JPH10130153A - Anti-malignant tumor agent useful for cancer selected from colon cancer, esophagus cancer and breast cancer - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an anti-malignant tumor agent slight in adverse effect and excellent in efficacy. SOLUTION: This anti-malignant tumor agent comprises a cyclic and straight- chain mixed poly L-lactic acid having 3-19 degree of condensation as a main component which is a fraction obtained by dehydrating and condensing L-lactic acid in a nitrogen gas atmosphere by reduction in pressure and heating by stages to give a reaction solution, drying soluble components of the reaction solution with ethanol and methanol under reduced pressure, carrying out a reversed phase ODS column chromatography, eluting the adsorbed substance with 25-50% aqueous solution of acetonitrile at pH2.0 and collecting a fraction prepared by elution with 100% acetonitrile at pH2.0.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an anti-neoplastic agent used for cancer selected from colon cancer, esophagus cancer and breast cancer in animals including humans.

[0002]

2. Description of the Related Art Conventionally, surgery, chemotherapy and radiation therapy have been the three major therapies for malignant tumors, but at present, satisfactory results have not yet been achieved. In particular, chemotherapeutic agents that are used for a long period of time generally have extremely strong side effects as well as indefinite antitumor effects, and there are few drugs effective for solid cancers such as colon cancer and esophageal cancer. .

[0003] For this reason, various combination drug therapies have been attempted with the expectation of enhancing the antitumor effect. However, overlapping and aggravation of side effects, new obstacles and phytotoxicity, etc. have been brought about, resulting in a remarkable quality of life (QOL). ), The exhaustion of physical strength and shortening of the life due to the treatment have occurred to a considerable extent.

[0004] Various immunotherapies have been proposed as new therapies for malignant tumors, but they are in the research stage, and the early realization of effective immunotherapeutics with few side effects is expected.

[0005] As for the administration route, injection into blood vessels or local tumors is mainly used, and at present, there is almost no highly useful oral anti-neoplastic agent. Further, L-lactic acid is heated in an atmosphere of an inert gas such as nitrogen gas under normal pressure or reduced pressure, and the resulting reaction solution is dissolved in methanol or ethanol while hot, and filtered. The filtrate is dried under reduced pressure and then acetonitrile is dried. Or a solution directly dissolved in acetonitrile is chromatographed on a reverse-phase ODS or DS column which has been equilibrated with a 25% acetonitrile aqueous solution of pH 2-3 beforehand, and a 30-50% acetonitrile aqueous solution of pH 2-3 is prepared. Eluted with an aqueous solution having an acetonitrile concentration of 70% or more at pH 2-3 and having a degree of condensation of 5-2.
Japanese Patent Application Laid-Open No. 5-310581 discloses an agent for inhibiting the growth of malignant tumor cells in animals including humans, comprising a mixture of a linear condensate of L-lactic acid 3 and a cyclic condensate of L-lactic acid having a degree of condensation of 2 to 15. ing. This is for human cervical cancer cell line, human nasopharyngeal cancer cell line, artificial cavity floor cancer cell line, mouse lung cancer cell line, rabbit liver cancer cell line, Yoshida sarcoma, human gastric cancer, thyroid cancer, lung cancer and uterine cancer Met.

[0006]

The inventor of the present invention has condensed degree 3
As a main mechanism of action of inhibiting the glycolytic activity of poly-19 L-lactic acid, which is significantly increased in malignant tumors, it exhibits strong antitumor effects against colon cancer, esophageal cancer and breast cancer, and pain associated with malignant tumors It has been found to have remarkable physical condition-improving effects, including relaxation. It is the first antineoplastic agent that has a tumor growth inhibitory effect by interfering with the metabolic system of the tumor without serious side effects.

[0007] It is an object of the present invention to provide poly L-lactic acid having a condensation degree of 3 to 19, which has newly found a characteristic effect, as an antineoplastic agent for animals including humans. Things.

[0008] This is to create a new treatment method for overlooked extremely malignant tumors, for which there is no reliable treatment method, and that the cure rate can be further increased by using it in combination with existing anticancer therapies. It contributes to improvement.

