JPS5953881B2 - anticancer drug - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to N-(2-ethylhexyl)-3-hydroxybutyramide (putoctamidof) or its half ester with dicarboxylic acid (particularly succinic acid ester or its alkali metal salt, alkaline earth metal salt). ) Regarding newly regulated cancer drugs whose main ingredients are regulated cancer drugs, various anticancer drugs have been developed to date, and although many of them have shown effective effects in inhibiting the growth of malignant tumors, many of them have serious side effects. Currently, it is used in conjunction with surgery and radiotherapy as an adjunct.

However, many of these conventional anticancer drugs have serious side effects on blood-forming organs and digestive organs, and may cause leukopenia, loss of appetite, nausea, and vomiting, so they must be used with caution. It is necessary to conduct tests and be careful, and therefore there is a demand for the development of anticancer agents that have excellent anticancer effects and are free of side effects. The inventors have determined that N-(2
As a result of various studies on N-(2-ethylhexyl)-3-hydroxybutyramide and its derivatives, we found that N-(2-
It has been found that ethylhexyl)-3-hydroxybutyramide and its half ester with dicarboxylic acid have anticancer activity. Conventionally, N-(2-ethelhexyl J-3-
Hydroxyptyramide, its ester succinate, its calcium salt, etc., do not exhibit anesthetic effects, add an active para-sleep-inducing effect, accurately provide a natural sleep effect, and have a mental stabilizing effect without a muscle relaxing effect. I don't know how to give
7 (Special Publication No. 18095, Special Publication No. 18095, Special Publication No. 18095)
18529, Japanese Patent Publication No. 48-18580), it is not known at all that these substances have anticancer effects, and as a result of extensive research based on this knowledge, the present inventors found that
N-(2-ethylhexyl)-3-hydroxybutyramide and its half ester with dicarboxylic acid exhibit reliable anticancer effects even when used in relatively small amounts, have almost no side effects, and are extremely effective as anticancer agents. This discovery led to the completion of the present invention. That is,
The present invention uses N-(2-ethylhexyl)-8-hydroxybutyramide or its half-esnar with dicarboxylic acid as the main component, and provides excellent anticancer effects even when administered in a relatively small amount, as well as toxicity. It has few side effects even after long-term administration, has no side effects especially on hematopoietic organs and digestive organs, can be used effectively without causing leukopenia, loss of appetite, etc., and can be easily used in various preparations. The purpose of the present invention is to provide a newly regulated cancer drug that can be easily prepared and handled in terms of storage.

The present invention will be explained in detail below. The substance used as a main component in the anticancer agent according to the present invention is N-(2-ethylhexyl)-3-hydroxyptyramide (butoctamide, hereinafter referred to as L-2) represented by formula (1).

), or formula (2) (where R is CnH, n (n≧O
, preferably a lower alkylene group represented by 1 to 4), or a functional group in which a part of the hydrogen group in this alkylene group is substituted with an amino group, an oxygen group, etc., and M is a hydrogen group, an alkali metal group, Or represents an alkaline earth metal group.

) A half-esdel of L-2 and a dicarboxylic acid formed by an esterification bond between the hydroxyl group of L-2 and the carboxyl group of one of the dicarboxylic acids, such as L-2 and malonic acid, succinic acid, glutaric acid, Adipic acid, glutamic acid,
Esterified products of malic acid, a-ketoglutaric acid with one carboxyl group, and salts thereof, especially formula (3) (however, R
represents a hydrogen group, an alkali metal group, or an alkaline earth metal group.

N-(2-ethylhexyl)-3-hydroxybutyramide semisuccinate (hereinafter referred to as ・M-2H・
It is called. ). Calcium salt (hereinafter, M-2H-
It is called Ca. ) such as N-(2-ethylhexyl)-
3-hydroxyptyramide semisuccinates. Note that L-2 is, for example, 2-ethylhexy [■■
Obtained after reacting 2-ethylhexylamine with diketene.

It can also be produced by reacting 2-ethylhexylamine with acid chloride of β-acyloxybutyric acid or its lower alkyl ester, and then hydrolyzing the resulting product.

