JPS6225679B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to water-soluble compounds that are effective as immunological adjuvants for promoting immune responses and also as preventive agents for bacterial infections. The present invention also relates to immunological adjuvants and bacterial infection preventive agents containing the above compounds as active ingredients. More specifically, the compounds of the present invention have the general formula (However, R ₁ is an alkoxyl group or an amino group represented by the general formula -OCnH _2o+1 , n is a positive number from 1 to 3, and R ₂ is an alkoxyl group represented by the general formula -OCpH _2p+1 . , p is a positive number from 0 to 3, and R ₁
is not 0 if is an amino group. ) Monoester or methyl of methyl, ethyl and propyl at the α-position of 2-(2-acetamido-2-deoxy-3-0-D-glucopyranosyl)-D-propionyl-L-alanyl-D-glutamic acid , ethyl and propyl diesters, or the methyl, ethyl and propyl esters of 2-(2-acetamido-2-deoxy-3-0-D-glucopyranosyl)-D-propionyl-L-alanyl-D-isoglutamine. Preferred groups of the compounds according to the invention are 2-(2
-acetamido-2-deoxy-3-0-D-glucopyranosyl)-D-propionyl-L-alanyl-D-glutamic acid mono (in the α-position) or dimethyl ester and 2-(2-
Methyl ester of acetamido-2-deoxy-3-0-D-glucopyranosyl)-D-propionyl-L-alanyl-D-isoglutamine, that is, in the above general structural formula, R ₁ is -OCnH _2o+1
When n of the group is 1, 2 or 3, p is 0 or 1, or when R ₁ is -NH ₂ group, p is 1. These compounds are hereinafter indicated by the following abbreviations. Mur―NAc―L―Ala―D―Glu―α―OCH ₃ Mur―NAc―L―Ala―D―Glu (OCH ₃ ) ₂ and Mur―NAc―L―Ala―D―iso―Gln
(OCH ₃ ) In order to produce the compound according to the present invention, the first
In the step, the peptide chain and the abbreviation Mur-
Equivalent derivatives of the fragments related to the peptide chain of the fragment represented by NAc are first synthesized with the functional groups that must not react protected, and in the second step, two derivatives of these fragments are synthesized. Coupling is done. The previously blocked group is released and the protecting group is finally removed. Of course, the synthesis of these compounds can be performed by following methods commonly used for peptide synthesis.
It is also possible to carry out the coupling of the derivative of L-alanine with the derivative of L-alanine separately and then to couple the compound thus obtained with the corresponding derivative of glutamic acid or isoglutamine. The present invention also relates to pharmaceutical compositions containing said agents, which improve the action of weakly immunological substances and which are also useful in the treatment of infectious diseases. Furthermore, especially
The present invention contains said agents for preventing or curing bacterial, viral and parasitic infections, or for combating tissue antigens of various organs of normal or pathological origin. It relates to compositions that can be used for the treatment or immunization of warm-blooded animals. One interesting fact of the novel composition according to the present invention is that it does not require any special media, in particular adjuvant action, on which the expression of pharmacological activity depends, and also the excipients that are combined therewith. has the sole purpose of facilitating the use of these products. especially,
When injecting these products, it is not necessary to use compositions containing an oil phase for this purpose. Furthermore, these compounds which can be used for their adjuvant or anti-infectious action can be administered orally or parenterally, especially by injection. In particular, the invention provides a product of the invention, especially
Mur―NAc―L―Ala―D―Glu―α―OCH ₃ ,
Mur―NAc―L―Ala―D―Glu― ((OCH ₃ ) ₂ or Mur―NAc―L―Ala―D―iso―Gln
( _OCH3 ) in combination with a pharmaceutically acceptable excipient. Particularly preferred compositions of this type are
