Gelatin matrix radionuclide carrier, gelatin matrix radionuclide loaded product, compositions, methods of preparation and use

Abstract

The application discloses a gelatin matrix radionuclide carrier, a gelatin matrix radionuclide product, a composition, a preparation method and application. The gelatin matrix radionuclide carrier is prepared from raw materials including modified gelatin and modified chelate; wherein the modified gelatin is selected from one or both of methacrylated gelatin and acrylated gelatin; the modified chelate is obtained by modifying a chelating agent through N-hydroxysuccinimide; the chelating agent is selected from one or more of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, 1,4,7-triazacyclododecane-1,4,7-triacetic acid, ethylenediaminetetraacetic acid and diethylenetriaminepentaacetic acid; and the mass ratio of the modified gelatin to the modified chelate is 1g:(10-200)mg. The gelatin matrix radionuclide carrier can load metal radionuclides and non-metal radionuclides, and the obtained gelatin matrix radionuclide product after loading radionuclides can be injected. The application belongs to the field of biological medicine.

Description

Technical Field

[0001] This invention relates to a gelatin-based nuclide carrier, a gelatin-based loaded nuclide product, a composition, a preparation method, and an application. Background Technology

[0002] Nuclear medicine is a medical discipline that uses radioactive nuclides (or nuclear rays) to diagnose and treat diseases and to study the mechanisms of disease development. Its core is the application of "radioactive nuclides," and it has the dual functions of imaging diagnosis and targeted therapy.

[0003] Nuclides are classified into metallic nuclides and non-metallic nuclides. Metallic nuclides primarily emit alpha and beta rays, which are high in energy and have a short range, allowing them to precisely target diseased cells (such as thyroid follicular cells and tumor cells). This ensures sufficient energy to destroy DNA while avoiding damage to surrounding normal tissues. Therefore, metallic nuclides are mainly used in radiotherapy. Furthermore, the use of metallic nuclides usually requires chelating agents to enhance stability and reduce the risk of expulsion within the body. Non-metallic nuclides primarily emit gamma rays and positrons, which are low in energy and have short half-lives. After injection into the human body, they are rapidly excreted through decay or physiological metabolism (such as respiration and urine). The radiation dose from a single tracer test is extremely low, and the risk to the individual and surrounding population is controllable. Therefore, non-metallic nuclides are mainly used in radiographic imaging. Moreover, the use of non-metallic nuclides does not rely on chelating agents; they can directly bind to biomolecules through covalent bonds without altering the molecular structure or physiological activity.

[0004] The matrix material used to deliver radionuclides is called a radionuclide carrier. CN118453920A discloses a polyaspartic acid targeted radionuclide carrier, its preparation method, and its application. This carrier system comprises two compounds: the first compound is RGD-modified polyaspartic acid, which has targeting properties; the second compound is chelating agent-modified polyaspartic acid, which has metal nuclide coordination function. The two compounds are mixed to self-organize into nanoparticles, which are then administered intravenously for targeted radionuclide therapy of tumors.

[0005] CN119613769A discloses a radionuclide dressing based on a photocrosslinked hydrogel. This photocrosslinked hydrogel has a crosslinked network structure composed of dopamine-modified methacrylated gelatin and o-nitrobenzyl sulfide phototriggered hyaluronic acid. The radionuclide is primarily immobilized within the hydrogel network through physical processes.

[0006] CN118846143A discloses an iodine-based polymer coating, specifically an iodine-131-gelatin protein film, which can be applied to the surgical wound during cancer resection. The iodine-131 on the coating releases beta rays to eliminate residual cancer cells in the surgical area or the remaining organs, as well as any residual cancer cells in nearby vascular and nerve spaces, achieving brachytherapy and effectively reducing tumor recurrence after cancer surgery. The gelatin protein film is a glutaraldehyde-crosslinked gelatin protein film.

[0007] CN117338960A discloses a radionuclide-labeled tumor imaging agent, which is prepared from a diagnostic drug precursor and a radionuclide; the radionuclide is selected from gallium-68, copper-64, and zirconium-89; the diagnostic drug precursor is prepared from velipanib and a macrocyclic ligand chelating agent; the macrocyclic ligand chelating agent is selected from NHS-DOTA and NHS-NOTA.

[0008] To date, there have been no reports on the preparation of gelatin-based nuclide carriers, gelatin-based loaded nuclide products, preparation methods, and applications of acrylamide gelatin and modified chelates. Summary of the Invention

[0009] In view of this, one object of the present invention is to provide a gelatin-based nuclide carrier that can stably load both metallic and non-metallic nuclides to meet different clinical needs; it is injectable and facilitates local administration after loading the nuclide.

[0010] Another object of the present invention is to provide a method for preparing the above-mentioned gelatin matrix nuclide carrier, which has a stable process.

[0011] Another object of the present invention is to provide a gelatin matrix-loaded nuclide product, which is obtained by loading nuclides onto the aforementioned gelatin matrix nuclide carrier.

[0012] Another object of the present invention is to provide a method for preparing a gelatin matrix loaded with radionuclides.

[0013] Another object of the present invention is to provide a composition for radiographic imaging or radiotherapy.

[0014] Another object of the present invention is to provide the use of a composition for radiographic imaging or radiotherapy in the preparation of a radiotherapeutic drug or radiographic imaging agent.

[0015] On the one hand, the present invention provides a gelatin-based nuclide carrier, which is prepared from raw materials including modified gelatin and modified chelates;

[0016] The modified gelatin is selected from one or both of methacrylamide gelatin and acrylamide gelatin;

[0017] The modified chelate is obtained by modifying the chelating agent with N-hydroxysuccinimide; the chelating agent is selected from one or more of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, 1,4,7-triazacyclo-1,4,7-triacetic acid, ethylenediaminetetraacetic acid and diethyltriaminepentaacetic acid;

[0018] The mass ratio of modified gelatin to modified chelate is 1g:(10-200)mg.

[0019] According to the gelatin matrix nuclide carrier of the present invention, preferably, the modified gelatin is methacrylamide gelatin; the chelating agent is 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid; and the mass ratio of modified gelatin to modified chelate is 1g:(15-150)mg.

