JPH06336427A - Malignant tumor cell proliferation inhibitor for animal including human - Google Patents

Abstract

PURPOSE:To obtain a malignant tumor cell proliferation inhibitor for animals including human, having slight side effect and excellent pharmacological effect. CONSTITUTION:This malignant tumor cell proliferation inhibitor comprises a mixture of a straight-chain condensate having 5-23 condensation degree and a cyclic condensate having 2-15 condensation degree which are a fraction obtained by heating L-lactic acid under normal pressure or reduced pressure in an atmosphere of an inert gas such as nitrogen gas, dissolving the prepared reaction solution in methanol or ethanol while being held hot, allowing to stand body cooling, filtering, drying the filtrate under reduced pressure and dissolving the dried substance in acetonitrile or directly dissolving the filtrate in acetonitrile to give a solution, equilibrating the solution with 25% acetonitrile aqueous solution at pH2-3, subjecting the solution to column chromatography by reversed-phase ODS or DS column, eluting with 30-50% acetonitrile aqueous solution at pH2-3 and eluting with >=70% acetonitrile aqueous solution at pH2-3.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to an agent for suppressing the growth of malignant tumor cells in animals including humans.

[0002]

2. Description of the Related Art Up to now, a malignant tumor cell growth inhibitor for animals including various humans and a method for producing the same have been proposed.
Many of them are produced by chemical synthesis methods or manufacturing methods that utilize ecosystems, and many of them have strong side effects or have low productivity and cannot be put to practical use. The current situation is that no malignant tumor cell growth inhibitor has been proposed.

For example, an agent for suppressing malignant tumors of animals including humans and a method for producing the same have been proposed in JP-A-59-33223 and JP-A-60-28930.

[0004]

SUMMARY OF THE INVENTION In view of these circumstances,
The present inventor has conducted extensive studies on a malignant tumor cell growth inhibitor for animals including humans, which has few side effects and has an excellent pharmacological effect. As a result, L-lactic acid low condensation product suppresses growth of malignant tumor cells for animals including humans. It was discovered that there is a drug action.

Based on this finding, it is an object of the present invention to provide a malignant tumor cell growth inhibitor for animals, including humans, which has few side effects and excellent pharmacological effects.

[0006]

According to the present invention for achieving the above object, L-lactic acid is heated under atmospheric pressure or reduced pressure in an atmosphere of an inert gas such as nitrogen gas, and the resulting reaction solution is treated with methanol or Dissolve in ethanol while hot, then cool and filter, and dry the filtrate under reduced pressure and dissolve in acetonitrile, or directly dissolve in acetonitrile and equilibrate the solution with a 25% acetonitrile aqueous solution having a pH of 2.0 to 3.0. Reversed phase Column chromatography using a system ODS or DS column
After elution with 30-50% acetonitrile aqueous solution of ~ 3,
Fractions eluted with an aqueous solution of H2-3 having a concentration of acetonitrile of 70% or more, L-lactic acid linear condensate having a condensation degree n of 5 to 23 and L-lactic acid having a condensation degree of 2 to 15 A malignant tumor cell growth inhibitor for animals including humans, which is characterized by comprising a mixture with a cyclic condensate.

The heating is carried out at an arbitrary temperature of 120 ° C. or higher and 200 ° C. or lower, or the temperature of the reaction system is kept constant and the pressure of the reaction system is successively reduced to obtain L-lactic acid low condensate as a reaction solution.
Next, this L-lactic acid low condensate is dissolved and suspended in methanol, equilibrated in an atmosphere of constant temperature, and allowed to cool.

In the reversed phase ODS or DS column, stepwise elution is performed by sequentially increasing the concentration of acetonitrile.

In the above-mentioned production method, the lactic acid low-condensate is heated at a low vapor pressure of L-lactic acid monomer, for example, at a temperature of 145 ° C. under normal pressure to remove coexisting water.
Reduce the pressure to 0-200 mmHg, then 150-160 ° C,
Keep at 10 ~ 20mmHg and finally 180 ~ 200 ℃, 3
~ 2mmHg within 2 hours to remove residual monomer and other low boiling point substances, 160 ~ 170 ℃, 100 ~
It was produced by heating under the condition of 200 mmHg, and the amount of residual monomer was small due to the upper limit cut with methanol 2,3,4,5,6, ... 18,19,20,2
It becomes a low condensate having a continuous degree of condensation with 1, 22, 23.

