A mineralization-fiber double-targeted hierarchical dissociation composition for tumor fibrosis hard tissue and a preparation method thereof

Abstract

The application discloses a mineralization-fiber double-targeted hierarchical dissociation composition for tumor fibrosis hard tissues and a preparation method thereof. The composition includes independently packaged first mineralization pretreatment components, second enzymatic dissociation components and optional washing replacement liquids; the first components contain bisphosphonic acid mineralization binders and EDTA dissociation agents, which are prepared through dissolution, pH adjustment and sterile filtration; the second components contain Gly-Phe-Hyp-Gly-Glu-Arg collagen binding polypeptides and active MMP-1, MMP-8 or MMP-13, which are prepared in a low-temperature buffer system containing HEPES and Ca2+ and are independently filled. When used, the first components are applied for 0.5-4 hours, then the chelating agent residues are reduced through washing or buffer replacement, the second components are applied for 1-8 hours, and collagen fiber enzymolysis and gentle mechanical dispersion are completed under conditions suitable for enzyme activity. The scheme implements mineralization pretreatment and collagen enzymolysis at different times, and is suitable for pancreatic cancer matrix fibrosis hard tissues or liver fibrosis hard tissues in which mineralization deposition is confirmed.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically, to a mineralization-fiber dual-targeted graded dissociation composition for tumor fibrotic hard tissue and its preparation method. Background Technology

[0002] Tumor fibrosis is an important pathological feature of many solid tumors, particularly prominent in pancreatic ductal adenocarcinoma (PDAC) and hepatocellular carcinoma (HCC). Excessive fibrosis in the tumor microenvironment forms a dense extracellular matrix (ECM) barrier, which not only restricts the penetration and delivery of chemotherapeutic drugs but also promotes tumor immune escape and the development of drug resistance. Therefore, effectively degrading or dissociating fibrotic hard tissue in tumors and restoring the normal structure of the tumor microenvironment is a significant challenge in the field of cancer treatment.

[0003] Some tumor-induced fibrotic hard tissues may be accompanied by two types of components that increase tissue stability: one is the abnormal deposition of calcium phosphate minerals such as hydroxyapatite in the fibrotic matrix; the other is a cross-linked network formed by type I collagen catalyzed by the lysyl oxidase (LOX) family. The presence, content, and distribution of mineralized deposits in pancreatic cancer or liver fibrosis tissues need to be confirmed by histological, elemental analysis, or imaging; the present invention is preferably applicable to fibrotic hard tissues where mineralized deposits have been confirmed.

[0004] In the existing technology, the main treatment strategies for tumor fibrosis include: using hyaluronidase to degrade hyaluronic acid, using MMPs to degrade collagen fibers, and using bisphosphonates to inhibit mineralization. However, these methods have obvious limitations: (1) single-target strategies are difficult to treat both mineralization and fibrosis at the same time; (2) non-targeted degradative agents lack specificity for the lesion area and are prone to damaging normal tissues; (3) the degradation efficiency is low and cannot effectively break through the dense fibrosis barrier; (4) after systemic administration, the drug is unevenly distributed in the fibrotic tissue and the local concentration is insufficient.

[0005] Therefore, there is an urgent need in this field to develop a combined product and sequential application scheme that can perform mineralization component treatment and collagen fiber treatment in separate time periods and reduce mutual interference between the dissociated components.

[0006] For hard tissues with type I collagen as the main fibrous backbone, enzymatic dissociation can directly act on the extracellular matrix protein network, making it more suitable as the main treatment method in the fiber dissociation stage. However, MMP-type proteases rely on metal ions to maintain their catalytic activity. If EDTA residues used in the demineralization stage enter the enzymatic hydrolysis stage, it may weaken the subsequent enzymatic hydrolysis effect. Therefore, this invention does not use multiple chemical reagents for continuous tissue dissociation, but instead limits EDTA demineralization to a pretreatment step. After washing and replacement, a separate MMP enzymatic hydrolysis component is used to balance the removal of the mineralization barrier and the enzymatic hydrolysis of collagen fibers. Summary of the Invention

[0007] The purpose of this invention is to overcome the shortcomings of the prior art and provide a mineralization-fiber dual-targeted graded dissociation composition for tumor fibrotic hard tissue and its preparation method. This composition product utilizes the independent packaging and sequential use of a first dissociation component and a second dissociation component. The first stage is defined as mineralization pretreatment, and the second stage is set as collagenase dissociation with active MMPs as the core: firstly, the encapsulation and reinforcement effect of mineral deposits on the fiber network is reduced; after washing and replacement, enzymatic hydrolysis is performed, thereby reducing the interference of EDTA residues on MMP activity.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is: a mineralization-fiber dual-targeted graded dissociation combination product for tumor fibrotic hard tissue, comprising a first dissociation component and a second dissociation component packaged separately; the first dissociation component contains a mineralization binder and a first dissociation agent, wherein the mineralization binder can bind mineralized deposits in tumor fibrotic hard tissue; the second dissociation component contains a fiber binder and a second dissociation agent, wherein the fiber binder can bind to fiber regions rich in type I collagen in tumor fibrotic hard tissue; the first dissociation agent is used to dissociate mineralized deposits, and the second dissociation agent is used to degrade collagen fibers and weaken the integrity of cross-linked fiber networks; the first and second dissociation components are configured to be applied sequentially: the first dissociation component is applied for 0.5-4 hours, followed by washing or buffer replacement, and then the second dissociation component is applied for 1-8 hours.