[0020] On the other hand, the present invention also provides a method for preparing the gelatin-based nuclide carrier as described above, comprising the following steps:

[0021] 1) Provide modified chelates;

[0022] 2) Provide modified gelatin;

[0023] 3) Dissolve the modified gelatin in a polar organic solvent, then add the modified chelate and alkaline reagent, mix and react to obtain a reaction solution; dialyze the reaction solution, filter the dialyzed solution and freeze-dry it to obtain the gelatin matrix nuclide carrier;

[0024] The polar organic solvent is selected from one or more of dimethyl sulfoxide, dimethylformamide, and dimethylacetamide; the basic reagent is selected from one or more of triethylamine, tributylamine, diisopropylethylamine, and pyridine.

[0025] Steps 1) and 2) are not in any particular order.

[0026] According to the preparation method of the present invention, preferably, the modified gelatin is methacrylamide gelatin obtained by reacting initial gelatin with methacrylic anhydride; the molecular weight of the initial gelatin is less than 50 kDa; and the chelating agent is 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid.

[0027] According to the preparation method of the present invention, preferably, the modified chelate is prepared by the following steps:

[0028] (a) 1,4,7,10-tetraazacyclododecane was reacted with tert-butyl haloacetate under alkaline conditions to obtain compound 1 as shown in formula (1);

[0029] (b) Compound 1 was reacted with methyl haloacetate under alkaline conditions to give compound 2 as shown in formula (2);

[0030] (c) Hydrolyze compound 2 to obtain the intermediate product shown in formula (3); react the intermediate product with N-hydroxysuccinimide in the presence of a dehydrating agent to obtain compound 4 shown in formula (4);

[0031] (d) Hydrolyze compound 4 under acidic conditions to obtain compound 5 as shown in formula (5), i.e., the modified chelate;

[0032] (1); (2);

[0033] (3); (4);

[0034] (5).

[0035] According to the preparation method of the present invention, preferably, the degree of substitution of the methacrylamide gelatin is 10-90%; the content of the modified chelate group, calculated as DOTA, is 2-10 wt% based on the total mass of the gelatin matrix nuclide carrier; and DOTA is an abbreviation for 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid.

[0036] In another aspect, the present invention also provides a gelatin matrix loaded with nuclides, which is obtained by reacting the gelatin matrix nuclide carrier as described above with nuclide ions, wherein the nuclide ions contain nuclides selected from metallic nuclides and / or non-metallic nuclides.

[0037] According to the gelatin matrix-loaded nuclide product of the present invention, preferably, the metal nuclide is selected from... 177 Lu、 90 Y、 67 Ga、 64 Cu、 99m Tc and 89 One or more of Sr; the nonmetallic nuclide is selected from... 125 I, 131 I, 18 F and 32 One or more of P.

[0038] In another aspect, the present invention provides a composition for radiographic imaging or radiotherapy comprising a gelatin matrix-loaded radionuclide product and a photoinitiator as described above; which can be cured to form a gel under light irradiation at a wavelength of 385–450 nm.

[0039] In another aspect, the present invention also provides the use of the composition described above for radiographic imaging or radiotherapy in the preparation of radiotherapeutic drugs or radiographic imaging agents.

[0040] The gelatin-based radionuclide carrier of this invention is obtained by modifying acrylamide gelatin with a modified chelate. It can stably load both metallic and non-metallic radionuclides, meeting diverse clinical needs. The gelatin-based radionuclide product of this invention is obtained by loading metallic and / or non-metallic radionuclides onto a gelatin-based radionuclide carrier. When mixed with a photoinitiator, it can be used for local injection and can rapidly solidify into a gel under light irradiation at a wavelength of 385–450 nm, thereby performing local radiotherapy or imaging functions and avoiding the drawbacks of systemic administration. Attached Figure Description

[0041] Figure 1 The proton NMR spectrum of compound 5 (i.e., DOTA-NHS) prepared for Preparation Example 1.

[0042] Figure 2 SPECT images of a composition comprising a gelatin-based loaded radionuclide product and a photoinitiator at different days after injection into the dura mater of SD rats and followed by photocuring are shown. Detailed Implementation

[0043] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto.

[0044] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although similar or equivalent methods and materials may be used in the implementation or testing of this invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. In case of any conflict, this specification and its included definitions shall prevail. Furthermore, materials, methods, preparation examples, and embodiments are exemplary only and are not intended to be limiting.

[0045] Gelatin-based nuclide carrier

[0046] The gelatin-based nuclide carrier is prepared from raw materials including modified gelatin and modified chelates. The modified gelatin is selected from one or both of methacrylamide gelatin and acrylamide gelatin; the modified chelate is obtained by modifying a chelating agent with N-hydroxysuccinimide; the chelating agent is selected from one or more of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, 1,4,7-triazacyclo-1,4,7-triacetic acid, ethylenediaminetetraacetic acid, and diethyltriaminepentaacetic acid; the mass ratio of modified gelatin to modified chelate is 1 g:(10–200) mg. The resulting gelatin-based nuclide carrier can stably load both metallic and non-metallic nuclides, meeting diverse clinical needs; it is injectable, facilitating local drug delivery.

[0047] In this invention, the modified gelatin is obtained by acrylating initial gelatin. The degree of substitution of the modified gelatin is 10-90%, preferably 10-50%, and more preferably 10-30%. The molecular weight of the initial gelatin is less than 50 kDa. The modified gelatin is preferably methacrylated gelatin.

[0048] In this invention, the mass ratio of modified gelatin to modified chelate can be 1g:(10-200)mg, preferably 1g:(10-180)mg, and more preferably 1g:(15-150)mg. This is beneficial on the one hand to form DOTA groups containing a specific amount to load nuclides within a specific content range, and on the other hand to facilitate the subsequent curing.

[0049] In this invention, the content of the modified chelate, calculated as a chelating agent, is 2 to 10 wt% based on the total mass of the gelatin matrix nuclide carrier.

[0050] According to a preferred embodiment of the present invention, the chelating agent is 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). That is, the modified chelate is obtained by modifying DOTA with N-hydroxysuccinimide (NHS). Based on the total mass of the gelatin matrix nuclide carrier, the content of the modified chelate group, calculated as DOTA, is 2–10 wt%, preferably 3–10 wt%, and more preferably 5–10 wt%. This is beneficial for the stable loading of the metal nuclide.

