Pharmaceutical composition containing insoluble antitumor active agent and preparation method thereof

文档序号：791729 发布日期：2021-04-13 浏览：36次中文

阅读说明：本技术 含有难溶性抗肿瘤活性剂的药物组合物及其制备方法 (Pharmaceutical composition containing insoluble antitumor active agent and preparation method thereof ) 是由吴翠栓程晓波王胜男李思明林家亮孙亚美张强于 2021-01-12 设计创作，主要内容包括：本发明涉及含有难溶性抗肿瘤活性剂的组合物及其制备方法。本发明的药物组合物包含难溶性抗肿瘤活性剂、稳定剂和冻干保护剂,其适用于癌症术后创面给药优选淋洒给药,用于术后癌症的治疗,减少术后肿瘤复发、转移和提高患者生存率。(The present invention relates to a composition containing a poorly soluble antitumor active agent and a method for preparing the same. The pharmaceutical composition comprises the insoluble antitumor active agent, the stabilizing agent and the freeze-drying protective agent, is suitable for the administration of the wound surface after the operation of cancer, preferably for the administration of shower, is used for treating the cancer after the operation, reduces the recurrence and the metastasis of the tumor after the operation and improves the survival rate of patients.)

1. A pharmaceutical composition containing a poorly soluble antitumor active agent, comprising a poorly soluble antitumor active agent, a stabilizer, and a lyoprotectant; wherein the stabilizer is selected from polyoxyethylene castor oils such as polyoxyethylene 35 castor oil, especially pure polyoxyethylene 35 castor oil; polyethylene glycol hydroxystearate such as polyethylene glycol 15-hydroxystearate, polyethylene glycol 12-hydroxystearate; polysorbate 80 and mixtures thereof; and the lyoprotectant is selected from sucrose.

2. The pharmaceutical composition according to claim 1, wherein the weight ratio of the poorly soluble antitumor active agent to the stabilizer is 10:2 to 10:12, preferably 10:2 to 10:6, and more preferably 10: 3.

3. The pharmaceutical composition according to claim 1 or 2, wherein the weight ratio of the poorly soluble antitumor active agent to the lyoprotectant is 10:80 to 10:300, preferably 10:90 to 10:200, more preferably 10: 100.

4. The composition according to any one of claims 1 to 3, wherein the poorly soluble anti-tumor active agent is selected from paclitaxel (paclitaxel), docetaxel (docetaxel), cabazitaxel (cabazitaxel), etoposide (etoposide), teniposide (teniposide), hydroxycamptothecin (hydroxycamptothecin), irinotecan (irinotecan), cisplatin (cispin), carboplatin (carboplatin), capecitabine (capecitabine), oxaliplatin (oxaliplatin), gefitinib (gefitinib), doxorubicin (doxorubicin), vinblastine (vinblastine), vincristine (vincristine); preferably, the poorly soluble anti-tumor active agent is selected from paclitaxel, docetaxel, cabazitaxel, etoposide, teniposide and hydroxycamptothecin; most preferably, the poorly soluble anti-tumor active agent is selected from paclitaxel, docetaxel, cabazitaxel.

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the pharmaceutical composition is a composition for wound administration after tumorectomy in a cancer patient, preferably a composition for wound shower administration.

6. The pharmaceutical composition according to any one of claims 1 to 5, which does not comprise poloxamer 188, more preferably poloxamer.

7. The pharmaceutical composition according to any one of claims 1 to 5, which does not comprise polysorbate 20, poloxamer, trehalose, and mannitol.

8. The pharmaceutical composition according to any one of claims 1 to 5, which does not comprise further stabilizers and lyoprotectants; preferably, the pharmaceutical composition consists of the poorly soluble antitumor active agent according to any one of claims 1 to 4, a stabilizer and a lyoprotectant.

9. The pharmaceutical composition according to any one of claims 1 to 5, consisting of paclitaxel, polysorbate 80 and sucrose, wherein the ratio of the amounts of paclitaxel, polysorbate 80 and sucrose is 10:3:100 (w/w); or the compound comprises docetaxel, 15-hydroxystearic acid polyethylene glycol ester and sucrose, wherein the dosage ratio of the docetaxel to the 15-hydroxystearic acid polyethylene glycol ester to the sucrose is 10:5:200 (w/w).

10. The pharmaceutical composition of any one of claims 1 to 9, which is a lyophilized formulation, such as a lyophilized powder.

11. Use of a pharmaceutical composition according to any one of claims 1 to 10 for the preparation of a suspension for postoperative administration to a wound, in particular to a wound shower, of a patient with a tumor.

12. Use according to claim 11, the suspension having an average particle size preferably between 100nm and 1000nm, more preferably between 100nm and 500nm, even more preferably between 100nm and 400 nm.

13. A process for the preparation of a pharmaceutical composition containing a poorly soluble antineoplastic active agent according to any one of claims 1 to 10, characterized in that it comprises the steps of:

(1) dissolving the stabilizer with injectable solvent (preferably water for injection) under stirring and optionally heating to obtain stabilizer solution; preferably, the mass ratio of the injectable solvent to the stabilizer is 500: 1-2: 1, such as 200: 1-5: 1, or 90: 1-15: 1;

(2) adding the insoluble antitumor active agent into the stabilizer solution obtained in the step (1), and heating and stirring until the system is a uniform suspension, preferably at a heating temperature of 30-90 ℃, for example, 40-80 ℃;

(3) wet grinding was performed as follows: grinding the suspension obtained in the step (2) for 0.5-10 h, such as 1-6 h or 1-4 h by using a wet grinding machine (the rotating speed is 2500-8000 rpm, such as 3500-8000 rpm and 4000-6000 rpm; the water inlet temperature is-5-30 ℃, such as 4-15 ℃, preferably, the diameter of grinding beads is 0.2-0.5 mm, and the loading is 50-60% (V/V)) to obtain a suspension; and

(4) diluting the suspension obtained in the step (3) with an injectable solvent (preferably water for injection) (preferably 10-500 times, for example, 50-100 times), adding sucrose, stirring for dissolving, optionally subpackaging in penicillin bottles, and freeze-drying in a freeze dryer.

14. A suspension obtained by diluting the pharmaceutical composition of any one of claims 1 to 10 with a vehicle, preferably a vehicle suitable for injection, such as water for injection, 5% dextrose injection or 0.9% sodium chloride injection.

15. An injection device prefilled with the pharmaceutical composition of any one of claims 1 to 10, or the suspension of claim 14, preferably said injection device is a disposable syringe.

Technical Field

The invention belongs to the field of pharmaceutical preparations, and particularly relates to a composition containing an insoluble antitumor active agent and a preparation method thereof.

Background

Tumors are one of the diseases with the highest lethality rate at present, and the clinical treatment effect and the prognosis survival period are not ideal. Data provided by the international cancer research institute show that there are about 1700 thousands of newly increased tumor cases and 950 thousands of tumor-associated death cases in 2018. Over the past few decades, advances in new therapeutic approaches, such as surgery, chemotherapy, radiation therapy, and molecular targeted therapy and immunotherapy, have greatly reduced morbidity and increased survival. Despite this progress, cancer remains the second leading cause of death following cardiovascular disease. Surgical resection remains the treatment of choice for most early and intermediate solid tumors. However, even with radical surgery that extends the scope of resection, biological radical treatment is difficult to achieve, and post-operative residual "invisible" tumor cells may still be present at the surgical margin and even into the systemic circulation, which increases the risk of tumor recurrence and metastasis. In many malignant tumors, local recurrence is an important index for marking clinical treatment failure, has high death risk, and seriously affects the survival period and the quality of life of tumor patients. Therefore, local control is crucial for a more reliable cure of cancer.

In recent years, implantation of a controllable drug delivery system at a surgical wound has been considered as a promising therapeutic strategy to suppress local recurrence and distant metastasis after tumor surgery. The treatment of postoperative tumors is mainly divided into local treatment and systemic treatment according to their mechanism of action and route of administration. Formulations and kits of gels, foams and fibers have been developed for topical administration.

Is the first local drug delivery system to treat recurrent malignant brain tumors and was approved by the FDA in 1995. It can increase median survival time from 4.6 months to 6.4 months (Attenulo F J, Mukherjee D, Datoo G, et al. use of Gliadel (BCNU) wafer in the scientific laboratory of cortical geomatan: a10-year insulation experiment [ J].Ann Surg Oncol,2008,15(10):2887-93.Brem H,Piantadosi S,Burger P C,et al.Placebo-controlled trial of safety and efficacy of intraoperative controlled delivery by biodegradable polymers of chemotherapy for recurrent gliomas.The Polymer-brain Tumor Treatment Group[J].Lancet,1995,345(8956):1008-12.Tamargo R J,Myseros J S,Epstein J I,et al.Interstitial chemotherapy of the 9L gliosarcoma:controlled release polymers for drug delivery in the brain[J]Cancer Res,1993,53(2): 329-33). In 2003, the U.S. food and drug administration also approved an upgraded product,in combination with BCNU (carmustine dichloroethyl nitrosourea) wafers, it can further extend survival of patients with Glioblastoma (GBM) from 11.6 months to 13.9 months (Westphal M, Hilt D C, Bortey E, et al. A phase 3 tertiary of local chemotherapy with biochemical able immune (BCNU) wafers (gliadel wafers) in patients with glioma [ J]Neuro Oncol,2003,5(2): 79-88). Recently, Mathios et al have shown a new version of BCNU wafers for controlled release that outperformed systemic chemotherapy in GBM mouse model (Mathios D, Kim J E, Mangraviti A, et al, anti-PD-1 anti-cancer or immunological enhancement by local and antigenic by systemic chemotherapy in GBM [ J]Sci Transl Med,2016,8(370):370ra 180). Topical chemotherapy and anti-PD-1 antibodies can induce hundreds of higher than anti-PD-1A more antigen-specific cell. In preclinical models, the benefit of prolonged survival is obtained by increasing tumor infiltrating immune cells and memory cells to suppress the tumor.

