TW202220687A - Use of carnosine nanoparticle for preparing medicine for colon cancer metastasis including administering 85 to 340 mg/kg of carnosine nanoparticles - Google Patents

Abstract

A use of a carnosine nanoparticle for preparing a medicine for colon cancer metastasis is disclosed. The use comprises administering an effective amount of carnosine nanoparticle (carnosine nanoparticles, C-NPs) and a pharmaceutically acceptable carrier to an individual. The carnosine nanoparticles with an effective dose of 85 to 340 mg/kg can effectively reduce liver function indicators of values of Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Aberrant crypt (AC) and Aberrant crypt foci (ACF), as well as expression levels of Caudal type homeobox 2 (CDX2) and Cytokeratin-20 (Krt20) to inhibit colon cancer metastasis. In this way, the use of carnosine nanoparticles has a good ability to inhibit colon cancer metastasis, thereby providing a reference for nanomaterials used in inhibiting colon cancer metastasis.

Description

Use of carnosine nanoparticles for preparing medicine for colon cancer metastasis

The present invention relates to a kind of carnosine nanoparticle for preparing the medicine of colon cancer metastasis The purpose of this study, especially involving a kind of carnosine nanoparticles (C-NPs), can reduce the generation of abnormal crypt (AC) and abnormal crypt foci (ACF) and homeobox in the large intestine tissue. The expression of protein 2 (Caudal type homeobox 2, CDX2) and cytokeratin (Cytokeratin-20, Krt20) can inhibit the continuous progression of colon cancer, and by reducing the incidence of liver and lung tumors and the expression of CDX2 and Krt20, to inhibit colon cancer Metastases to the liver and lungs.

According to the World Health Organization, about 9.6 million people died of cancer in 2018. Colon cancer ranks third in the list, and the cancer that causes death ranks second. The Ministry of Health and Welfare of the Republic of China announced in 2020 that among the top ten causes of death in Taiwan in 2019, malignant tumors (cancer) ranked first, among which the mortality rate of colon cancer reached 27.3%, ranking third among the top ten cancer causes of death (Ministry of Health and Welfare, 2020) Therefore, we know that colon cancer is a major threat to human health. In addition, cancer metastasis is one of the reasons for the death of cancer patients. In the past, literature pointed out that in the late stage of colon cancer, cancer cells often metastasize to the liver, lungs, bones and other organs through the lymphatic system or blood. 50% of patients with colon cancer die of colon cancer metastasis, so the development of effective and safe drugs for the treatment of cancer and cancer metastasis has become an important topic for scholars (Manfredi et al., 2006; Van Cutsem and Oliveira, 2009).

Carnosine is a combination of β-alanine and L-histidine The resulting double peptides are mainly found in vertebrate skeletal muscle and are known to have antioxidant, anti-inflammatory and anti-cancer effects. The applicant has investigated the effect of carnosine on AOM-induced colon cancer formation in ICR mice and the metastasis of human colon cancer HCT-116 cell line (Metastasis) and its regulatory mechanism. From the research results, it is known that carnosine administration can regulate the inflammatory response by regulating the inflammatory response. (Inflammation), cell proliferation (Proliferation), cell apoptosis (Apoptosis), autophagy (Autophagy) and angiogenesis (Angiogenesis) mechanisms inhibit colon cancer formation (Guo, 2019). HCT-116 cell line treated with 0.5, 1 or 5 mM carnosine for 48 hours can reduce its migration (Migration), invasion (Invasion), intravasation (Intravasation), adhesion (Adhesion) and extravasation by regulating NF-κB activity Extravasation can inhibit the transfer of HCT-116 (Hsieh et al., 2019; Wu et al., 2019a). It can be seen that carnosine has great potential for inhibiting colon cancer tumor metastasis.

With the vigorous development of biomedical research, many scholars began to combine samples with nanotechnology Experiments with nanomaterials are different from general materials in terms of their physical and chemical properties, unique size, and various shape changes, providing new methods for cancer treatment. Previous studies have found that treatment with 25 μM Nanostructured SLM encapsulated in micelles (Nano-SLM) for 48 hours can inhibit the survival rate of human colon cancer HT-29 cell line and significantly increase the relationship between apoptosis and necrosis of cancer cells. ratio (Mombeini et al., 2018). Dasgupta et al. (2018) treated HCT-116 cell line with 28.11 μg/mL silver nanoparticles (AgNPs) for 24 hours and found that AgNPs increased the proportion of apoptosis and cell cycle arrest at G2/M inhibit the proliferation of cancer cells. It is known from the above studies that nanomaterials have anticancer potential.

Due to the unique physicochemical properties of nanoparticles, they have received special attention in medical-related fields. It is often used in biomedical diagnostic imaging and disease treatment. For this reason, it is necessary to further develop a set of carnosine nanoparticles that can inhibit colon cancer metastasis and improve the aforementioned shortcomings of the prior art.

The main purpose of the present invention is to overcome the above-mentioned problems encountered in the prior art and to improve It can reduce the generation of AC and ACF and the expression of CDX2 and Krt20 in the large intestine, and inhibit the continuous deterioration of colon cancer; and by reducing the incidence of liver and lung tumors and the expression of CDX2 and Krt20, it can inhibit the metastasis of colon cancer to the liver and lung, and It was found that 85 mg/kg carnosine nanoparticle was the best carnosine nanoparticle for the preparation of a drug for colon cancer metastasis.

