JPS6054286B2 - immune enhancer - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an immune enhancer, particularly an immune enhancer containing a specific polyhydroxyanthraquinone gunphonic acid derivative such as latukaic acid, carminic acid or germesic acid as an active ingredient.

The emergence of drugs that can universally (non-specifically) increase immunity against infectious diseases and other diseases, thereby increasing resistance to those diseases, is extremely desirable for human health and disease treatment. That's true.

If the body's immune system is weakened, the body's resistance to infections and other diseases will naturally decrease, and recovery will be delayed or death will occur due to the induction of secondary infections and diseases rather than the original illness itself. . A typical example of drug-induced immunity reduction is the administration of most chemotherapy drugs and antibiotic anticancer drugs to cancer patients.

Because these drugs generally have strong immunosuppressive effects, patients often die from causes other than the original cancer. Furthermore, since conventional anticancer drugs are generally extremely toxic, there is a strong desire to develop anticancer drugs with lower toxicity. In recent years, a small number of cases of spontaneous cure of cancer have been reported, and the reason for this is thought to be enhancement of the immune system of the body against cancer cells.

Therefore, immuno-anticancer drugs that try to control cancer cells by increasing the body's immune system have been considered, and along this line, for example, polysaccharide of Sarunokoshike mushroom (PSK) and Picibanil (Picibanil8), a hemolytic streptococcus drug, have been developed. have already been commercialized, and as new research, the low molecular weight compounds Levamisole (Levamisole 8) and Bestatin (Bestatin 8) are currently under development.
The discovery of substances with immunoenhancing effects also suggests applications as anti-drug resistance agents.

That is, due to advances in chemotherapy triggered by the discovery of sulfonamide drugs, penicillin, and streptomycin,
Today, the development of treatment methods for microbial infections is remarkable, but on the other hand, the emergence of drug-resistant bacteria has become a serious problem.The development of drug-resistant bacteria makes treatment difficult and The development of means to suppress the emergence of resistant bacteria is one of the most important issues today, as it increases the chances of new infections caused by remaining resistant bacteria. In addition to natural mutation and selection due to use, infection and transmission of multidrug resistance factors (R plasmids), DNA
Transformation using bacteriophage and transduction using bacteriophage are considered.

Among these causes, the most frequent ones are natural mutation and selection, followed by the R factor. The former type of resistant bacteria that has developed in the body due to natural mutations survives even if antibiotics are continuously administered, so if the body's immune defense mechanisms (e.g., glucocorticoid action and cellular immunity) function normally (enhancement). If so, they will probably become extinct as a result. As mentioned above, the immune-enhancing effect, anti-cancer effect, and anti-drug resistance effect are mutually related to the body's inherent defense mechanism (e.g., glucophagic action or anti-cytophagic action) through the host. It is assumed that this will eventually serve a common therapeutic purpose.

Based on the above philosophy, the present inventor speculated that if it is highly effective as an immune enhancer, it may also be useful as an immunosuppressive agent or anti-drug resistance agent, and conducted experiments on many other compounds in addition to anthraquinone series compounds. As a result, it was found that kermesic acid, which belongs to 1,4-dihydroxyanthraquinonecarboxylic acid derivatives such as latsukaic acid and carminic acid, almost satisfies the intended purpose. Incidentally, the presence or absence and strength of immune-enhancing effects are the keys to screening based on the above assumptions, and for this reason, the inventor decided to treat Bacillus aeruginosa, which is still considered the most difficult to treat today.
Enteritidis (Gram-negative rodent bacteria), which is said to be difficult to respond to antibiotics because the bacteria invade the cells, and Smlthdiffuse strain (Gram-negative), a special strain of Staphylococcus that resists macrophages. positive cocci)
and multidrug-resistant E. coli were taken up as indicators. The gist of the present invention is to utilize latsukaic acid, carminic acid, and kermesic acid as immune enhancers.

In each of the above chemical substances, latsukaic acid (latsukaic acid;
Laccaicacid) is a Japanese scale insect (Ta
chardialacca. ．． This is a general term for the resin components secreted by Kerr.