[0051] Preparation method of gelatin matrix nuclide carrier

[0052] This invention also provides a method for preparing the gelatin-based nuclide carrier as described above, comprising the following steps: 1) preparation of the modified chelate; 2) preparation of the modified gelatin; 3) preparation of the gelatin-based nuclide carrier. Steps 1) and 2) are not sequential. A detailed description follows.

[0053] The modified chelate is obtained by modifying the chelating agent with N-hydroxysuccinimide. The modified gelatin is methacrylated gelatin or acrylated gelatin obtained by reacting initial gelatin with methacrylic anhydride or acrylic anhydride to achieve acrylation.

[0054] In some specific embodiments, the modified chelate is DOTA-NHS, which is obtained by modifying the chelating agent 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) with N-hydroxysuccinimide. The modified gelatin is methacrylated gelatin (GelMA) obtained by methacrylating the initial gelatin.

[0055] Preparation of modified chelate DOTA-NHS

[0056] The modified chelate is prepared by the following steps: (a) preparing compound 1; (b) preparing compound 2; (c) preparing compounds 3 and 4; and (d) obtaining compound 5, namely the modified chelate DOTA-NHS.

[0057] In step (a), 1,4,7,10-tetraazacyclododecane is reacted with tert-butyl haloacetate under alkaline conditions to obtain compound 1 as shown in formula (1).

[0058] (1);

[0059] The tert-butyl haloacetate can be tert-butyl bromoacetate or tert-butyl iodoacetate, preferably tert-butyl bromoacetate. Acetonitrile is used as the reaction solvent. Anhydrous sodium carbonate or anhydrous potassium carbonate is used to form alkaline conditions. The molar ratio of anhydrous sodium carbonate or anhydrous potassium carbonate to 1,4,7,10-tetraazacyclododecane is 3–6:1, preferably 4–6:1, more preferably 5–5.5:1. The molar ratio of tert-butyl haloacetate to 1,4,7,10-tetraazacyclododecane is 3–3.5:1, preferably 3.1–3.5:1, more preferably 3.2–3.3:1. This facilitates the BOC protection of the three amino groups.

[0060] Specifically, 1,4,7,10-tetraazacyclododecane is first dissolved in acetonitrile, then anhydrous sodium carbonate or anhydrous potassium carbonate is added, followed by dropwise addition of tert-butyl haloacetate under an ice-water bath (0–10°C), preferably at 0–5°C. After the addition is complete, the reaction is stirred at 20–35°C for 10–15 h. The preferred reaction temperature is 25–30°C. The preferred reaction time is 10–12 h. After the reaction is complete, the mixture is filtered to obtain a first filtrate. The first filtrate is concentrated to remove the solvent, yielding a concentrate. The concentrate is dissolved in a chlorinated hydrocarbon solvent, wherein the chlorinated hydrocarbon solvent is selected from dichloromethane or chloroform. The obtained chlorinated hydrocarbon solution is washed with saturated sodium dihydrogen phosphate, saturated disodium hydrogen phosphate, and saturated brine, respectively, and the organic phase is collected. The organic phase is dried with anhydrous sodium sulfate and filtered to obtain a second filtrate. The second filtrate is concentrated to obtain a crude product containing compound 1. The crude product containing compound 1 is subjected to column chromatography to obtain purified compound 1. The column chromatography used a dichloromethane and methanol system as the eluent, and gradient elution was performed. The volume ratio of methanol to the total volume of dichloromethane and methanol was 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, and 10%, respectively.

[0061] In step (b), compound 1 is reacted with methyl haloacetate under alkaline conditions to obtain compound 2 as shown in formula (2).

[0062] (2);

[0063] The haloacetic acid can be methyl chloroacetate, methyl bromoacetate, or methyl iodoacetate, preferably methyl bromoacetate. The reaction solvent is dichloromethane or chloroform, preferably dichloromethane. Anhydrous sodium carbonate or anhydrous potassium carbonate is used to form alkaline conditions. The molar ratio of anhydrous sodium carbonate or anhydrous potassium carbonate to compound 1 can be 3–6:1, preferably 4–6:1, more preferably 5–5.5:1. The molar ratio of haloacetic acid methyl ester to compound 1 can be 1–1.1:1, preferably 1.02–1.09:1, more preferably 1.05–1.08:1. This facilitates the formation of compound 2 and saves raw materials.

[0064] Specifically, compound 1 is first dissolved in dichloromethane or chloroform, then anhydrous potassium carbonate is added and stirred to mix. Then, methyl haloacetate is added dropwise under an ice-water bath to obtain a mixture. The mixture is stirred and reacted at 20–35°C for 10–15 h. The preferred reaction temperature is 25–30°C, and the preferred reaction time is 10–12 h. After the reaction, solid-liquid separation is performed to obtain a third filtrate. The third filtrate is concentrated to obtain a concentrate. Dichloromethane or chloroform is added to the concentrate to redissolve it, and then the solution is washed with saturated sodium dihydrogen phosphate, saturated disodium hydrogen phosphate, and saturated brine, respectively. The organic phase is collected, dried with anhydrous sodium sulfate, and filtered to obtain a fourth filtrate. The fourth filtrate is concentrated to obtain a crude product containing compound 2. The crude product containing compound 2 is subjected to column chromatography to obtain compound 2. The column chromatography used a dichloromethane and methanol system as the eluent for gradient elution. The volume ratio of methanol to the total volume of dichloromethane and methanol was 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, and 10%, respectively.

[0065] In step (c), compound 2 is hydrolyzed to obtain the intermediate product shown in formula (3) (denoted as compound 3); the intermediate product is reacted with N-hydroxysuccinimide in the presence of a dehydrating agent to obtain compound 4 shown in formula (4).