In order to achieve the above purpose, the present invention relates to a carnosine nanoparticle for the preparation of colon cancer cells. The use of the transferred drug, the drug comprises administering an effective amount of carnosine nanoparticles (C-NPs) and a pharmaceutically acceptable carrier to an individual, and the drug is administered in an effective dose of 85-340 mg Carnosine nanoparticles per kg can effectively reduce liver function indicators glutamate pyruvate transaminase (Alanine aminotransferase, ALT), glutamate phenylacetate transaminase (Aspartate aminotransferase, AST), abnormal gland crypt (Aberrant crypt). , AC) and the number of abnormal crypt foci (ACF), as well as the expression levels of colon cancer-specific markers Caudal type homeobox 2 (CDX2) and cytokeratin (Cytokeratin-20, Krt20) , and inhibit colon cancer metastasis.

In the above-mentioned embodiments of the present invention, the effective dose of the drug is administered at 85 mg/kg, 170 mg/kg or 340 mg/kg of carnosine nanoparticles.

In the above embodiments of the present invention, the carrier comprises excipients, diluents, thickeners, Fillers, binders, disintegrants, lubricants, oily or non-oily bases, surfactants, suspending agents, gelling agents, adjuvants, preservatives, antioxidants, stabilizers, colors, or flavors.

In the above embodiments of the present invention, the dosage form of the drug is a solution.

In the above-mentioned embodiments of the present invention, the carnosine used in the carnosine nanoparticles (Carnosin) It is (2S)-2-[(3-Amino-1-oxopropyl)amino]-3-(3H-imidazol-4-yl)propanoic acid, with a molecular weight of 226.23 and a density of 1.4.

In the above embodiment of the present invention, the molecular weight of the carnosine nanoparticle is 453.2, and the density is 453.2. The degree was 1.019, and the particle size was 3.9±0.8 nm.

In the above embodiments of the present invention, the drug is administered by azomethane (Azoxymethane, AOM)-induced colon lesions were treated for 45 weeks.

Please refer to "Figure 1 to Figure 10", which are respectively the carnosine nanoparticles of the present invention The schematic diagram of the specific experimental structure of the particles on colon cancer metastasis, the image quantitative analysis diagram of the target protein of the present invention, the schematic diagram of the effect of the carnosine nanoparticles of the present invention on the large intestine fossa lesions of AOM-induced ICR mice, the carnosine nanoparticles of the present invention on the induction of AOM Figure of the effect of the large intestine and liver tissue morphology of ICR mice, the effect of the carnosine nanoparticles of the present invention on the tissue morphology of the lungs and kidneys of the AOM-induced ICR mice, the effect of the carnosine nanoparticles of the present invention on the CDX2 and Krt20 immunity of the large intestine of the AOM-induced ICR mice The effect of tissue staining, the effect of carnosine nanoparticles of the present invention on the immunohistochemical staining of CDX2 and Krt20 in the liver of AOM-induced ICR mice, the effect of carnosine nanoparticles of the present invention on the immunohistochemical staining of CDX2 and Krt20 in the lungs of AOM-induced ICR mice Figure, the effect of carnosine nanoparticles of the present invention on the expression of CDX2 and Krt20 genes in the liver of AOM-induced ICR mice, and the effect of carnosine nanoparticles of the present invention on the expression of CDX2 and Krt20 genes in the lungs of AOM-induced ICR mice. As shown in the figure: the present invention relates to the use of carnosine nanoparticles for preparing a drug for colon cancer metastasis, the drug comprising administering an effective amount of carnosine nanoparticles (C-NPs) to an individual and pharmaceutically acceptable The accepted carrier, and the drug is administered with an effective dose of 85-340 mg/kg of carnosine nanoparticles to effectively reduce liver function indicators glutamate pyruvate transaminase (Alanine aminotransferase, ALT), glutamic acid Aspartate aminotransferase (AST), number of Aberrant crypt (AC) and Aberrant crypt foci (ACF), and Caudal type 2 (Caudal type), a specific marker for colon cancer homeobox 2, CDX2) and cytokeratin (Cytokeratin-20, Krt20) expression, and inhibit colon cancer metastasis.

The invention relates to the use of a carnosine nanoparticle for preparing a medicament for colon cancer metastasis, The provided carnosine nanoparticles can utilize the technology well known to those with ordinary knowledge in the technical field to which the present invention pertains. The carnosine nanoparticles provided in this case and at least one pharmaceutically acceptable carrier can be used to prepare a drug suitable for the present invention. In the dosage form, the drug can be administered to a subject intravenously, subcutaneously, or orally. The following examples are only examples for understanding the details and connotations of the present invention, but are not intended to limit the scope of the patent application of the present invention.

The above mentioned carriers include but are not limited to excipients, diluents, thickeners, fillers, Binder, disintegrant, lubricant, oily or non-oily base, surfactant, suspending agent, gelling agent, adjuvant, preservative, antioxidant, stabilizer, color, or flavor.

The above mentioned dosage forms include but are not limited to solutions, emulsions, suspensions, powders, lozenges, Oils, ointments, lozenges, or capsules and other similar or suitable dosage forms of the present invention.

When in use, the present invention uses carnosine nanoparticles for experiments with ICR mice as The experiment subjects used Azoxymethane (AOM) to induce colon cancer metastasis in ICR mice, and fed carnosine nanoparticles after colon cancer formation to explore whether carnosine nanoparticles could inhibit colon cancer metastasis to the liver and lung. The potential of carnosine nanoparticles for the treatment of colon cancer metastasis was analyzed to evaluate the therapeutic potential of carnosine nanoparticles for human colon cancer.

The following examples are only examples for understanding the details and connotations of the present invention, but not for limitation The scope of the patent application of the present invention

[Example 1] Experimental material - source of carnosine Carnosine ((2S)-2-[(3-Amino-1-oxopropyl)amino]-3-(3H-imidazol-4-yl) propanoic acid, Carnosine) used in this experiment was purchased from Sigma (Sigma). -Aldrich Co. USA), molecular weight It is 226.23 and the density is 1.4; the carnosine nanoparticles were provided by Mr. Xie Shuzhen from the Aerosol Science Research Center of National Sun Yat-Sen University, the molecular weight is 453.2, the density is 1.019, and the particle size is 3.9±0.8 nm.

