GB1584791A - Muromyl peptide immunological adjuvants - Google Patents

Description

(54) IMPROVEMENTS IN AND RELATING TO MURAMYL PEPTIDE IMMUNOLOGICAL ADJUVANTS (71) We, AGENCE NATIONALE DE VALORISATION DE LA RECHERCHE (ANVAR), a French body corporate, of 13, rue Madeleine Michelis, 92522 Neuilly-Sur-Seine, France, do hereby declare the invention, for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement:- The invention relates to new water-soluble agents which are efficient as immunological adjuvants which stimulate immunising responses in a host.

The adjuvant agents according to the invention are compounds of the general formula:

wherein R, is OH, NH2, NHCH3, N(CH3)2, SOCH3, OC2H5 or OC3H7 and R2 is OH, NHCH3, N(CH3)2, SOCH3, OC2Hs or OC3H7 for example 2 - 2 - acetamido - 2 deoxy - 3 - O - D - glucopyranosyl)- D - propionyl - L - seryl - D isoglutamine corresponding to the formula

(this will be subsequently denoted by the abbreviation Mur - NAc - L - Ser - D isoGln - OH and 2 - (2 - acetamido - 2 - deoxy - 3 - 0 - D - glucopyranosyl) D - propionyl - L - seryl - D - glutamic acid corresponding to the formula

(this will be subsequently denoted by the abbreviation Mur - NAc - L - Ser - D Glu - OH).

The invention also relates to medicinal compositions containing the adjuvant agents of formula (I) and serving in particular to increase the action of weak immunising substances. More especially, the invention relates to compositions containing the said agents and which can be used for the immunisation of men and other warm-blooded animals against bacterial, viral and parasitic infections, and against various organic tissue antigens of normal or pathological origin.

The very special interest of the products of the invention is that it is not necessary to use particular media for their administration, on which the manifestation of the adjuvant action would depend, and the vehicles with which one is led to associate them have only the object of facilitating the use of these products. In particular, when one of these products is injected, it is not necessary to use for this purpose a composition containing an oily phase.

In addition, the adjuvant agents according to the invention may be administrated orally or parenterally, especially by injection, scarification, or by application to the mucous membrane.

The invention also provides to medicinal compositions containing one of the adjuvant agents of formula (I) and in particular the Mur - NAc - L - Ser - D isoGln - OH, in association with a pharmaceutically acceptable vehicle.

Compositions of this type which are particularly preferred are constituted of injectable solutions containing an effective dose of one product of the invention.

Sterile solutions in an aqueous, preferably isotonic phase, such as isotonic saline solutions or isotonic solutions treated with glucose, are advantageously used for this purpose. This is of course not restrictive; a simple solution in distilled water may also be used. It is also possible to use injection media containing an oily phase, especially water-in-oil emulsions, in which the adjuvants of the invention are dissolved. Such emulsions are obtained in particular with metabolisable vegetable oils, such as are described in our U.K. Patent No. 1543783.

The medicinal compositions according to the invention may also be presented in various forms, by using for this purpose vehicles suitable for the selected method of administration. For example, compositions will be used in the form of cachets, compressed tablets or gelatine-coated pills, for oral administration, and aerosols or gels for application on the mucous membrane.

The adjuvant agents according to the invention can again be in lyophilised form in order to permit the extemporaneous preparation of injectable solutions or for scarification.

An advantageous pharmaceutical form for the extemporaneous preparation of injectable solutions comprises a dose of about 2 mg of one of the adjuvant products of the invention, in a lyophilised state, in association with about 10 mg of lactose as the vehicle.

The invention also provides medicinal compositions in which one of the products of the invention, notably the Mur - NAc - L - Ser - D - isoGln - OH, is associated with an immunising agent, especially a weak vaccinating antigen.

The invention also provides a process for the preparation of a compound of formula (I) in which the peptide chain L-Ser-D-isoGln, L-Ser-D-Glu, or an ester or amide derivative as defined above, is attached to N-acetyl-muramic acid whereby the reactive groups of the peptide chain other than -the amino group carried by the L- seryl moiety and the reactive groups of the N-acetyl-muramic acid other than its propionyl group have been previously blocked. After the formation of the peptide bond, the protected functions are freed.

According to the invention, the synthesis may also be carried out for example by fixing successively both aminoacids constituting the peptide chain. First an appropriate derivative of the L-serine is fixed to the N-acetyl-muramic acid, then the D-glutamic acid derivative is fixed on the L-serine by formation of a peptide linkage.

Other characteristics of the invention will appear from the description of an example of preparation of the Mur - NAc - L - Ser - D - isoGln - OH, as well as tests which show the pharmacological properties of this product.

Example of synthesis of the Mur - NAc - L - Ser - D - isoGln - OH This compound, of which the following name is the official nomenclature: 2 (2 - acetamido - 2 - deoxy - 3 - 0 - D - glucopyranosyl) - D - propionyl - L seryl - D - isoglutamine, is prepared in several stages. In a first stage, the peptide chain is synthesised, then, in a second stage, this chain is attached to the muramyl residue. During this synthesis, the functions which should not enter into reaction are protected by protective or blocking groups which are then removed.

