Smearing sustained-release preparation for adjuvant therapy of tumors as well as preparation method and application of smearing sustained-release preparation

Abstract

The invention provides a smearing sustained-release preparation and a preparation method and application thereof, the smearing sustained-release preparation comprises a chemotherapeutic drug component, an immune drug component and a sustained-release auxiliary material, and the smearing sustained-release preparation is in a transparent flowable sol shape. The sustained release preparation disclosed by the invention can be smeared and applied to a focus part in situ after a tumor operation. The application effect comprises the following steps: filling the treatment window period of the traditional postoperative adjuvant chemotherapy, and improving the local drug concentration of the focus through in-situ local slow release of the drug, thereby effectively removing residual tumor tissues and reducing the systemic toxicity of drug treatment; the metastasis and recurrence rate of tumors are further inhibited through the synergistic combined effect of chemotherapy-immune drugs in the preparation. The advantages show that the smearing sustained-release preparation provided by the invention has huge clinical potential in the aspect of tumor treatment.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and more specifically, discloses a smearable sustained-release preparation, a preparation method thereof, and its use in auxiliary treatment after tumor surgery, eliminating residual tumors after surgery, and inhibiting tumor recurrence. Background Art

[0002] Pancreatic cancer is a highly malignant, common digestive tract tumor with a five-year overall survival rate of less than 9%, the lowest among all cancer types. It is often called the "King of Cancer" in the oncology field. Clinical statistics show that pancreatic cancer exhibits "three highs and three lows" clinical characteristics: the "three highs" are a steadily increasing incidence rate, a high recurrence and metastasis rate, and a high mortality rate; and the "three lows" are a low rate of early diagnosis, low drug efficacy, and a low five-year survival rate. Therefore, the clinical diagnosis and treatment of pancreatic cancer is extremely challenging, making it the third leading cause of cancer mortality.

[0003] In domestic and international guidelines for the diagnosis and treatment of pancreatic cancer, surgical resection is the only effective way for pancreatic cancer patients to obtain a chance of cure and long-term survival. However, the successful implementation of pancreatic cancer surgery does not equate to the ultimate benefit for the patient. Due to the characteristics of pancreatic cancer such as neurotropism, long-distance infiltration and spread, and multiple metastatic pathways, the positive resection margin rate of resectable pancreatic cancer is as high as 50-75%, and some cases of occult metastasis have already occurred at the time of surgery. More than 80% of patients will experience local recurrence or metastasis after surgery. The "Guidelines for the Diagnosis and Treatment of Pancreatic Cancer (2022 Edition)" issued by the Chinese Society of Clinical Oncology (CSCO) and the "NCCN Clinical Practice Guidelines: Pancreatic Cancer (2024V1)" of the United States point out: "Compared with surgery alone, postoperative adjuvant chemotherapy has a clear treatment method that can prevent or delay tumor recurrence and improve long-term survival after surgery. Therefore, postoperative adjuvant chemotherapy is actively recommended", and postoperative adjuvant chemotherapy is included in the standard postoperative treatment plan for resectable pancreatic cancer as the main means to reduce the recurrence of pancreatic cancer after surgery. Currently, there is no clear optimal adjuvant chemotherapy regimen. Available options include gemcitabine, gemcitabine combined with albumin-paclitaxel, gemcitabine combined with erlotinib, FOLFIRINOX, and its modifications. These postoperative adjuvant chemotherapy regimens are systemic and lack targeted therapy, requiring a comprehensive assessment of the patient's postoperative performance status. Consequently, their overall applicability is very limited. Furthermore, pancreatic cancers, particularly pancreatic ductal adenocarcinoma (PDAC), have a dense fibrous stroma, low vascularity, and low perfusion of tumor vessels, further reducing the benefits of postoperative adjuvant chemotherapy.

[0004] In response to the unmet clinical needs of pancreatic tumors, a large number of targeted drugs are being actively tried for the treatment of pancreatic cancer, including approved targeted drugs such as Olaparib based on BRCA1 / 2 germline mutations (gBRCAm), NTRK inhibitors based on NTRK1 / 2 / 3 fusions, and erlotinib based on EGFR sensitive mutations. The development of new drugs targeting pancreatic cancer has also become an important area of drug development. Chinese invention patent CN108078956B discloses a nucleic acid polypeptide nanodrug targeting pancreatic cancer and a preparation method thereof. The pancreatic cancer targeting polypeptide self-assembles with miRNA to form a nanocomplex, providing technical support and preclinical research evidence for the clinical development of new pancreatic cancer targeted drugs. Chinese invention patent CN108623661B discloses a bispecific polypeptide molecular probe targeting pancreatic cancer tumor cells, which aims to improve the problem of limited targeting ability of a single target and enhance the sensitivity and specificity of detecting early micro pancreatic cancer. However, the development of targeted therapies for pancreatic cancer is fraught with challenges due to the limited number of interventional targets and low mutation rates. For example, the BRCA 1 / 2 gene mutation rate is only 4-7%, and the NTRK fusion mutation rate is less than 1%.

[0005] Existing treatment options often require consideration of the patient's postoperative physical condition. Adjuvant chemotherapy is typically not initiated until 1-2 months after surgery, resulting in a long postoperative treatment window. While adjuvant therapy may offer a survival benefit for patients with resectable pancreatic cancer who have positive margins or residual localized disease, its prognostic impact remains very limited. Therefore, new therapeutic agents are urgently needed to bridge this postoperative treatment window and inhibit pancreatic cancer metastasis and recurrence. Summary of the Invention

[0006] In response to the above-mentioned background technical problems, the purpose of the present invention is to provide a new sustained-release preparation that can fill the postoperative treatment window period and inhibit the postoperative metastasis and recurrence of pancreatic cancer. This sustained-release preparation is applied in situ to the lesion site after pancreatic tumor surgery. It can fill the treatment window period of traditional postoperative adjuvant chemotherapy. By locally releasing drugs in situ, it increases the drug concentration in the local lesion, effectively eliminates residual tumor tissue, and reduces the systemic toxicity of drug treatment; through the synergistic combination of chemotherapy and immune drugs in the preparation, it further inhibits the metastasis and recurrence rate of the tumor. Another purpose of the present invention is to provide a preparation method and use of the preparation.

