JPS6388136A - Preventive for canceration - Google Patents

Abstract

PURPOSE:To obtain a preventive for canceration caused by an N-nitroso compound and capable of essentially completely detoxicating carcinogenecity of a secondary amine, etc., by using hydrolyzed natural substance rich in proline and hydroxyproline and its thermal denaturation product as an active component. CONSTITUTION:The objective preventive for canceration caused by an N-nitro compound is produced by using hydrolyzed and/or enzyme-digested natural substance rich in proline and hydroxyproline and its thermal denaturation prod uct as an active component. A preventive for canceration having high safety can be produced at a low cost by the hydrolysis of collagen which is a natural substance rich in proline and hydroxyproline or of gelatin, casein, prolamine, etc., which is thermally denatured product of collagen.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a preventive agent for carcinogenesis such as liver cancer and gastric cancer caused by N-di)o compounds.

It is well known today that various N-nitroso compounds, typified by N-nitrosodimethylamine (NDMA), have strong carcinogenic effects even in trace amounts (It, Druck
rey et al. +2. Krebsforsh
+69103 (1967): J, H, Hotch
kiss et at,, J.

^5soc, ON, ^na1. CheIll,,
641037 (1981) and G, c, Gib
son et al. +′:5afety E
Valuation of old trostable Dr
B and Chemicals″Taylor an
d Francis Ltd, + London, 1
981 etc.). Furthermore, NDM, which has particularly strong carcinogenic properties,
N-nitrosamines such as A react with nitrites, which are converted in the body from nitrates, which are abundant in foods such as vegetables, and aliphatic amines, which are also contained in fish meat, in internal organs such as the stomach. It is also being elucidated in recent years that it is a substance that can be produced and can be a carcinogenic factor (Correa, P.
et al, Lancet II+ 58-60
(1975): Tannenbaum+ S, R
et at,, ^nnalsof New York
Academy of 5sciences 35526
7-277 (19SO): Tannenbaum,
S, R, et al, + Caneer Lett
, 14131-136 (1981)): D, l
I+ Fine etal, + Nature 265
753 (1977) and P, 5chlaHetal, +
Lancety April, 5727 (1980
L etc.).

Therefore, preventing the in vivo production of N-nitrosamines in advance is considered to be very significant in terms of reducing the risk of carcinogenesis.

However, to date, no such food or drug has existed, and ascorbic acid (vitamin C) is the only one that is said to be effective in preventing nitrosamine production, but it is unstable as a substance and cannot be used as a vitamin. Therefore, the intake amount is limited when taken alone, so it has not yet been put into practical use.

In order to prevent nitrosamines generated from aliphatic secondary amines and nitrites, the present inventors searched for a preventive substance. Reacts with nitrite in preference to aliphatic secondary amines to form non-carcinogenic nitrosoproline (NPRO), resulting in highly effective inhibition and reaction of carcinogenic N-nitroso compounds in vivo. The present invention was submitted in Japanese Patent Application Laid-open No. 164718/1983, as it is capable of rendering the entire mixture substantially harmless.

The present invention further develops the above-mentioned invention and provides a cheaper and safer product by hydrolyzing collagen, which is a natural product containing a large amount of proline and hydroxyproline, and its heat-denatured products, such as gelatin, casein, prolamin, etc. It provides a highly effective cancer prevention agent.

The raw materials, manufacturing method, pharmacological and biological effects, dosage and administration, toxicity, etc. of the agent of the present invention will be explained in detail below.

The raw material collagen and its thermally denatured products such as gelatin, casein, and prolamin are used.

Manufacturing method The above-mentioned raw materials are decomposed into amino acids to produce the agent of the present invention by hydrolysis or enzymatic digestion according to conventional methods.

A specific example of the hydrolysis method is shown in Experimental Example 1.

Pharmacological and Biological Effects As shown in the experimental examples below, the agent of the present invention has the effect of substantially completely detoxifying the carcinogenicity of secondary amines and nitrite-containing solutions. It can be said that it is extremely useful as a compound-derived carcinogenic preventive drug from the viewpoint of preventive medicine.

It has already been reported that the produced NPR○ is not carcinogenic, and its urinary excretion is almost complete and rapid (Chu + C, & Sagce,
P, N, Cancer Res, 413653
-3657 (1981); Dailey, R0E'
, et al., Toxicol, 323-2
8 (1975): Hirvish, S.
et al., J. Natl.

In5t, 641435-1442 (1980) and Ohshima, H & BarLsch, Il.
Cancer Res, 413658-3662
(1981).

etc).

Dosage and Administration Nitrate ingested from foods such as vegetables is converted to nitrite by microorganisms in the oral cavity, etc., and reacts with amines, which are components of the ingested food, mainly in the stomach to produce carcinogenic substances. has been done.