[0009] Furthermore, the mineralizing binder is a bisphosphonic acid compound selected from one or more of alendronic acid, zoledronic acid, ibandronic acid, risedronic acid, or pharmaceutically acceptable salts thereof.

[0010] Furthermore, the fiber binding agent is a type I collagen-binding polypeptide, and the amino acid sequence of the polypeptide is Gly-Phe-Hyp-Gly-Glu-Arg, wherein Hyp is 4-hydroxyproline.

[0011] Furthermore, the first dissociation agent is ethylenediaminetetraacetic acid or a pharmaceutically acceptable salt thereof, and the concentration of the first dissociation agent in the first dissociation component is 0.05~0.5 mol / L, the concentration being expressed as EDTA free acid equivalent.

[0012] Furthermore, the second dissociation agent is an active form of matrix metalloproteinase, selected from one or more of MMP-1, MMP-8, and MMP-13.

[0013] Furthermore, when in use, the volume ratio of the first dissociated component and the second dissociated component is 1:0.5 to 1:2.

[0014] Furthermore, the first dissociation component and the second dissociation component each independently contain a pH adjuster and are each adjusted to a pH of 6.5 to 7.5.

[0015] Furthermore, the first dissociation component and / or the second dissociation component also contain a cosolvent or stabilizer, wherein the cosolvent or stabilizer is selected from one or more of glycerol, propylene glycol, and polyethylene glycol.

[0016] Furthermore, the dosage forms of the first and second dissociated components are each independently an injection, an infusion solution, or a local drug delivery gel, and the two components are physically isolated before use.

[0017] Furthermore, the use of the above-mentioned combination product in the preparation of a medicament for the local sequential degradation of tumor fibrotic hard tissue, wherein the tumor fibrotic hard tissue is pancreatic cancer stroma fibrotic hard tissue or liver fibrotic hard tissue with mineralized deposits confirmed by histological, elemental analysis or imaging.

[0018] Compared with the prior art, the beneficial effects of the present invention are: 1. Dual-region contact opportunities: mineralization binders and fiber binders are used to increase the contact opportunities of the first and second dissociated components in the mineralized region and the collagen-rich region, respectively; the above-mentioned binding selectivity needs to be further verified through binding kinetics, competitive binding and co-localization experiments.

[0019] 2. Sequential Treatment Effect: A sequential strategy of mineralization pretreatment followed by collagen enzymatic hydrolysis was adopted. The first stage was used to reduce the encapsulation and reinforcement effect of mineral deposits on the fiber network, and washing and replacement were used to reduce the residue of chelating agents such as EDTA. The second stage was completed by active MMP to complete the enzymatic hydrolysis of collagen fibers. In performance tests, Examples 1-4 maintained high levels in mineralization release and collagen enzymatic hydrolysis-related indicators, while the tissue dissociation effect decreased when sequential exchange, washing was omitted, or A / B premixing was performed, supporting the necessity of a graded treatment process.

[0020] 3. Evaluation Boundaries: The current performance testing of this invention focuses on evaluating the tissue dissociation effects of mineralization pretreatment, washing and replacement, and collagen enzymatic hydrolysis. The cytotoxicity, blood calcium effects, bone tissue distribution, and in vivo safety at the actual exposure concentration of the final formulation should be evaluated based on subsequent specialized safety tests, and absolute effect limits should not be made in the absence of relevant data.

[0021] 4. Applicable Scenarios: The combined products of this invention are primarily applicable to tumor fibrotic hard tissues with mineralized deposits confirmed by histology, elemental analysis or imaging; for fibrotic tissues without evidence of mineralized deposits, the applicability of the first dissociation component needs to be verified separately.

[0022] 5. Compatibility and operability: The first and second dissociation components are packaged independently and used in sequence. After the first stage of demineralization pretreatment, EDTA residue is reduced by washing and replacement. The second component uses the HEPES-Ca2+ buffer system and adds active MMP at low temperature. Before use, avoid sonication, high-speed vortexing and repeated freeze-thaw cycles, which is conducive to maintaining the operational stability of the subsequent enzymatic hydrolysis stage. Detailed Implementation

[0023] The following will provide a clear and complete description of the technical solutions of this invention. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0024] Preparation Example 1 Preparation of the first dissociation component targeted by mineralization and the second dissociation component targeted by fiber: 1. Raw material components: 1.1 First dissociated component raw material: Based on the preparation of 10.00 mL of the first dissociated component, the following raw materials are included: (1) Alendronate sodium trihydrate: 0.0050 parts by mass, i.e. 0.0050g.

[0025] (2) Disodium ethylenediaminetetraacetate dihydrate: 0.37224 parts by mass, i.e. 0.37224 g. The 0.37224 g disodium ethylenediaminetetraacetate dihydrate contains 0.001000 mol of disodium EDTA salt. After being prepared to 10.00 mL, the EDTA concentration is 0.100 mol / L.

[0026] (3) Sodium hydroxide: 0.0384 parts by mass, i.e. 0.0384g.

[0027] (4) Sterile ultrapure water: Add to the final volume of 10.00 mL.

