, prolonging its residence time in tumor cells, solving the problem of BPA's high therapeutic dose due to boron neutron capture, further improving the concentration of the boron-containing drug at the tumor lesion site, and improving the therapeutic effect. Experiments on the distribution of boron-containing drug in subcutaneous brain gliomas in mice have shown that at a dose of 150 mg / kg, the boron concentration remains above 30 μg / g within 24 to 36 hours, with a therapeutic window of more than 12 hours. This eliminates the need for continuous infusion of the boron-containing drug during treatment, greatly enhancing patient treatment convenience.

[0035] Indications for the boron-containing drug in this embodiment include diseases such as head and neck tumors, lung cancer, peritoneal cancer, hepatocellular carcinoma, gastric cancer, melanoma, pancreatic cancer, brain glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, colorectal cancer, uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, B-cell lymphoma or leukemia, and preferably head and neck tumors, melanoma or brain glioma.

[0036] The boron-containing drug can be metabolized (biodegradable) in the body, and 48 hours after injection into the body, the drug concentration rapidly decreases until it is completely metabolized and eliminated.

[0037] This boron-containing drug can generate heat when irradiated with near-infrared light, has strong near-infrared absorption, and has high photothermal conversion efficiency. When irradiated with near-infrared light with a wavelength of 808 nm, the photothermal conversion efficiency of 0.1 mg / mL of the boron-containing drug is approximately 39%, making it possible to realize combined boron neutron therapy and photothermal therapy.

[0038] Regarding the selection of raw material components, the boron-containing compound may include, but is not limited to, one or more of boric acid, phenylalanine borate and its derivatives, proline borate, aspartic acid borate, tyrosine borate, cysteine ​​borate, methionine borate, serine borate, 5-amino-2,3-difluorophenylboric acid, 3-amino-5-cyanophenylboric acid, 3-amino-4-methylphenylboric acid, 3-aminophenylborate hydrochloride, and 4-carbamoylphenylboric acid.Preferably, it includes phenylalanine borate and its derivatives, and more preferably, 4-boron-10-acid-L-phenylalanine borate (BPA).

[0039] The dopamine and derivatives thereof include one or more of dopamine, 4-(2-aminopropyl)benzene-1,2-diol, noradrenaline (1-(3,4-dihydroxyphenyl)-2-aminoethanol), L-methyldopa (2-methyl-3-(3,4-dihydroxyphenyl)-L-alanine), clisib ((2S,3R)-2-amino-3-(3,4-dihydroxyphenyl)-3-hydroxypropionic acid), and 5-hydroxydopamine, and preferably dopamine, L-methyldopa (2-methyl-3-(3,4-dihydroxyphenyl)-L-alanine), or 5-hydroxydopamine.

[0040] Regarding the amounts of the raw material components used, the mass ratio of the dopamine and its derivatives to the boron-containing compound is (1 to 10):1, and preferably (3 to 4):1.

[0041] The boron content in the boron-containing drug is 0.1% to 1.0%.

[0042] The second embodiment of the present invention provides a method for producing a boron-containing drug, which involves mixing and reacting an aqueous solution of dopamine and its derivatives with an aqueous solution of a boron-containing compound, followed by solid-liquid separation to obtain a solid, which is then washed and freeze-dried.

[0043] The pH of the mixing reaction is 6 to 14, and preferably 8 to 11. The mixing reaction time is 0.5 to 48 hours, and the stirring speed during the reaction is 200 rpm or more.

[0044] The solvent for the above-mentioned mixed reaction is not particularly limited, and the reaction may be carried out in an aqueous solution as it is, or in methanol, ethanol, acetone, DMF, THF, or any mixed solution thereof.

[0045] The boron-containing drug according to the present invention has good resolubility after lyophilization, and the resolvents include, but are not limited to, one of sterile water for injection, sodium chloride injection, glucose injection, and PBS buffer solution.

[0046] The boron-containing drug and its performance will be described below through several specific examples. [Example]

[0047] Example 1 Preparation of boron-containing drugs (1) 80 mg of dopamine hydrochloride was weighed into a reaction flask, and 34 mL of 30% ethanol solution was added and stirred to dissolve.

[0048] (2) 20 mg of BPA was weighed into a centrifuge tube, 3 mL of water was added, and 0.2 mL of 2.5 mol / L NaOH solution was added until the BPA was completely dissolved.

[0049] (3) The solution of (2) was added to (1), the pH was adjusted to about 10 with NaOH, and the mixture was stirred at 300 rpm for 24 hours.