[0007] The first aspect of the present invention provides an in situ smearable sustained-release preparation comprising a chemotherapeutic drug component, an immunomodulatory drug component, and excipients.

[0008] The chemotherapy drug components include but are not limited to gemcitabine (Gem), S-1, fluorouracil anticancer agents, gemcitabine + albumin paclitaxel, gemcitabine + capecitabine (GAP), irinotecan, paclitaxel, flavopiridol, doxorubicin, idarubicin, vincristine, fluorouracil + mitomycin, fluorouracil + mitomycin + doxorubicin hydrochloride, gemcitabine + erlotinib, isatecan, doxorubicin hydrochloride + cisplatin + fluorouracil, mFOLFIRINOX (oxaliplatin combined with irinotecan, calcium folinate and 5-fluorouracil) and other clinical pancreatic cancer chemotherapy drugs and their combinations.

[0009] Preferably, the chemotherapy drug component is gemcitabine (Gem), gemcitabine + albumin paclitaxel.

[0010] The immunomodulatory drug components include, but are not limited to, non-specific immunostimulants, such as thymopentin, thymosin, thymosin, interferon gamma, interleukin, immunoglobulin, transfer factor oral solution; and immunosuppressive drugs, such as azathioprine, methotrexate, hydroxyurea, bleomycin, cyclophosphamide, chlorambucil, tacrolimus, tripterygium wilfordii, cyclosporine A; tacrolimus, imiquimod; compounds that enhance the immune response to tumors, such as inflammatory cytokines, such as antigenic peptides, immune checkpoint blocking peptides, interleukin-1 (IL-1) and tumor necrosis factor-α (TNF-α); histidine decarboxylase inhibitor peptides and defense peptides: LL-37, GCIR (SEQ ID NO1: sequence C 12 H 25 -IIRRIIRI-CONH2).

[0011] The histidine decarboxylase inhibitor is a histidine-phenylalanine dipeptide with a complete N-terminus, such as His-Phe, His-Phe-Leu, and His-Phe-Ile.

[0012] Preferably, the immunomodulatory drug components are thymopentin, thymosin, interferon γ, cyclosporine A, and LL-37.

[0013] More preferred are thymopentin and cyclosporine A.

[0014] The auxiliary material is the main body of the preparation to form the gel, and is selected from one or more of chitosan, chitosan quaternary ammonium salt, alginic acid, glucomannan, gelatin, starch, polyvinyl alcohol, cellulose, and collagen.

[0015] Preferred are chitosan, chitosan quaternary ammonium salt, cellulose and alginic acid; particularly preferred are chitosan, chitosan quaternary ammonium salt and hydroxypropyl cellulose.

[0016] The in situ smearable sustained-release preparation can be loaded with more functional components, giving the smearable sustained-release preparation richer diagnostic and therapeutic functions, including but not limited to one or more of fluorescent imaging probes, MRI radioactive imaging contrast agents, radionuclide probes, paramagnetic ions, metals, biological labels, fluorescent markers, chemiluminescent markers, and ultrasound contrast agents.

[0017] The fluorescent imaging probe is selected from indocyanine green, near-infrared fluorescent dyes (such as Cy5.5, FITC), fluorescent nanoparticles, fluorescently labeled antibodies and fluorescent probes.

[0018] The MRI imaging contrast agents include, but are not limited to, paramagnetic chelates, such as gadopentetate dimeglumine (Gd-DTPA), gadobutrol, clathrin-1 chelated gadolinium (Gd-PTP), gadolinium maltose liposomes, gadobenate dimeglumine, and gadoteric acid dimeglumine; superparamagnetic particles, such as superparamagnetic iron oxide particles (SPIO), such as AMI-25 and Resovist, which shorten the T2 relaxation time to a certain extent, manifesting as T2 hypointensity and improving image contrast; ferromagnetic particles, which shorten the T2 relaxation time and help improve image quality;

[0019] The nuclide labeling probe is composed of a targeting polypeptide chelated with a radioactive nuclide.

[0020] The radionuclide is selected from 67-68 Ga, 125 I. 18 F. 110-111 In, 67 Ga, 154-158 Gd, 64 Cu, 186 Re、 188 Re、 177 Lu or 90 One or more of Y.

[0021] The functional components mentioned above can be combined with cellular biomarkers targeting pancreatic cancer, such as mesothelin, urokinase plasminogen activator (uPA), insulin-like growth factor 1 receptor (IGF-1R), vascular endothelial growth factor receptor 2 (VEGFR2), mucin 1, zinc transporter 4 (ZIP4), carbohydrate antigen 19-9 (CA19-9), and the cytoskeletal scaffold protein Plectin-1.

[0022] In some embodiments, the cell biomarker is Plectin-1, and the targeting polypeptide is a PTP peptide selected from KTLLPTP, KTLLPTPGGSK, SAVEFLH, SKKDTHH, TMAPSIK, TQHQVTA, VNDRNVK and RGD peptides.

[0023] Preferably, the fluorescent imaging reagents are indocyanine green, Cy5, Cy7 and IRDye800CW;

[0024] Preferably, the radionuclide of the MRI imaging contrast agent is 154-158 Gd, 67-68 Ga;

[0025] In some embodiments, the functional component is PTP-FeO-IRDye800CW, wherein PTP-FeO-IRDye800CW is prepared according to the method described in the literature (eBioMedicine, 80, 2022, 104040; https: / / doi.org / 10.1016 / j.ebiom.2022.104040).