[0009]

In order to achieve the above object, the antineoplastic agent of the present invention is obtained by subjecting L-lactic acid to dehydration-condensation in a nitrogen gas atmosphere by stepwise decompression and heating to obtain the reaction. After the ethanol and methanol soluble components of the liquid were dried under reduced pressure, reverse phase ODS column chromatography was performed, eluted with a 25 to 50% acetonitrile aqueous solution of pH 2.0, and then fractionated with 100% acetonitrile of pH 2.0. The present invention utilizes an antineoplastic agent which is mainly composed of mixed cyclic and linear poly-L-lactic acid having a degree of condensation of 3 to 19 and is used for cancer selected from colon cancer, esophageal cancer and breast cancer.

[0010] Poly-L-lactic acid is composed of cyclic and linear condensates, and a kind of reversible equilibrium is established between the two, and the diversity of poly-L-lactic acid biological activity and Even in view of the fact that the anti-tumor effect is expressed by the combination of fractions having different degrees of condensation, the significance of using cyclic and linear condensates separately from each other is not significant.

For practical use, the condensation degree should be 3 to 1
After separating and purifying the poly-L-lactic acid of 9 and neutralizing with alkali,
The material dried under reduced pressure is used as an original powder, which is aseptically dissolved or suspended in an appropriate solvent so as to have a predetermined concentration, and filled into a vial or the like to prepare an injection.

Oral preparations are prepared by mixing the raw powder treated in the same manner as described above with a suitable dispersing agent, base, excipient, etc. so as to have a predetermined concentration, and formulating into the form of powders, capsules, liquids and the like. I do.

[0013]

【Example】

Production Example 500 ml of L-lactic acid was put into a separable flask housed in a mantle heater, nitrogen gas was introduced and stirred at 300 ml / min, and the distilled water was led to a flask equipped with a reflux condenser through a warmed down-type connecting pipe. After heating at 145 ° C. for 3 hours, further reducing the pressure to 150 mmHg and heating for 3 hours, 3 mmHg at 155 ° C. for 3 hours, and finally 3 mmHg
At 185 ° C. for 1.5 hours to obtain poly L-lactic acid as a reaction product.

The obtained poly L-lactic acid is kept at 100 ° C.
After 100 ml of ethanol and 400 ml of methanol were respectively added dropwise, the mixture was allowed to cool, and this was further added to methanol 5.
After the mixture was stirred well and allowed to stand, the mixture was filtered and purified. The filtrate was dried under reduced pressure and dissolved in acetonitrile to make a total volume of 200 ml (stock solution).

[0015] The above stock solution is reverse-phase ODS
Column (TSK gel ODS-80TM)
Elution stepwise with 30%, 50% and 100% acetonitrile (pH 2.0) containing 0.01 M hydrochloric acid,
Poly L-lactic acid as a 100% eluted fraction (condensation degree 3-1
9) was obtained. FIG. 1 shows the results of mass spectrometry of this substance.

As is apparent from the regular fragment ion peaks in this figure, poly-L-lactic acid is mainly composed of a cyclic low condensate, and is mixed with a linear low condensate. It is assumed that the cyclic low-condensate has the following chemical structural formula.

[0017]

Embedded image

Preparation Example for Injection The poly (L-lactic acid) obtained in the above preparation example is neutralized with 1N sodium hydroxide, and dried under reduced pressure to a concentration of 100 mg / ml. After dissolving in glycol and sterilizing by filtration with a 0.45 μm filter, 15 ml is aseptically dispensed and filled into vials,
An injection was prepared.

Oral Formulation Example In the same manner as described above, the poly-L-lactic acid obtained in the production example was neutralized with 1N sodium hydroxide, dried under reduced pressure, and added to 1 g of sorbitol so as to contain 250 mg. An oral powder was prepared.

