GMP (Good Manufacturing Practice) large-scale production method for synthesizing glucan-guanidine-bisphosphonate conjugate by one-pot method

Abstract

The present invention relates to a large scale GMP synthesis method of a modified glucan conjugate, and more particularly, to a glucan-guanidine-bisphosphonate conjugate wherein the bisphosphonate is an alendronate group. The large-scale GMP synthesis method related to the invention can be used for producing the glucan-guanidine-bisphosphonate conjugate with high purity and medicinal grade. The invention also relates to an application of the glucan-guanidine-bisphosphonate conjugate in a medicine for treating tumors.

Description

Technical Field

[0001] This invention relates to a method for synthesizing modified dextran conjugates, and more specifically, to a large-scale GMP synthesis method for obtaining pharmaceutical-grade dextran-guanidine-bisphosphonate conjugates. The invention also relates to the use of said dextran-guanidine-bisphosphonate conjugates in medicaments for treating tumors. Background Technology

[0002] Dextran-guanidine-bisphosphonate conjugate is a candidate drug for the treatment of bone-related cancer metastases accompanied by increased osteocyte activity. This conjugate demonstrated good safety and efficacy in clinical studies (Phase I and II) without any drug-related serious adverse events (Thellenberg-Karlsson et al. 2023).

[0003] Holmberg et al. (2010) and Holmberg et al. (2009) disclosed the biological effects of dextran-guanidine-bisphosphonate conjugates. The inventors' patents EP2131867B1 and EP2274018B1 describe the functions and properties of dextran-guanidine-bisphosphonate conjugates, as well as methods for the micro-preparation of said conjugates.

[0004] Clinical studies of dextran-guanidine-bisphosphonate conjugates require large-scale manufacturing processes that comply with Good Manufacturing Practice (GMP). GMP refers to processes designed, developed, and implemented according to GMP standards. GMP is a set of regulations, guidelines, and procedures mandated by regulatory agencies (such as the US FDA and the European EMA) to ensure that products, particularly pharmaceuticals, medical devices, and food, can be consistently and stably manufactured and controlled to meet quality standards.

[0005] The differences between laboratory synthesis and Good Manufacturing Practice (GMP) synthesis include scale, regulatory requirements, equipment and operational controls, and level of documentation. Scale-up production of drug candidates is challenging and complex. It is a multi-step, complex process involving technical and chemical challenges, while also requiring increased reactant feed amounts (APIs, the active pharmaceutical ingredient). It necessitates careful consideration, balancing, and optimization of API concentrations, reaction kinetics, safety, equipment design, API quality, cost, and regulatory compliance. Problems that arise in large-scale production often do not occur at small scale and can be unpredictable. Successful GMP manufacturing processes for drug candidates bear little resemblance to the original laboratory synthesis processes used to invent them, retaining only the principles and product consistency. The development of such GMP manufacturing processes can take several years.

[0006] The transition from laboratory-scale to GMP-scale operations often fails, for example, in scaling up from small-scale R&D to GMP standards where good experimental results cannot be reproduced. Due to insurmountable chemical / technical limitations and constraints, it may simply be impossible to mass-produce investigational drugs under GMP standards.

[0007] The translation of drug development from preclinical to clinical trials is subject to stringent regulatory requirements, particularly the production of drug candidates. This necessitates large-scale GMP-compliant synthesis methods that yield pharmaceutical-grade products. Every minute step of the synthesis process must strictly adhere to GMP standards. A complete Investigative Drug Application (IMPD) document is provided to regulatory agencies to obtain detailed information about the drug and for approval. The IMPD includes comprehensive chemical and pharmaceutical information, covering drug quality standards, analytical methods, preclinical information, clinical information, and regulatory and legal information. Passing the IMPD review is crucial and mandatory before obtaining regulatory approval to conduct clinical trials. This ensures that the investigational drug meets GMP standards before entering clinical trials with human subjects. Summary of the Invention

[0008] The purpose of this invention is to provide a large-scale GMP synthesis method, which is also an optimized and improved preparation method for dextran-guanidine-bisphosphonate conjugates, wherein the bisphosphonate group is preferably an alendronate group.

[0009] The present invention achieves the above-mentioned objective through the synthesis process described in the independent claim. Preferred embodiments of the present invention are described in the dependent claims.

[0010] The process is a sequential one-pot method, including the following steps: dextran activation, alendronate conjugation, aminoguanidine conjugation and reductive amination, followed by purification by tangential flow filtration (TFF) with optimized filtration cycle.