[0050] (4) The mixture was centrifuged at 10,000 rpm for 10 minutes and washed three times with pure water.

[0051] (5) The product was suspended in 6 mL of water and lyophilized to obtain the boron-containing drug.

[0052] The infrared spectrum of the boron-containing drug prepared in this example is shown in FIG. 1. The boron-containing drug has a BN bond and an infrared absorption at 1388 cm -1 The particle size distribution is shown in Figure 2, and the average particle size was 191.8 nm.

[0053] Example 2 Redissolution experiment of boron-containing drug after freeze-drying 5 mg of the boron-containing drug in Example 1 was taken, and 5 mL of a solvent was added, including but not limited to sterile water for injection, sodium chloride injection, glucose injection, and PBS buffer solution, with water being one of the dispersants. After manual mixing, SEM detection was performed, and the morphology was characterized as spherical, as shown in Figure 3.

[0054] Example 3: In vitro cytotoxicity evaluation of boron-containing drugs over 24-72 hours The evaluation period was 24 to 72 hours, and the detection method was the Cell Counting Kit-8 (CCK8), a rapid and sensitive detection kit for cell proliferation and cytotoxicity. High expression of three types of sialic acid was detected in the tumor cell lines HepG2, SKOV3, and H22.

[0055] The specific procedure is as follows: (1) Three types of cells, HepG2, SKOV3, and H22, which are in good growth condition, are cultured at a predetermined concentration (5 × 10 3 Prepare a cell suspension (cells / 100uL) and add 100uL to each well of a 96-well cell culture plate.

[0056] (2) 10 μL of the boron-containing drug of Example 1 at 0, 10, 20, 50, 100, 200, and 500 μg / mL was added to the culture wells, and the cells were cultured in an incubator at 37° C. for a certain period of time.

[0057] (3) Sampling was performed at 24 h, 48 h, and 72 h, and the absorbance was detected at a wavelength of 450 nm to calculate the cell viability. The cell viability at 72 h was over 80%, indicating the low cytotoxicity of boron-containing drugs. The specific results are shown in Figure 4.

[0058] Example 4: Biodistribution experiment of boron-containing drugs in a mouse subcutaneous brain glioma model The boron-containing drug from Example 1 was collected and injected into tumor-bearing mice via the tail vein at a dose of 150 mg / kg. 12, 24, and 36 hours after administration, the heart, liver, spleen, lungs, kidneys, blood, brain, tumor, and normal tissues at the tumor border were collected, weighed, disassembled, and the boron concentration in each organ measured by ICP-MS. 24 hours later, the boron concentration in the tumor reached 74 μg / g, with a T / N ratio of 70:1. The distribution of boron in the mice 12, 24, and 36 hours after administration is shown in Figure 5 and Table 1.

[0059] [Table 1]

[0060] This example studied the distribution of a subcutaneous brain glioma model animal. Mice were injected via the tail vein with a 150mg / kg dose of a boron-containing drug. At 12, 24, and 36 hours after administration, the boron concentrations in the heart, liver, spleen, lungs, kidneys, blood, brain, tumor, and normal tissues at the tumor border were measured using ICP-MS. At 24 hours, the boron concentration in the tumor reached a maximum of 74μg / g and remained above 30μg / g by 36 hours, resulting in a therapeutic window of more than 12 hours. Continuous infusion of the boron-containing drug was not required during the treatment process, and the T / N ratio reached 70:1 (Table 1). This indicates minimal damage to normal tissues during boron neutron capture therapy.

[0061] Example 5 Biodistribution experiment of boron-containing drugs in a mouse in situ brain glioma model The boron-containing drug from Example 1 was collected and injected into tumor-bearing mice via the tail vein at a dose of 100 mg / kg. 24 hours after administration, the mouse's heart, liver, spleen, lungs, kidneys, blood, right brain tumor region, right brain non-tumor region, and left brain were collected. Each tissue was weighed and disassembled, and the boron concentration in each organ was measured using ICP-MS. The boron concentration in the tumor region was all above 30 μg / g, with a T / N ratio of greater than 7:1. The distribution of boron in the mice 24 hours after administration is shown in Figure 6 and Table 2.

[0062] [Table 2]

[0063] As can be seen from the experimental results in Tables 1 and 2, the boron-containing drug prepared in the present invention is highly absorbed into tumor sites such as subcutaneous tumors and in situ cerebral gliomas, and can be concentrated in cerebral gliomas through the blood-brain barrier, with a T / N ratio of at least 7:1 (Table 2) and even as high as 70:1 (Table 1), thus overcoming the drawback of ordinary nano-drugs being unable to pass through the blood-brain barrier.