Toxicity Experiment 1 To confirm the safety of poly-L-lactic acid, male ICR mice were injected intravenously daily at 15, 30, and 60 mg / kg for two weeks. None of the treatment groups died during the treatment period, no changes in behavioral and physical conditions including motor coordination, defecation, and urination were observed, body weight was steady, and body weight gain during the treatment period was The low, medium and high dose groups ranged from 14.4 to 14.9 g versus 14.6 g for the group.

Toxicity Experiment 2 Assuming clinical application (intravenous infusion) by two people, 40 mg / kg of poly-L-lactic acid was intravenously infused daily (about 60 drops / min) to two dogs for 15 days. The safety was evaluated. There were no abnormal symptoms or death during the administration period, and physical conditions such as body temperature and heart rate were also normal. Hematology values are within normal limits, anemia,
No findings suggesting inflammation or impaired liver function and renal function were observed, and no pathological changes were confirmed in various organs and tissues. Table 1 shows the measurement results of the main items.

[Table 1]

Toxicity experiment 3 In order to confirm the safety by oral administration, ICR of male and female
Is the maximum dose of poly-L-lactic acid which is 200
A single oral dose of 0 mg / kg was observed for 2 weeks. No abnormal symptoms or death occurred after administration, and the lethal dose by oral administration was estimated to exceed 2000 mg / kg.
The body weight of the administered mice changed in the same manner as the vehicle control group, and no macroscopic lesion was observed at necropsy.

Immunostimulation and suppression of cancer metastasis B16 melanoma-transplanted mice were orally (500 mg / kg) or intravenously injected (10 mg / k) with poly-L-lactic acid for 7 days.
g), and the NK activity (cancer cytotoxicity of natural killer cells) was measured at the start and end of administration. As a result, in the control tumor-bearing mice, the NK activity was reduced to 30% of that at the start of administration, whereas in the oral and intravenous administration groups of poly-L-lactic acid, no decrease was observed in the NK activity, and the tumor growth was caused by cancer growth. It was confirmed that the decrease in immune ability was suppressed. Accordingly, the number of cancer colonies that metastasized to the lungs also decreased compared to the control group.

Analgesic Experiment Poly L-lactic acid was orally administered twice (500 mg / kg × 2) or subcutaneously (10 times) to mice injected with acetic acid intraperitoneally.
mg / kg) and pain induced by acetic acid (agonism)
The number of reactions was measured. Pain (writhing) due to administration of poly-L-lactic acid
The number of reactions decreased, especially in the subcutaneous injection group, to 52% of that in the control group, and a marked analgesic effect was observed.

Cancer Prevention Experiment Mice deficient in the tumor suppressor gene (p53) by genetic recombination technique were given 50 mg / kg of poly-L-lactic acid three times a week.
Oral administration was performed for 20 weeks to evaluate cancer incidence and mortality. In the control group, cancer death began to occur from the 8th week, and then gradually increased, and after 20 weeks, 1 out of 10 cases (10
%), Whereas the survival rate after 20 weeks was 50% in the group administered with poly-L-lactic acid, indicating that the group had the effect of preventing cancer development and prolonging life.

Influence of cancer cells on energy metabolism Since actively growing tumor cells rely on an anaerobic glycolysis system to maintain a large amount of energy supply, their key enzymes, pyruvate kinase and lactate, are The effect of poly L-lactic acid on dehydrogenase was examined. Poly-L-lactic acid markedly inhibits pyruvate kinase and lactate dehydrogenase activities of FM3A ascites tumor mouse supernatant derived from mouse breast cancer in vitro, and the 50% activity inhibitory concentration is 4 mg / ml for pyruvate kinase. 1. In the case of lactate dehydrogenase
It was 5 mg / ml. In particular, it was found that the lactate dehydrogenase activity of FM3A ascites cancer cells was more strongly inhibited than that of normal rabbit muscle. On the other hand, in the measurement of the anaerobic glycolysis, the amount of lactic acid produced was reduced to 50% at a concentration of 8 mg / ml of poly L-lactic acid. Effect of poly L on pyruvate kinase and lactate dehydrogenase activities and anaerobic glycolysis
-The effect of lactic acid is shown in Figures 2, 3 and 4.