[0011] Existing methods operate at the µL / mL level, which is unsuitable for large-scale (L) synthesis. This invention produces high-purity pharmaceutical-grade (GMP) dextran-guanidine-bisphosphonate conjugates, wherein one molecule of dextran is conjugated to approximately 5 to 8 alendronate molecules and approximately 40-45 aminoguanidine molecules. The method described in this invention is preferably a large-scale (L) process. The substitution relationship gives the conjugates an affinity for bone remodeling sites and tumor cytotoxicity, achieving a dual therapeutic effect.

[0012] Maintaining an acidic pH during dextran activation ensures efficient activation in a short time. The use of a buffer and pH control during alendronate conjugation ensures alendronate remains dissolved, preventing precipitation. Subsequent aminoguanidine conjugation and reductive amination are carried out without separation steps.

[0013] Purification is a crucial step in the GMP manufacturing process of pharmaceuticals. The purification method needs to ensure high purity, high recovery rate, reproducibility (small batch-to-batch variability), and reasonable process time. In addition, the method must be adaptable to large-volume production.

[0014] In this invention, optimized TFF ensures high purity and high synthesis yield (recovery) of the pharmaceutical product. The pH of the pharmaceutical solution is adjusted to pH 6.0–7.0 to ensure complete and continuous solubility of the pharmaceutical product. Under these conditions, the pharmaceutical product is stable and has an extended shelf life.

[0015] In this method, the obtained dextran-guanidinium-bisphosphonate product is diluted with sodium acetate and sodium chloride, the pH is adjusted to pH 6.0 - pH 7.0, and the product is filtered through a bacterial retention filter.

[0016] The inventors have discovered that when sodium acetate is used, it prevents the guanidinyl and alendronate groups from undergoing an electrostatic reaction to form byproducts, which could lead to precipitation, resulting in production batch failures and non-compliance.

[0017] The present invention also relates to the use of dextran-guanidine-bisphosphonate conjugates as tumor-killing therapy for cancers in bone.

[0018] This invention is the first and only method describing the GMP manufacturing process for this pharmaceutical product. Currently, there are no other GMP manufacturing methods for dextran-guanidine-bisphosphonate conjugates. This invention has not been previously published or described. Attached Figure Description

[0019] The present disclosure will be further described in detail below with reference to the accompanying drawings and preferred embodiments.

[0020] Figure 1 The main molecular structures of the dextran-guanidine-bisphosphonate conjugates obtained by the chemical method of the present invention are shown. Figure 2 The synthetic route for the dextran-guanidine-bisphosphonate conjugate is shown, comprising the following steps: a) + b) dextran activation, c) alendronate conjugation, d) aminoguanidine conjugation, e) reductive amination, f) purification; Figure 3The in vitro potency assay (FMCA) of the GMP batches after storage at 5°C is shown, with Gmp1 stored for 22 months, Gmp2 for 11 months, and Gmp3 for 9 months. The y-axis represents the percentage of cell viability, and the x-axis represents the drug concentration. The approved limit is IC50 ≥ 50% at 2–6 µM. Detailed Implementation

[0021] This invention relates to a method for synthesizing dextran-guanidine-bisphosphonate conjugates, characterized by comprising the following sequential steps: (a) Add periodate to an aqueous solution containing dextran, followed by the addition of sulfuric acid; (b) Add sodium dihydrogen phosphate to the solution obtained in step (a), and then add sodium hydroxide; (c) Add alendronate to the solution obtained in step (b); (d) Add aminoguanidine to the solution obtained in step (c); (e) Add sodium borohydride solution to the solution obtained in step (d) to produce a dextran-guanidine-bisphosphonate conjugate; (f) The dextran-guanidine-bisphosphonate conjugate obtained in step (e) is preferably purified by tangential flow filtration to produce a purified dextran-guanidine-bisphosphonate conjugate; (g) The purified dextran-guanidine-bisphosphonate conjugate obtained in step (f) is diluted with sodium acetate and sodium chloride to obtain a diluted dextran-guanidine-bisphosphonate conjugate. The pH of the diluted dextran-guanidine-bisphosphonate conjugate is adjusted to pH 6.0 - pH 7.0, and the diluted dextran-guanidine-bisphosphonate conjugate is filtered.

[0022] Preferably, the method is a one-pot GMP process.

[0023] More preferably, the method is a large-scale one-pot GMP process.

[0024] In step (a), periodate is added to the dextran solution. The dextran solution can be prepared by adding dextran to water in one or more batches to dissolve it, for example, 1, 2 or 3 batches.

[0025] In step (c), alendronate may be added in one or more batches, for example, in 2 or 3 batches.

