Drug conjugates for radiation protection and their preparation and use

Abstract

A drug complex for radiation protection and its preparation and use. In this drug complex, there is an osmotic pressure-driven bond between amifostine and natural microalgae, which is gradually decomposed in the intestinal tract after oral administration, covers the intestinal tract completely, slowly releases the drug, significantly improves the drug concentration in small intestinal tissue, fully exerts the radiation protection effect on intestinal tissue, and avoids the systemic toxic side effects of the drug. Furthermore, this drug complex can achieve the effects of nutritional supplementation and intestinal inflammation regulation, has good oral administration safety, is simple and easy to carry out the preparation process, is easy to store and take, overcomes the problems of low absorption efficiency and oral administration safety of drugs and their preparations prepared by conventional technology, and has a broad prospect of use in the field of intestinal radiation protection.

Description

[Technical field]

[0001] The present invention belongs to the technical field of drug formulation, specifically to a drug conjugate for radiation protection and its preparation method and use. [Background technology]

[0002] Malignant tumors are one of the diseases that seriously threaten the health and life of human beings, and surgery, chemotherapy and radiation therapy have always been the main forces against cancer. Radiation therapy (abbreviated as radiotherapy) is used in more than half of malignant tumor patients and plays a very important role in treating various types of different tumors. However, in radiation therapy, ionizing radiation to kill tumors inevitably damages healthy tissues adjacent to the tumor, thereby affecting the normal function of healthy tissues or organs. Here, in the radiotherapy of abdominal and pelvic solid tumors, the small intestine is highly sensitive to radiation and has a relatively large volume, which makes it highly susceptible to radiation damage, leading to radiation-induced enteropathy (or radiation enteropathy), which causes abnormalities such as impaired intestinal epithelial renewal, inflammatory cell infiltration, and disturbance of intestinal flora, followed by a series of intestinal toxicity symptoms, such as diarrhea, vomiting, intestinal bleeding, intestinal perforation, severe infection, and even death. According to estimates, the incidence of radiation-induced enteropathy symptoms among patients who receive pelvic or abdominal radiotherapy in the United States each year is as high as 60-80% (Hauer-Jensen, M. et al., Nat. Rev. Gastroenterol. Hepatol. 11, 470-479 (2014).), which seriously harms the quality of life of tumor patients. Therefore, the research and development of drugs, drug complexes, or preparations for the prevention of radiation-induced enteropathy (i.e., intestinal radiation protection) is of great importance.

[0003] Amifostine (chemical name is 3-aminopropylamine ethyl phosphorothioate, also called amifostine or etiophos, WR-2721, AMF, Amifostine) is a selective radiation / chemotherapy protectant for normal tissues approved for clinical use by the FDA (U.S. Food and Drug Administration). As a prodrug, it can be hydrolyzed by alkaline phosphatase in cells and converted into the active metabolite WR-1065, which can further protect cells from radiation damage by mechanisms such as free radical scavenging. This drug has selective protective effect on normal tissues without affecting radiation killing of tumor tissues. The protective mechanism is that normal tissues have higher alkaline phosphatase activity, higher pH value (favorable for alkaline phosphatase activity) and better vascular permeability than tumor tissues, so that normal tissues can concentrate more WR-1065 than tumor tissues, resulting in a specific protective effect (Smoluk, GDet al., Cancer Res. 48, 3641-3647 (1988).). At the same time, the disadvantages of amifostine are also very obvious. First, this drug is generally administered intravenously into the blood, where it is rapidly eliminated and metabolized, and cannot reach a sufficient effective concentration locally in the intestinal tissue. Increasing the intravenous administration concentration leads to a relatively high blood concentration, which in turn leads to systemic side effects such as low blood pressure, nausea, and vomiting (Praetorius, NP & Mandal, TKJ Pharm.Pharmacol.60,809-815(2008)). Direct oral administration of this drug results in a significant reduction in the amount of drug entering the intestinal tract due to partial inactivation of amifostine by the low pH environment of gastric acid, and effective protection of the intestinal tract cannot be achieved (Pamujula,S.et.al.,Int.J.Radiat.Biol.84,900-908(2008)). For this reason, some researchers are trying to realize oral administration and delivery of amifostine in the form of a drug carrier, but current reports often focus on nano-level carriers to study drug loading and release.