[0066] (3); (4);

[0067] Specifically, compound 2 is dissolved in methanol or ethanol (preferably methanol), followed by the addition of an aqueous solution of sodium hydroxide or potassium hydroxide (preferably sodium hydroxide solution), and the reaction is stirred at 20–35°C for 10–15 h. The reaction temperature is preferably 25–30°C, and the reaction time is preferably 10–12 h. The molar concentration of the sodium hydroxide or potassium hydroxide solution is 0.5–5 M, preferably 1–3 M, and more preferably 1–2 M. M refers to mol / L. The molar ratio of the added sodium hydroxide or potassium hydroxide solution to compound 2 can be 2.5–3.5:1, preferably 2.8–3.2:1, and more preferably 2.9–3.0:1. After the reaction is complete, the solution is concentrated to obtain a concentrated solution. The concentrated solution was adjusted to pH 2-3 with hydrochloric acid solution to precipitate the precipitate. After centrifugation, the lower precipitate was redissolved with dichloromethane or chloroform and then washed with saturated sodium dihydrogen phosphate, saturated disodium hydrogen phosphate, and saturated brine, respectively. The organic phase was collected, dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain a solid containing compound 3.

[0068] The solid product containing compound 3 was dissolved in dichloromethane or chloroform, and N,N'-dicyclohexylcarbodiimide (denoted as DCC) was added as a dehydrating agent, along with N-hydroxysuccinimide (denoted as NHS). The mixture was stirred at 20–35°C for 10–15 h. The preferred reaction temperature was 25–30°C, and the preferred reaction time was 10–12 h. The molar ratio of DCC to compound 3 was 1.1–1.5:1, preferably 1.15–1.3:1, and more preferably 1.2–1.21:1. The molar ratio of NHS to DCC was 1:1. After the reaction was complete, the mixture was allowed to stand, separated, and the organic phase was collected and concentrated to obtain a crude product containing compound 4. The crude product containing compound 4 was purified by column chromatography to obtain compound 4. The column chromatography used a dichloromethane and methanol system as the eluent, and gradient elution was performed. The volume ratio of methanol to the total volume of dichloromethane and methanol was 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, and 10%, respectively.

[0069] In step (d), compound 4 is hydrolyzed under acidic conditions to obtain compound 5 as shown in formula (5), i.e., the modified chelate;

[0070] (5);

[0071] Dichloromethane or chloroform is used as the reaction solvent, preferably dichloromethane. Trifluoroacetic acid (TFA) is used to create acidic conditions.

[0072] Specifically, compound 4 is dissolved in dichloromethane or chloroform, and then trifluoroacetic acid is added. The mixture is stirred at 20–35°C for 10–15 h. The preferred reaction temperature is 25–30°C, and the preferred reaction time is 10–12 h. After the reaction is complete, the mixture is concentrated to obtain a crude product containing compound 5. The crude product containing compound 5 is recrystallized from anhydrous diethyl ether to obtain compound 5, denoted as DOTA-NHS.

[0073] Preparation of methacrylamide gelatin

[0074] Initial gelatin (molecular weight <50 kDa) is dissolved in PBS solution to obtain an initial gelatin solution. The pH of the PBS solution is 8–9, preferably 8–8.5. The concentration of the initial gelatin solution is 0.005–0.25 g / mL, preferably 0.008–0.1 g / mL, more preferably 0.01–0.05 g / mL. The mass-to-volume ratio of initial gelatin to methacrylic anhydride can be 1 g: 0.2–2.3 mL, for example, 1 g: 0.25 mL, 1 g: 0.75 mL, 1 g: 1.25 mL, 1 g: 1.75 mL, or 1 g: 2.25 mL.

[0075] Specifically, methacrylic anhydride (MAAH) is added to the initial gelatin solution and reacted at 50–60°C for 3–5 hours. The preferred reaction temperature is 50–55°C, and the preferred reaction time is 3–4 hours. After the reaction, water (3–4 times the volume of the reaction water) is added to the reaction system to dilute it. Then, the solution is dialyzed for 3 days at room temperature using a dialysis bag with a capacity of 1800–2400 Da (e.g., 2000–2400 Da) to remove salts and methacrylic acid. The dialyzed solution is then vacuum filtered, and the filtrate is collected and freeze-dried at -30°C or below (preferably -50°C) to obtain methacrylamide gelatin (GelMA), i.e., modified gelatin. The degree of substitution of the obtained modified gelatin is 10–90%, for example, 10%, 30%, 50%, 70%, and 90%.

[0076] Preparation of gelatin-based nuclide carriers

[0077] Modified gelatin was dissolved in a polar organic solvent, then a modified chelate and a basic reagent were added and mixed to obtain a reaction solution. The reaction solution was dialyzed, and the dialyzed solution was filtered and freeze-dried to obtain a gelatin-based nuclide carrier. This method is beneficial for obtaining gelatin-based nuclide carriers that can stably load metal nuclides and / or non-metal nuclides.

[0078] The mass-to-volume ratio of modified gelatin to polar organic solvent can be 1g:8-20mL, preferably 1g:9-15mL, and more preferably 1g:10-12mL. The mass ratio of modified gelatin to modified chelate can be 1g:(10-200)mg, preferably 1g:(10-180)mg, more preferably 1g:(15-150)mg, and even more preferably 1g:(25-110)mg.

[0079] The polar organic solvent is selected from one or more of dimethyl sulfoxide, dimethylformamide, and dimethylacetamide, preferably dimethyl sulfoxide. The basic reagent is selected from one or more of triethylamine, tributylamine, diisopropylethylamine, and pyridine, preferably triethylamine. The reaction temperature can be 20–35°C, preferably 25–30°C. The reaction time can be 10–15 h, preferably 10–12 h.

[0080] The dialysis bag has a molecular weight cutoff of 1800–2400 Da, preferably 2000–2400 Da. The freeze-drying temperature is below -30°C, preferably below -50°C. The resulting gelatin-based nuclide carrier is injectable.

[0081] Gelatin-based loaded radionuclide products

[0082] This invention provides a gelatin-based loaded radionuclide product obtained by reacting a gelatin-based radionuclide carrier as described above with radionuclide ions, wherein the nuclear element in the radionuclide ions is selected from metallic radionuclides and / or non-metallic radionuclides. Metallic radionuclides are selected from... 177 Lu、 90 Y、 67 Ga、 64 Cu、 99m Tc and 89 One or more of Sr, preferably 177 Lu; nonmetallic nuclides are selected from 125 I, 131 I, 18 F and 32 One or more of P, preferably 125 I. The gelatin-based nuclide carrier of the present invention can stably load metallic and / or non-metallic nuclides. This gelatin-based nuclide-loaded product is injectable, enabling local drug delivery and avoiding the drawbacks of systemic administration.