It was also supported by cell morphology that the inhibition of the glycolysis system could cause a decrease in the function of cancer cells or suppression of growth. That is, in vitro (culture)
When poly L-lactic acid is applied to FM3A ascites cancer cells in vivo and in vivo (mouse intraperitoneal transplantation), degenerative findings such as loss of cytoplasmic processes of cancer cells, remarkable swelling of nuclei and reduction of chromatin, formation of vacuoles in cytoplasm, etc. Or findings suggesting apoptosis. Furthermore, the general condition was good in mice intraperitoneally injected with 4 mg / animal of poly L-lactic acid every other day after intraperitoneal transplantation of FM3A ascites cancer cells, and the survival time of control mice was about twice as long (14 to 16 days). A life extension effect of up to 2.5 times was observed.

Cultured human colon cancer DLD1 and human gastric cancer AZ
When 521 cells were cultured by adding poly-L-lactic acid, the MTT activity after exposure for 72 hours was 47 to 48% of the control at a concentration of 1.9 mg / ml, and the MTT activity was 7.5 to 7% at a concentration of 7.5 mg / ml. It was reduced to 12%, and a clear growth inhibitory activity corresponding to the glycolytic inhibitory effect was confirmed. Table 2 shows the effect of poly-L-lactic acid on cultured human cancer cells (MTT activity).

[Table 2]

Clinical treatment by intravenous drip Infusion and consent of patients, their families, etc. to about 50 severe patients with inoperable terminal cancer, postoperative residual cancer, metastatic cancer, recurrent cancer Based on the poly-L-
Anticancer therapy was performed by intravenous infusion of lactic acid injection. In the standard therapy, the intravenous drip infusion of 2000 mg / person / day of poly L-lactic acid was set as one course for 20 days.
ml / day was mixed and dissolved in 500 ml of an infusion solution (glucose solution, electrolyte solution, xylitol) and applied.

Among the various primary cancers treated with the poly-L-lactic acid injection, the most remarkable growth inhibitory effects were observed on colon cancer, esophageal cancer and breast cancer. Metastatic cancer was also suppressed. In particular, anticancer effects are remarkable for residual cancer seen in cancer patients (approximately 14) immediately after treatment by radiation therapy or surgical resection, and improvement is observed in 70 to 80% of patients. Of these, eight were judged to have almost cured clinically. Furthermore, this injection has not only an obvious antitumor effect, but also a marked improvement in nutrition and anemia, recovery from subjective symptoms such as malaise, a decrease in white blood cell count and side effects of radiation therapy and administration of anticancer drugs, and liver function. The early recovery of the disorder is a remarkable change, suggesting that a high anticancer effect can be expected in combination with existing therapies.

Substantially no side effects of poly-L-lactic acid were observed. Intravenous infusion was performed at 2000 mg × 2 / day, which is twice the standard dose, and continuously for a long period of time (about 3 times).
No special abnormalities occurred even when intravenous drip infusion was performed, indicating that the safety is extremely high. As a cancer patient-specific change that does not occur in healthy humans, fever may occur transiently, especially at the time of the first intravenous drip, but it is mild, and 100% of succisone (dehydrocortisone succinate) is used for the purpose of eliminating patient anxiety. Only 200 mg / time was used in combination.

Case 1: Colorectal cancer A 60-year-old female who had transverse colon cancer with invasion and adhesion to the pancreas, was subjected to surgical resection and bypass surgery, but was not completely resected and residual cancer was seen. Males were given an intravenous infusion of this injection over the standard therapy for about two years. As a result, suppression of cancer growth was confirmed by X-rays and ultrasonic echoes, and a clear inhibitory effect on metastasis and recurrence of cancer was recognized. Further, as a characteristic change by this injection, about 3 to 10 days after the start of treatment, reduction of physical symptoms such as pain, malaise, easy fatigue, nausea, recovery of appetite and improvement of nutritional status by weight gain are achieved. He was recognized, and his spirit recovered, and he was released from psychological anxiety and mental devastation, and his life activity was improved.