For Japanese people, the amount of nitrate intake from food is 218-4
08+ag/day, and the concentration of nitrate ions in saliva is about 11 to 50 ppm (several pl) [11 to 50 ppm (I
shiwajar H,et at,, Procee
diBs of the 3rd International
onal Conference on Enviro
nmental Mutagens+ Tokyo
5ept, 21-27. 1981 page
571-575), and nitrite concentration in gastric fluid is 0.
．． 1 to several tens of μg/ml (P, Schlageta
l, + Lancet, April 5727 (19
80)), the dose of the present agent is within the range of several 100 μg to several g of proline per 7 days per 50 kg body weight, more preferably from 1 mg to 1 g per day per 50 kg body weight, and is suitable for preventive medicine. From the point of view, it's actually a 5.
0 to 500 mg/day/50 kg body weight can exhibit a carcinogenic preventive effect.

Incidentally, the minimum carcinogenic amount of NDMA is approximately Ill]
It has been evaluated as DI, which corresponds to 50ff1g in a human body weight of 50kg (M, Arai et al.
, + Gann 70549 (1979), etc.).

Although the drug of the present invention is generally administered orally,
Even if you just take the dry powder after hydrolysis, you can add it to food and take it with it.

In addition, the dosage forms include aqueous solutions, powders, granules, tablets, capsules, sustained release microcapsules, etc., or in combination with a suitable carrier such as refined starch powder, a filler, a diluent, etc. They may be formulated separately without these and may be selected and implemented as appropriate.

The agent of the present invention can also be used in soy sauce, which is commonly used in daily use, including foods with high secondary amines and nitrate content.

Added in advance to foods such as seasonings such as sauces and mayonnaise,
Since it can be formulated and taken in a form that is naturally ingested, it can exhibit even greater preventive effects.

All of the toxic raw materials, such as fulladen, casein, prolamin, and gelatin, are found in ordinary foods and are completely non-toxic orally.

Incidentally, the agent of the present invention obtained by hydrolyzing gelatin was administered to ICR mice (average body weight 34.0±0.5 g at 166 weeks + 10 mice in each group) at a dose of 1 mg + 10m8 + 100+H.
The sample (dissolved in 0.5 ml of physiological saline) was administered intraperitoneally, and the test was carried out for 14 days for ¥1, and [! ehrens-Kmrb
LD calculated according to the er method. The value is 830mg7kg
It was more than my weight.

Next, the present invention will be explained in more detail with reference to an experimental example using q according to the present invention.

Experimental Example 1 Production from Gelatin Water-soluble gelatin (manufactured by Nitta Gelatin Co., Ltd.) was diluted with 6N hydrochloric acid for 4 hours.
A solution of approximately 0.00 g/A was prepared, and hydrolysis was carried out at 110° C. for 5 hours using a distillation reflux method. The solution after hydrolysis was treated in a rotary evaporator under reduced pressure at 50'C for 1 hour, and the remaining hydrochloric acid was further neutralized with 4N sodium hydroxide.

The generated sodium chloride was collected by electrodialysis (2 people).

1 hour). The desalted solution was immediately powdered by freeze-drying or spray-drying.

The proline content of the powder produced was 10%, and the powder was a brown, slightly sweet, odorless powder that was easily soluble in water after it had evaporated.

In addition, when ingesting it as food or medicine, we put safety first and conducted a mutagenicity test using the Al1es method, and found that the number of mutant Cohineys per gram of dry powder was approximately 2.
000 colonies appeared 6 Normally, what is considered mutagenic is the appearance of several thousand to tens of thousands of colonies at a few μg to several mg, and although there is no problem with mutagenicity, the following The operation was performed.

We believe that this mutagenicity is due to the brown humin produced during hydrolysis, and in order to remove it, we use the activated carbon adsorption method to absorb pJ for 1 hour at a ratio of 1 weight of activated carbon to 2 weights of the sample. ,
By stirring and adsorbing and filtering, the number of mutant colonies in the filtrate powder was reduced to 200 per gram, as shown in Figure 1. Furthermore, as shown in Table 1, the contents of proline and hydroxyproline were hardly affected, and the powder became a pale yellow powder, with no change in taste, odor, or solubility.

A 90-day subacute toxicity test of RfI was conducted on a dry powder with no mutagenicity.

ICR mice and Wistar rats (both 4 weeks old, male) were used, with 20 mice per group and 10 rats per group, each receiving 20B and 200B of dry powder per 8 body weight.
+ 2g in drinking water and administered daily, 9
As a result of l'iJl observation on day 0, mice.

No deaths were observed in either group, and no differences were observed between the control group and the control group in terms of weight gain and organ weight.

Example 3: Effect of the agent of the present invention [['' 1 sting pill ■ Dimethylnitrone amine production reaction solution Tsumethylamine hydrochloride... 69 mg/aa as the amount converted to Tsumethylamine
1 of the solution was prepared and 0.75 ml of it was used.

Sodium nitrite...Prepare a solution of 1106 Ifi/l111.

The value of 0.75 mN was used.

The above three are set to pl+3.4 in concHcj',
Further, an appropriate amount of distilled water was added to obtain a 3 ml reaction solution.

(2) Reaction solution for addition of the present invention agent Dimethylamine hydrochloride and sodium nitrite, each of which had the above formulation, were used.