A half ester between L-2 and one carboxyl group of a dicarboxylic acid is a dicarboxylic acid such as oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid, glutamic acid, malic acid, or anhydride thereof. It can be obtained by reacting with For example, M-2H can be obtained by heating L-2 with succinic anhydride, and M-2H-Ca
is prepared from M-2H by conventional methods. In this case, L
M-2H-C, which is a kind of salt of succinic acid ester of -2
a is water-soluble, and when administered orally, its calcium is removed in the stomach and absorbed as water-soluble M-2H, and lower acid esters of L-2, such as M-2H and M
-2H.Ca is present in the form of L-2 as succinic acid is gradually removed in blood and cerebrospinal fluid and metabolized to L-2, and this L-2 acts on cancer cells.

Therefore, CL-2 present in blood and cerebrospinal fluid in the form of CL-2 can be used as an anticancer agent. Na trigram, L-2
While M-2H and M-2H-Ca themselves are oily substances that are poorly soluble in water and generally difficult to crystallize, both M-2H and M-2H-Ca are easily formed as crystals and are easy to obtain as pure substances.
Succinic acid is a biological component and is naturally metabolized as an intermediate in the TCA cycle, so it is preferable from a safety standpoint, and from a pharmaceutical standpoint, it is preferable to use M-2H, M-2H-Ca. Particularly when used as an injection solution, it is preferable to use M-2H-Na (sodium salt of N-(2-ethylhexyl)-8-hydroxyptyramide semisuccinate), which is easily soluble in water. Then,
L-2 and L metabolized to L-2 in blood and cerebrospinal fluid
-2 and half-esdel of dicarboxylic acid (M-2H, M-2
H-Ca, etc.) acts on cancerous cells in its own 7-2 form,
It exhibits an effective anticancer effect, but especially when epinephrine or norepinephrine is combined with L-2 or a half ester of L-2 and a dicarboxylic acid, the half ester of L-2 or L-2 and a dicarboxylic acid Esdel and epinephrine or norepinephrine act synergistically,
The above-mentioned combination group exhibits a very remarkable anticancer effect, and acts specifically on cancerous cells to inhibit their growth, and hardly acts on normal cells and hardly inhibits their growth. It is preferable to blend epinephrine or nor-Lpinephrine with L-2 or its half ester with a dicarboxylic acid.

Next, the anticancer effect of L-2 will be explained with reference to the following experimental examples.

[Experimental Example 1] Mouse cancerous mast cells (Mast? YtO Peng P-815
) to 120 m1 of Maier medium and 8.
Add 0ml and leave it in a carbon dioxide incubator,
Culture was carried out for 40 hours, and the effect of L-2 on cell proliferation was examined, and the results shown in Table 1 and FIG. 1 were obtained.

Note that L-2 was added to the medium at a final concentration of 45 μg/ml. From the results shown in Table 1 and FIG. 1, it was confirmed that L-2 had a fairly strong growth-inhibiting effect on cancerous mast cells.

In addition, in the system to which L-2 was added, the number of dead cells increased over time, and the effect of L-2 was strongly apparent after about 1 cell cycle (the cell cycle was about 15 As shown in FIG. 1, a peculiar point was observed in the L-2 system, in which the degree of inhibition against cells at 40 hours was stronger than the degree of inhibition against cells up to 20 hours. [Experimental Example 2] Mouse cancerous mast cells (MastO(YtOmaP-81
5) was inoculated with 307f11VC culture medium, cultured with shaking in a carbon dioxide incubator for 40 hours, and the effect of L-2 on cell proliferation was examined, and the results shown in Table 2 and Figure 2 were obtained.

}, L-2 was added to the medium at a rate of 25 μg nod.
From the results in Table 2 and Figure 2, does L-2 reliably act on cells up to one cycle and promote cell proliferation? It was found that cell proliferation could be reliably suppressed by adding L-2 to each cell cycle.

[Experiment Example 3] Mouse cancerous cells (Mas°C0cyt0maP-815)
was inoculated into 25 ml of culture medium at a ratio of 1.0 x 104/ml, VCL-2 was added in the same amount, and static culture was performed at 37°C for 60 hours in carbon dioxide/air (5:95). The effect of the amount of L-2 added on cell proliferation was investigated and the results shown in Table 3 and FIG. 3 were obtained.