The mass analysis results of the thus obtained fraction of the L-lactic acid condensate of the present invention are shown in FIGS.
The analysis results of the 13 fractions are shown in FIG. As is clear from the figure, the lactic acid low condensate is in a state in which a linear condensate and a cyclic low condensate are mixed.

In the figure, Δ indicates a cyclic condensate of lactic acid, and other numerical values indicate a linear condensate of lactic acid.

In the above production, the degree of condensation of the low-condensation product of L-lactic acid can be easily controlled by appropriately adjusting the amount of water coexisting during the condensation reaction and the reaction temperature.

Generally, L-lactic acid (α-hydroxypropionic acid) is a liquid at room temperature and usually exists in a state where two molecules are hydrogen-bonded, and in its concentrated solution, 1-acticanh is present.
It is said that hydride (condensed with two molecules) is contained in an amount of 10 to 15%, and is easily dehydrated and condensed by heating, converted into a low condensate, and further easily polymerized and solidified.

The above-described agent for suppressing the growth of malignant tumor cells of animals including humans of the present invention (hereinafter referred to as an inhibitor) is produced by utilizing the above-mentioned properties of lactic acid, and converts lactic acid into a low-condensation product,
It is an agent obtained by fractionating it by column chromatography and collecting an active fraction having an inhibitory action on malignant tumor cells in animals including humans.

The agent of the present invention is a mixture of a linear low-condensate and a cyclic low-condensate according to mass spectrometry, but according to the results of animal experiments with rats, the one having a degree of condensation of 5 to 23 is optimal. Is. Then, after the intravenous administration of the inhibitor, the drug effect appears about 5 days later. The linear low-condensate and cyclic low-condensate of the condensate cannot be completely separated from each other at present. From the analytical point of view, a cyclic condensate having a condensation degree n of 4 or more (lactide) and a linear condensate can be roughly divided, but a lactide having a condensation degree n of 2 or 3 is a linear condensate. It cannot be separated from where it behaves. This suggests that not only the affinity is strong, but also a kind of reversible equilibrium relationship is established in the confined system.

The low-condensation product of lactic acid has strong adhesiveness and easily aggregates. Even if the solution seems to be transparent at first glance, it behaves as if it was not a single separation. This low condensate has a surface active action at a low concentration, and micelles or reverse micelles of a lactic acid low condensate are formed in an aqueous solution. It can be presumed that this acts as an inhibitor and facilitates passage of malignant tumor cells through the cell membrane, which is the first barrier layer, and enhances the pharmacological effect.

[0017]

Example 1 Production Example 1 500 ml of L-lactic acid was placed at a normal pressure in a separable flask equipped with a descending connecting pipe and a nitrogen gas introducing pipe, kept at 145 ° C. for 3 hours with a mantle heater, and then depressurized to 150 mmHg. For 2 hours, then 155 ℃, 10mmHg
After heating for 2 hours at 185 ° C., it was heated at 185 ° C. for 1.5 hours to obtain the desired low-condensate.

This low-condensation product was dispersed in twice the amount of methanol while still having fluidity, and the filtrate obtained by filtration was dried under reduced pressure to obtain an L-lactic acid low-condensation mixture with the upper limit cut. This mixture was dissolved in acetonitrile, and the reverse-phase ODS column (Chemco LC-sorbS) was previously equilibrated with 25% hydrochloric acid acidic solution (PH = 2.0) of acetonitrile.
P-C-ODS column), stepwise elution was carried out in order with 25%, 50%, 100% hydrochloric acid acidic solution of acetonitrile of PH2.0, and the elution fraction was neutralized and replaced with ethanol several times. It was dried under reduced pressure and dissolved in propylene glycol to obtain inhibitors 1, 2 and 3, respectively.

[0019]

Example 2 Production Example 2 L-lactic acid was stirred for 5 hours while introducing L-lactic acid at 200 mmHg at 160 ° C. in a nitrogen gas atmosphere at a descending connecting pipe of 200 to 300 ml / min. The obtained low condensate was dissolved in methanol, the filtrate was dried under reduced pressure, and then dissolved in acetonitrile. This is applied to a reverse phase ODS column equilibrated with a 25% acetonitrile hydrochloric acid acidic solution (PH = 2.0), and three-step stepwise elution is performed with 25%, 40%, 80% acetonitrile acidic hydrochloric acid solution. In the same manner as in Experiment 1, the elution fractions were sequentially added to inhibitors 1 ', 2', 3 '
And

[0020]

Example 3 Acute Toxicity Test 1 Male mouse was intravenously injected with inhibitor 2 obtained in Production Example 1,
The weight change was observed for one week. The results are shown in Table 1.