[0026] Similarly, in step (d), aminoguanidine can be added in one or more batches, for example, 2 or 3 batches.

[0027] According to one embodiment of the present invention, the bisphosphonate is an aminobisphosphonate, preferably alendronate-based.

[0028] According to another embodiment of the invention, in step (a), the pH is adjusted to pH 1.5 - pH 1.9, preferably pH 1.55 - pH 1.8, and more preferably pH 1.6 - pH 1.7. The acidic pH during dextran activation ensures efficient activation within a short reaction time. pH adjustment is achieved by slowly adding sulfuric acid (95-98%) to the reaction mixture and monitoring the pH until the desired pH is reached. The pH can be adjusted to 1.50, 1.55, 1.60, 1.65, 1.70, 1.75, or 1.80, or any pH within the range defined above.

[0029] According to another embodiment of this disclosure, in step (b), the pH is adjusted to pH 7-pH 7.7, preferably pH 7.3-pH 7.7, and more preferably pH 7.3-pH 7.5. pH adjustment is performed by adding NaOH (10 mol / L aqueous solution) to the reaction mixture and monitoring the pH to the desired value to achieve neutrality. The pH can be adjusted to 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, or 7.8, or any pH within the range defined above.

[0030] According to one embodiment of the present invention, steps (a), (c), or both (a) and (c) are performed by stirring the solution under light-protected conditions.

[0031] Step (a) takes 40 to 50 minutes, preferably 45 minutes. The time is calculated from the moment the sulfuric acid is added.

[0032] Step (c) requires 55 to 70 minutes, preferably 60 minutes. The time is calculated from the moment alendronate is added.

[0033] According to one embodiment of the invention, steps (d), (e), or both (d) and (e) involve stirring the solution at room temperature. The temperature can be from 15°C to 25°C. Specifically, the temperature can be 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, or 25°C, or a range defined by any two of these values.

[0034] Step (d) can be performed for 175 to 190 minutes, preferably about 180 minutes.

[0035] Step (e) can be carried out for 17 to 20 hours, preferably about 18 hours.

[0036] In one embodiment, the synthesis includes dextran activation in step (a), alendronate conjugation in steps (b) and (c), aminoguanidine conjugation in step (d), and reductive amination in step (e). Step (a) is dextran activation, wherein an aqueous solution of dextran (preferably dextran 40) is oxidized with sodium periodate at an acidic pH, and the pH is adjusted to pH 1.5–1.9, preferably pH 1.55–1.8, more preferably pH 1.6–1.7, until the desired pH is reached by adding sulfuric acid to the solution. Oxidation is carried out by stirring at room temperature for about 45 minutes under light-protected conditions.

[0037] "Dextran 40" refers to pharmaceutical-grade low-molecular-weight dextran with an average molecular weight of approximately 40,000 g / mol. Dextran is a polydisperse linear glucose polymer, primarily composed of glucose (C6H2O). 12 O6) is a repeating unit.

[0038] "Under dark conditions" should be understood as the reaction taking place in a room without windows and without light. In other words, when the reaction is carried out under "dark conditions," there is no light during the reaction process.

[0039] "Room temperature" should be understood as the reaction being carried out without heating or cooling in a room at a temperature of approximately 15-25°C, preferably 18-22°C, and more preferably 20-22°C.

[0040] After step (a) is completed, in step (b) the pH of the solution is adjusted to pH 7-7.8, preferably pH 7.2-7.7, and more preferably pH 7.3-7.5, using sodium dihydrogen phosphate and sodium hydroxide. In step (c), alendronate is added to the reaction mixture, and the reaction mixture is stirred at room temperature in the dark for about one hour.

[0041] After steps (b) and (c) are completed, aminoguanidine hydrochloride is added to the solution in step (d), and the reaction mixture is stirred at room temperature for about three hours.

[0042] After step (d) is completed, sodium borohydride solution is added to the reaction mixture in step (e), and the reaction mixture is stirred at room temperature for 14-15 hours.

[0043] Alendronate has poor water solubility and is clinically supplied in solid tablet form. In step (c), the pH of the alendronate conjugation is controlled by using a buffer, and in the presence of hydrophilic dextran, alendronate retains its water solubility and prevents precipitation. Subsequent aminoguanidine conjugation and reductive amination do not require separation steps.

[0044] According to one embodiment, in step (f), the dextran-guanidine-bisphosphonate conjugate is purified by tangential flow filtration. The product obtained in step (e) is concentrated by adding sodium chloride, and the concentrate is diafiltrated, a process that purifies the dextran-guanidine-bisphosphonate conjugate to high purity.