It consists of an injectable solution containing an effective amount of the product of the invention. Sterile aqueous solutions, preferably isotonic sterile solutions such as saline isotonic solutions or isotonic solutions treated with glucose, can be advantageously used for this purpose. Of course, the present invention is not limited to this, and a simple solution in distilled water can also be used. The adjuvant compositions of the invention can be presented in a variety of forms, using excipients suitable for this purpose for the chosen mode of administration. For example, for oral administration it is used in the form of a capsule, compressed tablet or gelatin-coated pill, and for mucosal administration it is used in the form of an aerosol or gel. The adjuvant can also be in a lyophilized state to allow for immediate dispensing of the adjuvant composition. Pharmaceutically advantageous forms are those containing a unit amount of about 100 to 800 μg, preferably about 400 μg, of the adjuvant product according to the invention. The invention also provides that the products of the invention are immunological agents,
It particularly relates to pharmaceutical compositions formulated with weak vaccinating antigens. Viewed from another point of view, the invention also provides products of the invention, in particular Mur-NAc-L-Ala-D-Glu-α
―OCH ₃ ,Mur―NAc―L―Ala―D―Glu
(OCH ₃ ) ₂ or Mur-NAc-L-Ala-D-iso
- This relates to a bacterial infection preventive agent containing Gln (OCH ₃ ). These products exhibit anti-infectious properties of the prophylactic and even curative type, in fact, when administered alone, ie without vaccine compositions, and in particular without weak immunizing agents. In other words, anti-infectious properties were observed when these products were administered at the same time that contamination was occurring or substantially occurring. These anti-infectious properties were completely unexpected even though it was known that the activity of the compounds could improve host resistance. Thus, certain species of Corynebacteria and Mycobacteria, and their “cords”
It is known that non-specific resistance to infection can be increased by pre-injecting different immune stimuli of bacterial origin, such as lipopolysaccharide (LPS) extracts of gram-negative bacteria or lipopolysaccharide (LPS) extracts of gram-negative bacteria. However, this protection is shown only in conditions related to a certain time interval between the administration of the immunostimulant and the moment of contamination. Thus, Klebsiella
In the case of rats infected with
About 14 days before for BCG, about 7 days before for Corynebacteria, 6 to 48 days before for LPS
It can be experimentally proven that the survival rate is better if done in advance. In all these cases, immune stimulation using these agents must occur prior to infection. For example, when LPS is injected at the same time or after the inoculum bacteria, it causes a "negative reaction" that tends to reduce host resistance, which would otherwise die after administration of the non-virulent bacterial strain. On the other hand, when administered under favorable conditions, these treatments significantly stimulate non-specific immunity, even for bacterial species that confer resistance to antibiotics by alteration or transposition of plasmids. However, these treatments are difficult or even impossible due to the side effects observed after administering large doses of Corynebacteria or BCG. This is not possible due to the toxicity of LPS to humans, which represents, inter alia, a toxic antigen of Gram-negative bacteria. Therefore, on the basis of the results obtained with adjuvants of bacterial origin, and in particular on the basis of the tests carried out with LPS, the above-mentioned synthetic adjuvants according to the invention may be useful within the scope of prophylactic immunization. In addition to these adjuvant properties used, it is surprising to see anti-infectious abilities demonstrated in prophylactic or even therapeutic methods even without being formulated with vaccine antigens. These products have no mitogen activity (toxic metastasis of lymphocytes). These are not antigenic. In fact, Freund complete adjuvant
In the case of guinea pigs sensitized using the adjuvant, they do not show any delayed sensitivity response. No abnormally high fever was observed in rabbits even when the dose was higher than that which caused an anti-infectious response. They test negative in the Limulus test and, by this procedure, can be injected into adrenalectomized rats, which are extremely sensitive to the lethal effects of endotoxins. These results indicate that these compounds are completely free of endotoxin properties. Although adjuvants such as LPS are only active when administered parenterally, they are effective against bacteria even in the absence of an oil phase, regardless of