[0083] Preparation method of gelatin matrix loaded with radionuclides

[0084] The method for preparing gelatin matrix-loaded radionuclide products of the present invention includes the following steps: dissolving the gelatin matrix radionuclide carrier in a buffer solution, then adding a salt containing the radionuclide, mixing and reacting, and after the reaction is completed, performing ultrafiltration and centrifugation purification to obtain the gelatin matrix-loaded radionuclide product.

[0085] According to a specific embodiment of the present invention, a gelatin-based radionuclide carrier (e.g., GelMA-DOTA) is dissolved in an ammonium acetate buffer solution with a pH of 4-5, and then added... 177 LuCl3 was vortexed at 40–50 °C for 1–1.5 h. The reaction solution was then purified by ultrafiltration centrifugation. An ultrafiltration membrane with a molecular weight cutoff of 2000–2200 Da was used, and centrifugation was performed at 3500–4500 r / min for 5–10 minutes. Under centrifugal force, unreacted molecules smaller than the ultrafiltration membrane's molecular weight cutoff were removed. 177 Lu ions and impurities such as buffer solution pass smoothly through the ultrafiltration membrane into the filtrate, while the loaded... 177 Lu's GelMA-DOTA was effectively retained within the ultrafiltration tube, thus obtaining a gelatin-supported matrix. 177 Lu nuclide products. The principle is to use the chelating groups in GelMA-DOTA to stably bind metal nuclides.

[0086] According to another specific embodiment of the present invention, a gelatin-based radionuclide carrier (e.g., GelMA-DOTA) is dissolved in phosphate buffer (PBS, pH=6-7), and Na is added. 125I and chloramine-T solution. Vortex reaction at room temperature for 1–1.5 h, then add sodium thiosulfate solution to terminate the reaction, followed by the addition of potassium iodide solution to provide cold iodine to compete with unreacted reactivity. 125 First, reduce its adsorption to the container. Then, purify the reaction solution using ultrafiltration centrifugation. Use an ultrafiltration membrane with a molecular weight cutoff of 2000–2200 Da, centrifuge at 3500–4500 r / min for 5–10 minutes. Under centrifugal force, unreacted raw materials and impurities such as buffer solution smaller than the ultrafiltration membrane's molecular weight cutoff pass smoothly through the membrane into the filtrate, while the loaded... 125 The gelMA-DOTA in I was effectively retained within the ultrafiltration tube, thus obtaining a gelatin-supported matrix. 125 I-type nuclide products. Among them, when loading non-metallic nuclides, the principle is to utilize the tyrosine residues on the main chain of the GelMA-DOTA molecule to stably bind the non-metallic nuclides.

[0087] According to another specific embodiment of the present invention, a gelatin-based radionuclide carrier (e.g., GelMA-DOTA) is dissolved in an ammonium acetate buffer solution at pH 4-5, and then added... 177 LuCl3 was vortexed at 40–50 °C for 1–1.5 h. The reaction solution was then purified by ultrafiltration centrifugation. An ultrafiltration membrane with a molecular weight cutoff of 2000–2200 Da was used, and centrifugation was performed at 3500–4500 r / min for 5–10 minutes. Under centrifugal force, unreacted molecules smaller than the ultrafiltration membrane's molecular weight cutoff were removed. 177 Lu ions and impurities such as buffer solution pass smoothly through the ultrafiltration membrane into the filtrate, while the loaded... 177 Lu's GelMA-DOTA was effectively retained within the ultrafiltration tube, thus obtaining a gelatin-supported matrix. 177 Lu radionuclide products, denoted as GelMA-DOTA- 177 Lu. Then, the gelatin matrix is ​​loaded. 177 Lu-based radionuclides (GelMA-DOTA-) 177 Lu) is dissolved in phosphate buffer (PBS, pH 6-7), and Na is added. 125 I and chloramine-T solution. Vortex reaction at room temperature for 1–1.5 h, then add sodium thiosulfate solution to terminate the reaction, followed by the addition of potassium iodide solution to provide cold iodine to compete with unreacted reactivity. 125 First, reduce its adsorption to the container. Then, purify the reaction solution using ultrafiltration centrifugation. Use an ultrafiltration membrane with a molecular weight cutoff of 2000–2200 Da, centrifuge at 3500–4500 r / min for 5–10 minutes. Under centrifugal force, unreacted raw materials and impurities such as buffer solution smaller than the ultrafiltration membrane's molecular weight cutoff pass smoothly through the membrane into the filtrate, while the loaded... 125I's GelMA-DOTA- 177 Lu is effectively retained within the ultrafiltration tube, thus obtaining a gelatin matrix loading. 177 Lu and 125 I-type nuclide products can be denoted as GelMA-DOTA- 177 Lu- 125 I.

[0088] Compositions for use in radiographic imaging or radiotherapy

[0089] This invention provides a composition for radiographic imaging or radiotherapy, comprising a gelatin-based loaded radionuclide product and a photoinitiator as described above; it can solidify into a gel under light irradiation at a wavelength of 385–450 nm. This facilitates local drug delivery via injection, resulting in better targeting.

[0090] The photoinitiator is selected from one or more of benzophenone, lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (LAP), and 2,4,6-trimethylbenzoyl-diphenylphosphine oxide, preferably LAP.

[0091] The mass ratio of the gelatin matrix loaded with nuclide product to the photoinitiator is 100:0.5 to 2.5, preferably 100:0.8 to 2, and more preferably 100:1 to 1.5.

[0092] In this invention, a gelatin-based loaded radionuclide product and a photoinitiator are dissolved in a solvent to form a mixed solution, wherein the solvent used is water. The mixed solution is then injected or sprayed onto the site to be imaged or treated, followed by irradiation with light of a wavelength of 385–450 nm for 5–10 seconds, causing it to solidify and form a gel (i.e., the gelatin-based loaded radionuclide product undergoes a polymerization reaction under the action of the photoinitiator). The preferred wavelength is 395–450 nm.