[0045] In the tangential flow filtration process, the number of filtration cycles, flow rate, and filter specifications (pore size) have all been optimized.

[0046] According to a preferred embodiment, the material obtained after step (e) is concentrated by about 3.5 times and percolated with 7 times the percolation volume of 0.9% sodium chloride solution through a tangential flow filtration (TFF) membrane with a nominal molecular weight cutoff of 1 kDa, preferably 1 kDa–5 kDa; the above process can purify the dextran-guanidine-bisphosphonate conjugate to high purity.

[0047] The term "tangential flow filtration (TFF)" refers to a separation method widely used in the biopharmaceutical industry. The tangential flow filtration process is also known as cross-flow filtration. After tangential flow filtration, the concentrated product is filtered through a bacterial retention filter. The concentrated product is sampled to determine the content of the active ingredient; this determination is based on the dry weight measurement of the sample after freeze-drying and subsequent additional drying in an oven.

[0048] In this invention, the term "pharmaceutical" refers to the final product obtained by this method—a dextran-guanidine-bisphosphonate conjugate.

[0049] To ensure the final product remains water-soluble and has a shelf life of 36 months, the final product's environment is optimized by adjusting pH, ionic strength, concentration, and buffer solution.

[0050] According to one embodiment, in step (g), the obtained dextran-guanidinium-bisphosphonate is filtered through a bacterial retention filter. According to a preferred embodiment, the product is diluted with sodium acetate and 0.9% sodium chloride to a concentration of 22-28 mg / mL, more preferably 25 mg / mL ± 10%, and the pH is adjusted to pH 6.0-7.0 before filtration through a Pall Kleenpak Supor EKV 0.2 µm bacterial retention filter. A 0.2 µm filter, such as a Pall Kleenpak Supor EKV 0.2 µm filter, is typically used. Complete protonation of the guanidinium side chain at pH 6.0-7.0 ensures the drug's solubility and stability. The drug typically has an extended approved shelf life, such as 36 months, and can therefore be safely stored in 10 mL vials and administered from vials.

[0051] The product concentration can be diluted to 22, 23, 24, 25, 26, 27, or 28 mg / mL, or to any concentration within the range defined by two of these values. The pH can be adjusted to 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, or 7.0, or to any concentration within the range defined by two of these values.

[0052] In one implementation, the product is sampled after filtration for quality control. Those skilled in the art are familiar with suitable quality control measures and can select the methods to use. Quality control involves a variety of validated analytical methods. If the analytical results meet predetermined criteria, the drug will be approved.

[0053] In a preferred embodiment of the present invention, by Figure 3 The presented FMCA testing method demonstrates batch-to-batch functional reproducibility. Tumor cell killing efficacy of production batches was tested using human prostate cancer cell lines in a tumor cell culture system. The FMCA test ensures that approved batches have consistent function and efficacy. Figure 3 This data shows the in vitro potency analysis of the current GMP batches after storage at 5°C; gmp1 was stored for 22 months, gmp2 for 11 months, and gmp3 for 9 months. The y-axis represents the percentage of cell viability, and the x-axis represents the drug concentration. The approval standard is: IC50 ≥ 50% at concentrations of 2-6 µM. Therefore, cell viability is ≤50% at 6 µM.

[0054] Some problems arose during the scale-up of the specific synthesis process, but they were eventually resolved by the applicant.

[0055] Precipitation in the reactor is addressed by optimizing the pH, ionic strength, and concentration of each step a)–g). Under these conditions, complete water solubility and the desired level of activation can be achieved. The activation level of the carbohydrate backbone—the number of hydroxyl residues modified and substituted by the two ligands—determines the water solubility of the product. An appropriate level of activation ensures complete water solubility of the product. The level of activation also determines the substitution capacity of the two ligands, thus determining the function of the final product.

[0056] The ratio of the ligand (alendronate-aminoguanidine) in the target product is approximately 5 to 45. This ratio is achieved by optimizing the sequential conjugation time.

[0057] Reductive amination generates bubbles during purification. By optimizing the incubation time for reductive amination, the number of bubbles was minimized.

[0058] To achieve the desired purity and molecular properties of the final product, the tangential flow filtration (TFF) purification process needed to be optimized. The number of filtration cycles, flow rate, and filter specifications (pore size) were all optimized.

[0059] By optimizing the final product environment by adjusting the pH, ionic strength, concentration, and buffer solution, stable water solubility and a shelf life of 36 months can be achieved.