Its main component is latsukaic acid A (or A1).
3,4,6-tetrahydroxy-2-[2″-hydroxy-5-(β-acetamidoethyl)-phenyl-anthraquinone-7,8-dicarboxylic acid (see the formula below), but in addition to this, phenyl at the 21-position Lacukaic acids B and C and laccaic acid D (with different terminal substituents at the 5″1 position of the group)
1,3,6-trihydroxy-8-meranthraquinone-7-carboxylic acid). Also, carminic acid (
Carminicacid is a type of insect that parasitizes prickly pear cacti, which is found in Central and South America.
7-α-D-glucopyranosyl-3,5,6,8-tetrahydroxy-1-methyl-2
-Has the structure of anthraquinone carboxylic acid (see formula below). Furthermore, Kermesic acid (Kermesic acid) is a type of insect that parasitizes the branches of the Kermes oak tree that grows in southwestern Europe.
．． It is contained in the body of female worms and has the structure of 1●3●4●6-tetrahydroxy-2-methylcarbonyl-8-methylanthraquinone-5-carboxylic acid (see the formula below).

(However, there is a theory that the keto group at the 21-position does not exist.) All of the above anthraquinone derivatives are natural products, and have been used since ancient times as red natural food pigments.

Therefore, as shown in the table below, these compounds are non-toxic or have extremely low toxicity.
Incubate for 2 m.

r thingELjζantiLlOA. Mayan et al. Therefore, it has the characteristic of being extremely safe as a drug.

As shown in the experiments below, these compounds exhibit immune-enhancing and anticancer effects by themselves, and also exhibit effects in combination with general constituents, vaccines, and anticancer agents. Furthermore, since it has the effect of defending against the invasion of multidrug-resistant bacteria, it is presumed that it should also have the effect of suppressing drug resistance in microorganisms, but the actual nature of these wide-ranging pharmacological actions is unclear. The discovery of such a pharmacological effect by a substance that itself has no antibacterial or bacteriostatic properties is an unprecedented and novel finding. The experimental facts that are the premise of the invention will be described below.
A Experimental method 1 Test bacterial species: Pseudomonas aeruginosa 70P■, Salmonella enteritidis NO. ll. Staphylococcus S
mithdlffuse and multidrug-resistant E. coli M strain 2 test animals Male mice, average weight 20 y. ddY system 3 inoculum amount and virulence Staphylococcus is
InfuslOn) was inoculated and cultured with shaking (37℃, 2 (
(transition) between).

(Use the seed cultured on blood agar) r+C,l
-1 side; 1101-+-0A5 drug (specimen) and administration method Among the specimens, water-soluble ones are dissolved in physiological saline to the required concentration and administered by subcutaneous injection or orally.

For those that are slightly dissolvable or insoluble in water, suspend them in a 1% CMC solution and administer by subcutaneous injection or orally.

5 days before, 3 days before and 1 day before (24 hours before) vaccination
Each specimen is subcutaneously injected or orally administered, and 2 hours later, the inoculum of each strain is suspended in 0.2 ml of physiological saline and inoculated intraperitoneally.

Control animals are injected subcutaneously with the same amount of physiological saline instead of the drug, and then similarly inoculated intraperitoneally with the test bacterial solution. For each experiment, the amount of bacteria diluted 2 times, 4 times, 8 times, 1 time, and 32 times (i.e., 112 amounts, 114 amounts, 11 times) was tested for virulence.
8, 1116 and 1132) were inoculated as controls.

Furthermore, for each infection experiment, the number of viable bacteria and the number of inoculated bacteria per 1 m1 are verified. 6. Method for determining efficacy The efficacy is determined by the survival rate of the sample-administered mouse group and the control mouse group.

The timing of determination is one week after inoculation in the case of Pseudomonas aeruginosa, Staphylococcus and multidrug-resistant E. coli, and 22 weeks after inoculation in the case of Salmonella Enteritidis. There were 5 to 10 mice in each group and at each stage.
Repeat the experiment several times to confirm its reproducibility. 7. Evolution of bacteria in the bodies of surviving mice.Death and surviving mice were dissected aseptically.