A solution having a proline content of 9311 g and 7 ml was prepared and added to dimethylamine in an amount of 1 molar equivalent.

When adding proline, mix dimethylamine and proline, add sodium nitrite, and add 1r in concHC1.
H was adjusted to 3.4, and an appropriate amount of distilled water was added to prepare a reaction solution. Intragastric conditions were set in which each tube was sealed tightly, shielded from light, and incubated at 37° C. for 3 hours.

1) ^mes test The mutagenicity suppressing effect of nitrosamines produced from the amine and nitrite of the agent of the present invention was measured by the Awes method.

Nitrosamines 0.5 + 1 + 1 per plate
．． In the present invention, the brightening agent was added in an amount of 1 molar equivalent.

The results are shown in Figure 2 as the number of reverting colonies, and A
B is a reaction solution for producing nitrosamine, and B is a reaction solution for adding the present invention agent.

2) Rat liver NAD level In response to DNA damage caused by carcinogen administration, NAD in the organ
It was reported that the amount of AD decreased and there was organ specificity [
Itamoto et al., 1981 Japanese Mutagen Society].

Therefore, the following experiment was conducted focusing on the liver specificity of trimethylnitronamine.

There were 34 Wistar male rats (5 weeks old) in each group, and in each test, one group was used as an untreated control group.

Oral administration was carried out using a sonde at a dose of 0.1 ml Q/100 g of animals.

Each reaction solution administration group was killed by decapitation 10 hours after administration.
After sufficient bloodletting, the abdomen was opened immediately and the IIF viscera was removed.
Approximately 1 g of liver per animal was accurately weighed to make four specimens, in order to minimize local measurement errors. Specimens were immediately frozen.

World J''D L Hetl+ods of Enzymajic Ana
lysis, + Vol 4+ 2045 (1974)
The measurement was carried out by an enzymatic method using ADH (alcohol Φ dehydrodenase, 1.2 B/l111) using ethanol as a substrate in accordance with the above. That is, after weighing, the frozen specimen was homodenized in a frozen state, and 5 mN of 0.6N -HCfO was added to it.

Add to remove protein. The sample is 3. OOOrpm
Centrifuge for 5 minutes to obtain supernatant, and add 2HPO1 to IM-
Add 0.2 mj' per supernatant 11111, 3N-KO
In step II, adjust the pH to a neutral value of 7.0 to 7.4. This was further centrifuged at 3,000 rpm for 5 minutes, and the KCt
) O, precipitate was removed to obtain supernatant 111, and 0.1M sodium birophosphate/semicalpants dovanofur hydrochloride (
ρ118.8) Add IJ, add 10 μl of ethanol, and after stirring, measure ○, D, at 340 nI11 (E,).

Furthermore, 10 μl of ADH was added and O and D were measured in the same way (
E2), E2-E, the difference (ΔE i + ollff
N A DB (μm.

le) was calculated.

The results are shown in Figure 3, where A is Funtrol, B is the nitrosamine production reaction solution, C is the reaction solution with the present agent added,
D is a group to which only the agent of the present invention was administered.

This shows that the agent of the present invention significantly suppresses the decrease in NAD amount caused by nitrosamines.

3) Anti-placental glutathione-8-) lance-7 erase (
GST-P) Effect based on antibody-positive cell foci as an indicator Since GST-P antibody staining-positive 1a Ha foci that appear early after administration of carcinogens are generally considered to be precancerous lesions in the liver, We applied an experimental system targeting the liver using the two-step theory of carcinogenesis as shown in Figure 4 using rats (5 male rats aged 4 weeks). D
50 mg/kg was administered once in the N cavity, and two weeks later, the test substances were dimethylamine alone, the amine and the preparation were mixed in drinking water, and sodium nitrite was mixed in feed, and the animals were given free ingestion. 273 Partial hepatectomy (pa) 1 week later
rtial hepato+B: PH) is applied,
After that, the drinking water and feed were given ad libitum for 7 weeks, and 8 weeks after administration of the test substance, an autopsy was performed, and the liver was stained with GST-P antibody. As a result of image analysis using an image processing device regarding the number of cells, as shown in Table @2, both the area and cell number were higher in the methylamine and sodium nitrite intake group than in the control group, and the number of cells was higher than that of the control group. group showed a significant suppressive effect.

The experimental system is schematically shown in FIG. 4, where A is the control group, B is the amine + nitrite intake group, and C is the inventive agent addition group.

Table 2

[Brief explanation of the drawing]

Figures 1 to 4 show Experimental Example 1.3-1). 3-2) and 3-3). Patent applicant: Advance Development Institute Co., Ltd. Kasui Makuji 9
Quantity (Kyukai, -〇(Fζ-8) Figure 3

Claims (1)

[Claims] (1) A carcinogenic preventive agent derived from an N-nitroso compound, characterized in that the active ingredient is a hydrolyzed and/or enzymatically digested natural product containing a high content of proline and hydroxyproline, and a heat-denatured product thereof.