[0004] Patents (WO2020258584A1, CN110200941B, CN109970987A) disclose several methods for preparing and using oral nano-carrier drugs, which use nanomaterials to package radiation protection drugs so that they can enter the intestinal tissue to exert their effect and achieve oral drug delivery, but there are inevitably some problems such as low absorption efficiency and oral administration safety, whereby the low absorption efficiency makes it difficult to achieve long-term storage and gentle decomposition of drugs in the intestinal tissue, because nanomaterials belong to the nanoscale and are extremely small in size, making it difficult to achieve long-term storage of drugs in the intestinal tract and the efficacy cannot be sustained, and on the other hand, the preparation process of nano drugs is relatively complicated and expensive, and is not suitable for large-scale production and use. Most importantly, most of the adjuvants of nano drugs are chemicals with a certain toxicity, such as organic reagents, and the safety of their preparations has not been reported. However, to date, a new type of intestinal radiation protective drug preparation or drug complex that simultaneously possesses multiple functions such as sustained efficacy, reliable safety when administered orally, and minimal side effects, and is low cost, has not yet been developed, so the research and development of related drugs and preparations has already become the most important point in the research issue of radiation therapy protective drugs. Summary of the Invention [Problem to be solved by the invention]

[0005] In view of the shortcomings of the prior art, the object of the present invention is to provide a drug complex for intestinal radiation protection, its preparation method and its use. This drug complex overcomes the shortcomings of the low absorption efficiency and low oral administration safety of traditional radiation protection drugs and related preparations, realizes the long-term storage and gentle decomposition of amifostine in intestinal tissue, strengthens the local protective effect of the drug on intestinal tissue, prevents intestinal radiation damage caused by low-dose radiation in abdominal / pelvic tumor radiotherapy or environment, and avoids the systemic toxic side effects caused by intravenous administration. The production flow is simple, highly feasible and easy to scale up, and this drug complex can also provide a small amount of protein, unsaturated fatty acids and trace elements required by the human body. [Means for solving the problem]

[0006] The first object of the present invention is to provide a drug complex for radiation protection, the drug complex comprising amifostine and natural microalgae, wherein the sizes of the natural microalgae and the drug complex are both micron-scale, the mass ratio of the natural microalgae to amifostine in the drug complex is 1:0.5-1:8 (i.e., SP:AMF is 1:0.5-1:8), and an osmotically-driven bond is formed between the amifostine and the natural microalgae, the osmotically-driven bond being driven by the action of osmotic pressure formed by different solutions inside and outside the microalgae, such that the amifostine molecule can bind to the surface of the microalgae or enter the interior through gaps in the water channels on the surface of the microalgae to form a drug complex, the drug complex having a characteristic peak in infrared detection, and the method for detecting the osmotically-driven bond is a method for detecting the osmotically-driven bond by using an infrared ray detection method of 400-4000 cm -1 Under infrared scanning conditions in the range of 758 and 3302 cm -1 The characteristic feature is that

[0007] Preferably, the drug complex further comprises a solvent.

[0008] Preferably, the solvent is at least one or more selected from sterile phosphate buffer, ultrapure water, distilled water, or physiological saline.

[0009] Preferably, the drug conjugate has long-term intestinal preservability and slow degradation, where long-term intestinal preservability and slow degradation refer to the fact that the drug conjugate can be detected in the intestinal tract even after 8 hours or more from administration after administration 4 hours before irradiation, and preferably, the detection method is to intragastrically administer a certain dose to a Balb / c nude mouse that has been fasted for 12 hours, and the fluorescence image after the action shows that the drug conjugate is still contained in the small intestine of the nude mouse, preferably, the certain dose is 120-600 mg / kg, more preferably, the certain dose is 360 mg / kg, and further preferably, the action time is 24 hours, and more preferably, the fluorescence image adopts Cy5.5, excitation wavelength 605 nm, emission wavelength 615-665 nm chlorophyll channel.

[0010] Preferably, the drug conjugate is orally safe, and the method for detecting this is to intragastrically administer a fixed dose to a Balb / c albino mouse, and after continuous administration once a day, the weight of the mouse remains unchanged and the hematological indices and liver and kidney functions are normal. Preferably, the fixed dose is 120-600 mg / kg, more preferably, the fixed dose is 360 mg / kg, and further preferably, the continuous administration is performed for 30 days.

[0011] Preferably, the natural microalgae is spirulina.

[0012] Preferably, the length of the spirulina is 100 to 500 μm.

[0013] A second object of the present invention is to provide a method for preparing a drug complex for radiation protection, the method including the preparation of an amifostine solution, natural microalgae and a drug complex, the method including the following steps:

[0014] (1) Preparation of natural microalgae powder and amifostine solution: Take micron-scale microalgae, culture, and centrifuge, remove the supernatant, wash and collect the precipitate, and after post-treatment obtain microalgae powder, weigh the amifostine solid, and prepare an amifostine solution with a concentration of 0.04 to 0.48 mg / mL. Preferably, the rotation speed and time of the centrifugation are 4500 rpm and 10 min, respectively.