whether administration is made parenterally or orally. It has the advantage of being active as an infection preventive agent. An important advantage of the anti-infectious use according to the invention of the above-mentioned compounds lies in the possibility of acting against pathogenic bacteria that are resistant to antibiotics by following treatment with traditional antibiotic methods. It must also be pointed out that the anti-infectious therapeutic abilities of these compounds were all remarkable and unexpected. This is because tests have shown that they have no bactericidal or bacteriostatic activity in vivo. Regarding compounds intended to promote an immune response, pharmaceutical compositions containing the above-mentioned methyl esters and used in anti-infectious therapy exhibit the properties of the product when administered orally or parenterally. Therefore, it can take various forms. In particular, the pharmaceutical compositions may be in the form of injectable solutions (particularly in the form of isotonic aqueous solutions), drinkable solutions, capsules, gelatin-coated pills, aerosols, gels, and the like. Other characteristics of the invention will become apparent from the examples of preparation of the products according to the invention described below as well as from the tests that characterize the pharmacological properties of these products. In this description, the following abbreviations have the following meanings. Mur-NAc: 2-acetamido-2-deoxy-3-0-(D-2-propionyl)-D-
Glucopyranose Ala: Alanine Glu: Glutamic acid iso-Gln: Isoglutamine 4,6-0-bzi: 4,6-0-penzylidene β-bzl: β-benzyl BOC: t-butyloxycarbonyl OBzl: Benzyl ester OSu: Succin Imidoester Bzl: benzyl ether 2-(2-acetamido-2-deoxy-3-
Synthesis example a of t-butyloxycarbonyl-L-alanyl-D-glutamic acid α-benzyl ester () E. Schnabel ( Justus Liebigs Ann.Chem.
702 , 188 (1967)) was dissolved in 15 ml of tetrahydrofuran. This solution was prepared by S. Guttman and R.
A. Boissenas (Helv. Chim. Acta, 41 , 1864
(1958) of an aqueous solution of 3.3 g (14 mmol) of α-benzyl ester of D-glutamic acid and 1.4 g (14 mmol) of potassium bicarbonate.
dissolved in After standing overnight, the pH was adjusted to 8.5 and the reaction mixture was extracted with ethyl acetate. The liquid phase was acidified to PH3.5 with 4N hydrochloric acid solution at low temperature and extracted with acetic ether. The organic phase was then washed with water, dried and concentrated. 4.77 g of product was obtained, or a yield of 87.8%. Its physical properties range from melting point 68 to
The temperature was 70°C, and [α] _D ²⁵ =−12° (methanol). The results of elemental analysis were as follows. C ₂₀ H ₂₈ O ₇ N ₂ (408.45) C% H% N% Calculated value: 58.81 6.9 6.85 Actual value: 58.7 6.4 6.8 b t-Butyloxycarbonyl-L-alanyl-D-glutamic acid α-methyl ester, γ-
Benzyl ester () 408 mg (1 mmol) of the product () was dissolved in 100 ml of absolute methanol. Diazomethane (approximately 10
mmol) in ether was added over 15 minutes at 0°C. After 2 hours at ambient temperature, the reaction mixture was concentrated to a solid and collected in 25 ml of ethyl acetate. The organic phase was subsequently washed with a 10% solution of citric acid, then with a 1 molar solution of sodium bicarbonate and then with water until the pH was neutral. The ethyl acetate phase was dried over magnesium sulfate, filtered and concentrated. A non-crystalline oil was obtained (385 mg or 91% yield). c Hydrochloride salt of L-alanyl-D-glutamic acid α-methyl ester, γ-benzyl ester () 385 mg (0.91 mmol) of the product () were treated with 3 ml of 1N hydrochloric acid in glacial acetic acid for 30 minutes. The reaction mixture was concentrated to solidity and the resulting oil was dried (330 mg or 100% yield). d 2-(benzyl-2-acetamide-4,6
-0-Benzyldene-2-deoxy-3-0-
α-D-glucopyranosyl)-D-propionyl-L-alanyl-D-glutamic acid α-methyl ester, γ-benzyl ester () Flowers and RW Jeanloz (J.Org.Chem. 28 ,
Benzyl-2-acetamido-4,6-benzylidene-3-0-(D-carboxyethyl)- prepared by the method disclosed by 2983 (1963))
2-deoxy-α-D-glucopyranoside 473
mg (1 mmol) was dissolved in 5 ml of dimethylformamide cooled to -15°C. followed by 0.11 ml (1 mmol) of N-methylmorpholine and 0.13 ml (1 mmol) of isobutyl chlorocarbonate.
added. To this reaction mixture were added 330 ml (0.9 mmol) of the product () and N-methylmorpholine dissolved in 5 ml of dimethylformamide, which had been pre-cooled to -15°C.