Lipid nanocapsule-based hydrogel scaffolds (GemC12-LNC hydrogels) were constructed by Bastiancich et al and the therapeutic effect on in situ GBM mouse models was studied (Bastiancich C, Bianco J, Vanvarenberg K, et al. After 6 months of exposure, GemC12-LNC hydrogel showed good biocompatibility in mouse brain. The post-operative administration of GemC12-LNC hydrogel significantly delayed GBM recurrence and improved survival of mice compared to traditional intratumoral administration.

Wang et al synthesized an in situ forming Reactive Oxygen Species (ROS) reactive hydrogel loaded with Gemcitabine (GEM) and an immune checkpoint antibody, aPDL1, as a combination chemoimmunotherapy for treating melanoma and 4T1 breast cancer in mice (Wang C, Wang J, Zhang X, et al. in situ formed reactive oxygen species-reactive scaffold with a genetic binding and checkpoint inhibitor for combination therapy [ J ]. Sci Transl Med,2018,10 (429)). In another work, Shao et al developed a Sprayable, thermally sensitive Hydrogel PLEL loaded with Black Phosphorus (BP) nanoplates for post-operative Photothermal therapy (Shao J, Ruan C, Xie H, et al Black-phosphorous-Incorporated Hydrogel as a dispersible and Biodegradable Photothermal Platform for Postcosmetic Treatment of Cancer [ J ]. Adv Sci (Weinh),2018,5(5): 1700848). After surgery, the mice receiving photothermal therapy were completely cured, with no recurrence after 16 days, the lifespan of the latter was only 18-24 days and the recurrence rate was 80% compared to mice receiving surgery alone.

Flexible foams with sufficient coverage are expected to be used in surgical implant formulations due to the irregular shape of surgical wounds, and Ranganath et al investigated this approach in preclinical mouse models of GBM (Ong B Y, Ranganath S H, Lee L Y, et al. Patlitaxel delivery from PLGA foams for controlled release in post-surgical chemotherapy and against the surface of a vitreous biomaterial [ J ]. Biomaterials,2009,30(18): 3189-96). Biodegradable PLGA foams loaded with paclitaxel were implanted into the wound bed during surgical resection, and researchers showed that PLGA microporous foams exhibited low order but near zero order release with minimal initial burst and suppressed post-operative GBM recurrence.

Membranes with similar configurations have also been widely used in preclinical studies for post-surgical cancer treatment. For example, Liu et al developed a PGC-C18 membrane containing 10% paclitaxel and applied it to inhibit local tumor recurrence (Liu R, Wolinsky J B, Walpole J, et al, prediction of local tumor recurrence and perfusion utilization low-dose chemotherapeutic polymers [ J ]. Ann Surg Oncol,2010,17(4): 1203-13). The modification of stearic acid allows the membrane to release paclitaxel continuously (68.8% of drug is released cumulatively on day 50), thereby prolonging the drug effect. Furthermore, the paclitaxel concentration at the tumor resection site of the membrane implantation was 3000 times higher than that of the systemic injection within 10 days after the treatment. Only 22% of the mice injected systemically had no postoperative recurrence, but 83.3% of the mice receiving the membrane-implanted group had no tumor recurrence observed.

Sustained release to enhance therapeutic effect can also be achieved by micro/nano local delivery of therapeutic agents made of different materials. Chung C K et al established an albumin derived platform in a lymph node metastasis triple negative breast cancer model for image guided photothermal therapy (Chung C K, Da Silva C G, Kralisch D, et al combination therapy adapting nanoparticle-based cancer therapy with animal chemistry block for treatment of site-patient tissue recurrences [ J ]. J Control Release,2018,285: 56-66). Gd3+ and IR825 were conjugated to albumin, acting as T1-weighted Magnetic Resonance Imaging (MRI) and photothermal agents, respectively. By intratumoral injection, the nano-platform migrated into the nearby sentinel lymph node by dual mode MRI and fluorescence imaging. Furthermore, by applying NIR laser irradiation at the sentinel lymph node after resection of the primary tumor, tumor metastasis is significantly inhibited and the survival of the animals is improved. An in situ sprayed, bioreactive fibrin gel was developed by Chen Q et al to inhibit postoperative tumor recurrence and potential metastasis and was tested in the B16F10 melanoma mouse model (Chen Q, Wang C, Zhang X, et al in situ sprayed biological immunogenic gel for post-clinical cancer treatment [ J ]. Nat Nanotechnol,2019,14(1): 89-97). Calcium carbonate nanoparticles pre-loaded with anti-CD 47 antibody were immobilized in a bioreactive fibrin gel ([email protected] [email protected] fibrin) that scavenges H + to promote polarization of tumor-associated macrophages and retains immunosuppressive TME. The released anti-CD 47 antibody promotes enhanced recognition of cancer cells by antigen presenting cells and initiates the innate and adaptive immune system. Researchers have found that sprayed bioreactive gels show enhanced inhibition of tumor regeneration. Compared to the other groups, 50% of the mice had no detectable tumor and survived for at least 60 days without significant changes in body weight. Furthermore, it has been demonstrated that local treatment with spray gels can trigger a systemic immune response to suppress local tumor recurrence and reduce tumor growth in the opposite site.

Overall, the research and development of local drug delivery systems has shown exciting advances in post-operative cancer treatment, and the successful application of these technologies to the clinic has the potential to have profound effects on tumor surgical practice by reducing post-operative tumor recurrence, metastasis and increasing patient survival. However, the gel, foam and fiber formulations and kits that have been developed still have their problems:

the components such as gel, foam and fiber used in the existing local administration all have the problems of influencing wound healing, easily causing postoperative infection and inflammation, possibly causing local allergy and liponecrosis after postoperative curing, complicated preparation, high preparation cost and the like. For example, the most commonly used postoperative fibrin gel in clinical use is that a piece of gel is implanted under the skin of a wound, so that the thickness is increased, the healing speed is slower, and the suture part is more easily broken, so that the wound is slowly healed. In addition, fibrin glue on the market today has problems of not excessive quality.

Therefore, it is a great challenge to develop new pharmaceutical compositions and/or preparations suitable for administration to the wound after cancer surgery, which have the advantages of simple preparation, convenient administration, etc.

Based on the above background, the inventors have surprisingly found through a great deal of experimental studies that a pharmaceutical composition prepared by using a specific stabilizer, a lyoprotectant and a poorly soluble anti-tumor active agent has the advantages of good sedimentation stability, storage stability and the like, is suitable for treating postoperative cancer, prevents tumor recurrence, is easy and convenient to apply, does not have the above problems of existing local administration gels, foams, fibers and the like, can smoothly realize postoperative local administration of the poorly soluble anti-tumor active agent, and has an unexpectedly outstanding technical effect on tumor treatment, thereby satisfying unmet clinical needs.

Brief description of the invention

In one aspect, the present invention provides a pharmaceutical composition containing a poorly soluble antitumor active agent, characterized in that the poorly soluble antitumor pharmaceutical composition comprises a poorly soluble antitumor active agent, a stabilizer, and a lyoprotectant;

wherein the stabilizer is selected from the group consisting of polyoxyethylene castor oils such as polyoxyethylene 35 castor oil, especially pure polyoxyethylene 35 castor oil; polyethylene glycol hydroxystearate such as polyethylene glycol 15-hydroxystearate, polyethylene glycol 12-hydroxystearate; polysorbate 80 and mixtures thereof; preferably polysorbate 80; the lyoprotectant is selected from sucrose.

In another aspect, the invention provides a method of making a pharmaceutical composition described herein. The method has the advantages of simple preparation process, good reproducibility and easy industrialization.

In another aspect, the invention provides a pharmaceutical composition as described herein for use in postoperative wound administration, treatment of a neoplastic disease or prevention or reduction of tumor recurrence.

In another aspect, the invention provides a method of treating a neoplastic disease or preventing or reducing tumor recurrence in a patient, comprising administering to a post-tumoral wound of the patient an effective amount of a pharmaceutical composition described herein.

In another aspect, the invention provides the use of a pharmaceutical composition as described herein in the manufacture of a medicament for treating a neoplastic disease or preventing or reducing the recurrence of a neoplasm in a patient.

In another aspect, the present invention provides an injection device prefilled with a pharmaceutical composition as described herein.

Drawings

FIG. 1 shows the recurrence of tumors on day 60 after tumor resection in SK-OV-3 tumor-bearing nude mice

FIG. 2SK-OV-3 mean recurrent tumor weight of nude mice bearing tumors on day 60 after tumor resection (n ═ 8)

Detailed Description

A pharmaceutical composition containing a poorly soluble antitumor active agent, comprising a poorly soluble antitumor active agent, a stabilizer, and a lyoprotectant;

preferably wherein said stabilizer is selected from the group consisting of polyoxyethylene castor oils such as polyoxyethylene 35 castor oil, especially pure polyoxyethylene 35 castor oil; polyethylene glycol hydroxystearate such as polyethylene glycol 15-hydroxystearate, polyethylene glycol 12-hydroxystearate; polysorbate 80 and mixtures thereof; preferably polysorbate 80; the lyoprotectant is selected from sucrose.

In one embodiment, the weight ratio of the poorly soluble anti-tumor active agent to the stabilizer is 10:2 to 10:12, 10:2 to 10:10, 10:2 to 10:8, 10:2 to 10:6, 10:2 to 10:5, 10:2 to 10:4, or 10:2.5 to 10: 4; for example, 10:2, 10:3, 10:4, 10:5, 10:6, 10:7, 10:8, 10:9, 10:10, 10:11, preferably 10:2.5 to 10:4, more preferably 10: 3.

In one embodiment, the weight ratio of the poorly soluble anti-tumor active agent to the lyoprotectant is 10:80 to 10:300, for example 10:80 to 10:250, 10:90 to 10:200, 10:90 to 10:150, such as 10:90, 10:100, 10:120, 10:140, 10:160, 10:180, preferably 10:90 to 10:200, more preferably 10: 100.