[0021]

[Table 1]

[0022]

Example 4 Acute Toxicity Test 2 (ii) Inhibitor 3 was administered to rabbit arteries to give 1, 4, 7
Table 2 shows the results of weighing the body weight on the day. The results of administration of adreamycin are also shown as comparative examples.

[0023]

[Table 2]

[0024]

Example 5 Acute Toxicity Experiment 3 (1) The inhibitor 2 was administered to the blood vessels in the tail of 10 mice C57Black (8 weeks old) 10 times once a day for 10 consecutive times. As a result of observing 10 days later, there was no change in appearance and the mortality rate was 0. Dose: 50mg / ml 0.2ml solution (400m
g / kg) (2) Inhibitor 2 was administered subcutaneously to the dorsal region of 10 female 4-week-old nude mice once a day. After 10 days, the mortality rate was 0. Dose: 50mg / 0.5ml H ₂ O (about 2.5g
(Equivalent to kg / kg) (3) Beagle female 6 months old, weight 7 kg, inhibitor 3
As a result of continuous intravenous injection once daily, and observing for 10 days, no special abnormal reaction was observed in the external appearance. (No clinical change). Dose: 0.7g (100mg / kg)

[0025]

Example 6 Malignant Tumor Cell Proliferation Inhibition Test 1 Nude Mouse ICR NU / NU Female 4-week-old subcutaneously on the dorsal part of the back, Hela C as human malignant tumor cells (cell line).
ell (human cervical cancer cell line) and KB (human nasopharyngeal cancer cell line) were transplanted, and from the third day after transplantation, the inhibitor 1 was continuously administered to the experimental group once a day for a total of 11 times. Similarly, physiological saline was administered to the control group. After that, the administration was stopped, and the tumor was taken out and weighed 7 weeks after the transplantation. However, the suppression rate is given by the following equation. Inhibition rate = (1-tumor weight g of experimental group / tumor weight g of control group) × 100% The results are shown in Tables 3 to 4.

(1) Hela Cell (human cervical cancer cell line) Number of transplants: 1 × 10 ⁷ Administration method: Subcutaneous administration (sc) Dose: 30 mg 0.3 ml (from day 3) Results: Control group and The tumor weights of 5 cases in each of the experimental groups are shown below.

[0027]

[Table 3] (1) KB (human oral floor cancer cell line) Number of transplants: 1 × 10 ⁷ dose: 20 mg / 0.2 ml Results: The tumor weights of 5 cases in both the control group and the experimental group are shown below.

[0028]

[Table 4]

[0029]

Example 7 Malignant Tumor Cell Proliferation Inhibition Test 2 Mouse Lung Cancer Cell Mouse C57Blac male (8 weeks old) was transplanted (SC) with 1.0 × 10 ⁶ LLC (mouse lung cancer cell), and the same as in Example 5. By the method, it was administered 11 times from the day after the transplantation. The results are shown in Table 5.

[0030]

[Table 5]

[0031]

Example 8 Malignant Tumor Cell Suppression Test 3 Yoshida Sarcoma 2.2 m after formation of a tumor by transplanting Yoshida sarcoma into a rat
g / kg, 10 mmg / kg was administered intravenously continuously for 7 days.
Table 6 shows the results of measuring the tumor size (mm) on a daily basis.
The same amount of physiological saline was administered to the control group. The results using adriamycin as a positive control drug are also shown.

[0032]

[Table 6]

[0033]

Example 9 Malignant Tumor Cell Suppression Test 4 Rabbit Hepatoma-Derived Cell Line VX2 Rabbit Hepatoma-Derived Cell Line VX2 was Transplanted into Rabbit Liver,
Two weeks later, a tumor having a size of 10 × 10 mm to 20 × 20 mm was selected, and 7 days after the administration of adreamycin to the inhibitors 2, 3 and the control as an artery, the tumor was taken out and observed.

[0034]

[Table 7] As described above, the size of the cancer = the major axis of the cancer × the minor axis is barely significant in the mouse, but a remarkable pharmacological effect appeared in the larger animals such as rat and rabbit.

[0035]

Example 10 Clinical Example Method: Propylene glycol 1 with 100 mg of inhibitor 3
A solution (hereinafter referred to as inhibitor 3 solution) dissolved in ml was prepared, and 30 mg of inhibitor per 1 kg of body weight (inhibitor about 0.3
The amount (ml equivalent) was added to an intravenous drip solution such as a vitamin preparation and glucose and mixed, followed by intravenous drip infusion. Administration once a day 5 times 3 after administration
The day was discontinued, and administration was repeated 5 times daily from the 9th day, and thereafter the patient's condition was observed over time.