[0060] In a preferred embodiment, the present invention relates to a method for synthesizing dextran-guanidine-bisphosphonate conjugates, the method comprising the following steps in sequence: (a) Periodate is added to an aqueous solution containing dextran, followed by the addition of sulfuric acid to adjust the pH to 1.5-1.9, preferably 1.55-1.8, more preferably 1.6-1.7, and the solution is stirred at room temperature in the dark for about 45 minutes. (b) Sodium dihydrogen phosphate is added to the solution obtained in step (a), followed by sodium hydroxide, to adjust the pH to pH 7 - pH 7.8, preferably pH 7.2 - pH 7.7, and more preferably pH 7.3 - pH 7.5.

[0061] (c) Add alendronate to the solution obtained in step (b) and stir the solution at room temperature for about 1 hour under light-protected conditions. (d) Add aminoguanidine to the solution obtained in step (c) and stir the solution at room temperature for about 3 hours; (e) Add sodium borohydride solution to the solution obtained in step (d) and stir the solution at room temperature for 14-15 hours to produce dextran-guanidine-bisphosphonate conjugate; (f) The dextran-guanidine-bisphosphonate conjugate obtained in step (e) is purified, preferably by tangential flow filtration, to obtain the purified dextran-guanidine-bisphosphonate conjugate; (g) The purified dextran-guanidine-bisphosphonate conjugate obtained in step (f) is diluted with sodium acetate and sodium chloride to obtain diluted dextran-guanidine-bisphosphonate conjugate. The pH of the diluted dextran-guanidine-bisphosphonate conjugate is adjusted to pH 6.0 - pH 7.0, and the diluted dextran-guanidine-bisphosphonate conjugate is filtered.

[0062] In step (g), the concentration of the dextran-guanidinium-bisphosphonate conjugate is diluted with sodium acetate and sodium chloride to 22-28 mg / mL, preferably 25 mg / mL ± 10%, and the pH is adjusted to pH 6.0 - pH 7.0 before being filtered through a bacterial retention filter.

[0063] In another preferred embodiment, the present invention relates to a method for synthesizing dextran-guanidine-bisphosphonate conjugates, the method comprising the following sequential steps: (a) Add periodate to an aqueous solution containing dextran, followed by sulfuric acid, to adjust the pH to pH 1.6-1.7, and stir the solution at room temperature in the dark for about 45 minutes. (b) Add sodium dihydrogen phosphate to the solution obtained in step (a), followed by sodium hydroxide, to adjust the pH to pH 7 - pH 7.8, more preferably pH 7.3 - pH 7.5; (c) Add alendronic acid to the solution obtained in step (b) and stir the solution at room temperature for about 1 hour in the dark; (d) Add aminoguanidine to the solution obtained in step (c) and stir the solution at room temperature for about 3 hours; (e) Add sodium borohydride solution to the solution obtained in step (d) and stir the solution at room temperature for 14-15 hours to produce dextran-guanidine-bisphosphonate conjugate; (f) The dextran-guanidine-bisphosphonate conjugate obtained in step (e) is purified, preferably by tangential flow filtration, to obtain the purified dextran-guanidine-bisphosphonate conjugate; (g) The purified dextran-guanidine-bisphosphonate conjugate obtained in step (f) is diluted with sodium acetate and sodium chloride to obtain diluted dextran-guanidine-bisphosphonate conjugate. The pH of the diluted dextran-guanidine-bisphosphonate conjugate is adjusted to pH 6.0 – pH 7.0, and the diluted dextran-guanidine-bisphosphonate conjugate is filtered.

[0064] According to a preferred embodiment, the product is diluted to 22-28 mg / mL, more preferably 25 mg / mL ± 10%, using sodium acetate and 0.9% sodium chloride, the pH is adjusted to pH 6.0 - pH 7.0, and filtered through a Pall Kleenpak Supor EKV 0.2 µm bacterial retention filter. The product can be diluted to 22, 23, 24, 25, 26, 27, or 28 mg / mL, or to any concentration within the range defined by two of these values. The pH can be adjusted to 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, or 7.0, or to any pH within the range defined by two of these values.