[0015] Preferably, the solution for washing the precipitate is at least one selected from distilled water or a phosphate buffer solution.

[0016] Preferably, the precipitate is washed 3 to 5 times.

[0017] Preferably, the amifostine solution is prepared by adopting at least one of a phosphate buffer solution or distilled water and mixing uniformly.

[0018] (2) Preparation of drug conjugates The obtained microalgae powder and amifostine solution are mixed at a dosage ratio of 1:0.8 to 1:10 to produce a drug complex.

[0019] Preferably, the above-mentioned mixing and preparation refers to adding microalgae powder to an amifostine solution under a certain temperature and light-shielding condition, stirring, and centrifuging to collect the precipitate, washing 3 to 5 times, and then performing post-treatment to obtain a solid powder of the drug complex.

[0020] Preferably, the constant temperature is 2 to 8°C.

[0021] Preferably, the stirring speed is 60 to 200 rpm.

[0022] Preferably, the stirring time is 6 to 12 hours.

[0023] A third object of the present invention is to provide a pharmaceutical composition, comprising at least one active ingredient and at least one pharma- ceutically acceptable excipient, wherein the active ingredient is any one selected from the above-mentioned drug complex or the drug complex obtained by the above-mentioned preparation method.

[0024] Preferably, the additives include conventional diluents, excipients, fillers, adhesives, wetting agents, disintegrants, absorption enhancers, surfactants, adsorption carriers, lubricants, etc. in the pharmaceutical field, and flavors, sweeteners, etc. may be added as necessary.

[0025] A fourth object of the present invention is to provide a use of the above-mentioned drug conjugate, the drug conjugate obtained by the above-mentioned production method, or the above-mentioned pharmaceutical composition in the production of a protective drug related to tumor treatment.

[0026] Preferably, the tumor is one selected from solid tumors.

[0027] Preferably, the solid tumor is one selected from tumors associated with intestinal tissue.

[0028] Preferably, the tumor is at least one selected from abdominal and pelvic cavity solid tumors.

[0029] Preferably, the abdominal or pelvic solid tumor is one selected from pancreatic cancer, prostate cancer and colon cancer, and further, the abdominal or pelvic solid tumor is preferably colon cancer.

[0030] Preferably, after oral administration, the drug complex or pharmaceutical composition enters the intestinal tract, and is gradually divided and decomposed during digestion, slowly releasing the drug, which is distributed to cover the entire area of ​​the proximal end, middle and distal end of the small intestine, significantly increasing the drug concentration in the small intestinal tissue, fully exerting the protective effect on the small intestinal tissue and cells, and reducing the drug concentration in the blood to avoid systemic toxicity.

[0031] Preferably, the drug conjugate or pharmaceutical composition does not affect the tumor tissue killing / inhibition effect of X-rays in radiotherapy of cecal intraepithelial colon carcinoma.

[0032] Preferably, the drug conjugate or pharmaceutical composition is used for radioprotection by long-term oral administration in clinical practice, exhibits good biosafety, can effectively avoid the potential toxic side effects of amifostine, and is suitable for daily use in long-term oral administration.

[0033] Preferably, the intestinal radioprotective orally administered drug complex and pharmaceutical composition overcomes the drawbacks of the single intravenous administration mode of amifostine and the insufficient efficacy of the nano-drug complex in the intestinal tissue.

[0034] A fifth object of the present invention is to provide a use of the above pharmaceutical composition in intestinal regulation.

[0035] Preferably, the gut regulation comprises at least one of inflammation regulation or gut nutritional supplementation.

[0036] Preferably, the nutritional components are one or more selected from proteins, unsaturated fatty acids, carotenoids, vitamins, and a number of trace elements such as iron, iodine, zinc, or probiotics such as polysaccharides.

[0037] A sixth object of the present invention is to provide an oral formulation, the active ingredient of which comprises at least one of the above-mentioned drug conjugate, the drug conjugate obtained by the above-mentioned preparation method, or the pharmaceutical composition, and at least one pharma- ceutically acceptable carrier or excipient.

[0038] Preferably, the formulation is a solid formulation.

[0039] Preferably, the solid formulation is at least one selected from tablets, powders, granules, and capsules, and each of the above dosage forms of drugs can be manufactured by conventional methods in the pharmaceutical field.