[0026] In some embodiments, the targeted contrast agent component is CREKA-Tris(Gd-DOTA)3, wherein CREKA-Tris(Gd-DOTA)3 is prepared according to the method described in the literature (Biomaterials, 34(31)(2013), 7683-769; https: / / doi.org / 10.1016 / j.biomaterials.2013.06.057).

[0027] In some embodiments, the targeted contrast agent component is Gd-Cy7-PTP / RGD, wherein Gd-Cy7-PTP / RGD is prepared according to the method described in the literature (https: / / doi.org / 10.1016 / j.biomaterials.2018.08.048).

[0028] The concentration of the chemotherapy drug component is 1-40 mg / mL, the concentration of the immunomodulatory drug component is 0.5-20 mg / mL, the concentration of the excipient is 5-200 mg / mL, preferably 10-100 mg / mL; the remainder is water or physiological saline.

[0029] In order to achieve good adhesion to postoperative lesions and sustained release of drugs, the in situ smearable sustained-release preparation is in the form of a flowable transparent sol with a certain viscosity range. Specifically, its viscosity is 100-10000 mPa·s. Preferably, the viscosity of the in situ smearable sustained-release preparation is 200-5000 mPa·s; more preferably, the viscosity of the in situ smearable sustained-release preparation is 500-2000 mPa·s.

[0030] The in situ smearable sustained-release preparation is used for adjuvant treatment of pancreatic cancer after surgery.

[0031] In a second aspect, a method for preparing the above-mentioned in-situ smearable sustained-release preparation is provided. Since the present invention contains two or more functional components, its functional compatibility and preparation process are very critical, and mainly include the following steps:

[0032] 1) preparing a solution of a chemotherapy drug component and an immunomodulatory immune drug component;

[0033] 2) adding the formulated amount of the immunomodulatory drug component to the chemotherapeutic drug component solution and mixing them evenly to form preformed solution A;

[0034] 3) Add the prefabricated liquid A to an appropriate amount of aqueous medium, add an appropriate amount of excipients to the solution, and mix quickly and evenly. After standing and aging for a certain period of time, a multifunctional spreadable sustained-release preparation is obtained.

[0035] Among them, in the preformed solution A, the mass ratio of the chemotherapy drug to the immunomodulatory drug is 1:0.02-5;

[0036] The aqueous medium in step 3) is water, physiological saline or acetic acid aqueous solution; the static aging time is 4-48 hours.

[0037] In a third aspect, the present invention also provides a method for preparing a multifunctional smearable sustained-release preparation obtained by compounding and modifying the above-mentioned in-situ smearable sustained-release preparation as a carrier:

[0038] 1) preparing a solution of a chemotherapy drug component and an immunomodulatory immune drug component;

[0039] 2) adding the formulated amount of the immunomodulatory drug component to the chemotherapeutic drug component solution and mixing them evenly to form preformed solution A;

[0040] 3) preparing functional component solution B;

[0041] 4) The preformed solution A and solution B are added to an appropriate amount of aqueous medium at the same time, and an appropriate amount of excipients are quickly added to the solution, mixed quickly and uniformly, and allowed to stand for a certain period of time to obtain a multifunctional spreadable sustained-release preparation.

[0042] When the chemotherapy drug component contains two or more components, step 2) is adjusted to evenly mix the chemotherapy drug components to form prefabricated solution A; step 3) is adjusted to prepare a functional component solution and add it to the immune drug component solution to form solution B.

[0043] The functional component solution B is one or more of a fluorescent imaging probe, an MRI radioactive imaging contrast agent, a radionuclide probe, a paramagnetic ion, a metal, a biological tag, a fluorescent marker, a chemiluminescent marker, and an ultrasound contrast agent; and the added amount is 0.01-1 mg / mL by mass volume concentration;

[0044] The aqueous medium in step 4) is water, physiological saline or acetic acid aqueous solution; the static aging time is 4-48 hours.

[0045] In some embodiments, centrifugation can be used to remove air bubbles from the sustained-release formulation.

[0046] In a fourth aspect, a method for using the above-mentioned in situ smearable sustained-release preparation in adjuvant drug treatment of tumors is provided.

[0047] It is particularly suitable for postoperative adjuvant treatment of operable pancreatic tumors.

[0048] Specifically, during pancreatic cancer surgery, after the resection of resectable tissue is completed and before the surgery is completely sutured, a therapeutically effective amount of the in situ application sustained-release preparation is directly applied to the surgical lesion.

[0049] In some embodiments, the pancreatic cancer is selected from the group consisting of: pancreatic head cancer, pancreatic body cancer, pancreatic tail cancer, and pancreatic cancer.

[0050] In other embodiments, the pancreatic cancer is selected from the group consisting of pancreatic ductal adenocarcinoma, pleomorphic pancreatic cancer, adenosquamous carcinoma, pancreatic mucinous carcinoma, pancreatic mucoepidermoid carcinoma and signet ring cell carcinoma, pancreatic ciliated cell carcinoma, acinar cell carcinoma, pancreatic small gland carcinoma, and pancreatic small cell carcinoma.

[0051] As used herein, the terms "treatment window period" and "window period" refer to the period after a patient undergoes surgery and is recovering from surgery. Due to the patient's physical condition not yet recovered, any form of treatment is suspended and the patient enters a period of waiting and observation.

[0052] The term "adjuvant" treatment refers to drug treatment after most of the tumor has been removed by surgery.

[0053] The term "treatment" refers to measures taken to achieve positive results such as tumor regression, tumor growth inhibition, prevention or inhibition of metastasis formation, prolongation of patient survival, and improvement of patient quality of life.