(1) Heart, liver, and spleen for protection against Pseudomonas aeruginosa infection. If necessary, remove the entire kidney aseptically and place each organ in a homogenizer. Add 2 ml of sterile broth and grind. The homogenate obtained by
@ Dilute with bouillon, its undiluted solution and Part 10-1
, 10-2, 10-3, 10-4, and 10-5. Collect 0.1 ml of each diluted solution and place them on ordinary agar medium or modified Drigalsky for Pseudomonas aeruginosa, enterobacteria, and multidrug-resistant E. coli. For staphylococci, apply to blood agar medium, brain heart infusion agar medium, or ordinary agar medium, mix well with a Conlage rod, and spread over the entire surface (this operation is also performed aseptically, of course).

). After that, each medium is cultured in a 3TC incubator for 24 to 30 hours, and the number of colonies is counted to calculate the number of bacteria per organ. Anticancer effect and combination effect with anticancer drugs Ascites from mice transplanted with sarcoma 180A obtained from the National Cancer Center was collected aseptically, and 0.05 ml of it was injected into ICR/
JCL1 is transplanted subcutaneously into the inguinal region of a 5-week-old male mouse to form a solid tumor.

1 m9, 0.1 m9 and 0.0 m9 of each drug after 2 hours of transplantation
Inject 1 m9 each (however, levamisole is highly toxic, so 0.1 m9 and 0.01 m9 each) intraperitoneally once a day.

30 days after transplantation, all solid tumors are taken out, their size and weight are measured, and the average values of individual mice in each group and each administration group are compared with that of the control, and the inhibition rate is calculated using the following formula.

(1) MLD means minimum lethal dose. Therefore -2ML.
D means twice the minimum lethal dose (the same applies below). (2
) Effect on preventing infection with Salmonella Enteritidis (3) Effect on preventing infection with Staphylococcus (4) Effect on preventing infection with multidrug-resistant Escherichia coli As shown by the above experimental results, carminic acid (or carmine), latukaic acid, and kermesic acid are effective against Pseudomonas aeruginosa. , S. Enteritidis ,
It shows excellent infection prevention effects against both staphylococcus and multidrug-resistant E. coli infections, and its results are superior to that of levamisole, a known representative immunostimulating drug.

The behavior of the surviving mice showed no difference at all from healthy mice. Including a large number of non-example anthochyranone compounds tested by the inventor, three drugs including carminic acid (or carmine) show excellent effects; It may show better effects than acids (or carmine).

Although the efficacy of kermesic acid is generally inferior to the first two, it is still superior to levamisole, and this trend is the same even when the drug is administered orally. Regardless of the amount of inoculation, the effect is greater if the amount of drug administered is 1 more, 1W1
No side effects were observed even after 9 doses (approximately 50 mg/kg body weight). However, 0.01 m9 (approximately 0.0 m9 per body weight K9)
It is noteworthy that even a microdose administration of 5m9) may show results comparable to those of the 0.1m9 administration group. 1
In the following experiments, all data are shown for carminic acid, but since carmine also shows exactly the same effect, carmine was included and exemplified as carminic acid.

In the case of carmine, it is hardly soluble, so if necessary,
It was suspended in %CMC and used. ■ Infection prevention in vivo bacteria evolution and drug resistance prevention effect in surviving mice (1) Pseudomonas aeruginosa infection prevention in vivo bacteria infection prevention experiment, 1 to 1 Pi2. Lethal dose inoculation group and 1 to 0
．． In each of the 01m9 administration groups, the number of viable bacteria in each organ (heart, liver, tiles) of mice that survived 7 days was examined.

Each lethal dose and each dose showed similar trends, so
The results for 10 animals are shown in Figure 1 (3).

As shown in the figure, 70% of the cells became completely sterile after 7 days, and even if the bacteria survived, they were all below 101/V.

The average number of viable bacteria is 10! It was demonstrated that the average number of viable bacteria was less than 10-5 to 10-6 around 10-1 CP/y, compared to 107-1 CP/y for control mouse 3S that died.
In other words, in this experiment, CA or LA activated and strengthened the body's defense mechanism, and as a result, 7
0% becomes sterile in 7 days and 100% 4C becomes sterile in 14 days.

Even if there are any remaining bacteria, it is estimated that they will be extinct after 7 days, as they will be in extremely small numbers.