[Example 2] Experimental Architecture and Design The present invention uses carnosine nanoparticles on the effect of AOM-induced colon cancer metastasis in ICR mice, and its experimental structure is shown in Figure 1. After one week of domestication and acclimatization, the experimental animals were divided into cages according to their body weight, 2 per cage, and randomly divided into 9 groups of 6-8 animals, respectively the control group (Control) and the AOM group (AOM: 5 mg/kg). ), 85 mg/kg carnosine nanoparticles combined with AOM group (AOM+85 mg/kg C-NPs), 170 mg/kg carnosine nanoparticles combined with AOM group (AOM+170 mg/kg C-NPs), and 340 mg/kg carnosine nanoparticles combined with AOM group (AOM+340 mg/kg C-NPs). The experiment was carried out for 45 weeks. During the experiment, the feed conversion rate and water intake were recorded and converted, and analyzed after sacrifice, including the relative weight of mouse organs, serum biochemical indicators, count of abnormal crypt lesions in the large intestine, and histomorphological observation (liver). Gene expression analysis of colon cancer markers (CDX2, Krt20), lung, kidney, large intestine), liver and lung, to explore the mechanism of carnosine nanoparticle anti-cancer metastasis through the above experiments.

The animal experimental model used in the present invention refers to the colon cancer metastasis in the past laboratory The establishment of animal models and related research, to explore the effect of carnosine on the formation of colon cancer in mice induced by AOM, and use the results of its animal experiments to know that AOM-induced ICR mice will develop colon cancer at 27 weeks; the present invention uses phosphate buffered saline ( Phosphate buffered saline, PBS) was used as the control group, and the concentration of carnosine nanoparticles was used to refer to the results of the cell model transfer test. HCT-116 cell line was treated with 0.25, 0.5 or 1 mM carnosine nanoparticles for 96 hours, which can inhibit the cancer metastasis marker MMP 2. The expression levels of MMP 9 and MMP 13 genes were calculated from the blood volume of mice at 1, 2 and 4 times the low concentration of 0.25 mM, which were 85, 170 and 340 mg/kg, respectively. The control group was injected with PBS once a week by intraperitoneal injection from week 0 to week 5; the AOM group and all doses of carnosine nanoparticles combined with AOM group were injected by intraperitoneal injection weekly from week 0 to week 5 A single dose of 5 mg/kg AOM was administered, and colon cancer was confirmed at week 27, and 85, 170, and 340 mg/kg of carnosine nanoparticles were administered orally during weeks 28 to 45, respectively.

[Example 3] Experimental method (1) Experimental animal strains and breeding environment 4-week-old male mice of ICR strain were used in this experiment, purchased from Lesco Biotechnology Co., Ltd. (Taipei, Taiwan). Standard diet (LabDiet 5001) was given, and the feed and sterile drinking water were supplied by ad libitum rearing method. The rearing temperature was maintained at 25±2°C, and the day-night cycle was 12 hours.

(2) Animal sacrifice and sample processing After fasting for 12 hours on the day before sacrifice, the experimental mice were sacrificed by carbon dioxide asphyxiation, and the heart was harvested. Blood was collected, and the organs were taken out and weighed. A part of the liver, lungs and large intestine was immersed in 10% formalin for use as tissue staining sections. The remaining part and other tissues were snap-frozen in liquid nitrogen and stored. Store in -80°C refrigerator.

3.飼料轉換率（Feed conversion ratio, FCR） 飼料轉換率計算式如下：

(3) Analysis of the growth characteristics of mice During the experiment, the feed intake, water intake and weight gain rate of the mice were recorded twice a week, and other growth characteristics of the mice were observed. 1. Food and water intake (Food and water intake) In this experiment, the daily average intake and daily average water intake of mice for 45 weeks were recorded. 2. Weight gain The formula for calculating the weight gain is as follows:

3. Feed conversion ratio (FCR) The feed conversion ratio is calculated as follows:

2.肝臟腫瘤發生率（Liver tumor incedence） 肝臟腫瘤發生率計算式如下：

3.肺臟腫瘤發生率（Lung tumor incedence） 肺臟腫瘤發生率計算式如下：

(4) Analysis of pathological characteristics of mice 1. Colorectal tumor incidence (Colon tumor incidence) The calculation formula of colorectal tumor incidence is as follows:

2. Liver tumor incidence The calculation formula of liver tumor incidence is as follows:

3. Lung tumor incidence The calculation formula for the incidence of lung tumors is as follows:

(5) Relative body weight and weight analysis of mouse organs After being sacrificed by carbon dioxide asphyxiation, the heart, liver, spleen, lung, kidney and large intestine were taken out and weighed, and the weights were recorded in detail to calculate the relative weight of the organs. The calculation formula is as follows: [Organ weight (g)/final body weight (g) ]×100%

(6) Analysis of serum biochemical indicators in mice After 4 hours at room temperature, blood samples were centrifuged at 4°C and 2438 × g for 15 minutes. After centrifugation, serum was removed and stored at 4°C. The Shangjie Medical Laboratory was entrusted to analyze the serum biochemical indicators such as AST, ALT, blood urea nitrogen (BUN) and serum creatinine (Creatinine).

(7) Analysis of abnormal crypt lesions in mouse large intestine tissue Referring to the method of Tudek et al., the whole large intestine was extracted, washed with PBS until there was no fecal residue, the colon was about 1 cm in size and cut open, flattened and fixed on a weighing dish with a needle, and immersed in 10% neutral In formalin solution, cover with plastic wrap and fix the tissue for 24 hours, then take it out and wash it twice with PBS, add 0.5% methyl blue for staining for 5 minutes, wash it twice with PBS, spread it on a glass slide, observe and calculate with a microscope The number of AC and ACF in the large intestine of mice.