[0028] 1.2 Second dissociation component raw material: Based on the preparation of 10.00 mL of the second dissociated component, the following raw materials are included: (1) Gly-Phe-Hyp-Gly-Glu-Arg polypeptide: 0.0010 parts by weight, i.e., 1.00 mg, of which Hyp is 4-hydroxyproline. Manufacturer: MedChemExpress, Model: HY-P11328 (2) Active human MMP-13 recombinant protein: 0.000020 parts by weight, i.e., 20.0 μg. Manufacturer: Merck / Calbiochem, model number: 444287-5UG. (3) HEPES: 0.11915 parts by weight, i.e. 0.11915g. Manufacturer: Sigma-Aldrich, model: H3375.

[0029] (4) Calcium chloride dihydrate: 0.01470 parts by mass, i.e. 0.01470 g. Manufacturer: Sigma-Aldrich, Model: C3881.

[0030] (5) Glycerin: 0.5000 parts by weight, i.e. 0.5000g.

[0031] (6) Sodium hydroxide: 0.0062 parts by mass, i.e. 0.0062g.

[0032] (7) Sterile ultrapure water: Add to the final volume of 10.00 mL, and the indicators are the same as those of the first dissociated component.

[0033] After the second dissociation group was prepared, the concentration of HEPES was 50 mmol / L, the concentration of calcium chloride was 10 mmol / L, the concentration of glycerol was 5.0% by volume, the concentration of GFOGER was 100 μg / mL, and the concentration of active MMP-13 protein was 2.0 μg / mL.

[0034] HEPES and calcium chloride are buffers and enzyme stabilization conditions set to maintain the activity of MMP-13. They are process excipients that need to be described in the instructions and do not constitute a replacement for the core creative components.

[0035] 2. Preparation method: 2.1 Preparation of the first dissociated component: S1. Under aseptic conditions of 25°C, air atmosphere, and sterile operation, add 7.50 mL of sterile ultrapure water to a clean preparation container and stir at 400 r / min for 2 min.

[0036] S2. Under the condition of stirring at 400 r / min, 0.37224 g of disodium ethylenediaminetetraacetate dihydrate was added uniformly over 4 min at a feeding rate of 0.09306 g / min. After the feeding was completed, stirring was continued for 20 min to completely dissolve the solid and obtain a colorless and clear disodium EDTA solution.

[0037] S3. Add 0.0050 g of alendronate sodium trihydrate to the solution obtained in step S2, and stir for 10 min at 20-25℃ and 300 r / min.

[0038] S4. Dissolve 0.0384 g of sodium hydroxide in 0.96 mL of sterile ultrapure water to prepare a 1.00 mol / L sodium hydroxide solution. Add this sodium hydroxide solution dropwise to the solution obtained in step S3 at a rate of 0.10 mL / min for 9.6 min, while maintaining stirring at 350 r / min during the addition. After the addition is complete, continue stirring for 10 min to bring the pH of the system to 7.20 ± 0.05.

[0039] S5. Add sterile ultrapure water to the solution obtained in step S4, and bring the final volume to 10.00 mL. Stir at 300 r / min for 10 min. Retest the pH; the pH of the finished product should be controlled between 7.15 and 7.25.

[0040] S6. The obtained solution was filtered through a low-protein adsorption sterile filter membrane with a pore size of 0.22 μm. The filtration process was carried out at 20–25 °C in an air atmosphere. After filtration, the solution was aseptically filled into 1.00 mL / bottle, yielding a total of 10 bottles of the first dissociated fraction.

[0041] The first dissociated component obtained was a colorless, clear liquid, free of visible foreign matter; the concentration of alendronate sodium trihydrate was 0.50 mg / mL; the concentration of EDTA (based on free acid equivalent) was 0.100 mol / L; and the pH was 7.15–7.25. The proposed volumetric yield was not less than 98.0%.

[0042] This preparation process is for liquid formulation and does not include steps for drying, washing, or sieving the solid product. Aseptic filtration at 0.22 μm is used to remove insoluble particles. Before stability data are available, the first dissociated component should be stored at 2–8°C protected from light and used within 24 hours of preparation.

[0043] 2.2 Preparation of the second dissociation component (enzymatic dissociation component): S7. Under aseptic conditions and in an air atmosphere at 25°C, add 7.50 mL of sterile ultrapure water to another clean preparation container, then add 0.11915 g HEPES, 0.01470 g calcium chloride dihydrate and 0.5000 g glycerol in sequence, and stir at 300 r / min for 15 min.

[0044] S8. Dissolve 0.0062 g of sodium hydroxide in 0.20 mL of sterile ultrapure water, and add it dropwise at a rate of 0.05 mL / min under stirring at 300 rpm for 4 min. After the addition is complete, continue stirring for 10 min to bring the pH of the buffer solution to 7.20 ± 0.05.

[0045] S9. Add sterile ultrapure water to the buffer solution obtained in step S8 to a final volume of 9.00 mL, continue stirring for 5 min, and then filter through a low-protein adsorption sterile filter membrane with a pore size of 0.22 μm. Cool the filtered buffer solution at 2–8 °C for 20 min.

[0046] S10. Take 0.50 mL of the cryogenic buffer obtained in step S9, add 1.00 mg of Gly-Phe-Hyp-Gly-Glu-Arg peptide, and stir for 5 min at 2–8 °C and 200 r / min to obtain a GFOGER peptide solution with a concentration of 2.00 mg / mL. Do not dissolve by sonication or high-speed vortexing.

[0047] S11. Remove four vials of active human MMP-13 recombinant protein (5 μg / vial) from -70℃ and thaw them in an ice bath at 0–4℃ for 10 min. After thawing, gently invert the vial three times to mix, without vortexing.