0.1 ml (0.9 mmol) was added. After standing overnight at -15℃, potassium bicarbonate 2.5N solution 1
Added ml. After 30 minutes, the product was precipitated by adding 40 ml of distilled water, filtered and dried. 667 mg of product were obtained, ie a yield of 95.5. e 2-(2-acetamido-2-deoxy-3
-0-D-glucopyranose)-D-propionyl-L-alanyl-D-glutamic acid α-methyl ester () 305 mg (0.39 mmol) of the product () was dissolved in 50 ml of glacial acetic acid solution for 15 hours at 5% on charcoal. palladium
Hydrogenated in the presence of 300 mg. After filtering off the catalyst, the acetic acid was concentrated to dryness and the product was precipitated from methanol-acetone-ether and then centrifuged. 140 mg was obtained, i.e. yield 70
It was %. Product name from BIORAD company
It was purified by chromatography on a column (2 x 10 cm) packed with a commercially available ion exchange resin (acetic acid type) named AGIX-2. Washed with 0.2 molar solution of acetic acid, combined and lyophilized, 117 mg
was recovered with a yield of 83.5%. The optical rotation of this product was [α] _D ²⁵ =+39° (methanol). SEPHADEX by PHARMACIA UPSARA
Ion exchange resin (acetic acid
column (2 x 80
cm) and then lyophilized to obtain the final product. 94 mg of final product () was obtained, yield 80
It was %. The optical rotation [α] _D ²⁵ =+39° (methanol), and the results of elemental analysis were as follows. C ₂₀ H ₃₃ O ₁₂ N ₃ , 1H ₂ O (525.50)
C% H% N% Calculated value: 45.7 6.7 7.99 Actual value: 44.93 6.32 7.91 2-(2-acetamido-2-deoxy-3-
Synthesis example of dimethyl ester of 0-D-glucopyranose)-D-propionyl-alanyl-D-glutamate In the first step, Mur-NAc-L-Ala-D
-Glu is prepared by the following method. a Preparation of benzyl ester (A) of BOC-L-alanyl-D-glutamic acid t-Butyloxycarbonyl whose amino group is protected by a t-butyloxycarbonyl group (BOC-L-Ala-OSu) While stirring 2.3 g (8 mmol) of the succinimide ester of L-alanine, a solution of 4.5 g (9 mmol) of p-toluene-sulfate of the benzyl diester of D-glutamic acid in dimethylformamimide and 1 ml (9 mmol) of N-methylmorpholine is added. mmol) was added. The reaction mixture was left at room temperature for 12 hours. It was then concentrated to dryness and the dried compound was taken up in 50 ml of ethyl acetate and washed successively with 10% citric acid solution, water, 1N sodium bicarbonate solution and finally with water. The ethyl acetate phase was dried over magnesium sulfate, filtered and concentrated. Recrystallize from a mixture of ethyl acetate and hexane,
2.50 g (67.5% yield) of product was obtained, the physical properties of which were as follows: Melting point: 105-106°C α _D ²⁵ = +7.3° The results of elemental analysis were as follows. C ₂₇ H ₃₄ O ₇ N ₂ (498.5) C% H% N% Calculated value: 65 6.9 5.6 Actual value: 64.85 7.0 5.5 b 2-(2-benzyl-2-acetamide-
4,6-Benzylidene-2-deoxy-3-0
Preparation of the benzyl diester of -D-glucopyranosyl)-D-propionyl-(0-benzyl)-L-alanyl-D-glutamic acid (B) 500 mg of compound (A) were treated with 5 ml of 1N hydrochloric acid in glacial acetic acid. After 30 minutes, the reaction mixture was concentrated to dryness. The resulting oil was taken up with 25 ml of acetonitrile-dimethylformamide mixture (2/1, volume ratio). The mixture was cooled to 0 °C and triethylamine