[0054] The term "patient" or "subject" refers to an individual who has symptoms of pancreatic cancer or other malignancies or is at risk for pancreatic cancer or other malignancies. A patient can be human or non-human and can include, for example, animal strains or species used as "model systems" for research purposes, such as the mouse models described herein. Patients may include adults or adolescents (e.g., children). The term "patient" further refers to any organism, preferably a mammal (e.g., human or non-human), who may benefit from the administration of the compositions contemplated herein.

[0055] Compared with the existing technical solutions, the present invention has the following beneficial effects:

[0056] 1) The sustained-release preparation of the present invention is applied directly to the lesion site immediately after tumor surgery, thereby reducing the treatment window period of postoperative adjuvant chemotherapy.

[0057] 2) The present invention increases the drug concentration in the local lesion through in situ local sustained release of drugs, effectively removes residual tumor tissue, and reduces the systemic toxicity of drug treatment.

[0058] 3) The preparation of the present invention contains chemotherapy and immune drugs, and the synergistic combination of the two has a better effect of inhibiting tumor metastasis and recurrence. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 The figures are actual photos of the formulations obtained in Example 1 (left) and Example 7 (right);

[0060] Figure 2 The figures are actual photos of the formulations obtained in Example 2 (left), Example 3 (center) and Example 4 (right);

[0061] Figure 3 Cryoscan photographs of the spreadable sustained-release formulations prepared in Example 1 (left) and Example 2 (right);

[0062] Figure 4 Cryoscan photographs of the smearable sustained-release formulations prepared in Example 5 (left) and Example 6 (right);

[0063] Figure 5 Subcutaneous pancreatic cancer model, tumor BLI monitoring results after postoperative adjuvant treatment with sustained-release preparation;

[0064] Figure 6 Subcutaneous pancreatic cancer model, changes in tumor volume after postoperative adjuvant therapy in each experimental group;

[0065] Figure 7 Flowchart of the construction and treatment of orthotopic pancreatic cancer models;

[0066] Figure 8 Schematic diagram of postoperative adjuvant treatment with in situ application of sustained-release gel;

[0067] Figure 9 Orthotopic pancreatic cancer model, survival of mice in each group;

[0068] Figure 10 After treatment, the number of CD4 + IL-4 + T cells and CD8+IFN-γ + T cell differentiation;

[0069] Figure 11 After treatment, CD3 + CD4+ T cells and CD3 + CD8 + T cell differentiation. DETAILED DESCRIPTION

[0070] The following describes the implementation method of the present invention in conjunction with specific specific examples. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this description. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention. Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing specific specific embodiments, not for limiting the scope of protection of the present invention. The present invention does not limit the source of the raw materials used. Unless otherwise specified, the reagents or instruments used in the present invention that do not indicate the manufacturer are conventional products that can be purchased through regular channels.

[0071] Formulated preparations

[0072] Example 1

[0073] This embodiment provides a gemcitabine (Gem) smearable sustained-release chitosan functional gel, the preparation method of which is as follows:

[0074] 1) Prepare a 20 mg / mL volume-to-mass ratio of gemcitabine aqueous solution and a 10 mg / mL thymopentin (TP5) saline solution;

[0075] 2) Add 2 mL of thymopentin saline solution to 5 mL of gemcitabine aqueous solution and mix well to form preformed solution A;

[0076] 3) preparing a fluorescence imaging functional component solution B: an aqueous solution of indocyanine green (ICG) with a volume-to-mass concentration of 1 mg / mL;

[0077] 4) The preformed solution A and 1 mL of solution B were simultaneously added to 2 mL of water, and 500 mg of chitosan quaternary ammonium salt was quickly added to the solution system. The mixture was quickly mixed and allowed to stand at 4°C for 4 hours to obtain a sol-like preparation with a certain fluidity. The viscosity values are listed in Table 1.

[0078] Figure 1 (Left) is an optical photograph of the sustained-release preparation prepared in this example. Figure 3 (Left) is a cryo-scanning electron micrograph of the sustained-release formulation prepared in this example. It can be seen that the formulation in the dry state is porous and serves as a base for the sustained-release of the composite drug.

[0079] Example 2

[0080] This embodiment provides a gemcitabine-albumin paclitaxel (Gem+NabP) smearable sustained-release chitosan functional gel, the preparation method of which is as follows:

[0081] 1) Prepare a 40 mg / mL volume-to-mass concentration of gemcitabine in saline, a 5 mg / mL albumin-paclitaxel suspension in saline (paclitaxel concentration is 0.5 mg / mL), a 10 mg / mL thymopentin (TP5) solution in saline, and a 1 mg / mL indocyanine green solution.

[0082] 2) Add 2 mL of gemcitabine saline solution to 20 mL of albumin-paclitaxel saline suspension and mix well to form preformed solution A;

[0083] 3) Add 6 mL of the indocyanine green aqueous solution to 12 mL of the thymopentin saline solution and mix well to form functional component B;

[0084] 4) The preformed solution A was added to the functional component B solution, and 1000 mg of chitosan quaternary ammonium salt was quickly added to the solution system. The mixture was quickly mixed and allowed to stand at 4°C for 48 hours to obtain a sol-like preparation with a certain fluidity. The viscosity values are listed in Table 1.

[0085] Figure 2 (Left) is an optical photograph of the sustained-release preparation prepared in this example. Figure 3 (Right) is a cryo-scanning electron micrograph of the sustained-release preparation prepared in this example.

[0086] Example 3

[0087] This embodiment provides a gemcitabine-albumin paclitaxel (Gem+NabP) smearable sustained-release chitosan functional gel, the preparation method of which is as follows:

[0088] 1) Prepare a 40 mg / mL volume-to-mass concentration of gemcitabine in saline, a 5 mg / mL albumin-paclitaxel suspension in saline (paclitaxel concentration is 0.5 mg / mL), a 5 mg / mL GCIR solution in saline, and a 1 mg / mL indocyanine green solution.