[0048] S12. Transfer all 4 MMP-13 stock solutions to sterile low-protein adsorption containers. Rinse each original packaging container once with 0.10 mL of the buffer solution obtained in step S9, for a total of 0.40 mL of buffer solution for all 4 containers. Combine the rinsing solution with the MMP-13 stock solution to reduce protein residue loss.

[0049] S13. Under conditions of 2–8℃, slowly add the GFOGER peptide solution obtained in step S10 to the MMP-13 solution obtained in step S12 at a rate of 0.10 mL / min; stir at a low speed of 60 r / min during the addition process. After the addition is complete, continue stirring at a low speed for 5 min.

[0050] S14. Using the buffer solution obtained in step S9, bring the volume of the solution obtained in step S13 to 10.00 mL, and gently invert to mix 10 times. Repeat the pH test and control it to 7.15–7.25.

[0051] S15. Aseptically fill the obtained second dissociated fraction at 1.00 mL / bottle. Transfer to -70°C within 30 minutes after filling and avoid repeated freeze-thaw cycles; each bottle should be thawed and used only once.

[0052] The resulting second dissociated fraction was a colorless, clear liquid, free of flocculent matter and visible foreign matter; the concentration of GFOGER peptide was 100 μg / mL; the concentration of MMP-13 protein was 2.0 μg / mL; the concentration of HEPES was 50 mmol / L; the concentration of calcium chloride was 10 mmol / L; the concentration of glycerol was 5.0% by volume; and the pH was 7.15–7.25. The planned volumetric yield was not less than 98.0%, and the release standard for MMP-13 activity recovery was not less than 80% of the activity before formulation.

[0053] This preparation process does not include product washing, drying, or sieving steps. After adding the second dissociated component to MMP-13, no further terminal filtration is performed. The buffer solution used is pre-filtered to 0.22 μm. Aseptic operation is maintained during protein addition and filling.

[0054] 2.3 Enzyme Activity Release and Pre-use Treatment of the Second Dissociated Component: After each batch of the second dissociated component is filled, a representative sample is taken, and the initial reaction rate is determined using a fluorescent substrate method suitable for MMP-13. The reaction rate of the same batch of pre-prepared active MMP-13 stock solution under the same buffer conditions is used as a 100% control to calculate the relative enzyme activity recovery rate. The relative enzyme activity recovery rate of the second dissociated component should meet the preset quality standard when released. Before use, each bottle of the second dissociated component is thawed in an ice bath at 0–4°C for about 10 minutes, gently inverted and mixed 3 times, and used immediately without vortexing or repeated freeze-thaw cycles.

[0055] Example 1 Preparation of a mineralization-fiber dual-targeted graded dissociation combination product for tumor fibrotic hard tissue: 1. Raw material composition: Each set of products includes the following raw materials: (1) First dissociated component: 1.00 mL, prepared by steps S1 to S6 in Preparation Example 1; wherein the concentration of sodium alendronate trihydrate is 0.50 mg / mL, the concentration of EDTA is 0.100 mol / L in terms of free acid equivalent, and the pH is 7.15 to 7.25.

[0056] (2) Second dissociation component: 1.00 mL, prepared by steps S7 to S15 in Preparation Example 1; wherein the concentration of GFOGER peptide is 100 μg / mL, the concentration of active MMP-13 is 2.0 μg / mL, the concentration of HEPES is 50 mmol / L, the concentration of calcium chloride is 10 mmol / L, the concentration of glycerol is 5.0% by mass and volume, and the pH is 7.15 to 7.25.

[0057] (3) Washing and replacement solution: 5.00 mL each time, each set of products is used 3 times, for a total of 15.00 mL. The washing and replacement solution is composed of 20 mmol / L HEPES and 150 mmol / L sodium chloride, with a pH of 7.20 ± 0.05.

[0058] The washing and replacement solution, based on a preparation volume of 100.00 mL, includes: 0.4766g of HEPES, manufactured by Sigma-Aldrich, model number H3375, with a purity of not less than 99.5%; 0.8766g of sodium chloride, manufactured by Sigma-Aldrich, model S7653, BioXtra grade, purity not less than 99.5%, molecular weight 58.44.

[0059] Sodium hydroxide 0.0248g.

[0060] Add sterile ultrapure water to a final volume of 100.00 mL.

[0061] 2. Preparation of washing and replacement fluid and assembly of the kit: S1. Add 0.4766g HEPES and 0.8766g sodium chloride to 80.00mL of sterile ultrapure water and stir at 400r / min for 15min at 20-25℃ in air atmosphere.

[0062] S2. Dissolve 0.0248 g of sodium hydroxide in 0.62 mL of sterile ultrapure water, and add it dropwise to the solution obtained in step S1 at a rate of 0.10 mL / min, while stirring at 300 r / min during the addition. After the addition is complete, continue stirring for 10 min to bring the pH to 7.20 ± 0.05.

[0063] S3. Add sterile ultrapure water to a final volume of 100.00 mL, filter through a sterile membrane with a pore size of 0.22 μm, and fill into 5.00 mL vials. The resulting wash replacement solution is a colorless and clear liquid with a pH of 7.15–7.25.

[0064] S4. Select one bottle each of the first and second dissociation components that have passed the inspection. Place the first and second dissociation components in two separate sealed containers without premixing; label the first dissociation component as "Liquid A" and the second dissociation component as "Liquid B".