0.141 ml (1 mmol) was added. Benzyl-2-acetamide-4,6-benzylidene-3-0-(D-1
-carboxyethyl)-2-deoxy-p-D-
472 mg (1 mmol) of glucopyranoside and acetonitrile-dimethylformamide mixture (2/
1. Triethylamine dissolved in 25 ml (volume ratio)
It was formed from 1.41 mg (1 mmol) and poured into a suspension injection prepared 1.5 hours earlier. The mixture was left at room temperature for 12 hours, then concentrated and
The residue was precipitated with 10% citric acid solution. The precipitate was filtered, thoroughly washed with water, and dried. product 800
mg (94% yield) was obtained. Melting point: 198-199°C α _D ²⁵ =4.92° (dimethylformamide) After recrystallization from ethanol, the melting point was 220°C. The results of elemental analysis were as follows. C ₄₇ H ₅₃ O ₁₂ N ₃ (851.96) C% H% N% Calculated value: 66.26 6.27 4.93 Actual value: 66.34 6.45 4.92 c 2-(2-acetamido-2-deoxy-3
-0-D-glucopyranosyl)-D-propionyl-L-alanyl-D-glutamic acid (C) or Mur-NAc-L-Ala-D-Glu 700 mg of compound (B) was heated on a boiling water bath for 1 hour. acetic acid
Treated with 40ml of 60% solution. The reaction mixture was then concentrated to dryness (then dried over magnesium sulfate). The residue was taken up with 1 ml of a chloroform-methanol mixture (3/3, by volume) and placed on a silica column (35 g) previously equilibrated with the same solvent mixture. The product fractions contained were collected and concentrated to dryness (their physical properties were tested by thin layer chromatography on silica gel in the same mixture of solvents). 185 mg (yield 30%) of the derivative was obtained. 76 mg of this derivative was dissolved in 15 ml of glacial acetic acid and hydrogenated in the presence of 5% palladium on charcoal. After filtration, the mixture was concentrated to dryness and diluted with methanol.
Precipitated with an acetone-ether mixture. 40mg
(yield 92%) product was obtained. Melting point: 150-155°C α _D ²⁵ =+33° (glacial acetic acid) The results of elemental analysis of this product were as follows. C ₁₉ H ₃₁ O ₁₂ N ₃・1H ₂ O (511.48)
C% H% N% Calculated value: 44.6 8.2 6.5 Actual value: 44.7 8.1 6.4 In the second step, previously prepared Mur-NAc-
L-Ala-D-Glu was esterified. 100 mg (0.2 mmol) of Mur-NAc-L-Ala-D-Glu was dissolved in 10 ml of absolute methanol. Within 15 minutes, 10 ml of an ethereal solution of diazomethane (approximately 0.7 mmol/ml) was added. After 90 minutes, 1 drop of acetic acid was added and the reaction mixture was concentrated to dryness. The obtained residue was dissolved in silica gel (1×16
cm) purified on a column. The pure fractions were concentrated together. The product was precipitated with a methanol-acetone-ether mixture. 83 mg of product (80% yield) was obtained. Melting point: 137-142°C α _D ²⁵ = +31.6° (glacial acetic acid) The results of elemental analysis were as follows. C ₁₂ H ₃₅ O ₁₂ N ₃ (521.53) C% H% N% Calculated value: 48.36 6.76 8.57 Actual value: 48.0 7.0 8.2 2-(2-acetamido-2-deoxy-3-
Synthesis example of 0-D-glucopyranose)-D-propionyl-L-alanyl-D-isoglutamine methyl ester.
Mur obtained by the method described in No. 7422909
Use NAc-L-Ala-D-iso-Gln. Mur―NAc―L―Ala―D―iso―Gln1mg (0.2
mmol) in the same manner as described above in the section on esterification of Mur-NAc-L-Ala-D-Glu.