Table 3: Results of the division time (Generation Ontime) Table 3: Figure 3 depicting the relationship between the L-2 amount and the number of cells (point A in the figure represents the number of cells at the start of culture.

) As is clearer, L-2, 50 μg/7f1I!
It was found that the addition of 20% almost certainly suppresses cell proliferation. [Experimental Example 4] Mouse oily mast cells (MastOcytOmaP-81
5) was inoculated into a medium at a rate of 2.08 x 104/d, L-2 was added in various amounts, and carbon dioxide/air (5:
95), the cells were statically cultured at 37° C. for 40 hours, and the effect of L-2 on cell proliferation was examined.

Furthermore, in addition to adding L-2 in various amounts, 1 μg/Tllll of epinephrine was added to each, and the effects of the epinephrine combination group were investigated. The results obtained are shown in Table 4 and Figure 4. Table 4 and Table 4 showing the relationship between L2 amount and cell number.
(In the figure, point A represents the number of cells at the start of culture, and L-2+Ep represents a combination group in which 1 μg/ml of Ep was mixed with L-2.

) As is clear, the addition of L-2 at 25 μg/d has a significant cell proliferation inhibiting effect, and by adding 1 μg/Mj of epinephrine to this, epinephrine itself has no inhibitory effect on cell proliferation. It was found that L-2 and epinephrine act synergistically to provide an excellent cell proliferation inhibiting effect, although the amount is almost negligible. [Experimental Example 5] Rat normal mast cells suspended in PH14 phosphate buffer
Add L-2, 2.8mM or L-2, 2.3mM with 270μM of epinephrine to peritOneal hidden Stll and mouse cancerous mast cells (MastOcytanaP-815), respectively, and shake gently at 37C. After culturing for 30 minutes, microscopic observation was performed.

The results are shown in the micrographs shown in Figures 5 to 10 (40 magnification).
times). FIG. 5 shows rat normal mast cells, and FIG. 6 shows the normal cells treated with L-2, showing almost no abnormality and no dislodgement. FIG. 7 shows a normal cell treated with L-2 and epinephrine, and although granule dislodgement is observed, the cell membrane is not destroyed. Figure 8 shows cancerous mast cells, and Figure 9 shows cancerous mast cells treated with L-2.The action of L-2 causes cells to swell even when cultured for only 30 minutes. This indicates that cancerous cells are in the process of destruction.

Furthermore, FIG. 10 shows cancerous cells treated with L-2 and epinephrine, and it is shown that the cancerous cells are being destroyed and disappear. Therefore, from the above results, L-2 definitely acts on cancerous cells, and shows particularly excellent effects when combined with epinephrine. However, it has been found that it has little effect on normal cells. [Experiment example
6] DdY mice (male, 7 weeks old, 5 to 8 mice per group) were subcutaneously inoculated with 8 x 105 Ehritzg ascites cancer cells/mouse, and L-2 was inoculated at the rate shown in Table 5 from 24 hours after cancer cell transplantation. It was administered intraperitoneally 7 times, and the mice were sacrificed on the 14th day after cancer implantation.
The weight of the cancer was measured and compared with the control group, and the anticancer effect was determined, and the results shown in Table 5 were obtained.

In addition, physiological saline was used as a control [Experimental example]
7] Mice (BDF, 7-week-old male and CDFl, 15-week-old male, 5 mice per group) VC. Mouse cancerous mast cells (Mast
Ocyt (RrlaP-815) 1X106/0.25
mj/mouse was implanted by intraperitoneal injection, and 24 hours after the cancer cells were transplanted, L-2 was intraperitoneally administered 6 times per day at a rate of 0.5η/mouse, and after transplantation. On the 8th day, mice were killed with Edel anesthesia and 9f. The cells were washed three times with 10 ml of phosphate buffered saline (PBS), and the number of cancer cells was determined by hemocytometer and compared with the control group/ζ.