[0089] 2) Add 2 mL of gemcitabine saline solution to 20 mL of albumin-paclitaxel saline suspension and mix well to form preformed solution A;

[0090] 3) Add 6 mL of the indocyanine green aqueous solution to 12 mL of the GCIR saline solution and mix well to form functional component B;

[0091] 4) The preformed solution A was added to the functional component B solution, and 4000 mg of chitosan quaternary ammonium salt was quickly added to the solution system. The mixture was quickly mixed and allowed to stand at 4°C for 24 hours to obtain a sol-like preparation with a certain fluidity. The viscosity values are listed in Table 1. Figure 3 (Middle) is an optical photograph of the sustained-release preparation prepared in this example.

[0092] Example 4

[0093] This embodiment provides a smearable sustained-release chitosan functional gel containing gemcitabine-albumin paclitaxel (Gem+NabP), the preparation method of which is as follows:

[0094] 1) Prepare a 40 mg / mL volume-to-mass concentration of gemcitabine in saline, a 5 mg / mL albumin-paclitaxel suspension in saline (paclitaxel concentration is 0.5 mg / mL), a 5 mg / mL GCIR solution in saline, and a 1 mg / mL indocyanine green solution.

[0095] 2) Add 2 mL of gemcitabine saline solution to 20 mL of albumin-paclitaxel saline suspension and mix well to form preformed solution A;

[0096] 3) Add 6 mL of the indocyanine green aqueous solution to 12 mL of the GCIR saline solution and mix well to form functional component B;

[0097] 4) 400 mg of chitosan was added to a 1% acetic acid solution. After complete dissolution, the preformed solution A and the functional component B solution were sequentially added to the chitosan acetic acid solution and mixed rapidly. After aging at 4°C for 1 day, a sol-like preparation with a certain fluidity was obtained. Its viscosity values are listed in Table 1. Figure 2 (Right) is an optical photograph of the sustained-release preparation prepared in this example.

[0098] Example 5

[0099] This embodiment provides a smearable sustained-release chitosan functional gel containing gemcitabine-albumin paclitaxel (Gem+NabP), the preparation method of which is as follows:

[0100] 1) Prepare a 40 mg / mL volume-to-mass ratio of gemcitabine in saline, a 5 mg / mL albumin-paclitaxel suspension in saline (paclitaxel concentration is 0.5 mg / mL), a 5 mg / mL TP5 in saline, and a 0.5 mg / mL PTP-FeO-IRDye800CW aqueous solution;

[0101] 2) Add 2 mL of gemcitabine saline solution to 20 mL of albumin-paclitaxel saline suspension and mix well to form preformed solution A;

[0102] 3) Add 1 mL of PTP-FeO-IRDye800CW solution to 7 mL of TP5 saline solution and mix well to form functional component B;

[0103] 4) 300 mg of chitosan was added to 10 mL of 1% acetic acid solution. After complete dissolution, the preformed solution A and the functional component B solution were added to the chitosan acetic acid solution in sequence and quickly mixed. After aging at 4°C for 1 day, a sol-like preparation with a certain fluidity was obtained. Its viscosity values are listed in Table 1. Figure 4 (Left) is a cryo-scanning electron micrograph of the sustained-release preparation prepared in this example.

[0104] Example 6

[0105] This embodiment provides a smearable sustained-release chitosan functional gel containing gemcitabine-albumin paclitaxel (Gem+NabP), the preparation method of which is as follows:

[0106] 1) Prepare a 20 mg / mL gemcitabine saline solution, a 5 mg / mL albumin-paclitaxel saline suspension (paclitaxel concentration is 0.5 mg / mL), a 5 mg / mL GCIR saline solution, and a 0.1 mg / mL PTP-FeO-IRDye800CW aqueous solution.

[0107] 2) Add 4 mL of gemcitabine saline solution to 20 mL of albumin-paclitaxel saline suspension and mix well to form preformed solution A;

[0108] 3) Add 2 mL of PTP-FeO-IRDye800CW solution to 14 mL of GCIR saline solution and mix well to form functional component B;

[0109] 4) After mixing the preformed solution A and the functional component B solution, 1000 mg of chitosan quaternary ammonium salt was added to the mixed solution and mixed rapidly. After aging at 4° C. for 1 day, a sol-like preparation with a certain fluidity was obtained. Its viscosity values are listed in Table 1. Figure 4 (Right) is a cryo-scanning electron micrograph of the sustained-release preparation prepared in this example.

[0110] Example 7

[0111] This embodiment provides a smearable sustained-release chitosan functional gel containing gemcitabine (Gem), and the preparation method thereof is as follows:

[0112] 1) Prepare a 20 mg / mL volume-to-mass ratio of gemcitabine in saline and a 5 mg / mL volume-to-mass ratio of TP5 in saline.

[0113] 2) Add 2 mL of TP5 saline solution to 20 mL of gemcitabine saline suspension and mix well to form preformed solution A;

[0114] 3) preparing a targeted MRI imaging contrast agent functional component solution B, i.e., a 0.5 mg / mL Gd-Cy7-PTP / RGD solution;

[0115] 4) 350 mg of chitosan was added to 7 mL of 1% acetic acid solution. After complete dissolution, the preformed solution A and 1 mL of solution B were added simultaneously to the chitosan acetic acid solution, mixed rapidly, and allowed to stand at 4°C for 2 days to obtain a sol-like preparation with a certain fluidity. The viscosity values are listed in Table 1. Figure 1 (Right) is an optical photograph of the sustained-release preparation prepared in this example.