In one embodiment, the poorly soluble anti-tumor active agent is selected from paclitaxel (paclitaxel), docetaxel (docetaxel), cabazitaxel (cabazitaxel), etoposide (etoposide), teniposide (teniposide), hydroxycamptothecin (hydroxycamptothecin), irinotecan (irinotecan), cisplatin (cispin), carboplatin (carboplatin), capecitabine (capecitabine), oxaliplatin (oxaliplatin), gefitinib (gefitinib), doxorubicin (doxorubicin), vinblastine (vinblastine), and vincristine (vincristine); preferably, the poorly soluble anti-tumor active agent is selected from paclitaxel, docetaxel, cabazitaxel, etoposide, teniposide and hydroxycamptothecin; most preferably, the poorly soluble anti-tumor active agent is selected from paclitaxel, docetaxel and cabazitaxel.

In one embodiment, the pharmaceutical composition is a composition for wound administration after tumor resection, for example of a tumor in a cancer patient, preferably for wound shower administration.

In one embodiment, the pharmaceutical composition does not comprise a poloxamer (e.g., poloxamer 188).

In one embodiment, the pharmaceutical composition does not comprise polysorbate 20, a poloxamer (e.g., poloxamer 188), trehalose, and/or mannitol.

In one embodiment, the pharmaceutical composition does not comprise other stabilizers.

In one embodiment, the pharmaceutical composition does not comprise other stabilizers and lyoprotectants.

In one embodiment, the pharmaceutical composition does not comprise other excipients.

In one embodiment, the pharmaceutical composition consists of the poorly soluble anti-tumor active agent, a stabilizer, and a lyoprotectant.

In one embodiment, the pharmaceutical composition consists of paclitaxel, polysorbate 80 and sucrose, preferably wherein the ratio of the amounts of paclitaxel, polysorbate 80 and sucrose is 10:3:100 (w/w).

In one embodiment, the pharmaceutical composition consists of docetaxel, polyethylene glycol 15-hydroxystearate and sucrose, preferably wherein the ratio of docetaxel, polyethylene glycol 15-hydroxystearate and sucrose is 10:5:200 (w/w).

In one embodiment, in the pharmaceutical composition, the poorly soluble anti-tumor active agent has a particle size between 100nm and 4000nm, such as between 100nm and 1000nm, between 120nm and 800nm, between 150nm and 600nm, between 100nm and 500nm, between 100nm and 400nm, between 200nm and 300nm, preferably between 100nm and 400 nm; preferably, the poorly soluble antitumor active agent has the following particle size dispersion coefficient (PDI): 0.1-0.7, such as 0.1-0.5, 0.1-0.4, 0.1-0.3 or 0.1-0.2.

In one embodiment, the pharmaceutical composition is a lyophilized formulation, such as a lyophilized powder.

In one embodiment, the pharmaceutical composition is a lyophilized formulation, which upon reconstitution with a vehicle gives a suspension having an average particle size of between 100nm and 4000nm, e.g. between 100nm and 1000nm, between 120nm and 800nm, between 150nm and 600nm, between 100nm and 500nm, between 100nm and 400nm, between 200nm and 300nm, preferably between 100nm and 400nm, preferably the suspension has the following particle size dispersion coefficient (PDI): 0.1-0.7, such as 0.1-0.5, 0.1-0.4, 0.1-0.3 or 0.1-0.2.

In another aspect, the present invention provides the use of a pharmaceutical composition as described herein for the preparation of a suspension for administration to a wound, particularly to a wound shower, after a tumor resection, preferably for the treatment of a tumor disease or for the prevention or reduction of tumor recurrence.

In one embodiment, the suspension has an average particle size of between 100nm and 4000nm, preferably between 100nm and 1000nm, more preferably between 100nm and 500nm, and even more preferably between 100nm and 400 nm.

In another aspect, the present invention provides a suspension of a poorly soluble anti-tumor active agent prepared by reconstituting or diluting the above lyophilized formulation.

In one embodiment, the suspension has an average particle size of between 100nm and 4000nm, for example between 100nm and 1000nm, between 120nm and 800nm, between 150nm and 600nm, between 100nm and 500nm, between 100nm and 400nm, between 200nm and 300nm, preferably between 100nm and 400 nm.

In one embodiment, reconstitution or dilution of the lyophilized formulation is carried out with a vehicle, which is preferably an injectable vehicle, such as water for injection, 5% dextrose injection, or 0.9% sodium chloride injection.

In one embodiment, the lyophilized formulation is reconstituted by addition of a vehicle in an amount of 0.05mg to 20mg of the poorly soluble anti-tumor active agent per ml of vehicle (e.g., 0.1 to 10mg/ml, 0.2 to 5mg/ml, 0.3 to 4mg/ml, 0.4 to 3mg/ml, 0.5 to 2 mg/ml).

In another aspect, the present invention provides an injection device prefilled with a pharmaceutical composition or suspension as described herein.

In one embodiment, the injection device is a disposable syringe.

In another aspect, the present invention provides a method of preparing a pharmaceutical composition as described herein, characterized in that the method comprises the steps of:

(3) wet grinding was performed as follows: grinding the suspension obtained in the step (2) for 0.5 to 10 hours, such as 1 to 8 hours, 1 to 6 hours, 1 to 4 hours, 2 to 4 hours, such as 2 hours, 2.5 hours, 3 hours and 4 hours, by using a wet grinding machine (the rotating speed is 2500rpm to 8000rpm, such as 3500rpm to 8000rpm, 4000rpm to 6000rpm, or 4000rpm to 5000 rpm; the water inlet temperature is-5 to 30 ℃, such as 4 to 15 ℃, preferably, the diameter of a grinding bead is 0.2 to 0.5 mm; the loading is 50 to 60% (V/V); so as to obtain a suspension; and

(4) diluting the suspension obtained in the step (3) with injectable solvent (preferably water for injection) (preferably 10-500 times, such as 20-400 times, 30-300 times, 40-200 times, 50-100 times, or 60-80 times), adding sucrose, stirring for dissolving, optionally packaging in penicillin bottles, and freeze-drying in a freeze dryer.

It is to be understood that, unless otherwise indicated or clearly contradicted, the amounts of the ingredients in the pharmaceutical composition in the method of preparing the pharmaceutical composition are as described above in the section on the embodiments of the pharmaceutical composition.

In another aspect, the present invention provides a pharmaceutical composition as described herein for administration to a wound, particularly to a wound shower, of a patient having a tumor after resection of the tumor, for treating the patient's tumor disease or for preventing or reducing tumor recurrence.

In another aspect, the invention provides a method of treating a neoplastic disease or preventing or reducing tumor recurrence in a patient, comprising administering to the wound following tumor resection of the patient an effective amount of a pharmaceutical composition described herein.

In one embodiment, the medicament is for administration to a wound, particularly wound shower administration, after a tumor resection in a patient with a tumor.

Compositions of the invention

The inventor discovers that the slightly soluble antitumor active agent, such as paclitaxel, can be prepared into a stable composition by using a stabilizing agent and a specific type of freeze-drying protective agent through research on the basic physicochemical properties of the slightly soluble antitumor active agent, the composition is a freeze-drying preparation, a proper solvent for injection, such as sterile injection water, 5% glucose injection or 0.9% sodium chloride injection, is used for diluting before use to form a nano suspension, and then the nano suspension is administered by the postoperative wound administration of a tumor patient, particularly by the wound spraying mode. In addition, the lyophilized preparation powder cake is complete and has no collapse, and the suspension obtained after dilution is a suspension with uniform and stable particle size.

Therefore, the invention provides a composition containing a slightly soluble anti-tumor active agent for drug delivery by wound surface spraying, which is characterized in that the slightly soluble anti-tumor drug composition comprises a slightly soluble anti-tumor active agent, a stabilizer and a freeze-drying protective agent;

the stabilizer is selected from polyoxyethylene 35 castor oil, pure polyoxyethylene 35 castor oil, 15-hydroxystearic acid polyethylene glycol ester, polysorbate 80 and a mixture thereof; preferably polysorbate 80; the lyoprotectant is selected from sucrose.

The weight ratio of the insoluble antitumor active agent to the stabilizer is 10: 1-10: 8, preferably 10: 2-10: 6, and more preferably 10: 3; the weight ratio of the insoluble antitumor active agent to the freeze-drying protective agent is 10: 80-10: 300, preferably 10: 90-10: 200, and more preferably 10: 100.

The composition containing the insoluble antitumor active agent administered by wound surface spraying is a freeze-dried preparation and has good physical and chemical stability. The preparation is placed at room temperature (25 ℃) for 24 months, and the appearance, the content and related substances of the preparation are not significantly changed;

the composition containing an insoluble antitumor active agent administered by wound surface showering of the present invention is a lyophilized preparation which can be diluted with a suitable solvent to form a suspension. The vehicle may be a vehicle suitable for injection (e.g., sterile water for injection, 5% dextrose injection, 0.9% sodium chloride injection, etc.). The suspension of the composition containing a poorly soluble antitumor active agent administered by drenching a wound surface of the present invention after dilution with a solvent has an average particle diameter of 100 to 4000nm, preferably 100 to 1000nm, more preferably 100 to 500nm, and still more preferably 100 to 400 nm. Can be used for postoperative wound surface drenching administration of tumor patients. For example, the composition of the present invention, specifically the composition containing paclitaxel, is diluted with 5% glucose injection to form a suspension with an average particle size of 210nm and a particle size distribution (PDI) of 0.1-0.7.

The compositions of the invention not only have good stability in themselves, but also the suspensions obtained from their dilution. For example, a suspension formed by diluting the composition of the present invention, specifically the composition containing paclitaxel, with a 5% glucose injection is placed at room temperature for 8 hours, the particle size is uniform, no significant change occurs, the stability is good, and the pH value is stable between 4.0 and 7.0.