The results are shown in Table 6. From the results in Tables 5 and 6, it was found that administration of L-2 effectively inhibited W ulcer. Next, L-2, M-2H, M-2H-Ca
The results of the toxicity test are shown below. [1] Acute toxicity (i) Acute toxicity of L-2 L-2 was suspended in gum Arapia solution and administered orally and intraperitoneally to DdY mice to test for acute toxicity.

Table 7 Acute toxicity test results for L-2 (by Litchfieid & WiicOxOn method)
(1;) Acute toxicity of M-2H, M-2H-Ca M-2
For H,M-2H-Ca, DdN mice (5-6
(8 weeks old, 10 rats per group) and Wista strain rats (8 weeks old,
Acute toxicity was tested using 10 animals per group).

The drug solution was administered as a suspension in 5% gum arabic solution in the case of M-2H, and as an aqueous solution in the case of M-2H-Ca. 8th
Table M-2} LM-2H-Caf) LD, O (normal ~) Results (by Lltchfield & YllcOxOn method) In addition, the histopathological finding C is that abnormal images that are thought to be drug-dependent are observed in all organs examined (stomach). , small intestine, liver, pancreas, brain, lungs,
It was not detected at all in the heart, tiles, mesenteric lymph nodes, thoracic line, pituitary gland, thyroid, paragluteal, buttocks, testis, or ovary).

[H] Subacute toxicity, chronic toxicity (;) Subacute toxicity of M-2H Wistar strain rats (5 weeks old, 110 to 120 g for males, 5 females for 90 to 100 g, 10 animals in 11 groups) were treated with 5 (suspended in L gum arabic). M-2H was administered orally and intraperitoneally for one month to test for acute toxicity.

〔result〕

A slight decrease in body weight was observed in females in the oral administration group of 600 η/Kg compared to the control group, and one case had disturbances in hepatocellular cytoplasm, and another case showed nuclear condensation in a part of the periphery of the hepatic lobule. Other changes were observed, including body weight gain rate, feed intake, blood (red blood cells, white blood cells, white blood cell percentage, blood cell volume, hemoglobin content), urine, organ appearance, wet weight changes of organs, histopathology. Findings (liver, buttocks, tiles, accessory buttocks, heart, fallopian tubes, pancreas, testes, other organs)
No abnormalities were observed, and no animals died during the above period.

The oral dose 3007!9/Kg and 150η/Kg groups showed similar results to the control group, and no abnormality was observed in each of the above test items.

In addition, the results of testing the intraperitoneal dose group of 80 ~/kg showed no abnormality in each of the above test items.

In all groups to which M-2H was applied, central depressant effects such as drowsiness and droopy eyelids were observed in parallel with the dose.

(1i) Chronic toxicity of M-2H 250 W/Kg (2 dogs) of M-2H, 10
07f1f/Kg (2 dogs), control (2 dogs) once daily,
A chronic toxicity test was conducted by orally administering the drug 6E3/week for 6 months.

M-2H was dissolved in linoleic acid and encapsulated in soft capsules 3, and a control group was administered soft capsules containing only linoleic acid. In addition, M-2H3001r9/Kg (3 dogs), 100η was applied to 6-month-old American male vehicle dogs.
/Kg (3 animals), 50mI! /Kg (3 animals), control (3
Chronic toxicity tests were conducted by orally administering the drug (head) once a day, 5 days a week for 1 year.

The specimen was filled into a hard capsule and used as a control, and silicic anhydride was used as an excipient. [Results] The results of the 6-month chronic test showed that the 250rf9/Kg group
Abnormalities were observed in some liver lobule in one case, and hemorrhage in the nodules was observed in one case and one case in the control group, but there were no other abnormalities, and in all other cases. are weight gain, feed intake, blood (red blood cells, white blood cells, hemoglobin, blood cell volume, white blood cell percentage) transaminase activity (GOT,
GPT), alkaline phosphatase activity, organ appearance, changes in organ wet weight, and histopathological findings were all normal.

In addition, in a 1-year chronic study, all cases, including the control group, were found to have blood stasis just below the capsule of the tile, and one case each in the 800~/Kg group and the control group had mild cell carcinoma in the liver. Infiltration was observed, and in one case in the 100η/K9 group, infiltration of small round cells in the pancreas and abnormal spermatogenesis were observed.