[0093] application

[0094] This invention also provides the use of a composition for radiographic imaging or radiotherapy in the preparation of radiotherapeutic drugs or radiographic imaging agents. The composition for radiographic imaging or radiotherapy can be injected, enabling local administration and avoiding the drawbacks of systemic administration. Irradiating the composition with light of a wavelength of 385–450 nm can solidify it into a gel, which can then be used for radiotherapy or radiographic imaging.

[0095] Test methods

[0096] When performing structural characterization on compound DOTA-NHS, the following methods were used: 1 ¹H NMR (Bruker AVANCE III 400), with deuterated DMSO as solvent.

[0097] In the following preparation example 1, the eluent used in column chromatography was a dichloromethane and methanol system, and gradient elution was adopted. The volume ratio of methanol to the total volume of dichloromethane and methanol was 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, and 10%, respectively.

[0098] The chloramine T solution used in the following examples was prepared by dissolving sodium N-chloro-4-toluenesulfonamide in PBS solution at pH=7 to obtain a chloramine T solution with a concentration of 1 mg / mL.

[0099] Preparation Example 1 - Preparation of Modified Chelates

[0100] Step (a), Preparation of Compound 1

[0101] Reaction equation:

[0102]

[0103] 1,4,7,10-tetraazacyclododecane (1.0 g, 5.8 mmol) was dissolved in 20 mL of acetonitrile, followed by the addition of anhydrous sodium bicarbonate (2.4 g, 29.0 mmol), and the mixture was stirred at room temperature for 15 minutes. Then, tert-butyl bromoacetate (2.7 mL, 18.5 mmol) was added dropwise at 0 °C to obtain a mixture. The mixture was stirred overnight at 30 °C. After the reaction was complete, the mixture was filtered to obtain a first filtrate. The solvent acetonitrile was removed from the first filtrate by rotary evaporation to obtain a concentrate. Dichloromethane was added to the concentrate and redissolved to obtain a dichloromethane solution. The dichloromethane solution was washed with saturated sodium dihydrogen phosphate, saturated disodium hydrogen phosphate, and saturated brine, respectively, and the organic phase was collected. The organic phase was dried over anhydrous sodium sulfate and then filtered to obtain a second filtrate. The solvent dichloromethane was removed from the second filtrate by rotary evaporation to obtain a crude product containing compound 1. The crude product containing compound 1 was purified by silica gel column chromatography to obtain compound 1.

[0104] Step (b), Preparation of Compound 2

[0105] Reaction equation:

[0106]

[0107] Compound 1 (1.0 g) prepared above was dissolved in 20 mL of dichloromethane, followed by the addition of anhydrous potassium carbonate (1.3 g, 9.7 mmol), and stirred at room temperature for 15 minutes. Then, methyl bromoacetate (0.2 mL, 2.1 mmol) was added dropwise at 0 °C to obtain a mixture. The mixture was stirred overnight at 30 °C. After the reaction was complete, the mixture was filtered to obtain a third filtrate. The third filtrate was rotary evaporated to obtain a concentrated rotary evaporator. The concentrated rotary evaporator was redissolved in dichloromethane to obtain a redissolved solution. The redissolved solution was washed with saturated sodium dihydrogen phosphate, saturated disodium hydrogen phosphate, and saturated brine, respectively. The organic phase was collected, dried over anhydrous sodium sulfate, and filtered to obtain a fourth filtrate. The fourth filtrate was concentrated by rotary evaporation to obtain a crude product containing compound 2. The crude product containing compound 2 was purified by silica gel column chromatography to obtain compound 2.

[0108] Step (c), Preparation of Compound 4

[0109] Reaction equation:

[0110]

[0111] Compound 2 (1.0 g) prepared above was dissolved in 10 mL of methanol, followed by the addition of 5 mL of 1 M sodium hydroxide aqueous solution. The mixture was stirred overnight at 30 °C. After the reaction was complete, the organic solvent was removed by rotary evaporation to obtain a concentrated solution. The pH of the concentrated solution was adjusted to 2-3 with 1 M hydrochloric acid solution, and a precipitate was formed. The precipitate was centrifuged, and the lower precipitate was redissolved in dichloromethane. The solution was then washed with saturated sodium dihydrogen phosphate, saturated disodium hydrogen phosphate, and saturated brine, respectively. The organic phase was collected, dried over anhydrous sodium sulfate, filtered, and the filtrate was rotary evaporated to remove the organic solvent, yielding a solid containing compound 3.

[0112] The solid containing compound 3 (1.0 g) was dissolved in 20 mL of anhydrous dichloromethane. N,N'-dicyclohexylcarbodiimide (0.4 g, 1.9 mmol, denoted as DCC) was added as a dehydrating agent, along with N-hydroxysuccinimide (0.2 g, 1.9 mmol, denoted as NHS). The mixture was stirred overnight at 30 °C, allowed to stand, separated, and the organic phase was collected. The organic solvent was removed by rotary evaporation to obtain the crude product containing compound 4. The crude product containing compound 4 was purified by silica gel column chromatography to obtain compound 4.

[0113] Step (d), preparation of compound 5 (i.e., DOTA-NHS)

[0114] Reaction equation:

[0115]

[0116] Compound 4 (1.0 g) prepared above was dissolved in 10 mL of dichloromethane, and then 10 mL of trifluoroacetic acid (TFA) was added dropwise. The mixture was stirred overnight at 30 °C. After the reaction was completed, the organic solvent was removed by rotary evaporation to obtain a crude product containing compound 5. The crude product containing compound 5 was recrystallized from anhydrous diethyl ether to obtain compound 5, denoted as DOTA-NHS.

[0117] The structure of compound 5 (DOTA-NHS) was characterized. 1 The H NMR results are shown in […]. Figure 1 .