(8) Evaluation of the damage degree of liver, lung, kidney and large intestine in mice 1. Hematoxylin and eosin (H&E) staining analysis After the mice were sacrificed, the length of the dissected large intestine was measured and photographed and recorded. The large intestine was washed with PBS until there was no fecal residue. The colon was then excised about 0.5 to 1 cm in size and soaked in 10% neutral formalin to fix the tissue. Entrusted Ceders Biomedical Technology Co., Ltd. to carry out tissue sectioning and H&E staining, and finally cover the slides. This experiment refers to the conventional experimental method (Enders and Peebles (1954)) for H&E staining. Hematoxylin is a blue-violet substance and binds to nucleic acids in the nucleus; eosin is an orange-red substance and binds to proteins in the cytoplasm. In this way, the tissue morphology was observed and photographed. 2. Immunohistochemistry (IHC) staining analysis After the mice were sacrificed, the colons of about 0.5 to 1 cm in size were taken, immersed in 10% neutral formalin to fix the tissue, and then entrusted to Ceders Biomedical Technology Co., Ltd. for tissue sectioning and IHC staining, and finally the slides were mounted. . This experiment refers to the conventional experimental method (refer to Harvey et al. (1999)), through the chemical reaction of antigen-antibody on the tissue, the specific antibody is labeled with a chromogenic agent to develop color on the tissue, thereby observing and photographing with a microscope. . The images of immunohistostaining were imaged in mouse organ sections (×400) by the imaging software ImageJ (National Institutes of Health, USA), as shown in Figure 2. First quantify the entire tissue range (Figure 2(A)), and then quantify the antibody color range (Figure 2(B)), and calculate the target protein index as (antibody color range B/whole tissue range A) × 100% (%).

(IX) Gene expression analysis of cancer metastasis-related indicators 1. Extracted ribonucleic acid All instruments were sterilized by high temperature and high pressure to remove residual ribonucleic acid (RNase). After HCT-116 cells were sampled, the cells were collected into a 1.5 mL microcentrifuge tube with 1 mL of TRIzol reagent. After thorough mixing and shaking, 200 μL of chloroform (Chloroform) was added and shaken up and down. , 16,200 × g for 15 minutes, take the supernatant to another microcentrifuge tube and add an equal amount (1:1) of isopropanol, shake and mix well, ice bath for 5 minutes to precipitate ribonucleic acid (RNA). ) at 4°C, 16,200 × g for 15 minutes, the supernatant was removed, and the precipitate was RNA. Add 1 mL of 75% alcohol, centrifuge at 4°C, 16,200×g for 10 minutes, remove the upper layer of alcohol, and then centrifuge at 4°C, 16,200×g for 1 minute, remove the remaining alcohol, and air dry at room temperature for 15 minutes , and finally redissolved in an aqueous solution of diethyl pyrocarbonate (DEPC-H ₂ O), and stored at -20°C after quantification. 2. Quantitative ribonucleic acid The nucleic acid and protein analysis system is used for RNA quantification. Before the measurement, 2 µL of DEPC-H2O must be taken in the sample tank for blank correction. After wiping with 75% alcohol, take 2 µL of each sample into the sample holding tank for testing. Repeat the above actions until the sample testing is completed. After the measurement is completed, it must be wiped with 75% alcohol and calculated by computer software (Gen 5). Sample RNA concentration and RNA quality (OD260/OD280). 3. Synthesized complementary Deoxyribonucleic acid (Synthesized complementary Deoxyribonucleic acid) This experiment was carried out with reference to the method suggested by the manufacturer (Promega). Add 1 μL of Oligo dT 0.125 μg/μL to a 1.5 mL microcentrifuge tube , 1 μL deoxynucleoside triphosphate (dNTP) 10 mM, and 1 μg RNA sample, then make up the volume with sterilized water to 20 μL, dry bath at 65°C for 5 minutes, and place on ice for 5 minutes. Then add 1 μL murine leukemia virus reverse transcriptase (M-MLV Reverse transcriptase), 1 μL RNase inhibitor (RNAsin), and 5 μL M-MLV RT 5X buffer in sequence, mix all the reagents by shaking, and store them at 37°C. C dry bath for 1 hour to obtain cDNA solution, which is stored at -20 °C for future use. 4. In the real-time polymerase chain reaction (Real-time polymerase chain reaction, Real-time PCR) real-time fluorescence quantitative detection system (Real-time Quantitative PCR Detecting System, Real-time PCR), by SYBR green I dye and The double-stranded DNA of the sample is bound, and the fluorescent light is excited under a halogen lamp. When the cyclic reaction increases the synthesis of the target gene, the binding amount of the SYBR green I dye to the double-stranded DNA will also increase. Therefore, as the target gene is amplified, more SYBR green I fluorescence can be detected. Real-time PCR mainly uses the different concentrations of each target gene in the sample. Under the same PCR reaction conditions, the reaction of the target gene template with a higher content will reach the geometric phase faster. At this time, DNA synthesis will take two steps. Double to double. In the definition, reaching the critical cycle number in the Geometric phase is called the cycle threshold (Cycle threshold, CT). In order to confirm the operation method and the presence or absence of contamination in the reaction process, a group of negative control (Non-template control, NTC) without genes was added, and in order to compare each quantitative result with each other, to avoid the deviation caused by the difference of PCR reaction efficiency , so each quantitative analysis will add more than one internal control group (Internal control), and use the concentration converted from the reaction of the specific primer and the internal control group to confirm the accuracy of each quantitative analysis. Before performing Real-time PCR quantitative analysis, 2.5 μL of double-distilled water (ddH2O), 5 μL of 2X SYBR Fast MM, 0.5 μL of 20X Target primer, and 2 μL of 10 ng of cDNA should be added in sequence to a 96-well plate dedicated to PCR. The final volume is to 10 μL. It was put into a real-time polymerase chain reaction machine (LightCycler® 96 Real-Time PCR System, Roche Life Science, Basel, Switzerland) to react, and the conditions of Real-time PCR were set as shown in Table 1. Table I program 1 95°C 120 sec × 1 cycle program 2 95°C 5 sec × 40 cycles 60°C 30 sec Procedure 3 95°C 10 sec × 1 cycle 65°C 60 sec 97°C 1 sec