[0065] According to another preferred embodiment, the present invention relates to a method for synthesizing dextran-guanidin-bisphosphonate conjugates, characterized in that the method is a large-scale one-pot GMP process, the method comprising the following steps: (a) Add periodate to an aqueous solution containing dextran, followed by the addition of sulfuric acid. (b) Add sodium dihydrogen phosphate to the solution obtained in step (a), followed by sodium hydroxide; (c) Add alendronic acid to the solution obtained in step (b); (d) Add aminoguanidine to the solution obtained in step (c); (e) Add sodium borohydride solution to the solution obtained in step (d) to produce a dextran-guanidine-bisphosphonate conjugate; (f) The dextran-guanidine-bisphosphonate conjugate obtained in step (e) is purified, preferably by tangential flow filtration, to obtain the purified dextran-guanidine-bisphosphonate conjugate; (g) The purified dextran-guanidine-bisphosphonate conjugate obtained in step (f) is diluted with sodium acetate and sodium chloride to obtain diluted dextran-guanidine-bisphosphonate conjugate. The pH of the diluted dextran-guanidine-bisphosphonate conjugate is adjusted to pH 6.0 - pH 7.0, and the diluted dextran-guanidine-bisphosphonate conjugate is filtered.

[0066] According to another preferred embodiment, the present invention relates to a method for synthesizing dextran-guanidin-bisphosphonate conjugates, characterized in that the method is a large-scale one-pot GMP process, the method comprising the following sequential steps: (a) Add periodate to an aqueous solution containing dextran, followed by the addition of sulfuric acid, wherein the dextran solution is prepared by adding dextran to water in one or more batches to dissolve it, preferably in one batch, more preferably in two batches, and most preferably in three batches.

[0067] (b) Add sodium dihydrogen phosphate to the solution obtained in step (a), followed by sodium hydroxide. (c) Add alendronate to the solution obtained in step (b), wherein alendronate may be added in one or more batches, preferably in one batch, more preferably in two batches, and most preferably in three batches. (d) Add aminoguanidine to the solution obtained in step (c), wherein alendronic acid may be added in one or more batches, preferably in one batch, more preferably in two batches, and most preferably in three batches. (e) Add sodium borohydride solution to the solution obtained in step (d) to obtain a dextran-guanidine-bisphosphonate conjugate. (f) Purification of the dextran-guanidine-bisphosphonate conjugate obtained in step (e), preferably by tangential flow filtration, to obtain the purified dextran-guanidine-bisphosphonate conjugate. (g) The purified dextran-guanidine-bisphosphonate conjugate obtained in step (f) is diluted with sodium acetate and sodium chloride to obtain diluted dextran-guanidine-bisphosphonate conjugate. The pH of the diluted dextran-guanidine-bisphosphonate conjugate is adjusted to 6.0-7.0, and the diluted dextran-guanidine-bisphosphonate conjugate is filtered.

[0068] The improved method of the present invention can prepare dextran-guanidine-bisphosphonate conjugates, wherein the conjugate is a compound with a guanidine group and a bisphosphonate group attached to the dextran, and contains at least one free amino group. The bisphosphonate and the guanidine group are covalently bonded to the activated hydroxyl group of the hydroxy polymer, and the conjugate has tumor cell killing activity.

[0069] In one embodiment, the guanidinyl group is guanidine butylamine or aminoguanidine.

[0070] In one embodiment, the bisphosphonate group is an aminobisphosphonate, preferably alendronate.

[0071] Preferably, this improved method can prepare dextran-guanidine-bisphosphonate conjugates, wherein the conjugate is a compound with a guanidine group and an alendronate group attached to the dextran. The guanidine group and the alendronate group are covalently bonded to the activated hydroxyl group of the hydroxy polymer, and the conjugate has tumor cell killing activity.

[0072] The present invention also relates to dextran-guanidine-bisphosphonate conjugates obtained by the method described herein.

[0073] According to one embodiment of the present invention, one molecule of dextran-guanidine-bisphosphonate conjugate contains about 5 to 8 alendronate molecules and about 40 to 45 aminoguanidine molecules.

[0074] One molecule of dextran-guanidinium-bisphosphonate conjugate may contain five (5), six (6), seven (7) or eight (8) alendronate molecules.

[0075] One molecule of dextran-guanidine-bisphosphonate conjugate may contain 40, 41, 42, 43, 44, or 45 aminoguanidine molecules.

[0076] One molecule of glucan guanidino Bisphosphonate conjugates may contain any combination of five (5), six (6), seven (7) or eight (8) alendronate molecules and 40, 41, 42, 43, 44 or 45 aminoguanidine molecules.

[0077] The present invention also relates to a pharmaceutical formulation comprising the dextran-guanidine-bisphosphonate conjugate of the present invention and its pharmaceutically acceptable salt or solvate, and at least one pharmaceutically acceptable excipient.

