KR20020013987A - Novel use of cyclocreatine - Google Patents

Abstract

PURPOSE: Provided is a novel application of cyclocreatine to a prophylaxis and therapeutic composit Áon for hepatitis and hepatic carcinoma. CONSTITUTION: The cyclocreatine represented by the chemical formula in figure 1 is a kind of non-digestible nitrogen compounds and contained in a tissue of a mammal in an amount of 0.3-0.4%. It inhibits hepatic carcinoma or hepatitis in vivo thus has prophylaxis and therapeutic effects thereon.

Description

Novel use of cyclocreatine

The present invention relates to the use of cyclocreatine in the inhibition of novel hepatic cancer lesions. More specifically, the present invention relates to the inhibitory, therapeutic and prophylactic effects of cyclocreatinine on hepatocellular carcinoma or hepatitis on a hepatospecific chemical carcinogenic model of a carcinogenic model red test ( in vivo ).

The liver, one of the most important organs in the body, plays a central role in intermediate metabolism in the body, including the breakdown, synthesis, storage and secretion of nutrients, and the detoxification and excretion of toxic substances. Liver disease develops and progresses to liver cancer. Hepatocellular carcinoma (HCC) is one of the most common cancers in Korea, with about 1,500,000 deaths worldwide. Especially, it is the top 10 cancers among Koreans. (Ministry of Health and Welfare). The cause of liver cancer is not yet clear, but statistically, the incidence of liver cancer is higher in men than in women, mainly in their 40s and 60s, with 80% of patients with cirrhosis, and 80% of hepatitis B viruses. Carriers, 10% are characterized by hepatitis C. In addition, patients with chronic liver disease such as cirrhosis and chronic hepatitis have a high rate of liver cancer by themselves regardless of hepatitis B or C. Alcohol and smoking are also known to increase the incidence of liver cancer by 6-8 times in patients with chronic hepatitis and cirrhosis.

Surgical surgery for resection of cancer is known to be most effective in the treatment of liver cancer, but less than 20% of all liver cancer patients are targeted. Depending on the condition of the liver cancer, for example, liver cirrhosis is too severe. . In this case, the anticancer agent is administered, but it is hardly effective by oral administration or a general injection method, and a method of injecting an anticancer agent directly into the liver cancer tissue through the hepatic artery or a method of occluding a blood vessel connected to a carcinoma after the anticancer agent is frequently used. Doing. In addition, treatment methods such as transarterial chemoembolization and ethanol injection are known, but their therapeutic effects are not very helpful for advanced cancer. Since most liver cancers lead to death, prevention of hepatitis and liver cancer should be prioritized over the treatment of already developed liver cancer. In addition, Korea has the world's number one mortality rate in the 40s due to hepatic insufficiency due to hepatitis and fatty liver formation, which has a strong correlation with hepatocellular death due to chemical carcinogenesis and alcohol attack of acetaldehyde. It has been proved and, therefore, prevention of hepatitis and liver cancer is a top priority for promoting national health and well-being.

Creatine is one of the non-digestible nitrogen compounds and contains about 0.3-0.4% of mammalian muscle tissue and some creatine is formed by interaction with ATP. Present in the form of creatine (phosphocreatine, PCr).

Cyclocreatine, as a substrate of creatine, has been reported to show significant anticancer activity and efficacy in reducing free radicals [Martin KJ., Winslow ER and Kaddurah-Daouk R., Cancer Research 54: 5160]. -5165 (1994); Lillie JW., O'Keefe M., Valinski H., Hamlin HA Jr., Varban ML and Kaddurah-Daouk R: Cancer Research 53: 3172-3178 (1993)]. Cohn M et al. Reported the extent of solid cancer development in animals transplanted with solid cancer in rats [Miller EE., Evans AE and Cohn M: Proc Natl Acad USA 90: 3304-3308 (1993)]. Lesion inhibition has not been reported to date. Phosphorylated Phosphocreatine (PCr) is deposited in the cells at high concentrations after cyclocreatinine has been degraded by creatine, suggesting that this secondary metabolite, phosphocreatine, has direct anticancer activity in chemical carcinogenic models. It became. The best known function of cyclocreatin in the defense mechanism is that phosphocreatine, produced by degradation and phosphorylation by creatine kinase, acts as a high energy supply form in cells or as an anticancer substance. Is that. Recently, the efficacy of phosphocreatin in protecting brain cells, protecting breast cancer cells and treating breast cancer has been demonstrated [Matthews RT., Yang L., Genkins BG., Ferrante RJ., Rosen BR., Kaddurah-Daouk R and Flint Beal M, J. Neuroscience 18: 156-163 (1998); Nimrod M., Degani H., Rushkin E., Sherry ADand Cohn M, Mechanism of activity, Am J. Physiol 277: C708-C716 (1999)].