Example 1: Gastric cancer A patient (60-year-old man) with a hen egg-sized tumor and a bleeding was mixed with 15 ml of the inhibitor 3 solution in 500 ml of glucose drip solution and administered by the above method. The medicinal effect appears,
He stopped bleeding, had an appetite and recovered his physical condition. Furthermore,
A radiographic examination followed the drug's efficacy and the tumor was found to shrink. This was also confirmed by a gastroscope.

Example 2: Thyroid cancer To a patient (50-year-old man) with an infiltrating tumor with hypertrophy and blood infiltration and lymph node transfer, 15 ml of the inhibitor 3 solution was mixed with 500 ml of glucose drip solution, and intravenous drip was performed in the same manner. I made a note. From the 6th day after administration, the moistening of blood and the like stopped, and the enlarged tumors and lymph nodes decreased with the passage of time. At the same time, he recovered his physical strength and showed sufficient significance for drug efficacy.

Example 3: Lung cancer A patient (55 year old) who also had a bronchial cancer that had grown large was mixed with 15 ml of the inhibitor 3 solution in 500 ml of glucose drip solution,
It was administered in a similar manner. The effect appeared on the 5th day after administration,
Blood in the sputum disappeared, cancer cells were significantly reduced, and general condition was improved. Two months later, an X-ray examination showed that the tumor had shrunk and that the physical strength of the tumor had recovered significantly.

Example 4 Uterine Cancer To a cervical cancer patient with bleeding, 12 ml of the inhibitor 3 solution was mixed with 500 ml of glucose drip solution and administered in the same manner. 4-5 days after administration, bleeding stopped and improvement of symptoms was observed.
No bleeding after months. Two months later, a CT scan confirmed the reduction of the tumor mass. Two months later, X-ray examination showed that the tumor had shrunk, and at the same time, a marked recovery of physical strength was observed.

[0040]

EFFECTS OF THE INVENTION The present invention shows that, as is clear from Example 1, a suppressive effect begins to appear within a few days after administration, and malignant tumor cells do not grow after the administration of 10 times. Indicates the period validity. The inhibitor of the present invention is used for human cervical cancer, human oral floor cancer, mouse lung cancer, Yoshida sarcoma, rabbit liver cancer, human gastric cancer,
Although it is effective against thyroid cancer, lung cancer, uterine cancer, etc., the effect on the cell line VX2 cancer derived from rabbit liver cancer is particularly remarkable, and when the cancer cells were transplanted into the liver for 7 days, it was administered to rabbit arteries. Invasion and proliferation of hepatoma-derived cells VX2 were suppressed, and most of the cancer cells died. Further, as shown in Table 6, the effect on Yoshida's sarcoma does not show the pharmacological effect of adreamycin which is a positive control drug, but it has a sufficient cancer suppressing effect. Furthermore, it can be seen that the inhibitor of the present invention is an oligomer of L-lactic acid present in a living body and has no side effect on the living body as seen in Examples 3 and 4. Considering the comprehensive evaluation including these, it is considered that a higher evaluation than the above-mentioned adriamycin can be obtained.

[Brief description of drawings]

FIG. 1 is a mass spectrum diagram of an inhibitor 2 according to an example of the present invention.

FIG. 2 is a mass spectrum diagram of another inhibitor 3 according to an example of the present invention.

FIG. 3 is a mass spectrum diagram of condensation degree n = 13 collected by concentration gradient elution by ODS column chromatography (condensation degree n = 13 of a linear condensate to condensation degree n = 11 of a cyclic condensate). Existing.).

Claims (1)

[Claims] 1. L-lactic acid is heated under an atmospheric pressure or a reduced pressure in an atmosphere of an inert gas such as nitrogen gas, and the resulting reaction solution is dissolved in methanol or ethanol while hot and allowed to cool, followed by filtration and filtration. Is dried under reduced pressure and then dissolved in acetonitrile, or a solution obtained by directly dissolving it in PH 2.0 to 3.0
Column chromatography with a reverse-phase ODS or DS column equilibrated with 25% aqueous acetonitrile solution, and after eluting with 30-50% aqueous acetonitrile solution of PH2-3, 70% or more acetonitrile concentration of PH2-3 L which has a degree of condensation of 5 to 23
-A malignant tumor cell growth inhibitor for animals including humans, which comprises a mixture of a lactic acid linear condensate and an L-lactic acid cyclic condensate having a condensation degree of 2 to 15.