[0040] Preferably, the additives include conventional diluents, excipients, fillers, adhesives, wetting agents, disintegrants, absorption enhancers, surfactants, adsorption carriers, lubricants, etc. in the pharmaceutical field, and flavors, sweeteners, etc. may be added as necessary. Effect of the Invention

[0041] The beneficial effects are as follows:

[0042] 1. The present invention is the first to produce a drug complex having an osmotically driven bond formed from a radioprotective drug, amifostine, and natural microalgae in a specific mass ratio (SP:AMF=1:0.5-1:8), and has a 400-4000 cm -1 Under infrared scanning conditions in the range of 758 and 3302 cm, the drug complex exhibited -1 It has a characteristic peak at 100 nm, overcomes the single-administration method of intravenous injection of the radiation protection drug amifostine, and after use, it can maintain a relatively high drug activity level and safety for oral administration, and can achieve long-term storage stability and gentle decomposition in intestinal tissue.

[0043] 2. The present invention overcomes the limitation that the radioprotective drug amifostine cannot enter intestinal tissue to provide global radiation protection. After use, the drug complex is distributed to cover the entire area of ​​the proximal, middle and distal ends of the small intestine, significantly improving the drug concentration in the small intestinal tissue, fully exerting the protective effect on small intestinal tissue and cells, while reducing the drug concentration in the blood and avoiding systemic toxicity, making it suitable for patients who require long-term radiation therapy.

[0044] 3. The drug complex of the present invention has osmotically driven binding and has specific characteristic peaks (FTIR detection), and is a micron-scale complex. Compared with nano-material drug preparations or complexes, the drug complex of the present invention not only exhibits long-term radiation protection function and intestinal inflammation regulating effect, but also shows the rule that the drug complex will gradually break down and decompose after use, which indicates that amifostine can be sufficiently released and that the drug complex is easily decomposed in the digestive system, thereby avoiding toxic side effects caused by long-term storage in the body.

[0045] In the present invention, the term "SP" refers to spirulina.

[0046] In the present invention, the term "AMF" refers to the intestinal radioprotective drug amifostine.

[0047] In the present invention, the term "SP@AMF" refers to a drug complex.

[0048] In the present invention, the term "SGF" refers to artificial gastric fluid. [Brief description of the drawings]

[0049] [Figure 1] Optical microscopy (bright field and chlorophyll fluorescence channel) and scanning electron micrographs of drug conjugates, scale = 20 μm. [Diagram 2] This is a positive correlation diagram between the SP / AMF input ratio in the manufacturing process and the SP / AMF mass ratio of the drug complex, and the corresponding relationship is y=1.3820x-0.0427, R2=0.9983, where x is the SP / AMF input ratio in the manufacturing process and y is the SP / AMF mass ratio in the drug complex obtained by manufacturing. [Diagram 3] FIG. 1 shows the Fourier transform infrared spectra (FTIR) of the drug complex (SP@AMF), amifostine (AMF) and spirulina (SP). [Figure 4]FIG. 1 shows the drug release curves of the drug complexes after treatment with simulated gastric fluid (SGF) for 0, 1, and 2 hours (ordinate is the percentage of cumulative released drug relative to the total drug loading). [Diagram 5] FIG. 1 is a trend diagram of the effect of drug complexes on the pH value of artificial gastric fluid (SGF). [Figure 6] FIG. 2 shows fluorescence imaging of biodistribution at different time points (0-24 h) after oral administration of drug conjugates. [Figure 7] Morphological picture (scanning electron microscope pseudocolor photograph) of small intestinal intervilli after oral administration of drug complex, scale=20 μm. [Figure 8] Morphological diagrams (scanning electron micrographs) of the drug complex in each stage of the digestive tract after oral administration, scale=20 μm. [Figure 9] Schematic diagram of the protective effect of the drug conjugate against crypt proliferation disorder in each stage of the small intestine (duodenum, jejunum, and ileum) caused by abdominal radiation (Ki67 immunohistochemical staining; black dotted lines indicate intestinal crypts with normal cell proliferation). Scale=100 μm. [Figure 10] Schematic diagram of the protective effect of the drug conjugate against fibrosis in each stage of the small intestine (duodenum, jejunum, and ileum) following abdominal radiation (stained with Masson's trichrome, fibers stained blue), scale=100 μm. [Figure 11] Figure showing that the drug complex reduces the content of inflammatory factor IL-1β in small intestinal tissue (*, p value < 0.05, **, p value < 0.01, ***, p value < 0.001, black * represents the p value between each group and the radiation exposure group, red * represents the p value between the radiation exposure + AMF group and the radiation exposure + SP@AMF group). [Figure 12] FIG. 1 shows that the drug complex reduces the content of the inflammatory factor IL-6 in small intestinal tissue (*, p-value <0.05; **, p-value <0.01; ***, p-value <0.001; black * represents the p-value between each group and the radiation exposure group; red * represents the p-value between the radiation exposure + AMF group and the radiation exposure + SP@AMF group). [Figure 13]Figure showing that the drug complex reduces the content of the inflammatory factor TNF-α in small intestinal tissue (*, p value < 0.05, **, p value < 0.01, ***, p value < 0.001, black * represents the p value between each group and the radiation exposure group, red * represents the p value between the radiation exposure + AMF group and the radiation exposure + SP@AMF group). [Figure 14] Schematic diagram of tumor volume and weight in nude mice bearing intraepithelial tumors of the cecum after abdominal radiation therapy (*, p value < 0.05; **, p value < 0.01; ***, p value < 0.001). [Figure 15] The blood general test index and liver and kidney function index after oral administration of the drug conjugate for 30 consecutive days (*, p value < 0.05; **, p value < 0.01; ***, p value < 0.001). [Figure 16] FIG. 1 is a schematic of body weight monitoring during 30 consecutive days of oral administration of drug conjugates (*, p-value <0.05; **, p-value <0.01; ***, p-value <0.001). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0050] The present invention will be further described below in conjunction with the following figures and examples, but the present invention is not limited to the following examples.