[0116] Example 8

[0117] This embodiment provides a smearable sustained-release chitosan functional gel containing gemcitabine (Gem), and the preparation method thereof is as follows:

[0118] 1) Prepare a 20 mg / mL gemcitabine saline solution, a 10 mg / mL CGIR saline solution, a 1 mg / mL indocyanine green solution, and a 0.1 mg / mL CREKA-Tris(Gd-DOTA)3 solution by volume-to-mass ratio.

[0119] 2) Add 5 mL of CGIR saline solution to 20 mL of gemcitabine saline suspension and mix well to form preformed solution A;

[0120] 3) Add 2 mL of CREKA-Tris(Gd-DOTA)3 solution to 3 mL of indocyanine green aqueous solution and mix well to form functional component B;

[0121] 4) After mixing the preformed solution A and the functional component B solution, 1500 mg of chitosan quaternary ammonium salt was added to the mixed solution and mixed rapidly. After aging at 4° C. for 1 day, a sol-like preparation with a certain fluidity was obtained. Its viscosity values are listed in Table 1.

[0122] Example 9

[0123] This embodiment provides a smearable sustained-release chitosan functional gel containing gemcitabine-albumin paclitaxel (Gem+NabP), the preparation method of which is as follows:

[0124] 1) Prepare a 40 mg / mL gemcitabine saline solution, a 5 mg / mL albumin-paclitaxel saline suspension (paclitaxel concentration is 0.5 mg / mL), a 5 mg / mL CGIR saline solution, a 1 mg / mL indocyanine green solution, and a 0.1 mg / mL CREKA-Tris (Gd-DOTA)3 solution by volume-to-mass ratio.

[0125] 2) Add 2 mL of gemcitabine saline solution to 20 mL of albumin-paclitaxel saline suspension and mix well to form preformed solution A;

[0126] 3) 1 mL of a 1 mg / mL indocyanine green aqueous solution and 1 mL of a CREKA-Tris(Gd-DOTA)3 solution were sequentially added to 12 mL of a CGIR saline solution and mixed well to form functional component B;

[0127] 4) After mixing the preformed solution A and the functional component B solution, 500 mg of hydroxypropyl cellulose was added to the mixed solution and mixed rapidly. After aging at 4°C for 2 days, a sol-like preparation with a certain fluidity was obtained. Its viscosity values are listed in Table 1.

[0128] Example 10

[0129] This embodiment provides a smearable sustained-release chitosan dual-function gel containing gemcitabine-albumin paclitaxel (Gem+NabP), the preparation method of which is as follows:

[0130] 1) Prepare a 40 mg / mL gemcitabine saline solution, a 5 mg / mL albumin-paclitaxel saline suspension (paclitaxel concentration is 0.5 mg / mL), a 1 mg / mL Gd-Cy7-PTP / RGD aqueous solution, and a 0.1 mg / mL CREKA-Tris(Gd-DOTA)3 aqueous solution;

[0131] 2) Add 2 mL of gemcitabine saline solution to 20 mL of albumin-paclitaxel saline suspension and mix well to form preformed solution A;

[0132] 3) Mix 1 mL of Gd-Cy7-PTP / RGD saline solution and 1 mL of CREKA-Tris(Gd-DOTA)3 solution to form functional component B;

[0133] 4) After mixing the preformed solution A and the functional component B solution, 500 mg of chitosan quaternary ammonium salt was added to the mixed solution and mixed rapidly. After aging at 4° C. for 1 day, a sol-like preparation with a certain fluidity was obtained. Its viscosity values are listed in Table 1.

[0134] Comparative Example 1

[0135] The TP-5 saline solution in Example 2 was replaced with an equal volume of saline solution to obtain an in situ application sustained-release preparation of a chemotherapy drug without immunomodulatory drugs, which was used in subsequent animal evaluations.

[0136] Comparative Example 2

[0137] The chitosan quaternary ammonium salt component in Example 2 was removed to obtain a drug-complex solution mixture system, which was used for subsequent animal evaluation to investigate the therapeutic effect of the preparation without sustained-release conditions.

[0138] Table 1: Summary of viscosity of sustained-release gels prepared in Examples 1-10

[0139]

[0140]

[0141] All animal experiments were conducted according to protocols approved by the local ethics committee and in compliance with Chinese laws on experimental animals. Male C57BL / 6 mice, 10–12 weeks old, were acclimated for ten days and housed in an SPF animal facility.

[0142] Example 11: Construction of a subcutaneous tumor model and evaluation of the postoperative adjuvant therapy effect of a sustained-release formulation

[0143] Establishment of subcutaneous tumor model: Luciferase-labeled mouse pancreatic cancer Pan02-luc cell suspension was mixed with Matrigel matrix gel at a volume ratio of 1:1 to obtain cell inoculation solution (5×10 7 Cell mL -1). 30 μL of cell inoculation solution was injected into the subcutaneous part of the upper right thigh of the mouse. After waiting for about 30 seconds, the tumor cell Matrigel solution became solid, and the needle was withdrawn to avoid spillage of tumor cells. 7 days after tumor cell inoculation, the size of the subcutaneous tumor was monitored by a vernier caliper until the tumor grew to an appropriate size. The mice with successfully established the above-mentioned subcutaneous pancreatic tumor model were divided into eight groups so that there was no substantial difference in the average tumor size of the mice in each group. All groups of mice underwent surgical resection, and 10% of the tumor tissue remained. Among them, except for the blank group in which the surgical wound was directly sutured after surgery, the other experimental groups applied 100 μl of the test sample prepared in each embodiment or comparative example to the affected area after surgical resection; 2.5 mg / kg buprenorphine was injected intraperitoneally twice a day for two days after surgery for postoperative analgesia.