(2) Prevention of Infection with Salmonella Enteritidis In the case of Infection with Salmonella enteritidis, 60%
% is completely sterile.

In the remaining 40%, although still a small number, viable bacteria of around 1σ/y survive in each organ. CA,. The same tendency is observed with LA, with the least amount surviving in the heart and relatively more in the liver and tiles.

(3) Prevention of staphylococcal infection In the case of staphylococcal bacteria, as shown in Figure 3, 40% of the mice become completely sterile within 7 days, but the remaining 60% of the mice are 1σ /y bacteria remain in the living body.

The Smithdiffuse strain used in this experiment has a capsule and is highly resistant to macrophages, so it can kill mice with about 1 Cf3, but SmithcO
Among pact strains and normal strains, there are generally many strains that cannot be killed even with 1 Cf)/mouse.

In this experiment, 40% became sterile in 7 days and 60% had only about 1σ/y bacteria remaining, indicating that CA or LA significantly enhanced defense mechanisms such as macrophages in vivo. (The number of bacteria in control dead mice is 107-1018/y).

(4) Multidrug-resistant Escherichia coli infection prevention In vivo fate of surviving mice The multidrug-resistant Escherichia coli used in this experiment was resistant to seven drugs: ampicillin, cephalothin, cephalozin, kanamycin, streptomycin, tetracycline, and curramphenicol. As a highly resistant strain, these drugs are clinically ineffective against this E. coli infection.

In the case of multidrug-resistant E. coli, as shown in Figure 4, the trend is almost the same as that of Pseudomonas aeruginosa, with 80% becoming sterile within 7 days and only 20% remaining viable bacteria. The real number is as small as about 1σ/y.

In dead mice (control), it was still 107~1σ/y.
It is.

In other words, bacteria proliferate in the bodies of mice that die, but by administering drugs (CA, LA, etc.) in mice that survive, the bacteria decrease and 80% become completely sterile, and after 7 days they are no longer present in the body. The remaining bacteria is only about 1σ/y.

This experiment showed that CA or LA not only synergistically enhances the therapeutic effects of chemotherapeutic agents and antibiotics in infectious diseases, but also inhibits this bacterium that has mutated and become resistant in vivo. This suggests that multi-drug resistant bacteria such as Bacteria can be universally and non-specifically exterminated by the enhanced defense mechanism of the body by the single administration of this drug.

76■ Immunity-enhancing effect when used in combination with a vaccine It is generally said that the immune effect of Salmonella Enteritidis is low unless it is a live bacterial vaccine.

Therefore, the immunological effect of killed bacteria vaccine, which is said to have little immunological effect, is due to CA. ．． How much does it increase when used in combination with LA?
．． I experimented to see if it would make me stronger. In this experiment, the amount of attacking bacteria was 8 times and the lethal dose.

(1) Immune effect of killed bacteria vaccine 100% Enteritidis No. 1 killed by heating for 30 minutes at C. ll2l
of bacterial cells and 1 per mouse (3 PllCfllO 0.
Suspended in 2 ml of physiological saline and subcutaneously injected n days, 18 days, and 14 days before the challenge date, and then intraperitoneally inoculated with 8 times the lethal dose and 1 times the lethal dose of the same bacteria, respectively. Observe their life and death for two weeks.

As shown in Figures 5 and 6, in the group inoculated with 1 CP/mouse, only 25% survived the challenge with an 8-fold lethal dose, and only 10% survived the challenge with a 1-fold lethal dose. .

At vaccine doses of 10i/mouse and 1CP/mouse, no surviving mice were observed in either the 8x or 16@lethal challenge. (2) Immune-enhancing effect of combined use with Cbe-LA Each group of mice pretreated as described above and injected subcutaneously with a killed vaccine were given CA,... 1, respectively, 5 days before, 3 days before, and 1 day before (24J1!f before) the LA attack date.
Tn9, 0.1 Tn9, and 0.01 mg each were injected subcutaneously, and these groups were challenged intraperitoneally with 0.2 ml of the same bacteria at 8 times the lethal dose and 16 times the lethal dose, and then their survival was determined for 2 weeks. Observed.