[0051] Example 1. Synthesis of SP@AMF A suspension of spirulina (SP) cultured under sterile conditions was taken, centrifuged (4500 rpm, 10 min), the supernatant was removed, washed three times with phosphate buffer to remove the residual medium, the precipitate was collected, and a solid powder was obtained after freeze-drying. A phosphate buffer solution containing 3.125 mg / mL amifostine (AMF) was prepared, and the above spirulina powder was added according to a mass ratio of spirulina to amifostine of 1:0.6, which was thoroughly dispersed in a 50 mL sterile centrifuge tube wrapped in tin paper, placed in a thermostatic shaker at 4°C, and gently shaken (60 rpm) for 12 hours. After centrifugation, the supernatant was removed, and the precipitate was collected and freeze-dried again to obtain a solid powder of the drug complex SP@AMF (Figure 1). The solid powder was then sealed, dried, and stored at 2-8°C in the dark, and a drug complex with an SP:AMF mass ratio of 1:1.25 was obtained. After post-processing, the drug complex powder was collected and photographed under a microscope and a scanning electron microscope. It was found that the drug complex powders with different mass ratios all formed a uniform suspension in liquid (distilled water), had a 3D spiral morphology, and had red fluorescent imaging properties (Figure 1).

[0052] According to the above-mentioned SP@AMF preparation method, drug complexes with different mass ratios (SP:AMF=1:0.5-1:8) were prepared with different input ratios (SP:AMF=1:0.8-1:10, i.e., SP / AMF=0.1-1.25). Electron microscopy showed that the morphology of the drug complexes with SP:AMF mass ratio of 1:1.25 was the same as that of the drug complexes with SP:AMF mass ratio of 1:1.25, all of which were 3D helical and had red fluorescent imaging properties. The infrared spectra of the SP@AMFs with different mass ratios (SP:AMF=1:0.5-1:8) showed that they exhibited the characteristic peaks of SP (1541, 1654 and 2926 cm -1 ) and the characteristic peaks of AMF (587, 956 and 1012 cm -1 ), and also has characteristic peaks (758 and 3302 cm ) that are unique to SP@AMF. -1 ) was formed.

[0053] In addition, due to the performance of SP itself and the limitation of the loading amount, the SP / AMF mass ratio in the drug complex corresponding to different SP / AMF input ratios is as shown in Table 1. It is found that there is a positive correlation between the SP / AMF input ratios in different manufacturing processes and the SP / AMF mass ratio in the drug complex obtained by manufacturing, as shown in Figure 2, where x is the SP / AMF input ratio in the manufacturing process, y is the SP / AMF mass ratio in the drug complex obtained by manufacturing, and R 2 is the correlation coefficient, R 2 The closer the value of R is to 1, the better the fit between the test data and the fitting function. 2 was 0.9983, indicating a high degree of compatibility between the test data and the fitting function.

[0054] Preferably, the drug complex having an SP:AMF mass ratio of 1:1.25 was prepared by adopting an input ratio of SP:AMF ≠ 1:1.7 in the mixed system (i.e., SP / AMF = 0.6, as shown in Figure 2). When the loading efficiency and production cost were analyzed together, this complex had the optimal AMF loading amount, and further, the drug complex having this mass ratio was used to conduct subsequent efficacy experiments.

[0055] In addition, the experiments in Examples 2 to 7 and Comparative Example were all carried out using a drug complex with an SP:AMF mass ratio of 1:1.25, and drug complexes with other mass ratios are all applicable to the present invention.