Purification was performed using a chloroform-methanol mixture (5/5,
(volume ratio). 80 mg of product (yield 80
%)was gotten. Melting point: 201°C α _D ²⁵ = +44.2° (glacial acetic acid) The results of elemental analysis of this product were as follows. C ₂₀ H ₃₄ O ₁₁ N ₄ (506.52) C% H% N% Calculated value: 47.52 6.76 11.06 Actual value: 47 6.5 10.3 Pharmacological properties 1 Toxicity The toxicity of the product according to the invention was determined parenterally in rats and rabbits. The investigation was carried out by targeted administration. The toxic amounts were found to be much higher than the amounts necessary for these products to exhibit their activity. Therefore,
These products are tolerated in rats in amounts equal to or greater than 100 mg/Kg of animal and in rabbits in amounts equal to or greater than 5 mg/Kg of animal. 2 Mur―NAc―L―Ala―D―Gln―α―
OCH ₃ ,Mur―NAc―L―Ala―D―Glu
(OCH ₃ ) ₂ and Mur-NAc-L-Ala-D-iso
- Adjuvant properties of Gln (OCH ₃ ) in aqueous solution phase In a series of tests, the results of which will be described later, the influence of the active ingredient according to the present invention on the ratio of anti-albumin antibodies was investigated under the following conditions. Eight 2-month-old Swiss rats were treated subcutaneously (SC) and orally (PO) with a test substance consisting of bovine serum albumin (BSA) or bovine serum albumin and a test substance in an isotonic saline solution. 0.5 mg of the antigen was administered. This large amount of antigen reaches a paralyzing threshold for the immune response, so that the control mice have a weak or no response to the antigen alone. Therefore, this represents a strict condition for the activity of an adjuvant substance. After 30 days, an additional dose of 0.1 mg of the same antigen was administered. The ratio of antigens is AAHirata and MWBrandiss
(J. Immunol., 100 , 641-648, 1968) using formalin-treated sheep red blood cells recovered from antigens studied by the method disclosed by
Determined by passive hemagglutination. Blood samples were taken 14, 28, 34 and 36 days after the first injection. For comparison, instead of the product according to the invention, designated by the symbol lipopolysaccharide (LPS) (extraction of Salmonella Enteritidis by the water-phenol method) or “WSA”, Adam et al. Infect.
Immun. (1973) 7, 855-861] was administered to rats. Control mice received antigen only. The results of these tests are shown in Table 1 below. The antibody ratio indicates the maximum serum dilution that will agglutinate a given amount of sheep red blood cells.

[Table] These results demonstrate that the product according to the invention, administered in an isotonic solution, significantly increases the proportion of antibodies formed, even when the adjuvant is administered orally. ing. The active ingredient according to the present invention is generally “LPS”
However, it is noteworthy that, contrary to the latter, it has no toxicity whatsoever. 3 Mur―NAc―L―Ala―D―Glu―α―
OCH ₃ ,Mur―NAc―L―Ala―D―Glu
(OCH ₃ ) ₂ and Mur-NAc-L-Ala-D-iso
- Adjuvant properties of C-ln (OCH ₃ ) in the presence of an oil phase. In these tests, the increase in the proportion of antibodies specific to a given antigen was determined using the antigen with or without the adjuvant compound according to the invention. We investigated the case of injection in the form of a water-in-oil emulsion. The test was carried out on 6 female Hartley guinea pigs weighing 350 g. Administration was by subcutaneous injection into the paw pad of the hind paw. Ovalbumin (constituting the antigen) 1mg or 0.5
The oil phase was supplemented with Freund's incomplete adjuvant (FIA) or 0.1 mg of whole cells of mycobacterial sebum (smegmatis) in the proportion of 0.0 mg of FIA.