[0118] Preparation Examples 2-6 - Preparation of Modified Gelatin

[0119] Reaction equation:

[0120]

[0121] 1.0 g of gelatin (i.e., initial gelatin, molecular weight <50 kDa) was dissolved in 100 mL of PBS solution at pH 8.5 to obtain the initial gelatin solution. A certain amount of methacrylic anhydride (MAAH) was added to the initial gelatin solution, and the reaction was carried out at 50 °C for 3 h. After the reaction was completed, 300 mL of deionized water was added to the reaction system for dilution, and then the solution was dialyzed at room temperature for 3 days using a 2000 Da dialysis bag to remove salts and methacrylic acid. The dialyzed solution was vacuum filtered, the filtrate was collected, and the filtrate was freeze-dried at -50 °C to obtain methacrylamide gelatin (GelMA), i.e., modified gelatin.

[0122] The amounts of gelatin, methacrylic anhydride (MAAH), and the degree of substitution of the modified gelatin are shown in Table 1 below.

[0123] The degree of substitution of modified gelatin was tested and calculated using the following method: the trinitrobenzenesulfonic acid (TNBS) method. This method utilizes the quantitative reaction of primary amino groups with TNBS. TNBS reacts with unmodified primary amino groups (i.e., free amino groups) on the gelatin matrix under weakly alkaline conditions to form an intermediate complex with chromophores. The number of free amino groups in both unmodified and methacrylamide gelatin was determined using a UV-Vis spectrophotometer, thereby calculating the degree of substitution at different amounts of methacrylic anhydride. A standard curve formed using glycine solution was used as an external standard curve. Preparation of the standard curve: The absorbance of glycine standard solutions with concentrations of 0 μg / mL, 2 μg / mL, 4 μg / mL, 8 μg / mL, 12 μg / mL, and 16 μg / mL was measured at 340 nm. A standard curve of absorbance versus concentration was plotted. The expression for the standard curve is y = 28.127x - 0.4594, R0. 2 =0.9946.

[0124] Formula for calculating degree of substitution:

[0125] .

[0126] Table 1

[0127]

[0128] Examples 1-5 - Preparation of gelatin-based nuclide carriers

[0129] Reaction equation:

[0130]

[0131] 1.0 g of GelMA (using the 10% substitution degree GelMA from Preparation Example 2) prepared as described above was dissolved in 10 mL of dimethyl sulfoxide to obtain a methacrylamide gelatin solution. A certain amount of the modified chelate (DOTA-NHS) and triethylamine (TEA) prepared in Preparation Example 1 were added to the methacrylamide gelatin solution, and the mixture was stirred overnight at 30°C. After the reaction was complete, the reaction solution was dialyzed for 3 days using a dialysis bag (2000 Da). The dialyzed solution was filtered and freeze-dried at -50°C to obtain the gelatin-based nuclide carrier, denoted as GelMA-DOTA.

[0132] The content of modified chelate groups (calculated as DOTA) in the gelatin matrix nuclide carriers corresponding to different feed ratios is shown in Table 2 below.

[0133] The DOTA content was tested and calculated using the following method: the trinitrobenzenesulfonic acid (TNBS) method. This method utilizes the quantitative reaction of primary amino groups with TNBS. TNBS reacts with unmodified primary amino groups (i.e., free amino groups) on the methacryloylated gelatin matrix under weakly alkaline conditions to form an intermediate complex with chromophores. The number of free amino groups in the DOTA-modified methacryloylated gelatin is determined using a UV-Vis spectrophotometer, thereby calculating the DOTA content at different reaction amounts. A standard curve formed using glycine solution was used as an external standard curve. The preparation and expression of the standard curve are described above.

[0134] The formula for calculating DOTA content is as follows:

[0135] .

[0136] Table 2

[0137]

[0138] The resulting gelatin-based nuclide carrier GelMA-DOTA can stably chelate metal nuclides using DOTA on its molecular side chain and stably bind non-metal nuclides using tyrosine residues on its molecular main chain.

[0139] Example 6 - Gelatin-based loaded nuclide products (loaded metal nuclides) 177 Lu)

[0140] Reaction equation:

[0141]

[0142] The specific steps are as follows: Dissolve the GelMA-DOTA (DOTA content 10wt%) prepared in Example 5 in 0.5 M ammonium acetate buffer solution at pH=4, and then add... 177 LuCl3 was vortexed at 50℃ for 1 h. The reaction solution was then purified by ultrafiltration centrifugation. Specifically, an ultrafiltration membrane with a molecular weight cutoff of 2000 Da was used, and centrifugation was performed at 4000 r / min for 5 minutes. Under the strong centrifugal force, unreacted molecules smaller than the ultrafiltration membrane's molecular weight cutoff were removed. 177 Lu ions and impurities such as buffer solution pass smoothly through the ultrafiltration membrane into the filtrate, while the loaded... 177 Lu's gelatin-based supported nuclide products were effectively retained within the ultrafiltration tube, achieving highly efficient separation and purification. The resulting supported... 177 Lu's gelatin-based loaded radionuclide products can be denoted as GelMA-DOTA- 177 Lu.

[0143] Example 7 - Gelatin-based loaded nuclide products (loaded with non-metallic nuclides) 125 I)

[0144] Reaction equation:

[0145]

[0146] The gelatin-based nuclide carrier GelMA-DOTA has tyrosine residues on its molecular backbone, which can stably bind non-metallic nuclides. The specific steps are as follows: GelMA-DOTA (10 wt% DOTA content) prepared in Example 5 was dissolved in 0.05 M phosphate buffer (PBS, pH=7), and Na... 125 I and chloramine-T solution. After vortexing at room temperature for 1 h, the reaction was terminated by adding sodium thiosulfate solution, followed by the addition of potassium iodide solution to provide cold iodine to compete with unreacted reactivity. 125 I. Reduce its adsorption to the container. Then purify the reaction solution using the ultrafiltration centrifugation method described in Example 2. Obtain the loaded solution. 125I's gelatin-based nuclide-loaded product is designated GelMA-DOTA- 125 I.

[0147] Example 8 - Gelatin-based loaded nuclide products (simultaneously loaded with metal nuclides) 177 Lu and nonmetallic nuclides 125 I)

[0148] Reaction equation:

[0149]

[0150] First, following the method of Example 6, the GelMA-DOTA (DOTA content 10wt%) prepared in Example 5 was dissolved in 0.5 M ammonium acetate buffer at pH=4, and then... 177 LuCl3 was vortexed at 50 °C for 1 h. The reaction solution was then purified by ultrafiltration and centrifugation to achieve GelMA-DOTA loading. 177 Lu, the resulting product is denoted as GelMA-DOTA- 177 Lu.