[0056] Table 1 Table showing the relationship between the SP / AMF input ratio and the SP / AMF mass ratio in the drug complex JPEG0007649505000001.jpg69170

[0057] Example 2. Verification of drug loading performance 400-4000 cm using a Fourier transform infrared spectrometer (FTIR) -1The infrared spectra of SP@AMF, SP, and AMF (Figure 3) were detected within the range of 1541, 1654, and 2926 cm -1 The AMF has characteristic peaks at 587, 956 and 1012 cm -1 The infrared spectrum of SP@AMF showed that it had the characteristic peaks of SP (1541, 1654 and 2926 cm -1 ) and the characteristic peaks of AMF (587, 956 and 1012 cm -1 ), and also has characteristic peaks specific to SP@AMF (758 and 3302 cm -1 ) was formed, proving that AMF was successfully loaded into the SP.

[0058] Example 3. Detection of drug release performance in vitro 1 mg of the synthesized drug complex was added to 1 mL of artificial gastric fluid (SGF), and shaken (180 rpm) on a shaker at 37 °C for 0, 1, and 2 hours, respectively, then centrifuged, the supernatant was removed, and the precipitate was transferred to 5 mL of phosphate buffer, shaken (180 rpm) on a shaker at 37 °C, and the concentration of AMF in the supernatant was detected after 0.5, 1.5, 3, 6, 12, and 24 hours, respectively, and the in vitro release curve of the drug (Figure 4) was drawn. The in vitro release curve proved that the drug complex has the characteristic of slowly releasing AMF, and even if it is pretreated with artificial gastric fluid for 1 to 2 hours, more than 50% of the drug is guaranteed to be released, indicating that SP can protect most of the AMF from entering the intestinal tract and slowly release it. At the same time, the detection of the pH value of the artificial gastric fluid containing different concentrations of the drug complex (Figure 5) demonstrated that the SP@AMF-drug complex has a certain gastric acid neutralization ability, which can reduce the acidity of the gastric fluid and is favorable for protecting the activity of the drug.

[0059] Example 4. Detection of distribution and degradation in vivo after oral administration Utilizing the fluorescence imaging function of chlorophyll contained in SP, the distribution law in the body after oral administration was monitored by a bioimaging device. The above drug complex was resuspended in distilled water, and 360 mg / kg of SP@AMF (AMF is approximately 200 mg / kg) was intragastrically administered to Balb / c nude mice that had been fasted for 12 hours. The nude mice were anesthetized 0, 0.5, 1, 2, 4, 6, 8, and 24 hours after intragastric administration, and fluorescence images (Figure 6) were taken in the chlorophyll channel of SP (channel Cy5.5, excitation wavelength 605 nm, emission wavelength 615-665 nm) using a bioimaging device. Three to four hours after oral administration of SP@AMF, the middle section of the small intestine was excised, the contents were slightly washed, and the inner surface of the small intestine was flattened to prepare a scanning electron microscope sample. The morphology of the material between the small intestinal villi (Figure 7) was observed, and the contents of each segment of the digestive tract were taken, slightly washed, and the morphological changes of the material were observed with a scanning electron microscope (Figure 8). The results show that the fluorescence of the material after oral administration always gathers in the abdominal area, and can maintain a relatively high fluorescence intensity within 0-6 hours, and has a relatively long intestinal distribution time, which is favorable for the concentration accumulation of the drug in the intestinal tissue. Scanning electron microscopy (Figure 7) shows that due to its spiral morphology and length close to the intestinal villi, the drug complex is widely distributed among the intestinal villi and directly contacts with the intestinal villi, which is favorable for the sufficient absorption of the drug by the intestinal epithelial cells. Figure 8 shows that from the proximal end (stomach) to the distal end (large intestine) of the digestive tract, the morphology of the drug complex exhibits the law of gradual fracture and decomposition, which is not only favorable for the sufficient release of the drug, but also shows that the drug complex is easily decomposed in the digestive system and avoids long-term storage in the body.

[0060] Example 5. Protective effect of drug complexes against intestinal radiation damage By analyzing the distribution law of SP@AMF in the digestive system, we found that it was possible to achieve full coverage and drug distribution in the small intestine about 4 hours after oral administration. At this point, we irradiated the animals' abdomens with X-rays and analyzed the protective effect of the material against X-ray-induced small intestinal damage through pathological examination. The drug complex was resuspended in distilled water and 360 mg / kg of SP@AMF (containing about 200 mg / kg of AMF) was intragastrically administered to Balb / c white rats that had been fasted for 12 hours. Four hours after intragastric administration, the animals were anesthetized and X-rayed to the abdomen at 12 Gy (dose rate = 8.415 Gy / min). The control group was administered equal amounts of distilled water, SP, and AMF. Three days after irradiation, the animals were euthanized, small intestinal tissues were taken, pathological sections were prepared, and immunohistochemical (Ki67) staining was performed to evaluate the degree of short-term radiation damage in the small intestine (proliferative crypts) (Figure 9). Thirty days after irradiation, small intestinal tissue was taken, pathological sections were prepared, and stained with Masson's trichrome (Figure 10) to evaluate the degree of long-term radiation damage (fibrosis) of the small intestine. A portion of the small intestinal tissue was taken out and prepared into tissue homogenate, and the contents of inflammatory factors IL-1β, IL-6, and TNF-α were detected (Elisa kit, Boster Biological Technology Co. Itd) (Figures 11, 12, 13). The results showed that the duodenum, jejunum, and ileum of the SP@AMF group all maintained near-normal crypt proliferation activity, the degree of final fibrosis was relatively mild, and the level of inflammatory factors was relatively low, indicating that all of these indicators were significantly superior to those of other treatment groups, including the AMF group, indicating that the drug complex significantly improved the radioprotective effect of AMF on intestinal tissue.