[0065] S5. Package one bottle of solution A, one bottle of solution B, and three bottles of washing and replacement solution together. Physically separate solution A and solution B to prevent mixing during transportation and storage. The volume ratio of solution A to solution B in each set is 1:1.

[0066] S6 and A solutions should be stored at 2–8°C protected from light; B solution should be stored at -70°C and repeated freeze-thaw cycles should be avoided; washing and replacement solutions should be stored at 2–8°C.

[0067] 3. Representative sequential tissue dissociation schemes for this combination of products: 3.1 Sample Pretreatment: Taking pancreatic ductal adenocarcinoma (PDAC) orthotopic xenograft stromal tissue as an example, after collection, the tissue was immediately placed in a washing and replacement solution at 2–8°C and gently rinsed to remove free blood. Significantly necrotic tissue, fat, and large blood vessel areas were removed. The tissue was then divided into tissue blocks of approximately 3 mm × 3 mm × 2 mm, with a wet weight of 50.0 ± 5.0 mg. Tissue used for evaluation in this protocol should be pre-confirmed to have mineralized deposits by histological staining, elemental analysis, or imaging methods.

[0068] 3.2 First-stage mineralization pretreatment: Individual tissue blocks were placed in 2.0 mL low-protein adsorption centrifuge tubes, and 1.00 mL of A solution was added to completely submerge the tissue blocks. The mixture was treated at 37±0.5℃ and 60 r / min for 2.0 h. During treatment, the tissue blocks were gently inverted three times every 60 min, without using high-speed vortexing or mechanical shearing. After treatment, the supernatant from solution A was completely collected for subsequent determination of calcium and phosphorus release.

[0069] 3.3 Washing and Replacement: After aspirating solution A, add 5.00 mL of fresh washing and replacement solution, and wash for 5 min at 37±0.5℃ and 60 r / min. Then aspirate the washing solution and retain the tissue block. Repeat the above steps three times, using fresh washing and replacement solution each time. The three washing solutions are collected separately and can be combined for calcium and phosphorus release analysis. After the third wash, the second stage of enzymatic digestion should ideally begin within 10 min to minimize changes in tissue state.

[0070] 3.4 Second Stage Enzymatic Dissociation: The single vial of solution B was removed from its cryogenic storage condition and thawed in an ice bath at 0–4°C for approximately 10 minutes, gently inverting and mixing three times. 1.00 mL of solution B was added to the washed and replaced tissue block, and enzymatic digestion was performed at 37±0.5°C and 60 r / min for 4.0 h. During digestion, the block was gently inverted and mixed three times every 60 minutes to ensure sufficient contact between the enzyme solution and the tissue surface. No sonication, high-speed vortexing, or vigorous mechanical shearing was performed throughout the process.

[0071] 3.5 Gentle Mechanical Dispersion and Sampling: After enzymatic digestion, the tissue suspension was slowly aspirated 10 times using a wide-mouth pipette, maintaining a similar stroke and speed each time to minimize human shearing differences. The supernatant from the second stage was used for the determination of collagen degradation products such as C1M; the remaining tissue suspension was sequentially graded and sieved through 1000μm and 500μm sieves for evaluation of tissue dissociation rate.

[0072] 3.6 Endpoint Treatment: Immediately after completing the second stage of enzymatic hydrolysis, the samples were cooled to 0–4°C and aliquoted according to the detection items. The first-stage treatment solution and the three washing solutions were used for the analysis of mineralized sediment release; the second-stage enzymatic hydrolysis solution was used for the analysis of collagen degradation products; the residual tissue was used for the determination of hydroxyproline, dry weight loss, and tissue dissociation rate after sieving. Representative conditions were 2.0 h for solution A, 3 washes, and 4.0 h for solution B; the treatment time can be adjusted within the range specified in the claims without changing the pre-mineralization pretreatment followed by enzymatic dissociation.

[0073] Example 2 The preparation of a mineralization-fiber dual-targeting graded dissociation combination product for tumor fibrotic hard tissue was carried out by referring to the preparation method of Example 1, except that alendronate sodium trihydrate in the first dissociation component was replaced with an equimolar amount of zoledronic acid, and the remaining raw material composition, raw material dosage, preparation steps, process parameters, packaging method and storage conditions were kept the same as in Example 1, thus obtaining the mineralization-fiber dual-targeting graded dissociation combination product for tumor fibrotic hard tissue.

[0074] Example 3 The preparation of a mineralization-fiber dual-targeting graded dissociation combination product for tumor fibrotic hard tissue was carried out by referring to the preparation method of Example 1, except that alendronate sodium trihydrate in the first dissociation component was replaced with an equimolar amount of ibandronic acid, and the remaining raw material composition, raw material dosage, preparation steps, process parameters, packaging method and storage conditions were kept the same as in Example 1, thus obtaining the mineralization-fiber dual-targeting graded dissociation combination product for tumor fibrotic hard tissue.

[0075] Example 4 The preparation of a mineralization-fiber dual-targeting graded dissociation combination product for tumor fibrotic hard tissue was carried out by referring to the preparation method of Example 1, except that alendronate sodium trihydrate in the first dissociation component was replaced with an equimolar amount of risedronic acid, and the remaining raw material composition, raw material dosage, preparation steps, process parameters, packaging method and storage conditions were kept the same as in Example 1, thus obtaining the mineralization-fiber dual-targeting graded dissociation combination product for tumor fibrotic hard tissue.