Since the relative quantification needs to select the constant expression characteristic gene possessed by humans as the necessary calculation value for normalization, the test method of the present invention selects mouse β-actin (β-actin) as the control group, Real-time The specific primers for each gene of RCR are shown in Table 2. Table 2 Gene Primer sequence (5-3) Reference (NCBI GenBank) β-actin F-ATGTGCAAGGCCGGCTTC NM001101.3 R-GAATCCTTCTGACCCATGCC CDX2 F-TGAGCTGGCTGCCACACTT NM007673.3 R-TGGCTCTGCGGTTCTGAAAC Krt20 F-CCGAGCACCATCCGAGACTA NM023256.1 R-ACGCACTGGGCATTTTGTACT

(10) Statistical analysis The experimental results were analyzed and compared within the group using the computer statistical analysis software SPSS12.0 (SPSS, USA) program One-way ANOVA, and then Duncan's test or Dukay's significance test was used. (Tukey's test) to compare different concentrations of the same sample. Then, the comparison between different samples was carried out by Dukai's test of significance, and the degree of significant difference was set as P<0.05.

[Example 4] Experimental Results - Effect of Carnosine Nanoparticles on Colon Cancer Metastasis Induced by AOM in ICR Mice (1) Effect of Carnosine Nanoparticles on the Growth Characteristics of Colon Cancer Metastasis in ICR Mice The effect of the growth characteristics of cancer metastasis, as shown in Table 3, the weight gain rate and feed conversion rate of the AOM group were significantly lower than those of the control group, indicating that AOM induction would reduce the weight gain rate by reducing the feed conversion rate of mice; giving 85, 170 Compared with the AOM group, the weight gain rate and feed conversion rate of the 340 mg/kg carnosine nanoparticle group had no significant difference, indicating that the administration of 85, 170 and 340 mg/kg carnosine nanoparticles did not affect the weight gain of the mice. rate and feed conversion rate. There was no significant difference between the AOM group and the control group in intake and water intake, indicating that AOM induction did not affect the intake and water intake of mice; administration of 85, 170 and 340 mg/kg carnosine nanoparticles in water intake was significantly higher than that of AOM However, the intake of 340 mg/kg carnosine nanoparticles group was significantly higher than that of AOM group, indicating that administration of 85, 170 and 340 mg/kg carnosine nanoparticles would increase the intake and water intake of mice. In addition, the percentage of lung weight in the AOM group was significantly higher than that in the control group, indicating that AOM would increase the lung weight of mice; there was no significant difference between the administration of 85, 170 and 340 mg/kg carnosine nanoparticles compared with the AOM group, indicating that administration of 85 , 170 and 340 mg/kg carnosine nanoparticles did not improve lung weight. Taking the above results together, AOM induction reduced the rate of body weight gain and feed conversion rate, but increased lung weight, while administration of 85, 170, and 340 mg/kg carnosine nanoparticles increased water intake in mice, while 340 mg/kg Carnosine nanoparticles increase uptake in mice. The data are expressed as mean ± SD (n=6~8), and Duncan's analysis was used to evaluate the significance of the difference. Different superscript letters a~d in the table indicate that they are statistically different from each other (P<0.05). ). Table 3 control group AOM group AOM group+85 mg/kg C-NPs Weight gain rate (%) 98.2±1.0a 56.8±1.3b 56.1±2.6b Intake (g/mouse/day) 7.9±0.6ab 7.4±0.8b 7.4±0.7b Feed conversion ratio (body weight/g food) 1.1±0.2a 0.7±0.2b 0.7±0.1b Water intake (mL/day) 11.8±0.9d 11.4±1.0d 14.1±0.4c heart(%) 0.41±0.06 0.43±0.03 0.45±0.02 liver(%) 3.70±0.25 3.53±0.30 3.74±0.11 spleen(%) 0.20±0.03 0.20±0.02 0.19±0.02 lung(%) 0.43±0.02b 0.50±0.03a 0.50±0.01a kidney(%) 1.55±0.08 1.59±0.09 1.58±0.10 the large intestine(%) 1.01±0.08 0.92±0.06 1.00±0.06 AOM group+170 mg/kg C-NPs AOM group+340 mg/kg C-NPs Weight gain rate (%) 57.4±5.3b 59.9±2.7b Intake (g/mouse/day) 6.9±0.8b 8.4±0.4a Feed conversion ratio (body weight/g food) 0.9±0.1ab 0.8±0.1b Water intake (mL/day) 16.0±1.4b 18.2±0.4a heart(%) 0.42±0.03 0.46±0.03 liver(%) 3.59±0.16 3.65±0.36 spleen(%) 0.20±0.03 0.19±0.03 lung(%) 0.47±0.04a 0.50±0.03a kidney(%) 1.49±0.07 1.61±0.04 the large intestine(%) 0.95±0.10 0.99±0.07