The "poorly soluble drug antitumor active agent" of the present invention refers to a known antitumor active agent that can be applied in the field of medicine, and has a low solubility in water relative to the effective dose thereof. More specifically, the "poorly soluble drug antitumor" of the present invention refers to a drug having a solubility of "slightly soluble" (solute 1g (ml) is soluble in 100 to less than 1000ml of a solvent), "very slightly soluble" (solute 1g (ml) is soluble in 1000 to less than 10000ml of a solvent), or "hardly soluble or insoluble" (solute 1g (ml) is not completely soluble in 10000ml of a solvent) as described in "general examples" of the chinese pharmacopoeia.

Examples of poorly soluble antitumor active agents of the present invention include, but are not limited to: paclitaxel, docetaxel, cabazitaxel, etoposide, teniposide, hydroxycamptothecin, irinotecan, cisplatin, carboplatin, capecitabine, oxaliplatin, gefitinib, doxorubicin, vinblastine, vincristine; preferably, the poorly soluble anti-tumor active agent is selected from paclitaxel, docetaxel, cabazitaxel, etoposide, teniposide and hydroxycamptothecin; most preferably, the poorly soluble anti-tumor active agent is selected from paclitaxel, docetaxel, cabazitaxel.

The most preferred compositions of the present invention are those given in example 3 of the present application, in particular the composition used in example 4.

The composition containing a poorly soluble antitumor drug to be administered by wound surface shower of the present invention described above may consist of only the following components: a poorly soluble anti-tumor active agent; a stabilizer; and a lyoprotectant which is sucrose.

Alternatively, the compositions of the invention comprising poorly soluble anti-tumour active agents for administration by wound showering as described above may also contain other components, such as pH adjusting agents and/or antioxidants. The pH regulator can be one or more selected from citric acid, citrate (such as sodium citrate), maleic acid, tartaric acid, hydrochloric acid, sodium hydroxide, acetic acid, acetate (such as sodium acetate), phosphoric acid, and phosphate (such as sodium monohydrogen phosphate, sodium dihydrogen phosphate, or sodium phosphate). The antioxidant can be selected from one or more of alpha-tocopherol succinate, ascorbyl palmitate, Butylated Hydroxyanisole (BHA) and Butylated Hydroxytoluene (BHT).

The inventor takes various insoluble antitumor active agents such as paclitaxel and the like as a model of the insoluble antitumor active agents, and examines main factors influencing the preparation formability, the physical and chemical stability, the physical stability of a suspension obtained after being diluted into the suspension and the drug effect of drug administration through a wound after a tumor operation in the composition containing the insoluble antitumor active agents in detail to obtain the composition. The composition can be popularized and applied to other insoluble antitumor active agents, is applied to treatment of postoperative cancers by postoperative administration of a tumor patient, particularly administration by wound spraying, has a remarkable effect of preventing tumor recurrence, is simple and convenient to operate, does not have the problems of existing local administration gel, foam, fiber and the like, can smoothly realize postoperative local administration of the insoluble antitumor active agents, and meets the unmet clinical requirements.

Definition of

In the context of the present application, the terms "composition comprising a poorly soluble anti-tumor active agent", "composition of the invention" and the like are used interchangeably and refer to a composition comprising a poorly soluble anti-tumor active agent, a stabilizer and a lyoprotectant, except where the context indicates otherwise.

The term "polyoxyethylated castor oil" as used herein refers to a material obtained by reacting varying amounts of ethylene oxide with castor oil. Examples of polyoxyethylated castor oils include, but are not limited to, polyoxyethylated 35 castor oil, pure polyoxyethylated 35 castor oil.

The term "polyoxyethylene 35 castor oil" as used in the present invention means a material obtained by reacting 1mol of glyceryl ricinoleate with 35mol of ethylene oxide, which contains, in addition to polyoxyethylene glyceryl triricinoleate, small amounts of polyethylene glycol ricinoleate and free ethylene glycol. Polyoxyethylene 35 castor oil is commercially available, for example under the tradenames kolliphor EL and kolliphor ELP from BASF, and the like

The term "pure polyoxyethylene 35 castor oil" as used herein refers to purified polyoxyethylene glycerol triricinoleate.

"polyethylene glycol 15-hydroxystearate" according to the present invention is commercially available, for example under the trade name kolliphor HS15 or Solutol HS-15 from BASF or Sigma-Aldrich.

The polysorbate is particularly polysorbate 80. Polysorbate 80 is commercially available, for example under the trade name tween80 from south beige wil pharmaceutical products limited.

By "reconstituted" is meant that the lyophilized formulation is diluted with a vehicle to reconstitute it into a suspension for administration.

"tumor" refers to a neoplasm formed by local tissue cell proliferation of the body under the action of various tumorigenic factors, including benign tumors and cancers, including but not limited to melanoma, glioblastoma, ovarian cancer, pancreatic cancer, prostate cancer, lung cancer, breast cancer, renal cancer, cervical cancer, thyroid cancer, metastases of secondary sites of primary solid tumors, myeloproliferative diseases, papillary thyroid cancer, non-small cell lung cancer, mesothelioma, hypereosinophilic syndrome, gastrointestinal stromal tumors, colon cancer.

Herein, "administration to a wound after surgery" or "administration to a wound after tumor resection" means that the drug is applied to the wound after the tumor resection, preferably by shower. "vehicle" means a solvent or solution suitable for the preparation of a pharmaceutical formulation, including purified water, double distilled water; in particular to injectable solvents, including but not limited to water for injection, 5% glucose injection or 0.9% sodium chloride injection.

The term "about" as used herein will float ± 10% of the value with which it is used. In the case of a scale, it is preferred,

the term "about" is used to define each number of the given ratio. For example, a ratio of about 1:1 means a ratio of 0.9 to 1.1:0.9 to 1.1.

The term "patient" as used herein refers to mammalian and non-mammalian subjects who may benefit from the poorly soluble anti-tumor active agents of the present invention. Mammal refers to any member of the mammalian family, including but not limited to: a human; non-human primates, such as chimpanzees and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, and pigs; domestic animals such as rabbits, dogs, and cats; laboratory animals, including rodents, such as rats, mice and guinea pigs; and so on. Examples of non-mammals include, but are not limited to, birds and the like. The term "patient" does not limit a particular age or gender. In some embodiments, the "patient" is a human.

Unless otherwise specified, "administration" and "administration" are used interchangeably herein.

Proportions referred to herein are by weight (w/w) unless otherwise determined by context or otherwise specifically stated.

Herein, "stabilizer" refers to a substance used as a stabilizer in a lyophilized formulation. Examples of lyophilized formulation stabilizers include, but are not limited to, polysorbate 20, polysorbate 80, polyoxyethylene 35 castor oil, neat polyoxyethylene 35 castor oil, polyethylene glycol 15-hydroxystearate, and poloxamer 188. Stabilizers suitable for use in the pharmaceutical compositions of the present invention include polyoxyethylene castor oils such as polyoxyethylene 35 castor oil, especially pure polyoxyethylene 35 castor oil; polyethylene glycol hydroxystearate such as polyethylene glycol 15-hydroxystearate, polyethylene glycol 12-hydroxystearate; polysorbate 80 or a mixture thereof.

By "lyoprotectant" herein is meant a lyoprotectant commonly used in lyophilized formulations, including but not limited to trehalose, sucrose, mannitol, and glucose. A suitable stabilizer for use in the pharmaceutical compositions of the present invention is sucrose.

"Mean Diameter" refers to the corresponding volume-to-volume Diameter of the particle formulation, as measured by a laser particle sizer.

Technical and scientific terms used herein that are not specifically defined have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs.

Detailed Description

Examples

The following examples are intended to illustrate the invention, but in no way limit the scope thereof as defined by the appended claims.

The meanings of abbreviations used in the examples are shown in Table 1.

TABLE 1 abbreviations

Example 1: screening of paclitaxel nanosuspension stabilizer

1. Prescription 1-5 selection of stabilizer species

TABLE 2-formulations containing different stabilizers (F68, Tween20, Tween80, ELP or HS15)

Components	Prescription 1	Prescription 2	Prescription 3	Prescription 4	Prescription 5
						Paclitaxel	10g	10g	10g	10g	10g
F68	3g	--	--	--	--
						Tween20	--	3g	--	--	--
Tween80	--	--	3g	--	--
						ELP	--	--	--	3g	--
HS15	--	--	--	--	3g
						Sucrose	100g	100g	100g	100g	100g
Total of	113g	113g	113g	113g	113g

The process comprises the following steps:

(1) preparing a stabilizer solution: weighing a prescription amount of the stabilizer into a 500mL beaker, weighing 100g of water for injection, adding the water into the beaker, and heating and stirring in a water bath to completely dissolve the stabilizer;

(2) preparing taxol bulk drug suspension: weighing 10g of paclitaxel raw material medicine, adding the paclitaxel raw material medicine into the stabilizer solution obtained in the step (1), heating in a water bath at 60 ℃, and stirring until the system is uniform suspension;

(3) and (3) wet grinding: in this order, a wet mill was installed (grinding bead diameter: 0.3 mm; loading: 55% (V/V); rotation speed: 4000 rpm; water feed temperature: 8 ℃ C.). And (3) pouring the suspension obtained in the step (2) into a grinding machine from a sample injection funnel, and grinding for 2.5 hours after starting up the machine to obtain the stock solution.

(4) And (3) freeze drying: diluting the stock solution obtained in the step (3) by 50 times with water for injection, adding sucrose in a prescribed amount into the stock solution, stirring and dissolving the mixture to obtain taxol suspension, subpackaging the taxol suspension in 7ml penicillin bottles, wherein each bottle contains 2ml, and freeze-drying the taxol suspension in a freeze-dryer to obtain taxol freeze-dried suspension;

the paclitaxel preparations obtained by the prescriptions 1-5 are all freeze-dried preparations, and all the powder cakes obtained after freeze-drying are complete and have no collapse; the resulting lyophilized preparation (1 in 2ml) was reconstituted with 4ml of 5% glucose injection, and the average particle size was measured with a laser particle sizer (us PSS, model nicomz 3000) and the stability was examined, and the results are shown in table 3.