However, until now, there have been few studies showing that cyclocreatin inhibits the formation of liver-specific precancerous lesions, and in particular, no studies have confirmed the effect of cyclocreatin on hepatocellular carcinoma lesions and hepatocyte protection.

In the present invention, induction of carcinogenesis by staining Glutathione S-transferase placental type (GST-P), a precancerous lesion detection factor of liver tissue, in order to investigate the inhibitory activity of hepatocarcinogenesis of cyclocreatin on hepatic cancer lesions by applying mid-term carcinogen In addition, the size, number, area of hepatic lesions, number of hepatocellular nodules, and apoptosis were measured to determine the degree of hepatoprotective activity of cyclocreatine on the chemical carcinogenesis model.

The present inventors intraperitoneally administered diethylnitrosamine (DEN), a cancer trigger, to fisher 344 rats, two weeks after the cancer promoter, 0.02% 2-acetylaminofluorene (2-acethylaminofluorene, 2). -AAF) was mixed with the feed to prepare the control group and at the same time the experimental group was prepared by mixing and administering 1% cyclocreatine (w / w) to the feed, and after 3 weeks a new cell cycle in the control and experimental groups In order to create a hepatic cancerous lesion, a partial hepatectomy was performed to excise about 70% of the liver to create an artificial carcinogenic model, and rats of all experimental and control groups 11 weeks after breeding the red for 10 weeks. Hepatic nodules on the surface of liver tissue and staining of Glutathione-S transferase placental type (GST-p), a marker of hepatic cancer lesion The area of the marine service area, measuring the apoptosis (apoptosis) statistical processing and apoptosis degree degree of number and compared on the tissue by confirming that it has markedly inhibited the precancerous lesion between the cycloalkyl creatine on in vivo, thereby completing the present invention.

Accordingly, an object of the present invention is to provide a composition for preventing hepatitis or liver cancer and inhibiting the development of liver cancer in view of the above facts using cyclocreatin as an active ingredient.

The above object of the present invention is carcinogenic by administering and feeding cancer trigger factor diethylnitrosamine (DEN) and cancer promoter 2-acetylaminofluorene (Fisher 344 rat) to cancer fisher 344 rats (Fisher 344 rat) Hematoxylin & eosin of GST-p, a marker of carcinogenicity, of GST-p, a visual observation of the degree of hepatocellular carcinoma in the experimental group prepared by mixing 1% cyclocreatine with feed in a portion of (hematoxylin & eosin) It was achieved by confirming the degree of inhibition of carcinogenicity of cyclocreatin in vivo by performing histopathological examination measured to the degree of staining and measuring the degree of cell death (apoptosis) using the tunnel method.

Hereinafter, the configuration of the present invention will be described in detail.

1 shows the chemical structural diagram of cyclocreatine.

2 is a schematic diagram of the process of the present invention.

Figure 3-A is a photograph showing the degree of inhibition of hepatic cancer lesions of the experimental rats to which cyclocreatine was added.

Figure 3-B is a photograph showing the extent of pre-cancerous lesions in control rats not added cyclocreatin.

Figure 3-C is a photograph showing the degree of staining of GST-p, a hepatic cancer lesion phagocytosis factor of experimental rats to which cyclocreatine was added.

Figure 3-D is a photograph showing the degree of staining of GST-p, a hepatic cancer lesion phagocytosis factor of control rats not added cyclocreatin.