[0078] In one embodiment, the dextran-guanidine-bisphosphonate conjugate obtained by the method of the present invention is administered to the desired subject in solution form, wherein the concentration of the conjugate in the solution is approximately 25 mg / mL, dissolved in an aqueous solution of 0.1 M sodium acetate and 0.9% NaCl. The molecular weight distribution of the polydisperse pharmaceutical component is typically 0.4–95 kDa (Mw, weight average molecular weight) or 0.3–90 kDa (Mn, number average molecular weight).

[0079] According to one embodiment of the present invention, the dextran-guanidine-bisphosphonate conjugate or its pharmaceutical formulation is used for the treatment of cancer.

[0080] According to one embodiment of the present invention, the dextran-guanidine-bisphosphonate conjugate or a pharmaceutical preparation thereof is used for the treatment of cancer located in the bone.

[0081] According to one embodiment, the dextran-guanidine-bisphosphonate conjugate or its pharmaceutical formulation is used to treat cancers that have metastasized to or originate in the bone, preferably any one of multiple myeloma, metastatic castration-resistant prostate cancer (mCRPC) with bone metastases, bone metastases of lung cancer, and bone metastases of breast cancer.

[0082] The dextran-guanidine-bisphosphonate conjugate of this invention is preferably administered intravenously after dilution. The conjugate is a dilutable, sterile concentrate, typically contained in a single glass vial dissolved in sodium acetate. The conjugate concentrate in sodium chloride solution.

[0083] In one embodiment, the dextran-guanidine-bisphosphonate conjugate concentrate comprises 10 mL of a 25 mg / mL conjugate solution dissolved in 0.10 M sodium acetate and 0.9% sodium chloride solution, the conjugate being available for intravenous administration after dilution.

[0084] Example Starting materials for synthesis Pharmaceutical-grade dextran 40 was supplied by Pharmacosmos AS of Denmark. Pharmaceutical-grade sodium alendronate and aminoguanidine (purity ≥98.0%) were supplied by Chemrio International Limited of China.

[0085] Raw materials for synthesis and formulation Sodium dihydrogen phosphate monohydrate, sodium hydroxide granules, sodium metaiodate, sodium borohydride, 37% hydrogen chloride spray, 95-98% sulfuric acid, 10 mol / L sodium hydroxide solution, sodium chloride, and sodium acetate trihydrate were supplied by Merck AG, Germany.

[0086] synthesis Pharmaceutical-grade dextran 40 was dissolved in 6 L of water in a glass reactor. The dextran was added in three batches (60g + 60g + 60g, 10 g / L), ensuring complete dissolution of each batch before adding the next. The solution was stirred at room temperature (250 rpm) for 14-20 hours to ensure complete dissolution. Sodium metaiodate (108g, 18g / L) was slowly added to the surface of the clear dextran solution while stirring (500 rpm) and in the dark. After the sodium metaiodate was completely dissolved, sulfuric acid (estimated to be 7 mL) was added under dark conditions to adjust the pH to 1.65 ± 0.5. The reaction was then stirred at room temperature for 45 minutes under dark conditions. Subsequently, sodium dihydrogen phosphate monohydrate (83g, 13.8g / L) was added, followed by 5M NaOH (estimated to be 170 mL) to adjust the pH to 7.5 ± 0.2.

[0087] Alendronic acid was added in three batches (50g + 50g + 50g, 8.33g / L), and evenly distributed on the surface of the reaction mixture. Each batch was completely dissolved before the next batch was added. The solution was then stirred for 30 minutes to ensure complete dissolution. The reaction mixture was stirred at room temperature (700 rpm) for 60 minutes.

[0088] Aminoguanidine was added in three batches (240 g + 240 g + 240 g, 80 g / L), ensuring it was evenly distributed on the surface of the reaction mixture and dissolved. The reaction mixture was incubated at room temperature with stirring (700 rpm) for 180 minutes.

[0089] The next step involves preparing the sodium borohydride solution as follows: Add 60 g of sodium borohydride (10 g / L) to 500 mL of 0.1 M NaOH (83.3 g / L) and stir for at least 30 minutes; the mixture may become cloudy. Slowly add the mixture to the reaction mixture while gently stirring (250 rpm). After complete addition and dissolution, gently stir (100 rpm) at room temperature for 14–20 hours, during which time the pH of the solution will rise to approximately 9.5.

[0090] purification The obtained dextran-guanidine-alendronate was purified by concentrating the solution 3.5 times, percolating it with 7 times the percolation volume of 0.9% sodium chloride solution through a tangential flow filtration (TFF) membrane with a nominal molecular weight cutoff of 1 kDa.