[0061] Example 6. Effect of drug conjugates on tumor killing during radiation therapy To further mimic the clinical process of tumor radiotherapy, an animal model of cecal intraepithelial colon carcinoma was constructed, followed by oral administration of radioprotective drug complexes and abdominal radiation therapy. Luciferase-transfected CT26-luci colon carcinoma cells were inoculated into the cecal intestinal wall of Balb / c nude mice to construct an animal model of cecal intraepithelial colon carcinoma, and tumor growth was monitored by detecting tumor fluorescent signals using a bioimaging device. When the tumor reached a major axis of 1-2 cm, the animals were fasted for 12 hours and intragastrically administered 360 mg / kg of SP@AMF. Four hours later, the animals were anesthetized and given 12 Gy abdominal X-ray irradiation. The two control groups were a no-radiotherapy group (sham radiotherapy + oral administration of an equivalent amount of distilled water) and an abdominal radiotherapy group (abdominal X-ray irradiation 12 Gy + oral administration of an equivalent amount of distilled water). After treatment, tumor growth was monitored weekly using a bioimaging device, and when the tumors reached a major axis of 10-12 cm, the animals were euthanized and the tumors were removed for volume measurement and weight measurement (Figure 14). The results showed that there was no significant statistical difference in tumor volume and weight between the abdominal radiotherapy group and the abdominal radiotherapy + SP@AMF group, indicating that the use of this drug conjugate had no radioprotective effect on tumor tissue and did not affect tumor killing by radiotherapy.

[0062] Example 7. Safety assessment of long-term oral administration of drug conjugates SP@AMF was resuspended in distilled water and then intragastrically administered to Balb / c rats at a dose of 360mg / kg. After continuous administration once a day for 30 days, the animals' blood was taken for blood general examination and liver and kidney function index detection (Figure 15), and the body weight change was monitored during that period (Figure 16). The control group was administered with equal amounts of distilled water, SP, and AMF. The results showed that the AMF group showed obvious weight loss, abnormal hematological indexes, and liver and kidney function disorders, while all the indexes after oral administration of SP@AMF were normal, indicating good biosafety. This is presumably because SP@AMF slowly releases AMF in the intestinal tract, improving the distribution of the drug in the intestinal tract and reducing the drug concentration in the blood, thereby avoiding the systemic toxicity caused by the wide distribution of AMF after direct oral administration.

[0063] Comparative Example The experimental steps were based on the reference patent CN110200941A.

[0064] Synthesis of nano-drug formulations (1) Arginine (0.867 g, 4.977 mmol) was dissolved in 40 mL of morpholinoethanesulfonic acid solution (25 mM, pH 5.0), and N-hydroxysuccinimide (2.291 g, 19.908 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (3.816 g, 19.908 mmol) were added successively to activate the mixture for 2 hours.

[0065] (2) A solution of chitosan dissolved in morpholinoethanesulfonic acid (1.0 g, 4.977 mmol) was added to the above mixture, and the mixture was allowed to react at room temperature for 24 hours with continuous stirring. Then, sodium hydroxide (0.1 M) was added to terminate the reaction.

[0066] (3) Amifostine (4.5 mg / mL, 10 mL) dissolved in a mixture of water and acetonitrile (v / v = 1 / 1) was slowly dropped into the above polymer solution (10 mg / mL, 100 mL), stirring was continued, nitrogen gas was passed through overnight, the acetonitrile was removed, and after centrifugation, the supernatant was collected and freeze-dried.

[0067] (4) The freeze-dried sample (20.0 mg) was transferred to a dopamine solution (2 mg / mL, 40 mL, pH 8.5), stirred at room temperature for 3 hours, washed with deionized water, centrifuged, and the supernatant was collected to obtain a nano-drug. The amount of amifostine loaded in this nano-drug is the same as that of the drug complex in the present invention having an SP:AMF mass ratio of 1:1.25.