TABLE 3 particle size and stability of paclitaxel lyophilized suspensions prepared with different stabilizers

Note: "- -" indicates that the particle size was not measured due to precipitation; "MD" means the average particle Diameter (Mean Diameter) in nm; "PDI" represents the particle size dispersion index (Polydispersity index).

Experimental results show that when the stabilizing agents are Tween20 and F68, the particle size and the dispersion coefficient of corresponding taxol suspension are large, the stability is poor, and a large amount of precipitates can be formed after about 3min after redissolution, so that the determination is not carried out; and when the stabilizer types are pure polyoxyethylene 35 castor oil, 15-hydroxystearic acid polyethylene glycol ester and polysorbate 80, the corresponding paclitaxel suspension has uniform particle size, good stability after being placed for 8 hours at room temperature, no significant change in particle size and no precipitation.

2. Prescription 6-8, screening of stabilizer dosage

TABLE 4 formulation of stabilizers with different amounts

The process comprises the following steps:

(1) preparing a stabilizer solution: weighing a prescription amount of the stabilizer into a 500mL beaker, weighing 100g of water for injection, adding the water for injection into the beaker, heating in a water bath and stirring to completely dissolve the stabilizer;

the paclitaxel preparations obtained by the prescriptions 6-11 are all freeze-dried preparations, and all the powder cakes obtained after freeze-drying are complete and have no collapse; the resulting lyophilized preparation (1 tube was reconstituted with 4ml of 5% glucose injection solution, and then the average particle size was measured by a laser particle sizer (us PSS, model nicomz 3000) and the stability was examined, and the results are shown in table 5.

TABLE 5 particle size and stability of suspensions formed after reconstitution of paclitaxel freeze-dried suspensions prepared with different stabilizer dosages

Example 2: screening of taxol nanosuspension freeze-drying protective agent

1. Prescription 3, prescription 12-14, screening of types of freeze-drying protective agents

TABLE 6 formulation containing different lyoprotectants

Components	Prescription 3	Prescription 12	Prescription 13	Prescription 14
					Paclitaxel	10g	10g	10g	10g
Tween80	3g	3g	3g	3g
					Sucrose	100g	--	--	--
Trehalose	--	100g	--	--
					Mannitol	--	--	100g	--
Glucose	--	--	--	100g
					Total of	113g	113g	113g	113g

The process comprises the following steps: same as example 1

The paclitaxel preparation obtained by the prescription 3 and the prescriptions 12-14 are freeze-dried preparations, and the powder cakes obtained by freeze-drying the prescription 3, the prescription 12 and the prescription 13 are complete and have no collapse; prescription 14 corresponds to a completely collapsed lyophilized formulation; the lyophilized preparation (1 lot) obtained in formulation 3 and formulations 12 to 14 was reconstituted with 4ml of 5% glucose injection, and the average particle size was measured with a laser particle sizer (us PSS, model nicomz 3000) to examine the stability, and the results are shown in table 7.

TABLE 7 particle size and stability of paclitaxel lyophilized suspensions prepared with different lyoprotectants

Experimental results show that when only the freeze-drying protective agent is cane sugar, the obtained preparation has uniform particle size and good stability. The corresponding taxol suspension has uniform grain diameter, good stability after being placed for 8 hours at room temperature, no obvious change of grain diameter and no precipitation.

2. Prescription 6-8, screening of dosage of freeze-drying protective agent

TABLE 8 formulation of different amounts of lyoprotectant

The process comprises the following steps: same as example 1

The paclitaxel preparations obtained by the prescription 3 and the prescriptions 16-19 are freeze-dried preparations, and the powder cakes obtained after freeze-drying are complete and have no collapse; the freeze-dried preparation corresponding to the prescription 15 has the problems of collapse, incomplete pressed powder and the like; therefore, the particle size measurement and stability investigation after redissolution are not carried out;

the lyophilized preparation (1 st 2ml) obtained by formulation 3 and formulations 16 to 19 was reconstituted with 4ml of 5% glucose injection, and then the average particle size was measured with a laser particle sizer (us PSS, model nicomz 3000) to examine the stability, and the results are shown in table 9.

TABLE 9 particle size and stability of paclitaxel lyophilized suspensions prepared with different amounts of lyoprotectant

Experimental results show that the particle size of the obtained preparation is uniform and the stability is good only when the weight ratio of the paclitaxel to the freeze-drying protective agent is 10: 80-10: 300. The corresponding taxol suspension has uniform grain diameter, good stability after being placed for 8 hours at room temperature, no obvious change of grain diameter and no precipitation.

Example 3: preparation of compositions containing poorly soluble antitumor active agents

Freeze-dried preparation 1

Components	Dosage of
		Paclitaxel	10g
Tween80	3g
		Sucrose	100g
Total of	113g

The process comprises the following steps:

lyophilized preparation 2

Components	Dosage of
		Docetaxel	10g
HS15	5g
		Sucrose	200g
Total of	215g

The process comprises the following steps:

(2) preparing docetaxel bulk drug suspension: weighing 10g of docetaxel bulk drug, adding the docetaxel bulk drug into the stabilizer solution obtained in the step (1), heating in a water bath at 50 ℃, and stirring until a system is in a uniform suspension;

(3) and (3) wet grinding: in this order, a wet mill was installed (grinding bead diameter: 0.3 mm; loading: 50% (V/V); rotation speed: 3500 rpm; water feed temperature: 7 ℃ C.). And (3) pouring the suspension obtained in the step (2) into a grinding machine from a sample injection funnel, and grinding for 2 hours after starting up to obtain the stock solution.

(4) And (3) freeze drying: diluting the stock solution obtained in the step (3) by 50 times with water for injection, adding sucrose in a prescribed amount into the stock solution, stirring and dissolving the mixture to obtain docetaxel suspension, subpackaging the docetaxel suspension in 7ml penicillin bottles, wherein each bottle contains 2ml, and putting the docetaxel suspension into a freeze dryer for freeze drying to obtain docetaxel freeze-dried suspension;

lyophilized preparation 3

Components	Dosage of
		Paclitaxel	10g
Tween80	2g
		Sucrose	100g
Total of	112g

The process comprises the following steps:

(3) and (3) wet grinding: in this order, a wet mill was installed (bead diameter: 0.2 mm; loading: 60% (V/V); rotation speed: 8000 rpm; water feed temperature: 4 ℃ C.). And (3) pouring the suspension obtained in the step (2) into a grinding machine from a sample injection funnel, and grinding for 3 hours to obtain the stock solution.

lyophilized preparation 4

Components	Dosage of
		Paclitaxel	10g
Tween80	8g
		Sucrose	80g
Total of	98g

The process comprises the following steps:

(2) preparing taxol bulk drug suspension: weighing 10g of paclitaxel raw material medicine, adding the paclitaxel raw material medicine into the stabilizer solution obtained in the step (1), heating in a water bath at 80 ℃, and stirring until the system is uniform suspension;

(3) and (3) wet grinding: in this order, a wet mill was installed (grinding bead diameter: 0.5 mm; loading: 50% (V/V); rotation speed: 3500 rpm; water feed temperature: 15 ℃ C.). And (3) pouring the suspension obtained in the step (2) into a grinding machine from a sample injection funnel, and grinding for 4 hours to obtain the stock solution.

lyophilized preparation 5

Components	Dosage of
		Paclitaxel	10g
Tween80	6g
		Sucrose	300g
Total of	316g

The process comprises the following steps:

(3) and (3) wet grinding: in this order, a wet mill was installed (grinding bead diameter: 0.3 mm; loading: 55% (V/V); rotation speed: 5000 rpm; water feed temperature: 7 ℃ C.). And (3) pouring the suspension obtained in the step (2) into a grinding machine from a sample injection funnel, and grinding for 1.2h after starting the machine to obtain the stock solution.

lyophilized preparation 6

Components	Dosage of
		Paclitaxel	10g
HS15	3g
		Sucrose	100g
Total of	113g

The process comprises the following steps:

lyophilized preparation 7

The process comprises the following steps:

(4) And (3) freeze drying: diluting the stock solution obtained in the step (3) by 50 times with water for injection, adding sucrose in a prescribed amount into the stock solution, stirring and dissolving the mixture to obtain docetaxel suspension, subpackaging the docetaxel suspension in 7ml penicillin bottles, wherein each bottle contains 2ml, and putting the docetaxel suspension into a freeze dryer for freeze drying to obtain docetaxel freeze-dried suspension;

lyophilized preparation 8

Components	Dosage of
		Cabazitaxel	10g
ELP	1g
		Sucrose	100g
Total of	111g

The process comprises the following steps:

(2) preparing a cabazitaxel bulk drug suspension: weighing 10g of cabazitaxel raw material medicine, adding the cabazitaxel raw material medicine into the stabilizer solution obtained in the step (1), heating in a water bath at 60 ℃, and stirring until a system is in a uniform suspension;

(4) And (3) freeze drying: diluting the stock solution obtained in the step (3) by 50 times with water for injection, adding sucrose in a prescribed amount into the stock solution, stirring and dissolving the mixture to obtain cabazitaxel suspension, subpackaging the cabazitaxel suspension in 7ml penicillin bottles, wherein each bottle contains 2ml, and putting the cabazitaxel suspension into a freeze dryer for freeze drying to obtain cabazitaxel freeze-dried suspension;

lyophilized preparation 9

Components	Dosage of
		Etoposide	10g
Tween80	8g
		Sucrose	100g
Total of	118g

The process comprises the following steps:

(2) preparing an etoposide bulk drug suspension: weighing 10g of etoposide raw material medicine, adding the etoposide raw material medicine into the stabilizer solution obtained in the step (1), heating in a water bath at 80 ℃, and stirring until the system is in a uniform suspension;

(4) And (3) freeze drying: diluting the stock solution obtained in the step (3) by 50 times with water for injection, adding sucrose in a prescribed amount into the stock solution, stirring and dissolving to obtain etoposide suspension, subpackaging the etoposide suspension in 7ml penicillin bottles, wherein each bottle is 2ml, and putting the etoposide suspension into a freeze dryer for freeze drying to obtain an etoposide freeze-dried suspension;

lyophilized preparation 10

Components	Dosage of
		Oxaliplatin	10g
Tween80	6g
		Sucrose	300g
Total of	316g

The process comprises the following steps:

(2) preparation of oxaliplatin bulk drug suspension: weighing 10g of oxaliplatin bulk drug, adding the oxaliplatin bulk drug into the stabilizer solution obtained in the step (1), heating in a water bath at 60 ℃, and stirring until the system is in a uniform suspension;

(4) And (3) freeze drying: diluting the stock solution obtained in the step (3) by 50 times with water for injection, adding sucrose in a prescription amount into the stock solution, stirring and dissolving to obtain oxaliplatin suspension, subpackaging the oxaliplatin suspension in 7ml penicillin bottles with 2ml per bottle, and freeze-drying in a freeze dryer to obtain oxaliplatin freeze-dried suspension;

lyophilized preparation 11

Components	Dosage of
		Gefitinib	10g
Tween80	3g
		Sucrose	100g
Total of	113g

The process comprises the following steps:

(2) preparing gefitinib raw material medicament suspension: weighing 10g of gefitinib raw material drug, adding the gefitinib raw material drug into the stabilizer solution obtained in the step (1), heating in a water bath at 60 ℃, and stirring until the system is in a uniform suspension;

(4) And (3) freeze drying: diluting the stock solution obtained in the step (3) by 50 times with water for injection, adding sucrose in a prescribed amount into the stock solution, stirring and dissolving the mixture to obtain gefitinib suspension, subpackaging the gefitinib suspension in 7ml penicillin bottles, wherein each vial contains 2ml, and putting the gefitinib suspension into a freeze dryer for freeze drying to obtain gefitinib freeze-dried suspension;

lyophilized preparation 12

Components	Dosage of
		Hydroxycamptothecin	10g
Tween80	3g
		Sucrose	100g
Total of	113g

The process comprises the following steps:

(2) preparing a hydroxycamptothecin raw material medicament suspension: weighing 10g of hydroxycamptothecin raw material drug, adding into the stabilizer solution obtained in the step (1), heating in a water bath at 60 ℃, and stirring until the system is uniform suspension;

(4) And (3) freeze drying: diluting the stock solution obtained in the step (3) by 50 times with water for injection, adding sucrose in a prescribed amount into the stock solution, stirring and dissolving the mixture to obtain hydroxycamptothecin suspension, subpackaging the hydroxycamptothecin suspension in 7ml penicillin bottles, wherein each vial contains 2ml of the solution, and putting the solution into a freeze dryer for freeze drying to obtain a hydroxycamptothecin freeze-dried suspension;

lyophilized preparation 13

Components	Dosage of
		Cis-platinum	10g
Tween80	3g
		Sucrose	100g
Total of	113g

The process comprises the following steps:

(2) cisplatin raw material medicine suspension preparation: weighing 10g of cisplatin raw material medicine, adding the cisplatin raw material medicine into the stabilizer solution obtained in the step (1), heating in a water bath at 60 ℃, and stirring until a system is in a uniform suspension;

(4) And (3) freeze drying: diluting the stock solution obtained in the step (3) by 50 times with water for injection, adding sucrose in a prescribed amount into the stock solution, stirring and dissolving the mixture to obtain cisplatin suspension, subpackaging the cisplatin suspension in 7ml penicillin bottles, wherein each bottle contains 2ml of cisplatin suspension, and freeze-drying the cisplatin suspension in a freeze dryer to obtain cisplatin freeze-dried suspension;

lyophilized preparation 14

The process comprises the following steps:

(2) preparing an etoposide bulk drug suspension: weighing 10g of etoposide raw material medicine, adding the etoposide raw material medicine into the stabilizer solution obtained in the step (1), heating in a water bath at 60 ℃, and stirring until the system is in a uniform suspension;

(4) And (3) freeze drying: diluting the stock solution obtained in the step (3) by 50 times with water for injection, adding sucrose in a prescribed amount into the stock solution, stirring and dissolving to obtain etoposide suspension, subpackaging the etoposide suspension in 7ml penicillin bottles, wherein each bottle is 2ml, and putting the etoposide suspension into a freeze dryer for freeze drying to obtain an etoposide freeze-dried suspension;

lyophilized preparation 15

Components	Dosage of
		Teniposide	10g
Tween80	3g
		Sucrose	100g
Total of	113g

The process comprises the following steps:

(2) preparing a teniposide bulk drug suspension: weighing 10g of teniposide bulk drug, adding into the stabilizer solution obtained in the step (1), heating in a water bath at 60 ℃, and stirring until the system is uniform suspension;

(4) And (3) freeze drying: diluting the stock solution obtained in the step (3) by 50 times with water for injection, adding sucrose in a prescribed amount into the stock solution, stirring and dissolving the mixture to obtain teniposide suspension, subpackaging the teniposide suspension in 7ml penicillin bottles, wherein each bottle contains 2ml, and putting the teniposide suspension into a freeze dryer for freeze drying to obtain teniposide freeze-dried suspension;

lyophilized preparation 16

Components	Dosage of
		Capecitabine	10g
Tween80	3g
		Sucrose	100g
Total of	113g

The process comprises the following steps:

(2) preparing capecitabine bulk drug suspension: weighing 10g of capecitabine bulk drug, adding the capecitabine bulk drug into the stabilizer solution obtained in the step (1), heating in a water bath at 60 ℃, and stirring until the system is uniform suspension;

(4) And (3) freeze drying: diluting the stock solution obtained in the step (3) by 50 times with water for injection, adding sucrose in a prescribed amount into the stock solution, stirring and dissolving the mixture to obtain capecitabine suspension, subpackaging the capecitabine suspension in 7ml penicillin bottles, wherein each vial contains 2ml of the capecitabine suspension, and freeze-drying the capecitabine suspension in a freeze dryer to obtain a capecitabine freeze-dried suspension;

lyophilized preparation 17

Components	Dosage of
		Carboplatin	10g
Tween80	3g
		Sucrose	100g
Total of	113g

The process comprises the following steps:

(2) preparing carboplatin crude drug suspension: weighing 10g of carboplatin raw material medicine, adding the carboplatin raw material medicine into the stabilizer solution obtained in the step (1), heating in a water bath at 60 ℃, and stirring until the system is uniform suspension;

(4) And (3) freeze drying: diluting the stock solution obtained in the step (3) by 50 times with water for injection, adding sucrose in a prescribed amount into the stock solution, stirring and dissolving the mixture to obtain carboplatin suspension, subpackaging the carboplatin suspension in 7ml penicillin bottles, wherein each bottle contains 2ml, and putting the suspension into a freeze dryer for freeze drying to obtain carboplatin freeze-dried suspension;

lyophilized preparation 18

Components	Dosage of
		Vincristine	10g
Tween80	3g
		Sucrose	100g
Total of	113g

The process comprises the following steps:

(2) preparing vincristine bulk drug suspension: weighing 10g of vincristine raw material medicine, adding into the stabilizer solution obtained in the step (1), heating in a water bath at 60 ℃, and stirring until the system is a uniform suspension;

(4) And (3) freeze drying: diluting the stock solution obtained in the step (3) by 50 times with water for injection, adding sucrose in a prescribed amount into the stock solution, stirring and dissolving the mixture to obtain vincristine suspension, subpackaging the vincristine suspension in 7ml penicillin bottles, wherein each bottle contains 2ml of vincristine, and putting the vincristine suspension into a freeze dryer for freeze drying to obtain vincristine freeze-dried suspension;

lyophilized preparation 19

Components	Dosage of
		Gefitinib	10g
Tween80	3g
		Sucrose	100g
Total of	113g

The process comprises the following steps:

(4) And (3) freeze drying: diluting the stock solution obtained in the step (3) by 50 times with water for injection, adding sucrose in a prescribed amount into the stock solution, stirring and dissolving the mixture to obtain gefitinib suspension, subpackaging the gefitinib suspension in 7ml penicillin bottles, wherein each vial contains 2ml, and putting the gefitinib suspension into a freeze dryer for freeze drying to obtain gefitinib freeze-dried suspension;

lyophilized preparation 20

Components	Dosage of
		Vinblastine	10g
Tween80	3g
		Sucrose	100g
Total of	113g

The process comprises the following steps:

(2) preparing a vinblastine bulk drug suspension: weighing 10g of vinblastine bulk drug, adding into the stabilizer solution obtained in the step (1), heating in a water bath at 60 ℃, and stirring until the system is in a uniform suspension;

(4) And (3) freeze drying: diluting the stock solution obtained in the step (3) by 50 times with water for injection, adding sucrose in a prescribed amount into the stock solution, stirring and dissolving to obtain vinblastine suspension, subpackaging the vinblastine suspension in 7ml penicillin bottles, wherein each bottle contains 2ml of vinblastine, and freeze-drying the vinblastine suspension in a freeze-drying machine to obtain a vinblastine freeze-dried suspension;

and (3) particle size measurement:

the results of measuring the average particle size of the formulations corresponding to 1 to 20 of the lyophilized formulations of example 3 (1 was reconstituted with 4ml of 5% glucose injection and then measured with a laser particle sizer (us PSS, model nicomz 3000) are shown in the following table.

Example 4: stability Studies of the compositions of the invention

According to the guiding principle of stability test of raw material medicines and pharmaceutical preparations in the general regulation 9001 of the 2015 edition of Chinese pharmacopoeia, the lyophilized preparation 1 in the example 3 is placed at the temperature of 25 +/-2 ℃ and the relative humidity of 60 +/-5 percent according to the market package, samples are taken at the end of 3, 6, 12 and 24 months, and the corresponding indexes are examined.

1. Sample information: lyophilized paclitaxel formulation prepared according to the method for preparing lyophilized formulation 1 of example 3, lot No. 02170901

2. Stability test lofting conditions:

3. analytical method

(1) Traits

The checking method comprises the following steps: visual inspection was carried out.