[0064] Examples 6-12 Preparation of other boron-containing drugs The production of Examples 6 to 12 was carried out in accordance with Example 1, and the amount of each component used in Table 3 is shown in parts by mass.

[0065] [Table 3]

[0066] When the dose was 150 mg / kg, the boron-containing drugs prepared in Examples 6 to 12 had a T / N ratio of 5:1 to 70:1 in a distribution experiment using a mouse subcutaneous brain glioma model. The SEM image of the boron-containing drug in Example 8 is shown in Figure 7.

[0067] Example 13 Photothermal Experiments of Boron-Containing Drugs The semiconductor laser subsystem emits an 808 nm near-infrared laser, with a laser irradiation spot area of ​​0.6 cm2 , and 0.1 mg / mL of the boron-containing drug aqueous solution of Example 1 was added to the quartz cell, and the power density was set to 1.5 W / cm 2 The solution was irradiated with a near-infrared excitation light source (wavelength 808 nm) for 15 minutes, and the solution temperature was measured every 30 seconds using a thermometer. The specific results are shown in Figure 8. The above value was finally calculated to be 39.46% of the photothermal conversion efficiency of the boron-containing drug.

[0068] Example 14 Photothermal treatment of mouse subcutaneous brain gliomas with boron-containing drugs Physiological saline and 150 mg / kg of the boron-containing drug of Example 1 were injected into the bodies of tumor-bearing mice via the tail vein, and then irradiated with a near-infrared excitation light source (wavelength 808 nm) for 30 minutes. The volume of the subcutaneous brain gliomas of the mice was measured on the 3rd, 5th, and 10th days. The specific results are shown in Table 4.

[0069] [Table 4]

[0070] Finally, it should be noted that the above embodiments are only for explaining the technical solutions of the present invention, and are not intended to limit the same. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art can still modify the technical solutions described in the above embodiments or make equivalent substitutions for some or all of the technical features therein, and these modifications or substitutions should not cause the essence of the corresponding technical solutions to depart from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A boron-containing drug obtained by a polymerization reaction between dopamine or a derivative thereof and a boron-containing compound.

2. The boron-containing compound includes, but is not limited to, any one or more of boric acid, phenylalanine borate and its derivatives, proline borate, aspartic acid borate, tyrosine borate, cysteine ​​borate, methionine borate, serine borate, 5-amino-2,3-difluorophenylboronic acid, 3-amino-5-cyanophenylboronic acid, 3-amino-4-methylphenylboronic acid, 3-aminophenylborate hydrochloride, and 4-carbamoylphenylboronic acid; The boron-containing drug according to claim 1, characterized in that:

3. The boron-containing drug according to claim 2, wherein the boron-containing compound is phenylalanine borate and its derivatives.

4. The dopamine and derivatives thereof include one or more of dopamine, 4-(2-aminopropyl)benzene-1,2-diol, noradrenaline, L-methyldopa, droxidopa, and 5-hydroxydopamine; The boron-containing drug according to claim 1, characterized in that it is preferably dopamine, L-methyldopa or 5-hydroxydopamine.

5. The boron-containing drug according to claim 1, wherein the mass ratio of the dopamine and its derivatives to the boron-containing compound is (1-10):

1.

6. The boron-containing drug according to claim 5, wherein the mass ratio of the dopamine and its derivatives to the boron-containing compound is (3-4):

1.

7. The boron-containing drug according to claim 1, characterized in that the boron content is 0.1% to 1.0%.

8. The boron-containing drug according to any one of claims 1 to 7, characterized in that the indications for the boron-containing drug include head and neck tumors, lung cancer, peritoneal cancer, hepatocellular carcinoma, gastric cancer, melanoma, pancreatic cancer, brain glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, colorectal cancer, uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, B-cell lymphoma or leukemia.

9. The boron-containing drug according to claim 8, characterized in that the indication of the boron-containing drug is head and neck tumor, melanoma or brain glioma.

10. 10. A method for producing a boron-containing drug according to any one of claims 1 to 9, comprising the steps of mixing and reacting an aqueous solution of dopamine and its derivatives with an aqueous solution of a boron-containing compound, performing solid-liquid separation to obtain a solid, washing the solid, and freeze-drying the solid.

11. The method for producing a boron-containing drug according to claim 10, characterized in that the pH of the mixed reaction is 6 to 14.

12. The method for producing a boron-containing drug according to claim 11, characterized in that the mixing reaction time is 0.5 to 48 hours, and the stirring speed during the reaction is 200 rpm or more.

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