[0068] Example: Testing radiation protection effects in vivo The above formulation was resuspended in distilled water, and the above nano-drug containing 200 mg / kg AMF was administered intragastrically to Balb / c white rats that had been fasted for 12 hours. Four hours after intragastric administration, the animals were anesthetized and X-rays were administered to the abdomen at 12 Gy (dose rate = 8.415 Gy / min). Three days after irradiation, the animals were euthanized, small intestinal tissue was taken, pathological sections were prepared, and immunohistochemical staining (Ki67) was performed to evaluate the degree of short-term radiation damage (proliferative crypts) in the small intestine. Thirty days after irradiation, small intestinal tissue was taken, pathological sections were prepared, and Masson's trichrome staining was performed to evaluate the degree of long-term radiation damage (fibrosis) in the small intestine. A portion of the small intestinal tissue was extracted and prepared as a tissue homogenate, and the contents of inflammatory factors IL-1β, IL-6, and TNF-α in it were detected. The results showed that the nanomaterial had a certain protective effect against crypt proliferation activity in the proximal end of the small intestine (duodenum) (about 50% of the normal control group), but the protective effect on the middle and distal ends of the small intestine, the jejunum and ileum, was relatively weak, with the number of surviving crypts only about 20% of the normal control, and other indicators, such as the degree of end-stage fibrosis and the levels of three types of inflammatory factors, all showed a similar pattern.The above results showed that the nanomedicine had a certain preventive effect against radiation enteritis, but its protective effect was still insufficient and could not cover the entire length of the small intestine.

[0069] The above examples and comparative examples are merely for illustrating the present invention, and are not intended to limit the scope of the present invention. It should be understood that after reading the contents described in the present invention, a person skilled in the art can make various changes or modifications to the present invention, and these equivalent forms are also included in the scope limited by the appended claims of this application.

Claims

1. 1. A drug conjugate for radiation protection comprising: The drug complex comprises amifostine and natural microalgae, Here, the sizes of the natural microalgae and the drug complex are both on the micron scale, In the drug complex, the mass ratio of the natural microalgae to amifostine is 1:0.5 to 1:8 (i.e., SP:AMF is 1:0.5 to 1:8); The method for detecting the osmotically-driven bond comprises detecting a binding force of 400 to 4000 cm between amifostine and a natural microalga. －１ Under infrared scanning conditions in the range of 758 and 3302 cm, the drug conjugate has both characteristic peaks of amifostine and native microalgae, and －１ It has a characteristic peak at The natural microalgae is spirulina. A drug complex for radiation protection, comprising:

2. The drug complex further comprises a solvent. The drug conjugate of claim 1 .

3. The solvent is at least one selected from the group consisting of sterile phosphate buffer, ultrapure water, distilled water, and physiological saline; The drug conjugate of claim 2 .

4. The length of the spirulina is 100 to 500 μm. The drug conjugate of claim 1 .

5. 1. A method for preparing a drug conjugate for radiation protection comprising the steps of: Preparation of natural microalgae powder and amifostine solution: (1) taking micron-scale microalgae, culturing, centrifuging, removing the supernatant, washing and collecting the precipitate, obtaining microalgae powder after post-treatment, weighing the amifostine solid, and preparing an amifostine solution with a concentration of 0.05-0.5 mg / mL; (2) preparing a drug complex by mixing the obtained microalgae powder and an amifostine solution at a dosage ratio of 1:0.8 to 1:10 to prepare a drug complex; The natural microalgae is spirulina.

2. A method for producing a drug complex for radiation protection, comprising:

6. In the step (1), the rotation speed and time of the centrifugation are 4500 rpm and 10 min, respectively; The method according to claim 5 .

7. In the step (1), the solution for washing the precipitate is at least one selected from distilled water and a phosphate buffer solution. The method according to claim 5 .

8. In the step (1), the precipitate is washed 3 to 5 times. The method according to claim 5 .

9. In the step (1), the amifostine solution is prepared by adopting and mixing at least one of a phosphate buffer solution or distilled water; The method according to claim 5 .

10. In the step (2), microalgae powder is added to the amifostine solution under a certain temperature and light-shielding conditions, and the mixture is stirred and centrifuged to collect the precipitate, which is washed 3-5 times and then post-treated to obtain a solid powder of the drug complex; The method according to claim 5 .

11. In the step (2), the constant temperature is 2 to 8°C. The method according to claim 5 .

12. In the step (2), the stirring speed is 60 to 200 rpm; The method according to claim 5 .

13. In the step (2), the stirring time is 6 to 12 hours. The method according to claim 5 .

14. The post-treatment includes drying. The method according to claim 5 .

15. The drying is freeze-drying. The method according to claim 14 .

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