Freund's Complete Adjuvant (FCA) was prepared in an emulsion of 0.1 mg of a salt isotonic solution made up of 1. The compound according to the present invention was administered at a dose of 0.1 mg in a suspension containing FIA. On the 18th day after immunization, animals were subcutaneously injected with 0.01 mg or 5 μg of ovalbumin to investigate delayed hypersensitivity reactions to the antigen, and 48 hours later, reactions at the injection site were observed. The diameter of the reaction thus occurring was measured in mm. Animals were bled 21 days after injection. For the collected serum, the amount of protein nitrogen contained in the precipitate of antibody-antigen complexes in the equivalence zone was determined according to the Folin method. The average values of antibody content are shown in Table 2. These values represent the amount of nitrogen in micrograms that can be precipitated by antigen per ml of serum. In some cases, antibody levels may be determined by passive hemagglutination (PHA) as described above.
It was also determined by The results of these tests are shown in Table 2 below.

[Table] These results show that the compound according to the invention administered in an oily emulsion affects the proportion of antibodies formed in response to injection of an antigen and causes a delayed hypersensitivity reaction for the same antigen. It is understood that it induces 4 Anti-infectious properties The following tests were conducted on Mur-NAc-L-Ala-D-Glu
―α―OCH ₃ ,Mur―NAc―L―Ala―D―Glu
(OCH ₃ ) ₂ and Mur-NAc-L-Ala-D-iso-
Demonstrates anti-infectious properties of Gln( _OCH3 ). In preliminary tests, an experimental procedure was established allowing for the anti-infectious properties of the product to be demonstrated. In this way, rats were injected intramuscularly with
10 ⁴ (Klebsiella pneumonia), it was demonstrated that in the week of carrying out the inoculation, the majority, but not all, of the animals were progressively reduced. After 8 days, remaining animals were finally identified. Survival rates of groups of mice inoculated under the conditions indicated above and treated with the methyl esters under consideration were followed. For comparison, a group of rats were treated with BCG and LPS.
It was treated with. The latter is well known to be a highly active immunostimulant when administered 24 hours before infection. For these tests, we used hybrid rats (C57B1/6×AKR) FL bred at the Pasteur Institute.
Variants grown in Orleans C, N, R, and S were used. endotoxin or
LPS is Salmonella enteritidis variant Danysz (NO,
5629, Institut Pasteur) by the phenol-water method. BCG is the bacterial species Mycobacterium var. bovis (Institut Pasteur NO.
1173p ₂ ) was cultured in Sauton medium,
% phenol solution. Infection with Klebsiella pneumonia, variant of capsular type 2, genetic factor d type, is carried out by culturing for 16 hours in medium for Streptococcus pneumoniae (Institut Pasteur NO, 53515). 0.2 for parenteral administration when prepared by injection at or before the moment of infection.
ml, 0.5 ml for oral administration, always diluted with athermic physiological solution; control rats received solution only. The results of the tests are shown in Tables 3 and 4, and the effects of the treatments were determined by varying the method, including the amount and time of administration of the product under study. % protection refers to the difference in % survival in a treated group of animals compared to the corresponding control group. From the results it is clear that the product under consideration has anti-infectious activity whether it is administered parenterally or orally.
In contrast, when LPS is administered orally,
It is inactive even with very large doses (100 μg LPS corresponds to 100 times the anti-infectious dose taken parenterally). Furthermore, the same results are obtained if the product is administered intramuscularly, whether 24 hours or 1 hour before injection of the infectious agent.

【table】

【table】

Table: In a series of other tests, the anti-infectious properties of the product were investigated in relation to highly severe infections caused by intravenous (not intramuscular) injection of 10 ³ Klebsiella pneumoniae. In these studies, rats were similarly inoculated24
I took action on time. The method, dosage and test results are shown in Table 5.

Table 1 From the results it is clear that there is a significant protection in mice treated with the product according to the invention. As detailed above, the present invention provides a new immunological adjuvant which is soluble in water and active without the presence of an oily phase, which is completely non-toxic and can be administered parenterally or orally. Furthermore, a drug for treating infectious diseases that is active against antibiotic-resistant disease factors is provided.