(2) Effects of carnosine nanoparticles on serum biochemical indexes of colon cancer metastasis in ICR mice The changes of carnosine nanoparticles on serum biochemical indexes of colon cancer metastasis in ICR mice are shown in Table 4. The AOM group showed the changes of liver function indexes AST and AST The ALT values were significantly higher than those in the control group, indicating that AOM induction would damage the liver function of mice; the ALT and AST values in the groups given 85, 170 and 340 mg/kg carnosine nanoparticles were significantly lower than those in the AOM group, indicating that the groups given 85, 170 and 340 mg/kg carnosine nanoparticles , 170 and 340 mg/kg carnosine nanoparticles can improve liver function damage in mice. There were no significant differences in renal function index BUN and serum creatinine values among the groups, indicating that AOM induction and carnosine nanoparticles did not affect BUN and serum creatinine. Based on the above results, AOM induction can increase AST and ALT in mice, while administration of 85, 170 and 340 mg/kg carnosine nanoparticles can reduce AST and ALT in mice. The data are expressed as mean ± SD (n=6~8), and Duncan's analysis was used to evaluate the significance of the difference. Different superscript letters a to c in the table indicate that there is a statistical difference between each other (P<0.05). ). Table 4 control group AOM group AOM group+85 mg/kg C-NPs AST(U/L) 54.7±11.6b 139.7±10.6a 75.5±6.4b ALT(U/L) 86.3±17.0b 157.5±9.2a 94.5±20.5b BUN(mg/dL) 25.6±6.9 25.7±2.6 22.1±2.3 Serum creatinine (mg/dL) 0.30±0.03 0.33±0.02 0.24±0.02 AOM group+170 mg/kg C-NPs AOM group+340 mg/kg C-NPs AST(U/L) 75.0±5.6b 69.5±12.2b ALT(U/L) 86.5±7.8b 110.0±19.8bc BUN(mg/dL) 26.2±3.3 22.2±2.8 Serum creatinine (mg/dL) 0.26±0.04 0.25±0.03

(3) The effect of carnosine nanoparticles on the colorectal tissue and metastatic characteristics of colon cancer metastasis in ICR mice. There were no significant differences in colon length, colon, liver and lung tumor numbers among the groups, indicating that neither AOM induction nor carnosine nanoparticles affected colon length, colon, liver and lung tumor numbers. The incidence of colorectal and lung tumors in the AOM group was 25% higher than that in the control group; administration of 85 and 170 mg/kg carnosine nanoparticles could reduce the incidence of tumors. Based on the above results, AOM induction and administration of 85, 170 and 340 mg/kg carnosine nanoparticles did not affect the colorectal tissue and metastasis characteristics, but could reduce the incidence of colorectal tumors and metastasis. Among them, the data are expressed as mean±SD (n=6~8), and the number of tumors=the total number of tumors/the number of examined mice. Tumor incidence (%) = (number of tumor-bearing mice/number of examined mice) × 100. Table 5 control group AOM group AOM group+85 mg/kg C-NPs Large intestine length (cm) 17.6±1.4 17.8±1.4 18.0±0.7 Colorectal tumor count 0.0±0.0 0.8±1.6 0.1±0.4 number of liver tumors 0.0±0.0 0.0±0.0 0.0±0.0 number of lung tumors 0.0±0.0 0.4±0.7 0.0±0.0 Incidence of colorectal tumor (%) 0 25 0 Incidence of liver tumor (%) 0 0 0 Incidence of lung tumor (%) 0 25 0 AOM group+170 mg/kg C-NPs AOM group+340 mg/kg C-NPs Large intestine length (cm) 18.1±0.8 17.8±0.9 Colorectal tumor count 0.0±0.0 0.1±0.4 number of liver tumors 0.0±0.0 0.1±0.4 number of lung tumors 0.0±0.0 0.0±0.0 Incidence of colorectal tumor (%) 0 13 Incidence of liver tumor (%) 0 13 Incidence of lung tumor (%) 0 0

(4) The effect of carnosine nanoparticles on colon cancer metastases in ICR mice with abnormal crypt lesions of the large intestine Figure 3 shows the effect of carnosine nanoparticles on AOM-induced ICR mice colon cancer metastasis and colorectal abnormal crypt foci, as shown in Figure 3, Figure (A) is the number of AC, Figure (B) is the number of ACF. The numbers of AC and ACF in the AOM group were significantly higher than those in the control group, indicating that AOM induction for 45 weeks would make the colon lesions persist in ICR mice; the numbers of AC and ACF were significantly lower than those given 85, 170 and 340 mg/kg carnosine nanoparticles. In the AOM group, administration of 85, 170 and 340 mg/kg carnosine nanoparticles can improve the condition of colorectal crypt lesions. Based on the above results, AOM induction can increase the AC and ACF in the large intestine of mice, while administration of 85, 170 and 340 mg/kg carnosine nanoparticles can reduce the AC and ACF in the large intestine of mice. The data are independent experiments, expressed as mean ± SD, and Duncan's analysis method was used to conduct statistical analysis to evaluate the significant differences in ACF. Different superscript letters a to d in the figure indicate significant differences in the number of ACFs (P < 0.05). .

(5) The effect of carnosine nanoparticles on the tissue morphology of colon, liver, lung and kidney with colon cancer metastasis in ICR mice The effect of carnosine nanoparticles on the tissue morphology of colon, liver, lung and kidney of AOM-induced ICR mice colon cancer metastasis is shown in Figures 4 and 5, of which Figure 4 (A) is the large intestine, and Figure 4 (B) ) is the liver, Fig. 5(A) is the lung and Fig. 5(B) is the effect of the histological observation of the kidney and other sections. The tissue was stained with hematoxylin-eosin and examined under a microscope (400 times magnification). observe below. In the morphological observation of the large intestine, liver, lung and kidney, compared with the control group, the AOM group had more severe damage to the large intestinal goblet cells and abnormal arrangement of mitoses at the bottom of the villi (as shown by the yellow arrow in Figure 4 (A). liver); liver interstitial blur and fat vacuoles are more serious (as shown by the yellow arrow in Figure 4 (B)); alveolar tissue damage is more serious (as shown by the yellow arrow in Figure 5 (A)); kidney There was no damage (as shown in Figure 5 (B)), and the damage to the large intestine, liver, lung and kidney tissue was improved after administration of 85, 170 and 340 mg/kg carnosine nanoparticles.