However, there were no other abnormalities, and in all cases, there were no abnormalities in weight gain rate, blood tests, blood tests, and urine tests, including the stomach, small intestine, large intestine, brain, lungs, heart, and mesentery. No abnormalities were observed in the lymph nodes, thoracic line, pituitary gland, thyroid, paragluteal, buttock, bladder, prostate, or bone marrow. Although a sleep effect was observed in the M-2H administration group, no unsteadiness on all four legs was observed after awakening, and no withdrawal phenomenon was observed after completion of administration.

[] M-2H-Caf) Acute toxicity and chronic toxicity Wistar strain rats (male 180 ± 20 g, female 140 ± 20 g
, 10 or 15 animals per group) were given 3 doses of M-2H-Ca aqueous solution.
Toxicity was tested by oral and intraperitoneal administration for 1 month (subacute) and 6 months (chronic).

Note that physiological saline was used as a control. [Results] Subacute toxicity: oral 400W9/Kg administration group, intraperitoneal 1
00~/I<g administration group showed almost the same growth as the control group, with no changes in body weight suppression, blood, liver function, or urinalysis.

Chronic toxicity: Both the oral 400 η/Kg administration group and the intraperitoneal 50 η/Kg administration group showed almost the same growth as the control group, and no significant changes were observed in red blood cell count, white blood cell count, liver function, or urinalysis. .

As is clear from the above-mentioned cancer-fighting effect experiments and toxicity test results, L-2 and esters of L-2 with dicarboxylic acids (M -2H,M-
2H-Ca, etc.) can be administered by intravenous injection, subcutaneous injection, oral administration,
It is administered by methods such as rectal administration using suppositories.

The dosage varies depending on the administration route and number of administrations, but L
In the case of M-2, the dosage for adults is preferably 100 to 1,500 watts, for example 400η, and in the case of M-2H, the dosage for adults is preferably 200 to 2,000 watts, for example 6001r9, within the above dosage range. It reliably exhibits anticancer effects without causing any side effects. In addition, when blending epinephrine or norepinephrine, the blending amount is less than V3η per one use, preferably 0.5 to 1
It is ~.

The preparation of L-2 and its half ester with a dicarboxylic acid into a formulation is carried out by conventional methods. Preparations for oral administration are provided for use in a conventional manner using any required pharmaceutical carriers or excipients. The formulation for oral administration is desirably provided in a form suitable for absorption from the gastrointestinal tract. Tablets and capsules (hard capsules, soft capsules) for oral administration are in unit dosage form and contain binders such as gum arabic, gelatin, zolpit, tragacanth, polyvinylpyrrolidone, lactose, sugar, corn starch, silicic anhydride, and avicel. ,
Using prescribed ingredients such as excipients such as linoleic acid and propylene glycol, lubricants such as magnesium sudeate and talc, disintegrants such as horse starch, and wetting agents such as lecithin, L-2 or its A preparation for oral administration containing a half ester with a dicarboxylic acid and, if necessary, epinephrine or norepinephrine is prepared by a conventional method. Also,
When preparing preparations for mucous membranes, especially suppositories, cocoa butter, lauric fat, polyethylene glycol, glycerogelatin, sodium suarate, or appropriate mixtures thereof are used as bases, and the suppositories prepared Melts and softens at body temperature, and AQI acts by dissolving in local moisture.

Injectables are also prepared by conventional methods.

In this case, since L-2, M-2H, etc. are poorly soluble in water,
Water-soluble ester salt of L-2, especially M-2H・Na
It is preferable to use Incidentally, no particular problem will occur with any of the above preparations if they are stored in an airtight container at room temperature.