(a) Effect of combination with CA When using 1σ/mouse killed vaccine in combination with CA,
m9, 0.1m9 and 0.01m9 all showed a 100% survival rate at an 8 times lethal dose.

When challenged with a lethal dose of 1 g, 90% even in the 0.01 m9 dose group.
The rescue rate was

101/When used in combination with killed mouse vaccine and CA,
Against 8x lethal attack, 90% for 1 instant and 0.1m9,
Even at 0.01m9, the rescue rate was 70%.

80%, 85%, and 7% for lethal attack, respectively.
The rescue rate is 0%.

The 1σ/mouse killed vaccine also showed a rescue rate of 80 to 50% in each administration group, although it was inferior to 10i/mouse.

The results of this experiment show that even with dead bacteria or bacterial species that do not have a strong immune effect, the combined use of CA and LA (described below) can significantly enhance the immune effect, and that even with a small number of bacteria, the immune effect is well exerted. ing.

(b) Effect of combination with LA Immunological efficacy of vaccine when used in combination with LA As shown in Figure 6, the increase in the result is due to the CA field. It is almost the same as the case.

In addition, from the above experiments, it was found that the present invention Drugs include not only Enteritidis vaccine but also Other cutins, such as polio vaccine, Measles vaccine, Japanese encephalitis vaccine, Fluenza vaccine, smallpox seedlings, whooping cough vaccine It is expected to have excellent combination effects with other drugs such as chin. while also reducing their usage. This will prevent vaccine paralysis, etc. It is estimated that it can prevent the occurrence of side effects. However, the present invention is based on further separate findings. However, even if this drug is used alone, it may cause influenza, etc. It has been shown that it may be effective against viral diseases such as I'm getting hints.

Effect of combined use with chemotherapeutic agents (antibiotics) The drug according to the present invention has the effect of synergistically increasing the efficacy of the former when used in combination with chemotherapeutic agents (antibiotics). has the potential to show excellent therapeutic efficacy when used in combination with the main drug in cases of refractory, chronic diseases, or weakened immune systems that are difficult to cure even with the administration of chemotherapeutic agents, antibiotics, etc.

FIG. 10 shows that mice inoculated with an 8-fold lethal dose (8MLD) of S. enteritidis were given tetracycline (TC) (1 m9 and 0.0 m9, respectively) once each day for 5 days starting 2 hours after inoculation.
5WL9, 0.1m9, 0.057n9, 0.01Tn
9, inject 0.005m9 or 0.001m9 subcutaneously,
An example in which life and death were observed on the 28th day is shown.

The survival rate (rescue rate) is 80% for 1m9 and 80% for 0.5m9:
60%, 0.1m9:50%, 0.05m9:30%,
0.01u9:20%, 0.005m9, 0.001m
g and control (no treatment or saline injection): 0%. Next, 0.1 mg administration group (survival rate 50%) and 0.01 m
9 administration groups (survival rate 20%) and 1T of CA or LA, respectively.
n9, 0.1m9, 0.01m9, or 0.001m9
was injected in combination with TC, and the survival and death were observed in the same manner.

As shown in the figure, CA is TCO. The survival rate was 100-80% for the lm9 administration group when combined with 1-0.01m9.
In addition, the combined use of 0.001 m9 also increased it to 60%. Similarly, TCO. Also for the Olm9 administration group, CA 1 to
The combined use of 0.001m9 is significantly superior to redundant alone, and 70
It showed a rescue rate of ~30%. The combined use of LA and TC showed almost the same tendency as LA, but it was even more effective, and TC
O. The combined use of lm9 and LAl~0.001m9 increased the survival rate to 100-65%, and the TCO. When used in combination with Olm9, the rescue rate increased to 65-40%. From the results of the above experiments, we found that the use of drugs such as antibiotics, which are difficult to administer in large quantities due to strong toxicity or side effects, or where there is a concern that side effects may occur due to long-term administration, can be reduced by combining small amounts of CA or LA. It is envisaged that the usual dose could be reduced to 1'10 or less.