[0091] The concentrated product was filtered through a Pall Kleenpak Supor EKV 0.2 µm bacterial interception filter and samples were taken for active ingredient detection. The detection method was to freeze-dry the sample and then further dry it in an oven before weighing the dry weight.

[0092] The obtained dextran-guanidine-alendronate was diluted to 25 mg / mL ± 10% with sodium acetate and 0.9% sodium chloride. The pH was adjusted to 6.0-7.0 with 0.1 M sodium acetate and filtered through a Pall Kleenpak Supor EKV 0.2µm bacterial retention filter. Subsequently, the active ingredient, nitrogen, phosphorus, free guanidine, free phosphorus, boron, iodine, and lead contents were determined, along with identification, molecular weight distribution, pH, endotoxin, and total aerobic microbial count (TAMC) / total yeast and mold count (TYMC).

[0093] References

Claims

1. A method for synthesizing dextran-guanidine-bisphosphonate conjugates, characterized in that, The method includes the following steps in sequence: (a) Add periodate to an aqueous solution containing dextran, and then add sulfuric acid; (b) Add sodium dihydrogen phosphate to the solution obtained in step (a), and then add sodium hydroxide; (c) Add alendronic acid to the solution obtained in step (b); (d) Add aminoguanidine to the solution obtained in step (c); (e) Add sodium borohydride solution to the solution obtained in step (d) to produce a dextran-guanidine-bisphosphonate conjugate; (f) The dextran-guanidine-bisphosphonate conjugate obtained in step (e) is preferably purified by tangential flow filtration to obtain the purified dextran-guanidine-bisphosphonate conjugate; (g) The purified dextran-guanidine-bisphosphonate conjugate obtained in step (f) is diluted with sodium acetate and sodium chloride to produce a diluted dextran-guanidine-bisphosphonate conjugate. The pH of the diluted dextran-guanidine-bisphosphonate conjugate is adjusted to 6.0-7.0, and the diluted dextran-guanidine-bisphosphonate conjugate is filtered.

2. The method according to claim 1, characterized in that, The bisphosphonate is an aminobisphosphonate, preferably alendronate.

3. The method according to claim 1 or 2, characterized in that, In step (a), the pH is adjusted to pH 1.5 - pH 1.9, preferably pH 1.55 - pH 1.8, and more preferably pH 1.6 - pH 1.

7.

4. The method according to any one of the preceding claims, characterized in that, In step (b), the pH is adjusted to pH 7 - pH 7.7, preferably pH 7.3 - pH 7.7, and more preferably pH 7.3 - pH 7.

5.

5. The method according to any one of the preceding claims, characterized in that, Steps (a), (c), or both (a) and (c) involve stirring the solution under light-protected conditions.

6. The method according to any one of the preceding claims, characterized in that, Steps (d), (e), or both (d) and (e) involve stirring the solution under light-protected conditions.

7. The method according to any one of the preceding claims, characterized in that, The dextran-guanidine-bisphosphonate conjugate in step (g) is filtered through a bacterial retention filter.

8. The method according to any one of the preceding claims, characterized in that, The method described is a large-scale one-pot GMP process.

9. A dextran-guanidine-bisphosphonate conjugate obtained by the method of any one of the preceding claims, characterized in that, One molecule of dextran-guanidine-bisphosphonate conjugate contains 5 to 8 alendronate molecules and 40 to 45 aminoguanidine molecules.

10. A dextran-guanidine-bisphosphonate conjugate, characterized in that, One molecule of dextran-guanidine-bisphosphonate conjugate contains 5 to 8 alendronate molecules and 40 to 45 aminoguanidine molecules.

11. A pharmaceutical preparation, characterized in that, The pharmaceutical formulation comprises the dextran-guanidine-bisphosphonate conjugate according to claim 8 or 9 and its pharmaceutically acceptable salt or solvate, and at least one pharmaceutically acceptable excipient.

12. The dextran-guanidine-bisphosphonate conjugate according to claim 8 or 9, or the pharmaceutical formulation according to claim 10, for the treatment of cancer.

13. The dextran-guanidine-bisphosphonate conjugate or pharmaceutical formulation according to claim 11, characterized in that, The cancer in question is a cancer located in the bone.

14. The dextran-guanidine-bisphosphonate conjugate or pharmaceutical formulation according to claim 11 or 12, characterized in that, The cancer is selected from cancers that have metastasized to the bone or cancers with bone as the primary site, preferably selected from multiple myeloma, metastatic castration-resistant prostate cancer (mCRPC), bone metastatic lung cancer, and bone metastatic breast cancer.

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