(6) The effect of carnosine nanoparticles on CDX2 and Krt20 immunohistochemical staining of colon cancer metastasis in colon, liver and lung of ICR mice Tissues were embedded in paraffin and detected by immunohistochemical analysis using CDX2 and Krt20 antibodies, shown as brown spots (400x magnification). The number of CDX2 and Krt20 labeling indices (%) was counted in different fields (400x magnification) of each mouse by the imaging software ImageJ.

Carnosine nanoparticles on colon cancer metastases in AOM-induced ICR mice on colonic immune tissue Staining, as shown in Figure 6, Figures (A) and (B) are the expression and quantitative results of CDX2, and Figures (C) and (D) are the expression and quantitative results of Krt20. The expression of CDX2 and Krt20 in the AOM group was significantly higher than that in the control group, indicating that AOM induction for 45 weeks would make colon cancer persist. The performance of Krt20 in the 170 and 340 mg/kg carnosine nanoparticle groups was significantly lower than that in the AOM group, indicating that administration of 170 and 340 mg/kg carnosine nanoparticles could inhibit AOM-induced colon cancer. Among them, Duncan's analysis was used to evaluate the significant differences in the expression of CDX2 and Krt20 proteins in the colon. Different superscript letters a to c in the figure indicated that there was a significant difference in the expression of CDX2 and Krt20 (P<0.05).

Carnosine nanoparticles on the liver immune tissue of AOM-induced ICR mice colon cancer metastasis Staining, as shown in Figure 7, Figures (A) and (B) are the expression and quantitative results of CDX2, and Figures (C) and (D) are the expression and quantitative results of Krt20. The expression of CDX2 and Krt20 in the AOM group was significantly higher than that in the control group, indicating that AOM induction for 45 weeks would cause colon cancer to metastasize to the liver. Carnosine nanoparticles at 170 and 340 mg/kg inhibited colon cancer metastasis to the liver. Among them, Duncan's analysis was performed to evaluate the significant differences in the expression of CDX2 and Krt20 proteins in the colon. Different superscript letters a to c in the figure indicated that there was a significant difference between the expression of CDX2 and Krt20 (P<0.05).

Carnosine nanoparticles on lung immune tissue of AOM-induced colon cancer metastasis in mice with ICR Staining, as shown in Figure 8, Figures (A) and (B) are the expression and quantitative results of CDX2, and Figures (C) and (D) are the expression and quantitative results of Krt20. The expression of CDX2 and Krt20 in the AOM group was significantly higher than that in the control group, indicating that AOM induction for 45 weeks would cause colon cancer to metastasize to the lung; the groups given 85, 170 and 340 mg/kg carnosine nanoparticles showed significantly lower CDX2 and Krt20 performance than AOM group, indicated that administration of 85, 170 and 340 mg/kg carnosine nanoparticles could inhibit colon cancer metastasis to the lung. Based on the above results, AOM induction can increase the expression of CDX2 and Krt20 in the large intestine, liver and lung tissue of mice, and administration of 85, 170 and 340 mg/kg carnosine nanoparticles can increase the expression of CDX2 and Krt20 in the large intestine, liver and lung tissue of mice. decrease, to achieve the result of inhibiting cancer metastasis. Among them, Duncan's analysis was performed to evaluate the significant differences in the expression of CDX2 and Krt20 proteins in the colon. Different superscript letters a to c in the figure indicated that there was a significant difference between the expression of CDX2 and Krt20 (P<0.05).

(7) Effects of carnosine nanoparticles on CDX2 and Krt20 in liver and lung metastases from colon cancer in ICR mice The effects of carnosine nanoparticles on the expression of CDX2 and Krt20 genes in the liver and lung of ICR mice are shown in Figures 9 and 10. Figure 9 (A) and (B) are the gene expression of CDX2 and Krt20 in the liver, respectively , the first Figure 10 (A) and (B) are the gene expression levels of CDX2 and Krt20 in the lungs, respectively. The expression levels of CDX2 and Krt20 genes in the AOM group were significantly higher than those in the control group, indicating that AOM induction would cause colon cancer to metastasize to the liver and lung. The gene expression level was significantly lower than that in the AOM group, and the 85 and 170 mg/kg carnosine nanoparticles administered the CDX2 gene expression in the lungs were significantly lower than those in the AOM group, indicating that 85, 170 and 340 mg/kg carnosine nanoparticles would increase the expression of CDX2 gene in the lungs. 85 and 170 mg/kg carnosine nanoparticles inhibited colon cancer metastasis to the lung by inhibiting the expression of CDX2 and Krt20 genes in the lung. The data are independent experiments, expressed as mean ± SD, and Duncan's analysis was used to conduct statistical analysis to evaluate the significant differences in the expression of CDX2 and Krt20 genes in the colon. Different superscript letters a to d in the figure represent CDX2 and Krt20 genes There was a significant difference in the amount of expression (P<0.05).