[0144] The inhibition and recurrence of tumors were determined by bioluminescence imaging (BLI) before surgery, 0.5 hours, 1 day, 3 days, 5 days, 21 days, and 28 days after surgery. The specific operation was as follows: 200 μL of PBS solution of luciferin potassium salt (15 mg / mL) was injected into the mouse via the abdominal cavity. After about 5 minutes, the mouse was anesthetized and placed in the imaging system for bioluminescence imaging monitoring. Luciferase in tumor cells catalyzes the oxidation reaction of luciferin potassium, causing tumor cells to emit light for imaging. The imaging results are shown in Figure 2. Figure 5 shown.

[0145] Monitoring of tumor volume changes: After treatment, the length and width of the subcutaneous tumors of each group of mice were measured using a vernier caliper. The tumor volume was calculated using the following formula:

[0146]

[0147] Where a is the length of the tumor and b is the width of the tumor.

[0148] The results of tumor volume changes in mice after treatment are as follows Figure 6 This result is consistent with the fluorescence monitoring result.

[0149] Example 12: Construction of an in situ tumor model and evaluation of the postoperative adjuvant therapy effect of applying a sustained-release formulation

[0150] Establishment of orthotopic tumor model: Luciferase-labeled mouse pancreatic cancer Pan02-luc cell suspension was mixed with Matrigel matrix gel at a volume ratio of 1:1 to obtain cell inoculation solution (5×10 7 Cell mL -1The mouse abdomen was opened midline, and 30 μL of cell inoculum was injected into the pancreatic tail. After approximately 30 seconds, the tumor cell Matrigel solution solidified, preventing tumor cell spillage. Following inoculation, the peritoneum and skin were sutured. Buprenorphine (2.5 mg / kg) was administered intraperitoneally twice daily for two days postoperatively for postoperative analgesia.

[0151] 14 days after tumor cell inoculation, the effect of tumor model establishment was determined by bioluminescence imaging (BLI). According to the bioluminescence imaging results, the mice with successfully established pancreatic tumor models were divided into five groups so that the average bioluminescence intensity of the mice in each group was comparable. Among them, except for the control group, the surgical wound was directly sutured. After surgery, 100 μl of the test sample prepared in each embodiment or comparative example was applied to the affected area in each experimental group. Specifically, tumor-bearing mice underwent median laparotomy under anesthesia, and general anesthesia was induced by fentanyl / midazolam / medetomidine (doses of 0.05 mg / kg; 5 mg / kg; 0.5 mg / kg). The supply blood vessels were ligated with Vicryl 6 / 0, and the pancreas with the tumor was carefully moved. The pancreas was opened and most of the tumor was removed, leaving 10% of the tumor tissue for treatment evaluation. After treatment, the same suturing operation was performed, and 2.5 mg / kg of buprenorphine was injected intraperitoneally twice a day for two days after surgery for postoperative analgesia. Figure 7 Flowchart of the construction and treatment of orthotopic pancreatic cancer model. Figure 8 Schematic diagram of postoperative adjuvant treatment with in situ application of sustained-release gel.

[0152] The mice were observed for more than 60 days after surgery, and the tumor suppression, recurrence, and survival of the mice were recorded by BLI. The complete remission rate (the percentage of mice with complete disappearance of all tumor lesions and no recurrence after treatment) is summarized in Table 2. The survival curves of the mice in each group are shown in Table 2. Figure 8 The results show that the in situ application sustained-release preparation provided by the present invention has obvious therapeutic advantages. In particular, Example 10, by using a combination of a specific chemotherapy drug and an immune drug to form a sustained-release preparation, further improved the complete remission rate.

[0153] Table 2: Complete remission rate of adjuvant chemotherapy with the prepared sustained-release gel

[0154] serial number Complete remission rate (CR) Blank group 16.7％ Comparative Example 1 50.0％ Comparative Example 2 33.3％ Example 2 83.3％ Example 10 100％

[0155] Evaluation of in vivo immunomodulatory effects

[0156] After treatment, the content of interleukin-4 and interferon-γ was measured: 28 days later, the spleen of mice was taken for erythrocyte lysis, centrifugation, washing and spleen cells were dispersed in culture medium and incubated for 24 hours. After the incubation, specific antibody staining was performed and the content of interleukin-4 and interferon-γ was measured by flow cytometry. Interleukin-4 was used to indicate the activation of CD4+ cells, and interferon-γ was used to indicate the activation of CD8+ cells. After treatment, the content of CD4 in the spleen cells of mice in each group was significantly decreased. + IL-4 + T cells and CD8 + IFN-γ + T cell differentiation Figure 10 shown.

[0157] The spleens of mice in different groups were removed and ground, sieved, and cell suspensions were obtained. The cell suspensions were placed in RPMI-1640 medium containing 10% FBS (v / v) and cultured for 24 hours, wherein the ambient temperature was 37°C, the CO2 content was 5%, and the humidity was 95%. After the culture, the cells were washed 3 times with PBS, and the extracellular membrane was stained using flow cytometry antibodies (PE-CD3, FITC-CD4, PerCP-Cy5.5) for 25 minutes. After the end, the cells were washed, fixed, and penetrated, and intracellular staining was performed using flow cytometry antibodies (APC-IFN-γ, PE-Cy7-IL-4) for 25 minutes. After washing, the cell suspension was tested using a flow cytometer, and 1×10 4 After treatment, the differentiation of CD3+CD4+T cells and CD3+CD8+T cells in mouse spleen cells was as follows Figure 11 shown.

[0158] The applicant states that the present invention is intended to illustrate the detailed methods of the present invention through the above-described embodiments, but the present invention is not limited to the above-described detailed methods, that is, it does not mean that the present invention must rely on the above-described detailed methods in order to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions for various raw materials in the products of the present invention, addition of auxiliary ingredients, and selection of specific methods, etc., are all within the scope of protection and disclosure of the present invention.