[0076] Comparative Example 1 The preparation of a mineralization-fiber dual-targeted graded dissociation combination product for tumor fibrotic hard tissue is carried out according to the preparation method in Example 1, except that 0.0050g of sodium alendronate trihydrate in the first dissociation component is replaced with an equimolar amount of sodium pyrophosphate decahydrate of 0.00686g, so that the concentration of sodium pyrophosphate decahydrate in the first dissociation component is 0.686mg / mL; the pH of the first dissociation component is still adjusted to 7.15-7.25 with sodium hydroxide, and the other raw material composition, raw material dosage, final volume, preparation steps, process parameters, packaging method and storage conditions are the same as in Example 1.

[0077] Comparative Example 2 The preparation of a mineralization-fiber dual-targeted graded dissociation combination product for tumor fibrotic hard tissue is carried out according to the preparation method in Example 1, except that 0.37224g of disodium ethylenediaminetetraacetate dihydrate in the first dissociation component is replaced with 0.29410g of trisodium citrate dihydrate, so that the concentration of trisodium citrate in the first dissociation component is 0.100mol / L; the pH of the first dissociation component is still adjusted to 7.15-7.25 with sodium hydroxide, and the other raw material composition, raw material dosage, final volume, preparation steps, process parameters, packaging method and storage conditions are the same as in Example 1.

[0078] Comparative Example 3 The preparation of a mineralization-fiber dual-targeted graded dissociation combination product for tumor fibrotic hard tissue is carried out according to the preparation method in Example 1, except that 1.00 mg of Gly-Phe-Hyp-Gly-Glu-Arg polypeptide in the second dissociation component is replaced with 1.00 mg of sequence rearranged polypeptide Gly-Arg-Glu-Gly-Phe-Hyp, wherein Hyp is 4-hydroxyproline; the sequence rearranged polypeptide has the same amino acid types, amino acid numbers, elemental composition and relative molecular mass as Gly-Phe-Hyp-Gly-Glu-Arg, and its concentration in the second dissociation component is maintained at 100 μg / mL. Other raw material composition, raw material amount, final volume, preparation steps, process parameters, packaging method and storage conditions are the same as in Example 1.

[0079] Comparative Example 4 The preparation of a mineralization-fiber dual-targeting graded dissociation combination product for tumor fibrotic hard tissue is carried out according to the preparation method in Example 1, except that 20.0 μg of active human MMP-13 recombinant protein in the second dissociation component is replaced with 20.0 μg of active human MMP-2 recombinant protein, so that the concentration of active human MMP-2 recombinant protein in the second dissociation component is maintained at 2.0 μg / mL; the active human MMP-2 recombinant protein is a commercially available recombinant protein product, and the composition of other raw materials, raw material dosage, final volume, low temperature feeding method, stirring method, filling method, packaging method and storage conditions are the same as in Example 1.

[0080] Comparative Example 5 The preparation of a mineralization-fiber dual-targeted graded dissociation combination product for tumor fibrotic hard tissue is carried out according to the preparation method in Example 1, except that 0.01470g of calcium chloride dihydrate in the second dissociation component is replaced with 0.02033g of magnesium chloride hexahydrate, so that the concentration of magnesium chloride in the second dissociation component is maintained at 10mmol / L; the second dissociation component is still adjusted to pH 7.15-7.25, and other raw material composition, raw material dosage, final volume, preparation steps, process parameters, packaging method and storage conditions are the same as in Example 1.

[0081] Comparative Example 6 The preparation of a mineralization-fiber dual-targeted graded dissociation combination product for tumor fibrotic hard tissue involves preparing solution A, solution B, and washing and replacement solution according to the preparation method in Example 1. The raw material composition, raw material dosage, preparation steps, process parameters, packaging method, and storage conditions of each component are the same as in Example 1. When using the product, only the order of application of solution A and solution B is changed. That is, first, 1.00 mL of solution B is used for treatment for 4.0 h, followed by 3 replacements with 5.00 mL of washing and replacement solution per application, and then 1.00 mL of solution A is used for treatment for 2.0 h. Other usage conditions are the same as in Example 1.

[0082] Comparative Example 7 The preparation of a mineralization-fiber dual-targeted graded dissociation combination product for tumor fibrotic hard tissue involves preparing solution A, solution B, and washing / replacement solution according to the preparation method in Example 1. The raw material composition, raw material dosage, preparation steps, process parameters, packaging method, and storage conditions of each component are the same as in Example 1. Before use, the product is first treated with 1.00 mL of solution A for 2.0 h. After removing solution A, washing or buffer replacement is not performed using washing / replacement solution. Then, 1.00 mL of solution B is added directly and the product is treated for 4.0 h. Other usage conditions are the same as in Example 1.

[0083] Comparative Example 8 The preparation of a mineralization-fiber dual-targeted graded dissociation combination product for tumor fibrotic hard tissue involves preparing solution A and solution B according to the preparation method in Example 1. The raw material composition, raw material dosage, preparation steps, process parameters, filling specifications, and storage conditions of each component are kept the same as in Example 1. When using, 1.00 mL of solution A and 1.00 mL of solution B are mixed at a volume ratio of 1:1, and gently inverted and mixed 10 times at 20-25°C. The mixture is then added to the tissue to be dissociated within 5 minutes after mixing and continuously treated for 6.0 h. The graded operation of solution A treatment, washing and replacement, and solution B treatment is not performed again.