Claims (1)

[Claims] 1. General formula (However, R ₁ is an alkoxyl group represented by the general formula -OC _o H _2o+1, n is a positive number of 1 to 3 or an amino group, and R ₂ is represented by the general formula -OC _p H _2p+1. 2-(2-acetamido-2-deoxy-3-O-D-glucopyranosyl)-D expressed as ( _2- (2-acetamido-2-deoxy-3-OD-glucopyranosyl)-D) ―
Ester of propionyl-L-alanyl-D-glutamic acid or 2-(2-acetamido-2-deoxy-3-O-D-glucopyranosyl)-D-propionyl-L-alanyl-D-isoglutamine. 2 Informal Mur-NAc-L-Ala-D-Glu-α-
2-(2-acetamide-2 represented by OCH ₃
-deoxy-3-O-D-glucopyranosyl)-
The ester according to claim 1, which is α-methyl ester of D-propionyl-L-alanyl-D-glutamic acid. 3 Informal Mur-NAc-L-Ala-D-Glu-
(OCH ₃ ) _2- (2-acetamido-
2-deoxy-3-O-D-glucopyranosyl)
The ester according to claim 1, which is a methyl diester of -D-propionyl-L-alanyl-D-glutamic acid. 4 Informal Mur-NAc-L-Ala-D-iso-Gln-
2-(2-acetamide-) represented by (OCH ₃ )
2-deoxy-3-O-D-glucopyranosyl)
The ester according to claim 1, which is a methyl ester of -D-propionyl-L-alanyl-D-isoglutamine. 5 The following general formula along with pharmaceutically acceptable excipients and/or immunizing agents: (However, R ₁ is an alkoxyl group represented by the general formula -OC _o H _2o+1, n is a positive number of 1 to 3 or an amino group, and R ₂ is represented by the general formula -OC _p H _2p+1. In the alkoxyl group, p is a positive number from 0 to 3, and R ₁
When is an amino group, p is not 0. ) 2-(2-acetamido-2-deoxy-3-O-D-glucopyranosyl)-D-
Immune containing an ester of propionyl-L-alanyl-D-glutamic acid or 2-(2-acetamido-2-deoxy-3-O-D-glucopyranosyl)-D-propionyl-L-alanyl-D-isoglutamine Scientific adjuvant composition. 6. An adjuvant composition according to claim 5, containing an effective amount of said ester and in the form of an injectable solution. 7. An adjuvant composition according to claim 5 or claim 6, which contains an effective amount of said ester and is in the form of a saline isotonic solution. 8. The adjuvant composition according to claim 5, which is formulated with an excipient for facilitating oral administration or an excipient for facilitating mucosal administration. 9. The adjuvant composition according to any one of claims 5 to 8, wherein the ester is blended with an immunizing agent. 10. An adjuvant composition according to any one of claims 5 to 9 useful as a human vaccine composition comprising an effective amount of the ester in addition to the immunizing agent. 11. The adjuvant composition according to any one of claims 5 to 9, which is useful as a home-stock vaccine composition containing an effective amount of the ester in addition to the immunizing agent. 12 General formula below (However, R ₁ is an alkoxyl group represented by the general formula -OC _o H _2o+1, n is a positive number of 1 to 3 or an amino group, and R ₂ is represented by the general formula -OC _p H _2p+1. In the alkoxyl group, p is a positive number from 0 to 3, and R ₁
When is an amino group, p is not 0. ) 2-(2-acetamido-2-deoxy-3-O-D-glucopyranosyl)-D-
An effective amount of ester of propionyl-L-alanyl-D-glutamic acid or 2-(2-acetamido-2-deoxy-3-O-D-glucopyranosyl)-D-propionyl-L-alanyl-D-isoglutamine as an active ingredient. A bacterial infection preventive agent containing as. 13. The bacterial infection preventive agent according to claim 12, which comprises the active ingredient mixed with an excipient for facilitating its oral administration. 14. The bacterial infection preventive agent according to claim 12, which is in the form of an injectable solution containing the active ingredient. 15. The bacterial infection preventive agent according to claim 12, wherein the active ingredient is dissolved in an isotonic physiological saline solution for injection.