For example, no change was observed in the M-2H bulk powder when stored at room temperature for 18 months, and there was no change in the ingredient content of the M-2H bulk powder formulation when stored at room temperature for 18 months. It's not affected either. In this case, changes were observed in the M-2H bulk powder after 2 months at 40°C, and when humidified to RH53%, 4υ°C (
The change when C was stored was larger than the change when stored at 40°C in non-column humidity, indicating that it was influenced by humidity, but according to thin layer chromatography, the state after the change was M-
There is a degree C in which spots of 2H and traces of succinic acid are observed, and even when M-2H is heated in water, M-2H is hydrolyzed to produce L-2 and succinic acid. It has been confirmed that this is the case, and in any case, no special problems arise in terms of preservation. As explained above, the anticancer agent of the present invention containing N-(2-diethylhexyl)-3-hydroxyptyramide (L-2) or its half-esal with a dicarboxylic acid can be used in a relatively small amount. It also exhibits excellent anticancer effects upon administration. In particular, when epinephrine and norepinephrine are combined, the synergistic action of the two provides an extremely good anticancer effect. In addition, it has low toxicity and has almost no side effects even when administered for a long period of time, and does not show any serious side effects, especially on hematopoietic organs or digestive organs, and there is no risk of causing white blood cell reduction, anorexia, nausea, vomiting, etc.
It is useful as a FyIjS agent as it does not have hemolytic or local irritating effects. It has a sleep effect, not an M1 effect.)
5.Furthermore, it can be easily prepared into the daily preparation A, and the preparation can be simply stored at room temperature in a sealed bag, so there are no special problems in storing the preparation, and it is easy to handle. Examples of formulations are shown below. [Formulation example 1] Soft capsules
After stirring and mixing 4L-2 (or M-2H) with safflower monofatty acid and vitamin E to form a homogeneous mixture, soft capsules were prepared according to a conventional method.

[Formulation Example 2] Hard capsule L-2 (or M-2H) is dissolved in acene, silicic anhydride is added and dispersed, and then the acetone is distilled off and granulated.

The particles were passed through a black 60 mesh sieve, magnesium stearate was added and mixed to make the particles smooth, and the mixture was filled into capsules to prepare hard capsules. [Formulation example 3]
Tablet L-2 (or M-2H) is dissolved in acetone, silicic anhydride is added and dispersed, and then the acetone is distilled off and granulated.

The particles were passed through a black 60 mesh sieve, and Avicel, corn starch, and magnesium stearate were added and mixed to form tablets of 400,719 (or 600 and above) each.
Cocoa butter is heated to 50°C to melt it, L2 is added to it to make it homogeneous, it is poured into a container, and it is cooled and solidified to make a suppository.

A suppository was prepared in the same manner using cacao butter, lauric oil, glycerogelatin, or polyethylene glycol.

The mixture containing M-2H-Na2OO7r9 was aseptically dispensed into vials and plugged to remove moisture and bacteria.

This was made into an injection by adding 2 ml of sterile distilled water at the time of use. In addition, when epinephrine or norepinephrine was used together, these epinephrine and norepinephrine were blended in M-2H-Na in advance.

[Brief explanation of the drawing]

Figures 1 and 2 are graphs showing the relationship between culture time after addition of L-2 and proliferation of mouse cancerous mast cells, respectively.
The figure is a graph showing the relationship between the amount of L-2 added and the proliferation of tumor-forming mast cells in mice. Figure 4 shows the relationship between the amount of L-2 added and the presence or absence of epinephrine combination and the proliferation of cancerous mast cells in mice. Figure 5 is a photomicrograph showing normal mast cells in rats, and Figure 6 is a photomicrograph showing normal mast cells treated with 2.3mM of L-2. FIG. 7 shows that the normal cells have L-
2.3mM and 270μM of epinephrine. Figure 8 is a micrograph showing a mouse cancerous mast cell. Figure 9 is a micrograph showing the cancerous cells treated with L-2, 2.3mM.
FIG. 10 is a micrograph of the cancerous cell VCL-2, 2.3mM and epinephrine 270μM when M was applied.
Here is a microscopic photo of the effect.

Claims (1)

[Scope of Claims] 1 An anticancer agent characterized by containing as a main component N-(2-ethylhexyl)-8-hydroxybutyramide or its half ester with a dicarboxylic acid. 2. Claim 1 in which the ester is a compound represented by the general formula ▲A mathematical formula, a chemical formula, a table, etc.▼ (where M represents a hydrogen group, an alkali metal group, or an alkaline earth metal group) The anticancer drug described in Section 1. 3. The anticancer agent according to claim 1 or 2, which contains epinephrine or norepinephrine.