(2) pH value

The lyophilized preparation (1 tube 2ml) was reconstituted with 4ml of 5% glucose injection and the pH was measured using a laser particle sizer (US PSS, model Nicomp Z3000); the pH value should be between 4.0 and 7.0

(3) Related substances

The detection method comprises the following steps: HPLC method

The experimental conditions are as follows:

a chromatographic column: c18 column (type: Zishengtang C18, 15cm long, 4.6mm inner diameter, filler diameter 3.0 μm)

Column temperature: 35 deg.C

A detector: UV detector (detection wavelength 227nm)

Mobile phase: mobile phase A: water-acetonitrile (3:2), mobile phase B: acetonitrile; the linear gradient elution was performed as follows:

flow rate: 1.2ml/min

Sample introduction volume: 10 μ L

Operating time: 75min

The system adaptability is as follows: the separation degree between the paclitaxel impurity e and the paclitaxel should be more than 1.2; the relative standard deviation of the peak areas of the main peak and each known impurity peak of the repeated sample injection is not more than 2.0 percent; the control solution should have a major peak signal to noise ratio greater than 10.

Specific experimental operations:

dissolving 1 part of the product in ethanol-water (3:2), and diluting to obtain solution containing 1mg per 1ml as test solution; an appropriate amount was precisely measured and diluted with ethanol-water (3:2) to contain about 12. mu.g of solution per 1ml, as a control solution. And taking a proper amount of paclitaxel, an impurity a, an impurity b, an impurity c, an impurity d, an impurity e and an impurity f as reference substances, dissolving the reference substances with acetonitrile, and diluting the reference substances with a mobile phase A to prepare a solution containing 1.2mg of paclitaxel, 9 mug of impurity a, 4.5 mug of impurity b, 9 mug of impurity c, 6 mug of impurity d, 6 mug of impurity e and 7 mug of impurity f in each 1ml, wherein the solution is used as a system applicability solution. Testing by high performance liquid chromatography (appendix V D) using octadecylsilane bonded and silica gel as packing (150 × 4.6mm, 3.0 μm column); water-acetonitrile (3:2) is taken as a mobile phase A, and acetonitrile is taken as a mobile phase B; the flow rate is 1.2 ml/min; the column temperature was 35 ℃; gradient elution was performed as follows. Respectively and precisely measuring 10 μ L of each of the reference solution and the system applicability solution, injecting into a liquid chromatograph, and recording chromatogram. The separation degree between the paclitaxel peak and the paclitaxel impurity e peak is more than 1.2, the relative standard deviation of the peak areas of the main peak of repeated sample injection and each known impurity peak is not more than 2.0%, and the signal-to-noise ratio of the main peak of the contrast solution is more than 10.

Precisely measuring 10 μ l of the reference solution, injecting into a liquid chromatograph, adjusting detection sensitivity to make the peak height of the main component chromatographic peak about 10-20% of the full scale, precisely measuring 10 μ l of each of the test solution and the reference solution, injecting into the liquid chromatograph, and recording chromatogram. In the chromatogram of the sample solution, impurity peaks (minimum peak area 1000counts) are present except the blank solvent peak, and the impurity content is calculated according to a 1% self-contrast method added with a correction factor, and the impurity limit is shown in the following table.

Calculating the formula:

the amount of each known impurity (%) - (Ai/Ar) × (1/Fi) × 100%

Other single impurity (%) ═ a_{Simple impurity}/Ar×100％；

The total amount of impurities (%). sigma.i/Ar. times.100%

Remarking: ai: peak areas of known impurities in the test sample solution;

ar: the major peak area of the control solution;

fi: a correction factor for each known impurity;

A_{simple impurity}: the single largest impurity peak area in the test sample solution (except for the known impurity peak);

A_{total miscellaneous}: the sum of the peak areas of the impurities in the test solution.

(4) Particle size

Dynamic light scattering, the lyophilized formulation (1 in 2ml) was reconstituted with 4ml of 5% glucose injection and the mean particle size and polydispersity index (PDI) was determined using a laser particle sizer (us PSS, model nicomz 3000);

(5) moisture content

ChP2015 type four-part general rule 0861 contains 5% (w/w) of water

(6) Content (wt.)

The detection method comprises the following steps: HPLC method

The experimental conditions are as follows:

a chromatographic column: 5-fluorophenyl column (type: FluoroSep-RP Phenyl, length 25cm, inner diameter 4.0mm, filler particle size 5 μm)

Column temperature: at room temperature

A detector: UV detector (detection wavelength 227nm)

Mobile phase: water-acetonitrile (11:9)

Flow rate: 1.5ml/min

Sample introduction volume: 10 μ L

Operating time: 15min

The system adaptability is as follows: injecting 10 μ l of system applicability solution under related substance into liquid chromatograph, wherein the separation degree of paclitaxel and impurity peak is greater than 1.0, and the relative standard deviation of repeated sample injection is not more than 1.5%.

Specific experimental operations:

a5-fluorophenyl column (model: FluoroSp-RP Phenyl, length of 25cm, inner diameter of 4.0mm, filler particle size of 5 mu m) is used as an analysis column, water-acetonitrile (11:9) is used as a mobile phase, the detection wavelength is 227nm, the flow rate is 1.5ml/min, and the relative standard deviation of the retention time of a main peak of repeated sample injection is not more than 1.5 percent.

Injecting 10 μ L of system applicability solution into liquid chromatograph, wherein the separation degree of paclitaxel and impurity peak should be greater than 1.0. Dissolving 1 part of the product with ethanol-water (3:2), diluting to obtain solution containing 0.1mg per 1ml, precisely measuring 10 μ L, injecting into liquid chromatograph, and recording chromatogram. Taking another appropriate amount of paclitaxel reference substance, measuring by the same method, and calculating by peak area according to external standard method.

Calculating the formula:

note: wr: weighing the reference substance (mg);

p: the indicated amount of the formulation (mg);

ar: area of the main peak of the reference;

as: the main peak area of the test sample;

dr: dilution volume of control;

ds: the dilution volume of the test article;

cr: purity of the reference.

4. Results of the experiment

TABLE 10 Long-term test (25 ℃. + -. 2 ℃ RH 60%. + -. 5%) of lyophilized formulation 1 of example 3 results (batch No.: 02170901)

The experimental results show that the freeze-dried preparation 1 is placed for 24 months under the conditions of the temperature of 25 +/-2 ℃ and the relative humidity of 60 +/-5 percent, all indexes meet the requirements, and the stability is good.

Experiments demonstrated that lyophilized formulations 2-6 of example 3 have similar stability to lyophilized formulation 1.

The inventor also carried out influence factor examination on part of the freeze-dried preparations (freeze-dried preparations 7, 8,10, 12 and 15) in example 3 under the condition of 40 ℃, sampling time of 0, 5, 10 and 30 days and content index, and the results are shown in the table below.

TABLE 11 influence factor test results for part of lyophilized formulations in example 3 at 40 ℃

Preparation	Day 0	5 days	10 days	30 days
					Lyophilized preparation 7	100％	100.2％	99.5％	97.8％
Lyophilized preparation 8	100％	100.3％	101.2％	96.3％
					Lyophilized preparation 10	100％	98.6％	97.9％	98.5％
Lyophilized preparation 12	100％	99.6％	97.7％	96.9％
					Lyophilized preparation 15	100％	100.6％	101.7％	98.5％

From the above results, it was found that the lyophilized preparation of example 3 was excellent in chemical stability.

Example 5: the invention relates to a pharmacodynamics research of animal in vivo of the wound surface shower administration of the paclitaxel suspension

1 cell line and experimental animal

Human ovarian cancer SK-OV-3 cells, purchased from the institute of basic medicine of Chinese academy of medical sciences.

Balb/c nude mice, female, 6-8 weeks old, purchased from the center of Beijing Wintolite laboratory animals, were raised under SPF-level conditions for one week before study, and were able to eat freely and water.

2 method of experiment

2.1 cell culture and inoculation

Preparing a complete cell culture solution: taking a bottle of MCCOY 'S5A culture solution, pouring 50ml of the MCCOY' S5A culture solution out, sealing the bottle for later use, adding 5ml of double antibody (penicillin-streptomycin solution 100X, namely double antibody, Shanghai Biyuntian biotechnology limited) and 50ml of FBS (fetal bovine serum, PAN, Beijing Hua Meilan Bo biotechnology limited) into the rest culture solution, mixing uniformly, sealing the bottle for later use, and storing the bottle at 4 ℃.

Cell recovery: taking out the frozen SK-OV-3 cells from the liquid nitrogen tank, thawing in water bath at 37 deg.C, transferring to 15ml centrifuge tube, adding 10 times of cell complete culture solution, centrifuging at 1000rpm for 3min, discarding supernatant, adding 1ml complete culture solution, blowing to make cell suspension, transferring to 25cm²Adding complete culture solution into culture bottle to completely cover the whole bottle bottom, screwing the bottle mouth, and transferring to CO₂The culture is carried out in an incubator at 37 ℃.

Cell liquid change: when the cell culture medium was observed to turn yellow from red, 25cm was transferred²The culture solution in the culture bottle is washed by PBS for 1-2 times, then proper amount of corresponding complete culture solution is added to completely cover the bottom of the bottle, and CO is added₂The incubator was incubated at 37 ℃ in a closed environment.

Cell passage: transferring the culture solution in a 25cm2 culture bottle after the bottom area of the cells is 90% full, cleaning the culture solution for 1-2 times by PBS, adding 1ml of pancreatin for digestion, placing the culture bottle for 2-3min, observing under a microscope, adding 3ml of complete culture solution to stop digestion when the cells are completely separated from the bottle wall and the cells are loosely connected, and gently blowing and beating the surface of the cell layer for a plurality of times to disperse the cells to form cell suspension. Transferring the cell suspension into a 15ml centrifuge tube, centrifuging at 1000rpm for 3min, discarding the supernatant, adding 3ml complete culture solution, blowing to obtain cell suspension, transferring to 75cm²The culture flask is filled with complete culture solution in CO₂The incubator was incubated at 37 ℃ in a closed environment.