In addition,. In addition to TC, it has been demonstrated that the antimicrobial effect of the main drug can be significantly enhanced when used in combination with synthetic penicillins such as ampicillin, cephalexin from the cephalosporin C family, gentamine from the aminoglycoside series, kanamycin, etc. Ta. ■ Anti-cancer effect and combined effect with anti-cancer drugs (1) Anti-cancer effect The inhibition rate against solid cancer of meat type 180A is 1 for CA.
m9 administration group: 41%; 0.1119 administration group: 36%;
It was 45% in the 0.01WL9 administration group.

In LA, the rate was 36% in the 11n9 administration group, 37% in the 0.1m9 administration group, 49% in the 0.01m9 administration group, and about 18-29% in KA. In both cases, administration of 0.01 m9 showed rather good results. (See Figure 7) (2) Effect of combination with adriamycin The inhibitory effect of adriamycin (Ad) alone is as follows:
Administration of 0.17FL9 and 0.017F19 (intraperitoneal injection) showed inhibition rates of 1% and 52%, respectively.

On the other hand, when CA and LA are used alone, 1119 and 0.
Around 40% for 0111L9, 10-2 for 0.1m9
The inhibition rate was around 5%. However, JAdO. Ol
In addition to mg administration, CA and LA 1 m9, 0.1 m9,
0.01W! In combination with 9, each? %, 96%
It showed some inhibition effect.

Also, CA or LAO. When used in combination with lTn9, the inhibition rate was around 74-80%, and when used in combination with 1m9, the inhibition rate was also around 82-89%. (See Figure 8)
. As detailed above, the drug according to the present invention brings about a synergistic effect with chemotherapeutic agents and antibiotics by activating the bactericidal action by mobilizing the in-vivo defense mechanism, and as a result, it reduces the development of drug resistance. Furthermore, it prevents the weakening of immunity caused by the repeated use of adrenocortical hormones and general anticancer drugs, and also prevents the bacterial replacement phenomenon or alternation caused by the repeated use of antibiotics, etc., and has an extremely beneficial relationship in many fields. bring about an effect.

Furthermore, as a cancer immunotherapeutic agent, it has a remarkable effect when used in combination with vaccines, anticancer drugs, etc. Based on its immune-enhancing effect, it has a wide range of potential clinical applications. It is also expected that the immunomodulatory effect will be effective against these diseases. The modified drug according to the present invention may be used alone or in the form of a mixture, in the form of an injection for intravenous, intramuscular or subcutaneous injection, or in the form of a liquid solution, elixir, capsule, tablet, granule, powder, troche, vagina, etc. It is used in the form of oral preparations or transmucosal (transdermal) preparations such as vaginal or rectal suppositories and even ointments.

For formulation, diluents, carrier excipients, binders, vehicles, etc. known in the art of formulation are used. Non-toxic vehicles such as liquid oils and oils are useful when it is desired to present the drug as a suspension for injection. The drug may also be transformed into a salt form with a physiologically acceptable base, if desired. Suitable bases include, for example, Li, . Na,. K. ．． Ca,
,M? An example is an alkaline substance that supplies ions. This drug also has Fe. ．． It may be converted into a salt or complex salt form with a metal such as CO or Cu. The appropriate dosage for this product is approximately 0.5 to 50m91k9, but it is difficult to state definitively because it varies depending on the administration method, dosage form, etc.

[Brief explanation of drawings]

Figure 1 is a graph showing the number of viable bacteria in the bodies of mice that survived the Pseudomonas aeruginosa infection experiment, and Figure 2 is a graph similar to Figure 1 showing the number of viable bacteria in the bodies of mice after the Pseudomonas enteritidis infection experiment. , 3rd
The figure is a graph similar to Figure 1 showing the number of viable bacteria in mice after an experiment of infection with Staphylococcus, and Figure 4 is the same as Figure 1 showing the number of bacteria in the body of mice after an experiment of infection with multidrug-resistant E. coli. FIG. 5 is a graph showing the effect of combined use of carminic acid on immunity by killed bacteria enteritidis vaccine, and FIG. 6 is a graph similar to FIG. 5 showing the combined effect of latukaic acid.

Claims (1)

[Claims] 1. An immune enhancer containing as an active ingredient a compound selected from the anthraquinone carboxylic acid compound group consisting of latukaic acid, carminic acid, and kermesic acid.