Case 2: Lung metastasis of colorectal cancer A 60-year-old man who was found to have extensive metastasis to the lung after surgical resection of colorectal cancer and was judged atelectasis-like to have a life expectancy of 3 to 4 months. Was treated similarly by intravenous infusion. Although the lung shadow remained, the pulmonary function was extremely good throughout the treatment period of about 3 years, and there was no finding of enlargement of the cancer lesion or metastasis to other organs, and a remarkable survival effect was observed. Also in this example, the appetite and nutritional state were good, without any physical symptoms such as pain, and the life activity was almost normal.

Case 3: Esophageal cancer A 50-year-old man who underwent surgical resection including total gastrectomy for esophageal cancer. Hemesis occurred 17 months later, complaining of anorexia and dysphagia, and endoscopy. A similar intravenous drip was administered to the patient in whom recurrent esophageal cancer (about 6 cm) and stenosis were found in the anastomosis of the surgical operation. After 10 days from the start of the treatment, the patient became able to ingest, and endoscopically confirmed that the cancer had been reduced and the gastrointestinal tract had been communicated, and that a good state was maintained in subsequent follow-up.

Case 4: Breast cancer Three years after surgery for breast cancer, complained of pain in the hip and ribs, and a 42-year-old woman in whom metastatic cancer was found on a bone scintigram was intravenously infused with the present injection. Treated. This patient is 4 after the discovery of cancer
She has been treated with anticancer drugs for months, and at the start of treatment, she has decreased white blood cell count (2,900) and has anemia (red blood cell count 3.84 million, hemoglobin level 10.7 g / d)
1) and liver damage (GPT value: 102 U) were observed. On the 4th day after the start of intravenous infusion, the white blood cell count recovered (8,100),
There was anemia and improvement of liver function (GPT value: 21 U), and on the seventh day, subjective symptoms such as malaise, chest pain and bone pain disappeared.
One course of intravenous infusion was completed, and two years later, there was no subjective symptom, and suppression or cessation of metastatic cancer growth was confirmed. The effects of the present injections tend to be relatively high for gynecological cancers, and there are relatively many cases of long-term survival or significant effects. In general, breast cancer patients complained of local discomfort during intravenous infusion, suggesting a direct effect on cancer, and local cancer therapy such as hepatic arterial infusion is one of the useful routes of this injection. Something was supported.

As expected from the metabolic and physiological actions of short-chain fatty acids such as lactic acid, the effect of improving metabolic abnormalities of sugar and lipid was also suggested, and findings such as diabetes and hyperlipidemia associated with this clinical case. Ie, elevated serum and urine sugars,
Serum cholesterol and triglyceride levels returned to normal. These were considered to be beneficial for the improvement of malignant tumors, in addition to the liver function improving effect of this injection.
In addition, as a result of attempting to use the intravenous drip and the oral powder prepared in Formulation Example 2 in combination, it was found that this injection was useful for maintaining and enhancing the effect.

Example of Clinical Treatment by Oral Administration A powdered oral preparation prepared in Formulation Example 2 was prescribed to a female patient whose liver metastasis was found by extirpation of breast cancer and was declared inoperable to have a life expectancy of 3 months. Daily intake (about 2400 mg / day as poly L-lactic acid) is being continued. Inspection after 7 months of ingestion has shown improvement in liver function and improvement in physical condition for completing work, and has clearly confirmed cancer growth suppression and life extension effects.

[0038]