Fig. 4-A is a photograph showing the degree of hematoxylin-eosin staining of cells undergoing apoptosis in experimental rats to which cyclocreatin was added.

4-B is a photograph showing the degree of hematoxylin-eosin staining of cells in the course of apoptosis of control rats without addition of cyclocreatin.

Fig. 4-C shows the degree of positive reaction as measured by the tunnel method for apoptosis of experimental rats to which cyclocreatine was added.

Fig. 4-D shows the degree of positive reaction as measured by the tunnel method for apoptosis of control rats without addition of cyclocreatin.

The present invention was administered to cancer fisher 344 rats (Fisher 344 rats) by the intraperitoneal injection of the cancer trigger factor diethylnitrosamine (DEN, 200mg / Kg) after two weeks of breeding 0.02-% 2-acetylamino as a cancer promoter Inducing carcinogenesis for 3 weeks by feeding 2-fluorethyl (2-acethylaminofluorene) to the feed; Breeding the cancer-induced red thread into an experimental group fed with a diet containing 1% cyclocreatine and a control fed with a normal diet for 10 weeks; 70% partial ablation of the liver of the test group and the control group to create a hepatic cancer lesion applying the new cell cycle; After the necropsy of the experimental group and the control group, a portion of the liver tissue was removed, and liver tissue sections were made through a conventional tissue treatment process, and hematoxylin & eosin staining was performed to stain the liver by immunohistochemical staining. Histopathological examination step of irradiating the size and number of surface nodule cells with an optical microscope; It consists of identifying using tunnel technique to determine the degree of programmed apoptosis.

Hereinafter, specific examples of the present invention will be described in detail with reference to Examples and Experimental Examples, but the scope of the present invention is not limited to these Examples and Experimental Examples.

Example 1 Induction of Precancerous Lesions Using Administration of Carcinogen and Cyclocreatine and Partial Hepatectomy in a Fisher 344 Rat Model

Stage 1: Administration of carcinogens and cyclocreatine in fisher 344 rats

The male and female fish rat 344 was selected as a test subject of the present invention, and was raised for 2 weeks after intraperitoneal injection of diethylnitrosamine (DEN) at a concentration of 200 mg / Kg. A control group was prepared by inducing the cancer by injecting a mixture of 0.02% 2-acetylaminofluorene (2-acethylaminofluorene, 2-AAF), which is a cancer promoter, to the reared red dogs. After 3 weeks, some rats were fed a diet containing 1% of cyclocreatine (w / w) for 10 weeks, and this group was used as the experimental group, and the carcinogenic red model was established using the model fed the normal diet as a control group. It was.

Stage 2: Induction of Precancerous Lesions Using Partial Hepatectomy

Partial hepatectomy was performed on the reds of the experimental group and the control group of the first stage to remove about 70% of the liver, and a hepatic cancer lesion was applied to which a new cell cycle was applied.

Example 2 Confirmation of the Inhibition of Carcinogenesis of Cyclocreatine

Experimental Example 1: Visual observation of nodules on the surface of liver tissue

Visual inspection of the experimental group and the control group of Example 1 was performed. Necropsy revealed no significant liver surface nodule in the experimental group treated with cyclocreatine, which could be reverse detected.

Liver necropsy showed that the production of hepatic nodules produced during regeneration of the liver was significantly increased in the control group, but completely suppressed in the experimental group treated with cyclocreatin, and clear borderline and pale tissue damage were observed in the liver nodules in the control group. Tissue microscopic examination showed hepatic phenomena and various necrotic findings. Consistent with gross findings, hepatic nodule in the experimental group was significantly reduced compared to the control group. Therefore, it can be seen that the experimental group fed 1% cyclocreatine significantly inhibited the degree of liver damage significantly compared to the control group.

The weight change of the experimental group and the control group was measured (Table 1).

Changes in Body Weight of Experimental and Control Rats group process Number of rats Final weight (g) I DEN + 2-AAF 9 171.2 ± 6.8 Ⅱ DEN + 2-AAF + CCr 9 152.0 ± 11.8 Ⅲ 0.85% NaCl + BD 9 199.0 ± 5.8 DEN: Diethylnitrosamine 2-AFF 2-AcethylaminofluoreneCCR: Cyclocreatine BD: Basic dietary significance: P <0.05

It can be seen that the weight of the experimental group is reduced compared to the control group, which suggests that cyclocreatin administration inhibits body fat gain without damaging tissues, thereby reducing the weight, and suggesting the possibility of development as a therapeutic agent for obesity.