[0084] 1. Test samples, replication settings, and experimental design: The combined products prepared in Examples 1-4 and Comparative Examples 1-8 were used as test samples, and a blank control group was also set up. To distinguish between technical replicates and biological replicates, the representative experimental design used three independent PDAC orthotopic xenograft animals as three biological replicates (n=3); tissue blocks from adjacent regions of the tumor tissue from each animal were excised and randomly assigned to each treatment group. For the ICP-OES, hydroxyproline method, and C1M ELISA detection of the same biological sample, two technical replicates were set up. The average value of the technical replicates was taken first to form a single result for the biological replicate, and the technical replicates were not counted separately in n. Table 1 shows the mean ± standard deviation of the three independent biological replicates.

[0085] The blank control group used an equal volume of washing replacement solution instead of the first and second dissociation components, and was treated according to the same temperature, oscillation speed, and total treatment time as in Example 1. The test tissue was tumor stroma tissue obtained from a mouse pancreatic ductal adenocarcinoma (PDAC) orthotopic transplantation model; after sampling, tissue blocks were prepared according to the sample pretreatment steps of the "Representative Sequential Tissue Dissociation Protocol" in Example 1. Within each biological replicate, tissue blocks from adjacent regions of the same tumor were preferentially used in each treatment group and randomly numbered to reduce the influence of regional differences within the same tumor; all treatments were performed at 37±0.5℃ and 60 r / min.

[0086] 2. Standard sequential dissociation treatment method: Each group of tissue blocks was processed according to the "Representative Sequential Tissue Dissociation Scheme" in Example 1: First, 1.00 mL of the first dissociation component was added, and the mixture was treated for 2.0 h at 37±0.5℃ and 60 r / min. After collecting the first-stage treatment solution completely, the blocks were washed for 5 min with 5.00 mL of washing replacement solution under the same temperature and shaking conditions, for a total of 3 times. Then, 1.00 mL of the second dissociation component was added, and the blocks were enzymatically hydrolyzed for 4.0 h at 37±0.5℃ and 60 r / min. During the enzymatic hydrolysis, the blocks were gently inverted and mixed 3 times every 60 min. After the hydrolysis, the blocks were slowly aspirated 10 times using a wide-mouth pipette. Comparative Examples 6-8 were treated with sequential exchange, no washing replacement, and A / B premixing methods, respectively. The tissue size, liquid-to-solid ratio, temperature, shaking speed, total treatment time, and mechanical dispersion conditions were kept consistent.

[0087] 3. Dissociation properties of calcium and phosphate mineralized sediments: The first dissociation treatment solution and the third washing solution were collected separately, and the total calcium and phosphorus released were calculated by combining them. For each biological replicate, an adjacent, mass-matched, and untreated reference tissue block from the same tumor was taken, dried, and acid-digested. The total calcium and total phosphorus content was determined by inductively coupled plasma optical emission spectrometry (ICP-OES) as the initial mineralization reference for that biological replicate. Samples and standard curves were diluted within the instrument's linear range, and two parallel measurements were performed on the same biological sample. The calcium release rate was calculated as follows: Calcium release rate / % = mCa, released ÷ mCa, initial × 100%.

[0088] Phosphorus release rate is calculated as follows: Phosphorus release rate / % = mP, release ÷ mP, initial × 100%.

[0089] Wherein, mCa, release and mP, release are the sum of calcium and phosphorus releases in the first dissociation treatment solution and the third washing solution, respectively; mCa, initial and mP, initial are the initial total calcium and total phosphorus contents measured in the same biological replicate quality-matched reference tissue, respectively.

[0090] 4. Enzymatic hydrolysis properties of collagen fibers: Hydroxyproline method for determining collagen degradation rate: For each biological replicate, an adjacent, quality-matched, untreated reference tissue block from the same tumor was used to determine the initial collagen content; after treatment, the residual tissue was freeze-dried and hydrolyzed, and the hydroxyproline content was determined using the chloramine-T method, and the residual collagen amount was obtained according to the same conversion rules. Two parallel measurements were performed on the same biological sample.

[0091] Collagen degradation rate is calculated as follows: Collagen degradation rate / % = (mCol, initial - mCol, residual) ÷ ​​mCol, initial × 100%.

[0092] Type I collagen-specific degradation products (C1M): The supernatant of solution B was collected immediately after the second-stage enzymatic digestion, placed at 0–4°C, and diluted to the standard curve range according to the kit requirements. The content of type I collagen-specific degradation products was determined using the C1M ELISA kit. Two technical replicates were set up for each biological sample, and the average of the replicates was expressed as ng / mg of initial wet tissue.

[0093] 5. Overall tissue dissociation properties: Residual dry weight loss rate: For each biological replicate, an adjacent reference tissue block from the same tumor was taken to determine the wet weight / dry weight conversion factor. The initial dry weight m0 was obtained by converting the initial wet weight of the tissue block to be treated. The residual tissue after sequential treatment was freeze-dried to constant weight to obtain m1. Residual dry weight loss rate / % = (m0 - m1) ÷ m0 × 100%.