Industrial Applicability According to the present invention, poly-L-lactic acid having a condensation degree of 3 to 19 exhibits particularly excellent growth suppression for colon cancer, esophagus cancer and breast cancer, and also inhibits metastasis and recurrence of these cancers. It has been clarified that it has a remarkable effect on improving physical condition, and indicates that intravenous infusion of the substance added to an infusion or the like can be an effective treatment for these malignant tumors. Since the poly-L-lactic acid is a low-condensation product of L-lactic acid derived from a biological component, the bio-compatibility is extremely high, and there are no side effects even under the most severe intravascular injection, and the subcutaneous or oral route. However, it is a major feature that the antitumor effect is recognized. From such characteristics, it can be applied to each stage from the early stage to the end of the disease stage and in the condition of a patient who is difficult to treat. It is possible to reduce or eliminate the side effects of existing anticancer therapies such as reduction and liver dysfunction. Furthermore, it makes safe and easy long-term continuous ingestion, and can be used for prevention of carcinogenesis experimentally suggested. It has been demonstrated that the antitumor effect of poly-L-lactic acid is related to an immunostimulatory effect and an improving effect on liver function and digestive function mainly on glycolipid metabolism. Furthermore, cancers with a high growth rate, unlike normal cells, have extremely high metabolic activity or energy requirements, and strongly depend on glycolysis to maintain them. It has been found that suppressing the anaerobic glycolysis can exert a cancer growth inhibitory effect. In particular, it strongly inhibits the activity of lactate dehydrogenase derived from cancer cells, suggesting that poly-L-lactic acid may be one of the main functions. These effects are different from those of existing antineoplastic drugs that show strong cytotoxic effects, and that poly-L-lactic acid contributes to a relatively broad anticancer spectrum without severe side effects. Inferred. Clinical experience shows that it has an excellent improvement effect on malnutrition including anorexia and weight loss, anemia, pain, malaise, and other physical symptoms, as well as reduced activities of life requiring mental and physical assistance. There was found. In particular, malignant tumors cause a decrease in immunity and physical strength, and have an effect of remarkably ameliorating anorexia or elimination, which is a crucial factor in death, and are cited as an important effect of the present drug. It is also of great significance in the treatment of intractable diseases, including palliative therapy for cachexia and anti-AIDS therapy associated with chronic diseases. Thus, poly L
-Lactic acid not only has an anticancer effect but also has an effect of improving the accompanying symptoms of malignant tumors, indicating that it is an effective means for improving the quality of life (QOL) of patients,
Overall, it can be said that it surpasses existing anti-cancer drugs.

[Brief description of the drawings]

FIG. 1 is a mass spectrum diagram of poly-L-lactic acid obtained in a production example of the present invention.

FIG. 2 shows the effect of poly-L-lactic acid on pyruvate kinase activity derived from FM3A ascites cancer cells (○) and pyruvate kinase activity derived from normal rabbit muscle (□). The vertical axis shows the activity when poly L-lactic acid is not present as 100%, and the activity when poly L-lactic acid at the concentration shown in the horizontal axis is shown as a relative activity.

FIG. 3 shows the effect of poly-L-lactic acid on lactate dehydrogenase activity derived from FM3A ascites cancer cells (細胞) and lactate dehydrogenase activity derived from normal rabbit muscle (□). The vertical axis shows the activity when poly L-lactic acid is not present as 100%, and the activity when poly L-lactic acid at the concentration shown in the horizontal axis is shown as a relative activity.

FIG. 4. Effect of poly-L-lactic acid on anaerobic glycolysis in FM3A ascites carcinoma cell supernatant. The amount of lactic acid produced in the absence of poly L-lactic acid was 10
The amount of lactic acid produced from glucose by FM3A ascites tumor cell supernatant was measured as 0%.

 ──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Shigeo Takada 517-12 Higashi Tomioka, Isehara-shi, Kanagawa Prefecture (72) Inventor Kiyotaka Sato 894-92 Kokura, Kiyama-cho, Miyoki-gun, Saga

Claims (1)

[Claims] 1. L-lactic acid is dehydrated and condensed in a nitrogen gas atmosphere by stepwise decompression and temperature elevation, and the ethanol and methanol-soluble components of the obtained reaction solution are dried under reduced pressure, followed by reverse phase ODS column chromatography. After elution with a 25 to 50% acetonitrile aqueous solution of pH 2.0, the mixture was adjusted to pH 2.0.
0 is a fraction eluted with 100% acetonitrile, and is mainly composed of cyclic and linear mixed poly L-lactic acid having a degree of condensation of 3 to 19, and is used for cancer selected from colon cancer, esophageal cancer and breast cancer. Tumor agents.