Claims

1. A smearable sustained-release preparation, characterized in that: The smearable sustained-release preparation comprises a chemotherapy drug component, an immune drug component and a sustained-release excipient, and the smearable sustained-release preparation is in a transparent and flowable sol state; The chemotherapy drug component is selected from gemcitabine, S-1, fluorouracil anticancer agents, gemcitabine + albumin paclitaxel, gemcitabine + capecitabine, irinotecan, paclitaxel, flavopiridol, doxorubicin, idarubicin, vincristine, fluorouracil + mitomycin, fluorouracil + mitomycin + doxorubicin hydrochloride, gemcitabine + erlotinib, isatecan, doxorubicin hydrochloride + cisplatin + fluorouracil, mFOLFIRINOX and combinations thereof; The immunomodulatory drug component is selected from thymopentin, thymosin, thymosin, interferon gamma, interleukin, immunoglobulin, transfer factor oral solution, azathioprine, methotrexate, hydroxyurea, bleomycin, cyclophosphamide, chlorambucil, tacrolimus, tripterygium wilfordii, cyclosporine A; tacrolimus, imiquimod, inflammatory cytokines, such as antigenic peptides, immune checkpoint blocking peptides, interleukin-1 (IL-1) and tumor necrosis factor-α (TNF-α), histidine decarboxylase inhibitor peptides and defense peptides: LL-37, GCIR (SEQ ID No. 1: sequence C 12 H 25 -IIRRIIRI-CONH2); The sustained-release excipient is selected from one or more of chitosan, chitosan quaternary ammonium salt, alginic acid, glucomannan, gelatin, starch, polyvinyl alcohol, cellulose, and collagen.

2. The smearable sustained-release preparation according to claim 1, wherein the concentration of the chemotherapeutic drug component is 1-40 mg / mL; the immunomodulatory drug component is 0.5-20 mg / mL; the concentration of the sustained-release excipient is 5-200 mg / mL, preferably 10-100 mg / mL; and the balance is water or saline.

3. The in situ smear sustained-release preparation according to claim 1, characterized in that The chemotherapy drug components are gemcitabine (Gem), gemcitabine + albumin paclitaxel; The immunomodulatory drug components are thymopentin, thymosin, interferon gamma, cyclosporin A, LL-37, GCIR (SEQ ID NO1: sequence C 12 H 25 -IIRRIIRI-CONH2), methotrexate; preferably thymopentin, cyclosporine A, methotrexate, GCIR (SEQ ID NO1: sequence C 12 H 25 -IIRRIIRI-CONH2); The sustained-release excipient is selected from one or more of chitosan, chitosan quaternary ammonium salt, cellulose and alginic acid.

4. The smearable sustained-release preparation according to claim 1, characterized in that The viscosity is 100-10000 mPa·s. Preferably, the viscosity of the in situ smearable sustained-release preparation is 200-5000 mPa·s; more preferably, the viscosity of the in situ smearable sustained-release preparation is 500-2000 mPa·s.

5. The smearable sustained-release preparation according to claim 1, characterized in that The smearable sustained-release preparation is further loaded with functional components, and the functional components are selected from one or more of fluorescent imaging probes, MRI radioactive imaging contrast agents, radionuclide probes, paramagnetic ions, metals, biological labels, fluorescent markers, chemiluminescent markers, and ultrasound contrast agents.

6. The in situ smear sustained-release preparation according to claim 5, characterized in that The functional component can be combined with a cell biomarker of pancreatic cancer, and the cell biomarker is selected from Plectin-1. Preferably, the functional component is selected from KTLLPTP, KTLLPTPGGSK, SAVEFLH, SKKDTHH, TMAPSIK, TQHQVTA, VNDRNVK and RGD peptide.

7. The method for preparing the spreadable sustained-release preparation according to any one of claims 1 to 4, characterized in that: The following steps are involved: 1) preparing a solution of a chemotherapy drug component and an immunomodulatory immune drug component; 2) adding the formulated amount of the immunomodulatory drug component to the chemotherapeutic drug component solution and mixing them evenly to form preformed solution A; 3) adding the preformed liquid A to an appropriate amount of aqueous medium, and simultaneously adding an appropriate amount of sustained-release excipient to the solution, and rapidly mixing the solution, and allowing it to stand for a certain period of time to obtain a multifunctional spreadable sustained-release preparation; Wherein, in the preformed solution A described in step 2), the mass ratio of the chemotherapy drug to the immunomodulatory drug is 1:0.02-5; The aqueous medium in step 3) is water, physiological saline or acetic acid aqueous solution; the static aging time is 4-48 hours.

8. The method for preparing the smearable sustained-release preparation according to claim 5, wherein: The following technical steps are included: 1) preparing a solution of chemotherapy drug components and an immunomodulatory immune drug component; 2) adding the formulated amount of the immunomodulatory drug component to the chemotherapeutic drug component solution and mixing them evenly to form preformed solution A; 3) preparing functional component solution B; 4) Adding the preformed solution A and solution B simultaneously to an appropriate amount of aqueous medium, and quickly adding an appropriate amount of excipients to the solution, mixing quickly and uniformly, and allowing to stand for aging to obtain a multifunctional spreadable sustained-release preparation; The functional component in step 3) is selected from one or more of fluorescent imaging probes, MRI radioactive imaging contrast agents, radionuclide probes, paramagnetic ions, metals, biological labels, fluorescent markers, chemiluminescent markers, and ultrasound contrast agents; and its solution concentration is 0.001-0.1%wt. The aqueous medium in step 4) is water, physiological saline or acetic acid aqueous solution; the static aging time is 4-48 hours.

9. The use of the smearable sustained-release preparation according to claims 1-8, characterized in that: The smearable sustained-release preparation is used for preparing auxiliary medicine after tumor tissue resection surgery.

10. Use of the smearable sustained-release preparation according to claims 1-9, characterized in that: The auxiliary drug is directly applied to the surgical lesion after the tumor tissue resection surgery and before the surgery is completely sutured.

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