Cell collection and seeding: transferred out by 75cm²The culture solution in the culture bottle is washed by PBS for 1-2 times, then 3ml of pancreatin is added for digestion, the culture box is placed for 2-3min, the cell is observed under a microscope, when the cell is completely separated from the bottle wall and the connection of the cell is loose, then 3ml of complete culture solution is added to stop digestion, and the cell is gently blown to the surface of the cell layer for a plurality of times, so that the cell is dispersed to form cell suspension. The cell suspension was transferred to a 15ml centrifuge tube, centrifuged at 1000rpm for 3min, the supernatant was discarded, the cells were resuspended in an appropriate volume of blank medium, and aspirated by a 1ml syringe for inoculation. Average two 75cm²The total amount of cells in the culture flask is inoculated under axilla of one Balb/c nude mouse, and 8 Balb/c nude mice are inoculated in total.

2.2 construction of model for recurrence after subcutaneous tumor excision

Tumor mass passage: when the tumor mass of 8 Balb/c nude mice inoculated with SK-OV-3 cells at the armpit grows to 100mm³On the left and right sides, the mouse cervical vertebra is dislocated and killed in a super clean bench, the oxter skin of the mouse is disinfected by alcohol, and the oxter skin is peeled off after being cutPlacing the whole tumor in a prepared blank cell culture solution, trimming with a sterile surgical blade to retain tumor tissue with vigorous growth and no ulceration at the edge, and cutting into 1.5mm³The left and right squares were inoculated subcutaneously in the axilla of 70 healthy Balb/c nude mice on the same side with a trocar.

Surgical excision of subcutaneous tumors: when the animal tumor grows to 100-250mm³Selecting 56 tumor-bearing nude mice with normal body type, weight of 18-22g and non-ulcerated tumor mass, performing intraperitoneal injection anesthesia with 4% chloral hydrate solution, placing in a super clean bench, sterilizing axillary skin with alcohol, cutting off most tumor tissue, and leaving about 2mm³The tumor mass with the size is used as a recurrence focus and is reserved for subsequent grouping experiments.

2.3 dose and group

A total of 3 inventive formulation experimental groups, 1 blank control group and 3 commercial formulation control groups were set, each group consisting of 8 animals, each group consisting of 4 animals, were housed in an SPF environment.

Among them, the experimental group of the formulation of the present invention used a suspension prepared by reconstituting lyophilized formulation 1 of example 3 with physiological saline.

Blank Control (Blank Control): the wound was closed immediately after tumor resection without treatment.

Low dose group (NCs Low) of the formulation of the invention: a pipette is used to uniformly sprinkle 0.012ml of the local paclitaxel in the physiological saline suspension of 0.06mg of the preparation of the invention on the wound surface after operation.

Dosage groups (NCs Medium) in the formulations of the invention: a pipette is used to uniformly spray 0.024ml of a physiological saline suspension containing 0.12mg of the preparation of the invention for local paclitaxel on the wound surface after operation.

The High dose group (NCs High) of the formulation of the invention: a pipette gun was used to uniformly spray 0.048ml of a physiological saline suspension containing 0.24mg of the formulation of the present invention containing topical paclitaxel over the postoperative wound.

Commercial formulation control group 1 (Taxol):(tail vein injection, starting on the third day after surgery, 10mg/kg every three days at a concentration of 1mg/ml for a total of 30 mg/kg).

Commercial formulation control group 2 (Abraxane):(tail vein injection, starting on the third day after surgery, 10mg/kg every three days at a concentration of 1mg/ml for a total of 30 mg/kg).

Commercial formulation control group 3(Taxol local): uniformly sprinkling 0.048ml of solution containing 0.12mg of paclitaxel on the wound surface after operation by using a pipette gunDiluted solution of sodium chloride (2.5 mg/ml).

Wherein, each dose group of the preparation of the invention and a contrast group 3 of the preparation sold in the market are administrated on the wound surface in the operation cavity immediately after tumor resection, the wound and the residual recurrent focus are ensured to be covered as much as possible, and the wound is sutured after the liquid infiltrates into the tissue and does not flow any more; commercial preparations of control groups 1 and 2 were administered by tail vein injection 3 times on days 3, 6, and 9 after tumor resection with immediate wound closure.

2.4 monitoring of recurrent tumor growth after drug administration

Wound healing and survival status of each experimental group were observed every 3 days from the day of operation, and the long diameter (length) and short diameter (width) of subcutaneously recurrent tumor were measured using a vernier caliper, and the tumor Volume (Volume) was calculated according to the following formula.

Animals were sacrificed on day 60 post-surgery, the tumors were peeled off and weighed and the average tumor weight was calculated while taking pictures of the ex vivo tumors. The treatment effect and in vivo toxicity of the preparations of each group on the tumor are comprehensively evaluated by combining indexes such as the volume of the recurrent tumor, the average tumor weight of each group, the number of recurrent animals in each group, the relative tumor increment rate, the weight change of mice and the like.

Tumor Volume [ length × (width)2]/2

2.5 relative tumor proliferation Rate

The relative tumor proliferation rate T/C% ═ TRTV/CRTV × 100%.

RVT ═ Vn/V0, Vn representing the volume of recurrent tumors on day n post-surgery; v0 represents the postoperative residual tumor recurrence focus volume. TRTV represents the treatment group RVT and CRTV represents the blank control group RVT.

Evaluation criteria: T/C% > 40% is invalid; T/C% is less than or equal to 40%, and P <0.05 is treated by statistics.

2.6 monitoring of animal body weight and preliminary assessment of medication safety

From the day of surgery, the body weight of the mice was weighed every 3 days, and a body weight-time change graph was plotted. And calculating the body weight change ratio of the mouse by taking the body weight before the operation as a reference value, and drawing a body weight change rate-time change graph.

The preliminary judgment of the safety of the administration focused on the examination of the rate of change of body weight of animals administered with the formulation of the invention: within 30 days after administration, if the weight of the animals is reduced by 10% or more, the safety of the administration form is considered to be poor; if the body weight is reduced to within 10%, the safety of the administration form is considered to be good. When the administration is carried out for more than 30 days after the administration, the weight change trends are different due to factors such as increase of the animal week age, natural weight increase or large volume of recurrent tumors, and therefore, the time range for determining the medication safety is not included.

3 results and analysis of the experiments

3.1 post-operative wound healing and mental status in animals

Within 2-3 hours after tumor resection, the anesthetized animals can be fully revived, and can normally take food, drink and exercise without obvious blood seepage of wounds; all animals recovered well after 1 day. On the 9 th day after operation, partial animal wounds in each group are completely healed and have no infection and suppuration; on day 15 of the experiment, all animal wounds had healed.

3.2 tumor recurrence and tumor volume changes

The rate of tumor recurrence in each group of animals by day 60 after surgery is shown in Table 11, and all of the nude mice in the placebo and commercial preparations control groups developed recurrent tumors, and one of the nude mice in the placebo group died at day 54. The relapse rate of 3 experimental groups of the preparation does not exceed 50 percent, and no relapse occurs in a high-dose group.

TABLE 11 tumor recurrence rate of SK-OV-3 tumor-bearing nude mice in experimental and control groups after tumor resection (n ═ 8)

In the drug effect investigation period of 60 days after operation, the SK-OV-3 cells grow faster. Some animals developed measurable recurrent tumors starting on day 12 post-surgery. The growth rate of recurrent tumors in the Taxol local group was similar to that in the non-administered Blank Control group; after the two groups of commercial preparations of Taxol and Abraxane are administrated through tail veins, the growth speed and the average volume of the recurrent tumor are superior to those of a blank group; the NCs Medium group and NCs High group showed significant recurrence-inhibiting effects, especially in the High dose group, animals did not develop recurrent tumors within 60 days.

The NCs Low group and NCs Medium group differ greatly between the two groups, both in the presence of relapsed animals and in the presence of relapsed animals with larger tumor volumes. The Taxol and Abraxane tail vein administration group showed the effect of inhibiting the growth of the recurrent tumor in the first 30 days, but the growth rate of the recurrent tumor in the later period was gradually increased.

After sacrifice, tumors were dissected out, as in fig. 1, and no undegraded residual drug was found in all local drug-administered groups. The Blank Control group had one death at day 54, and the recurrent tumors were taken into account for tumor volume and tumor weight but not presented in the figure; the Taxol local group had tumors with internal ulceration with pus, which was not intact solid when dissected out. The mean tumor weight results for each group are shown in figure 2. The low-high group of the preparation of the invention presents the treatment effect of inhibiting tumor recurrence in a dose-dependent manner, and is obviously superior to the contrast group of the preparation sold in the market.

3.3 relative tumor proliferation Rate and statistical Difference

The results of the relative tumor proliferation rates were calculated from the mean tumor volumes of the experimental groups at day 60 as shown in table 12.

TABLE 12 relative tumor proliferation rates of SK-OV-3 tumor-bearing nude mice administered to animals following tumor resection

Through statistical treatment of Student's t-test, the results show that P between the NCs Medium group and the NCs High group and the Blank Control group is less than 0.05, and the significant difference exists, so that the treatment is considered to be effective.

3.4 summary and discussion

The experiment verifies that the preparation can be conveniently and directly applied to the wound surface by spraying and administration by constructing an animal model of the recurrence of the nude mouse subcutaneous SK-OV-3 humanized ovarian cancer after tumor resection, and unexpectedly, the preparation can obviously and more effectively inhibit the tumor recurrence, is greatly superior to the existing preparation, has the effect of inhibiting the recurrence in a dose-dependent manner, and has the effect of greatly superior to the local administration of the traditional intravenous chemotherapy and the commercially available preparation.

The specific embodiments set forth herein are presented by way of example only and are not intended as limitations on the scope of the invention as defined by the claims. Equivalent variations of the technical solutions of the present application will be obvious to those skilled in the art on the basis of the disclosure of the present application, and these variations are also covered by the scope of the present application.

35页详细技术资料下载

Pharmaceutical composition containing insoluble antitumor active agent and preparation method thereof

相关技术

网友询问留言