As can be seen from the above, the present invention utilizes ICR mice to induce colon cancer transformation by intraperitoneal injection of AOM. After the colon cancer was confirmed at week 27, different doses (85 mg/kg, 170 mg/kg and 340 mg/kg) of carnosine nanoparticles were administered starting at week 28 to verify whether carnosine nanoparticles could Inhibition of colon cancer metastasis. The results showed that in the analysis of the growth characteristics of mice, AOM induction reduced the rate of body weight gain and feed conversion rate and increased lung weight, and administration of different doses of carnosine nanoparticles did not affect the growth characteristics. In serum biochemical analysis, AOM increased liver function indexes ALT and AST, and administration of different doses of carnosine nanoparticles could reduce AST and ALT. In the observation of intestinal pathological characteristics, AOM induction can increase the number of AC and ACF, the incidence of colorectal and lung tumors, and the damage of intestinal, liver and lung tissue, and administration of different doses of carnosine nanoparticles can reduce the number of AC and ACF, and colorectal tumors. and the incidence of metastasis and improve intestinal, liver and lung tissue damage. Analysis of colon cancer indicators, it was found that AOM induction can increase the expression of CDX2 and Krt20 proteins in the terminal large intestine, and the expression of CDX2 and Krt20 proteins and genes in the liver and lung. Administration of different doses of carnosine nanoparticles can reduce the expression of CDX2 and Krt20 in the large intestine, liver and lung. quantity. Based on the above results, carnosine nanoparticles have a good ability to inhibit colon cancer metastasis, which can provide a reference for nanomaterials in inhibiting colon cancer metastasis.

To sum up, the present invention is a kind of carnosine nanoparticle for preparing the medicine for colon cancer metastasis Carnosine nanoparticles (C-NPs) can effectively reduce the abnormal crypt (AC) and abnormal crypt foci (ACF) in the large intestine tissue. The production and expression of Caudal type homeobox 2 (CDX2) and cytokeratin (Cytokeratin-20, Krt20) inhibit the progression of colon cancer; and by reducing the incidence of liver and lung tumors and the expression of CDX2 and Krt20, It is found that the administration of 85 mg/kg carnosine nanoparticles to inhibit colon cancer metastasis to the liver and lung has the best effect, thus making the production of the present invention more advanced, more practical and more in line with the needs of users, which has indeed met the invention patent The requirements for application are to file a patent application in accordance with the law.

However, the above are only preferred embodiments of the present invention, and should not be limited to this The scope of implementation of the present invention; therefore, all simple equivalent changes and modifications made according to the scope of the patent application of the present invention and the contents of the description of the invention should still fall within the scope of the patent of the present invention.

none

Figure 1 is a schematic diagram of the specific experimental structure of the carnosine nanoparticles of the present invention for colon cancer metastasis. Figure 2 is an image quantitative analysis diagram of the target protein of the present invention. Fig. 3 is a schematic diagram showing the effect of carnosine nanoparticles of the present invention on AOM-induced ICR mouse colorectal fossa lesions. Figure 4 shows the effect of carnosine nanoparticles of the present invention on the morphology of large intestine and liver in AOM-induced ICR mice. Figure 5 shows the effect of carnosine nanoparticles of the present invention on the morphology of lung and kidney tissue in AOM-induced ICR mice. Figure 6 shows the effect of carnosine nanoparticles of the present invention on the immunohistochemical staining of CDX2 and Krt20 in the large intestine of AOM-induced ICR mice. Figure 7 shows the effect of the carnosine nanoparticles of the present invention on the immunohistochemical staining of CDX2 and Krt20 in the liver of AOM-induced ICR mice. Fig. 8 shows the effect of carnosine nanoparticles of the present invention on the immunohistochemical staining of CDX2 and Krt20 in the lungs of AOM-induced ICR mice. Figure 9 shows the effect of carnosine nanoparticles of the present invention on the expression of CDX2 and Krt20 genes in the liver of AOM-induced ICR mice. Figure 10 shows the effect of carnosine nanoparticles of the present invention on the expression of CDX2 and Krt20 genes in the lungs of AOM-induced ICR mice.

Claims (7)

Use of a carnosine nanoparticle for preparing a medicine for colon cancer metastasis, the medicine comprising administering an effective amount of carnosine nanoparticle (Carnosine nanoparticles, C-NPs) and a pharmaceutically acceptable carrier to an individual, and the medicine Carnosine nanoparticles with an effective dose of 85-340 mg/kg can effectively reduce liver function indicators glutamate pyruvate transaminase (ALT), glutamate phenylacetate transaminase (Aspartate aminotransferase) , AST), the number of abnormal crypt (Aberrant crypt, AC) and aberrant crypt foci (ACF), as well as the colon cancer-specific markers Caudal type homeobox 2 (CDX2) and cell angle Protein (Cytokeratin-20, Krt20) expression level, and inhibit colon cancer metastasis. Use of the carnosine nanoparticles described in item 1 of the scope of the application for preparing a drug for colon cancer metastasis, wherein the drug is administered with an effective dose of 85 mg/kg, 170 mg/kg or 340 mg/kg. Carnosine Nanoparticles. Use of carnosine nanoparticles described in item 1 of the scope of the application for preparing a drug for colon cancer metastasis, wherein the carrier comprises excipients, diluents, thickening agents, fillers, binders, disintegrating agents agents, lubricants, oily or non-oily bases, surfactants, suspending agents, gelling agents, adjuvants, preservatives, antioxidants, stabilizers, colors, or flavors. According to the use of the carnosine nanoparticle described in item 1 of the scope of application for preparing a medicine for colon cancer metastasis, the dosage form of the medicine is a solution. Use of the carnosine nanoparticle described in item 1 of the claimed scope for preparing a drug for colon cancer metastasis, wherein the carnosine used in the carnosine nanoparticle is (2S)-2-[(3- Amino-1-oxopropyl)amino]-3-(3H-imidazol-4-yl)propanoic acid with a molecular weight of 226.23 and a density of 1.4. According to the application of the carnosine nanoparticle described in item 1 of the scope of application for the preparation of a drug for colon cancer metastasis, the carnosine nanoparticle has a molecular weight of 453.2, a density of 1.019, and a particle size of 1.019. The diameter is 3.9±0.8 nm. The use of the carnosine nanoparticle described in item 1 of the scope of the application for preparing a drug for colon cancer metastasis, wherein the drug is administered to individuals with colon lesions induced by Azoxymethane (AOM) for 45 weeks .

Images