Experimental Example 2: Histopathological examination using GST-p staining, a carcinogenic marker

Histopathological examination of the experimental group and the control group of Example 1 was performed.

In order to detect the degree of staining of GST-p (Glutathione S-Transferase placental type), a precancerous lesion inspector, the liver of the experimental group and the control rats were immersed in 10% neutral formalin and fixed in paraffin and then embedded in paraffin. Hematoxylin & Eosin staining was performed to make 3-4 μm tissue sections, and the stained liver tissue sections were examined under an optical microscope. As a result of examining the degree of GST-p staining, the experimental group showed a significant reduction of GST-p positive hepatocellular lesions (Table 2, FIG. 3).

Size and number according to the positive response of GST-p, a pre-cancerous lesion of cyclocreatin group process GST-p positive hepatocellular lesion No./cm ² mm ² / cm ² I DEN + 2-AAF 31.28 ± 5.2 0.56 ± 0.07 Ⅱ DEN + 2-AAF + CCr 10.04 ± 6.0 0.17 ± 0.04 Ⅲ 0.85% NaCl + BD 0 0 Note: Numbers are mean and standard deviation (number of animals per group: 9) DEN: Diethylnitrosamine (CCr): cyclocreatine (BD): Basic dietary significance: P <0.05

Experimental Example 3 Identification of Cell Death by Tunneling Technique

In order to investigate the degree of cell death, a part of the liver of the test group and the control rats were immobilized in 10% neutral formalin and fixed. After normal tissue treatment, embedding was performed with paraffin, and 3-4 μm tissue fragments were made. after subjected to Proteinase K Evan response by a washing in distilled water and mixed with 3% hydrogen peroxide to remove endogenous peroxidase activity and then washed with a terminal deoxynucleotidyl transferase (TdT) buffer and at the same time in the buffer which contains the TdT enzyme 37 ^o C, After the reaction for 1 hour, the reaction was performed for 30 minutes at room temperature with peroxidase-attached streptoavidin and contrast staining with hematoxylin & Eosin was observed.

By using tunnel, a technique for detecting the degree of cell death, it was possible to detect a significant decrease in cell death in the experimental group (Table 3, Figure 4).

Degree of positive reaction of tunnel, apoptosis marker in experimental and control groups group process Individual number of the rat (n = 8) One 2 3 4 5 6 7 8 I DEN + 2-AAF 16.0 14.2 12.0 10.4 11.3 12.5 14.0 12.4 Ⅱ DEN + AAF + CCr 3.2 4.4 3.8 4.2 3.4 4.0 3.2 3.1 Ⅲ 0.85% NaCl + BD 2.4 2.1 1.7 2.6 1.8 2.0 1.8 2.4 Note: Degree of positive apoptosis: Average number of cells showing positive response observed on 100X optical microscope

Since the degree of cell death in the experimental group fed cyclocreatine was significantly reduced compared to the control group, it can be seen that cyclocreatin inhibits cell death. This indicates that the degree of cell death expression caused by DEN + 2-AAF in the carcinogenic model was markedly suppressed by cyclocreatin.

As evident from the above examples and experimental examples, the cyclocreatine of the carcinogenic inhibitory function of the present invention significantly reduces hepatic necrosis and the incidence of precancerous lesions caused by potent chemical carcinogens in vivo . Prophylactic and early stage treatment of liver cancer, as well as inhibiting the development of liver cancer, and the effect of being able to be added to the medicament in the treatment of already advanced liver cancer, and can also provide a glucose solution containing cyclocreatine directly in the form of the sap It is a very useful invention in biomedical industry, pharmaceutical manufacturing industry and health medicine because of its outstanding effect.

Claims (1)

A composition for preventing and treating hepatitis or liver cancer, comprising cyclocreatin as an active ingredient.