[0151] Then, following the method in Example 7, GelMA-DOTA- 177 Lu was processed using the chloramine-T method. 125 The loading of I was also purified using ultrafiltration and centrifugation, ultimately achieving simultaneous loading of gelatin-based nuclide carriers GelMA-DOTA. 177 Lu and 125 I. The resulting product is denoted as GelMA-DOTA- 177 Lu- 125 I.

[0152] Experimental Example

[0153] After anesthetizing SD rats (SD rats do not require modeling), a craniotomy was performed by drilling a hole in the skull, and the loaded substance was sprayed onto the dura mater in a single application. 125 I's GelMA-DOTA- 125 I (prepared according to the method of Example 7) and a mixed aqueous solution containing LAP photoinitiator (the mass of the photoinitiator is 1 wt% of the mass of the gelatin matrix loaded with radionuclide product) were irradiated for 10 s under a light source with a power of 10 W and a wavelength of 395 nm to solidify and form a gel. Then, the skull was closed, the skin was sutured, and the surgical site was disinfected. SD rats underwent SPECT (single-photon emission computed tomography) imaging on days 4, 16, and 60 postoperatively. The results are as follows: Figure 2 As shown, the radioactive signals at the administration site and thyroid gland were strong on days 4 and 16, but significantly weakened on day 60. The radioactive signals in major organs such as the kidneys, spleen, and liver remained at a weak level throughout, showing no significant trend.

[0154] Therefore, it can be seen that the composition formed by the gelatin matrix loaded with radionuclide product and photoinitiator of the present invention can achieve local drug delivery and can be used as a radioactive imaging agent after photocuring.

[0155] This invention is not limited to the above-described embodiments. Any modifications, improvements, or substitutions that can be conceived by those skilled in the art without departing from the essential content of this invention fall within the scope of this invention.

Claims

1. A gelatin-based nuclide carrier, characterized in that, It is prepared from raw materials including modified gelatin and modified chelates; Among them, the modified gelatin is methacrylamide gelatin; The modified chelate is obtained by modifying the chelating agent with N-hydroxysuccinimide; the chelating agent is 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid. The mass ratio of modified gelatin to modified chelate is 1g:(10-200)mg; The preparation method of this gelatin-based nuclide carrier includes the following steps: 1) Provide modified chelates; 2) Provide modified gelatin; 3) Dissolve the modified gelatin in a polar organic solvent, then add the modified chelate and alkaline reagent, mix and react to obtain a reaction solution; dialyze the reaction solution, filter the dialyzed solution and freeze-dry it to obtain the gelatin matrix nuclide carrier; The polar organic solvent is selected from one or more of dimethyl sulfoxide, dimethylformamide, and dimethylacetamide; the basic reagent is selected from one or more of triethylamine, tributylamine, diisopropylethylamine, and pyridine. Steps 1) and 2) are not in any particular order.

2. The gelatin-based nuclide carrier according to claim 1, characterized in that, The mass ratio of modified gelatin to modified chelate is 1g:(15-150)mg.

3. The method for preparing a gelatin-based nuclide carrier according to claim 1, characterized in that, Includes the following steps: 1) Provide modified chelates; 2) Provide modified gelatin; 3) Dissolve the modified gelatin in a polar organic solvent, then add the modified chelate and alkaline reagent, mix and react to obtain a reaction solution; dialyze the reaction solution, filter the dialyzed solution and freeze-dry it to obtain the gelatin matrix nuclide carrier; The polar organic solvent is selected from one or more of dimethyl sulfoxide, dimethylformamide, and dimethylacetamide; the basic reagent is selected from one or more of triethylamine, tributylamine, diisopropylethylamine, and pyridine. Steps 1) and 2) are not in any particular order.

4. The preparation method according to claim 3, characterized in that, The modified gelatin is methacrylated gelatin obtained by reacting initial gelatin with methacrylic anhydride; the initial gelatin has a molecular weight of less than 50 kDa.

5. The preparation method according to claim 4, characterized in that, The modified chelate is prepared by the following steps: (a) 1,4,7,10-tetraazacyclododecane was reacted with tert-butyl haloacetate under alkaline conditions to obtain compound 1 as shown in formula (1); (b) Compound 1 was reacted with methyl haloacetate under alkaline conditions to give compound 2 as shown in formula (2); (c) Hydrolyze compound 2 to obtain the intermediate product shown in formula (3); react the intermediate product with N-hydroxysuccinimide in the presence of a dehydrating agent to obtain compound 4 shown in formula (4); (d) Hydrolyze compound 4 under acidic conditions to obtain compound 5 as shown in formula (5), i.e., the modified chelate; （1）； （2）； （3）； （4）； （5）。 6. The preparation method according to claim 4, characterized in that, The degree of substitution of the methacrylamide gelatin is 10% to 90%; the content of the modified chelate group, calculated as DOTA, is 2 wt% to 10 wt% based on the total mass of the gelatin matrix nuclide carrier; DOTA is an abbreviation for 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid.

7. A gelatin-based loaded radionuclide product, characterized in that, It is obtained by reacting the gelatin matrix nuclide carrier as described in claim 1 or 2 with nuclide ions, wherein the nuclear element in the nuclide ions is selected from metallic nuclides and / or non-metallic nuclides; Wherein, the metal nuclide is 177 Lu; the non-metallic nuclide is 125 I.

8. The gelatin-based loaded radionuclide product according to claim 7, characterized in that, It is obtained by reacting the gelatin matrix nuclide carrier with non-metallic nuclide ions; wherein, the non-metallic nuclide in the non-metallic nuclide ions is... 125 I.

9. A composition for use in radiographic imaging or radiotherapy, characterized in that, It includes the gelatin matrix loaded with nuclide product as described in claim 8 and a photoinitiator; it can be cured to form a gel under light irradiation with a wavelength of 385-450 nm.

10. The use of the composition for radioimaging or radiotherapy according to claim 9 in the preparation of radiotherapeutic drugs or radioimaging agents.

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