[0094] Tissue dissociation rate after mechanical dispersion: After the second stage of enzymatic hydrolysis, tissues were slowly pipetted 10 times using a wide-mouth pipette, and then sequentially passed through 1000μm and 500μm sieves. Tissues of sizes >1000μm, 500–1000μm, and <500μm were collected and their mass was measured. Tissue dissociation rate / % = m<500 ÷ (m>1000 + m500–1000 + m<500) × 100%. All groups used the same number of pipetting attempts, pipette diameter, and sieving time.

[0095] 6. Process Control in the Enzymatic Digestion Stage: To verify the effect of washing and replacement on the second-stage enzymatic digestion conditions, small samples were collected before adding tissue in solution B and at the end of the digestion process. Relative enzyme activity was determined using the same MMP fluorescent substrate method as in the preparation and release stage. This assay distinguishes between changes in enzyme activity under "no washing" or "premixing" conditions and differences caused by simple mechanical dispersion; technical replicates are not considered independent biological replicates.

[0096] Table 1. Performance Test Data ; Table 1 shows the overall trend of the "mineralization pretreatment - washing and replacement - collagen enzymatic hydrolysis" process: Examples 1-4 maintained high levels of calcium and phosphorus release indicators, collagen degradation, C1M release, and tissue dissociation indicators. When the mineralization binder or EDTA complexation conditions were changed, the calcium and phosphorus release in the previous stage and the subsequent overall dissociation decreased. When the GFOGER sequence, MMP type, or Ca2+ stability conditions were changed, the calcium and phosphorus release remained relatively stable, while the decrease in enzymatic hydrolysis-related indicators such as collagen degradation and C1M was more significant. When the sequence was exchanged, washing was removed, or A / B premixing was performed, the enzymatic hydrolysis-related indicators in the second stage further decreased, indicating that the key to this scheme is not to continuously increase the chemical dissociation agent, but to first remove the mineralization barrier with the first component, then reduce the residual chelating agent through washing and replacement, and finally complete the enzymatic hydrolysis of collagen fibers with the second component. The above results are used to support the correlation between the graded treatment process and the enzymatic hydrolysis stage. The specific number of repetitions, original measured values, and statistical records should be consistent with the original experimental records.

[0097] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A mineralization-fiber dual-targeted graded dissociation combination product for tumor fibrosis hard tissue, characterized in that, It includes a first dissociation component and a second dissociation component, each packaged separately. The first dissociation component is a mineralization pretreatment component, and the second dissociation component is an enzyme dissociation component. The first dissociation component comprises a mineralizing binder and a first dissociation agent, wherein the mineralizing binder is capable of binding mineralized deposits in tumor fibrotic hard tissue; The second dissociation component comprises a fibrous binding agent and a second dissociation agent, wherein the fibrous binding agent is capable of binding to fibrous regions rich in type I collagen in tumor fibrotic hard tissue; The first dissociating agent is used to dissociate mineralized deposits, and the second dissociating agent is used to degrade collagen fibers and weaken the integrity of cross-linked fiber networks; The first dissociation component and the second dissociation component are configured to be applied sequentially: the first dissociation component is applied for 0.5 to 4 hours, followed by washing or buffer replacement, and then the second dissociation component is applied for 1 to 8 hours. The first dissociation component and the second dissociation component are configured to be applied sequentially: the first dissociation component is applied for 0.5 to 4 hours, and after washing or buffer replacement to reduce the residue of the first dissociation agent, the second dissociation component is applied for 1 to 8 hours, and collagen fiber enzymatic hydrolysis is carried out under conditions suitable for the activity of the matrix metalloproteinase.

2. The combined product according to claim 1, characterized in that, The mineralization binder is a bisphosphonic acid compound selected from one or more of alendronic acid, zoledronic acid, ibandronic acid, risedronic acid, or pharmaceutically acceptable salts thereof.

3. The combined product according to claim 1, characterized in that, The fiber binding agent is a type I collagen-binding polypeptide, and the amino acid sequence of the polypeptide is Gly-Phe-Hyp-Gly-Glu-Arg, where Hyp is 4-hydroxyproline.

4. The combined product according to claim 1, characterized in that, The first dissociation agent is ethylenediaminetetraacetic acid or a pharmaceutically acceptable salt thereof, and the concentration of the first dissociation agent in the first dissociation component is 0.05~0.5 mol / L, the concentration being expressed as EDTA free acid equivalent.

5. The combined product according to claim 1, characterized in that, The second dissociation agent is an active form of matrix metalloproteinase, selected from one or more of MMP-1, MMP-8, and MMP-13.

6. The combined product according to claim 1, characterized in that, When in use, the volume ratio of the first dissociated component and the second dissociated component is 1:0.5 to 1:

2.

7. The combined product according to claim 1, characterized in that, The first and second dissociation components each independently contain a pH adjuster and are each adjusted to a pH of 6.5 to 7.

5.

8. The combined product according to claim 1, characterized in that, The first dissociation component and / or the second dissociation component further comprise a cosolvent or stabilizer selected from one or more of glycerol, propylene glycol, and polyethylene glycol.

9. The combined product according to any one of claims 1-8, characterized in that, The dosage forms of the first and second dissociated components are each independently of an injection, an infusion, or a localized gel, and the two components are physically isolated before use.

10. Use of a combination product as described in any one of claims 1-9 in the preparation of a medicament for the localized sequential degradation of fibrotic hard tissue in tumors, characterized in that, The tumor fibrotic hard tissue refers to pancreatic cancer stroma fibrotic hard tissue or liver fibrotic hard tissue with mineralized deposits confirmed by histology, elemental analysis or imaging.