In the course of this account, the abbreviations used have the following meanings: Mur-NAc 2-acetamido-2-deoxy-3-O-(D-2-propionyl)-D-glucopyranose Glu . glutamic acid isoGln: isoglutamine Ser serine 4,6-O-bzi: 4,6-O-benzylidene p-bzl : A-benzyl BOC t-butyloxycarbonyl OBzl benzyl ester OSu succinimide ester Bzl : benzyl ether BOC-L-Ser(Bzl)-D-iso-Gln-OBzl (I) 1.48 g (5 mmoles) of BOC-L-Ser-OH (Bzl) [E. Wtinsch, A. Zwick, Chem. Ber.

97 (1964) 2497] are added with stirring to a solution in dimethylformamide of 1.64 g (6 mmoles) of the hydrochloride of the benzyl ester of D-isoglutamine [P.

Lefrancier, E. Bricas, Bull. Soc. Chim. France (1969) 3561] and of 0.66 ml (6 mmoles) of N-methylmorpholine. 1.24 g (6 mmoles) of NN'-dicyclohexylcarbodiimide are added, and the reaction mixture is left at the ambient temperature for 12 hours. It is then concentrated and taken up in 50 ml of ethyl acetate and the organic phase is washed successively with a 10% by weight solution of citric acid in water, an N solution of sodium bicarbonate, then with water until a neutral pH is obtained. The ethyl acetate phase is dried over MgSO4, then concentrated. The product is crystallised from a diethyl ether-petrol cum ether mixture. 1.40 g of product are obtained, i.e. a yield of 55%.

M.p. 65-660C aD5=+5.8 (absolute methanol) The elementary analysis of this product is C27H35O7N3(5l3.6) C% H% N% calculated: 63.1 6.9 8.2 found: 63.2 7.1 7.97 Hydrochloride of H-L-Ser(Bzl)-D-iso Gln-OBzl (II) 1.3 g (2 mmoles) of (I) are treated for 30 minutes with 5 ml of an approximately IN solution of hydrochloric acid in acetic acid. The reaction mixture is concentrated to dryness and the product is crystallised in a methanol-diethyl ether mixture. 765 mg of product are obtained, i.e. a yield of 85%.

M.p. 158--161"C aD5= +8.2" (absolute methanol).

Mur-NAc(/s-bzl-4,6-bzi)-L-Ser(Bzl)-D-iso-Gln-OBzl (111) A solution of 471.43 mg (1 mmole) of Mur-NAc(ss-bzl-4,6-bzi)-OH in a mixture of acetonitrile and dimethylformamide (2:1, v/v-l0 ml) containing 0.141 ml (1 mmole) of triethylamine is poured into a suspension, maintained at OOC, of N-ethyl5-phenylisoxazolium-3'-sulphonate (253 mg-I mmole) (reagent D of Woodward, Aldrich) in 10 ml of acetonitrile.

The mixture is stirred at OOC until a clear solution is obtained (about 90 minutes), then 450 mg (1 mmole) of (II) in solution in 10 ml of the acteonitriledimethylformamide mixture (2:1, v/v) containing 0.141 ml (1 mmole) of triethylamine are added. After stirring overnight at the ambient temperature, the solvents are evaporated under vacuum and the product is precipitated in the N,Ndimethylformamide-water mixture. 818 mg of product are obtained, i.e. a yield of 94.3%.

M.p. 180--1860C an26 170 (N,N-dimethylformamide).

Mur-NAc-L-Ser-D-isoGln-OH (I V) 768 mg of the compound (III) (0.88 mmole) are treated with 30 ml of a 60% by weight solution of acetic acid for an hour at 1000C. After cooling the solution, the acetic acid is evaporated under vacuum and the residue is chromatographed on a column of silica gel (35 g) (chloroform-methanol, 6:2, v/v). The pure fractions are combined, evaporated under vacuum, giving a residue chromatographically pure (plate of silica gel with chloroform-methanol, 6:2, v/v). 287 mg of product are obtained, i.e. a yield of 42%.

This product, taken up in 20 ml of glacial acetic acid, is then hydrogenated for 10 hours in the presence of palladium on carbon. After filtration of the catalyst and concentration to dryness of the reaction mixture, the product is obtained by precipitation in an ethanol-acetone-diethyl ether mixture. 140 mg of product are obtained, i.e. a yield of 74%. The rotatory powder is an25 = +47.10 (glacial acetic acid) The product is chromatographically homogeneous on plates of silica gel in the mixtures n-butanol-pyridine-acetic acid-water (30:20:6:24 v/v/v/v) and n-butanolacetic acid-water (4:1:5, v/v/v-upper phase).

The elementary analysis of this product is C19H32O,2N4 (508.5) C% H% N% calculated: 44.88 6.34 11.02 found: 44.8 6.3 11.1 Pharmacological properties 1) Determination of the harmlessness of the active principle according to the invention The harmlessness, or on the contrary the toxicity, of the Mur-NAc-L-Ser-DisoGln-OH is studied by intravenous injection on mice aged two months. By progressively increasing the doses administered, that for which the mortality of the mice of a single batch is established at 50% (LDso) is determined.

For the product studied, it was shown that the LD60 is superior to I g per kg of animal, which is much greater than the doses for which the product shows its adjuvant properties.

2) Adjuvant character in aqueous phase In the series of tests of which the results are indicated hereafter, the influence of the active principle according to the invention on the strength of the antialbumin antibodies has been studied under the following conditions.

Groups of 8 Swiss mice aged two months receive, by subcutaneous injection, 0.5 mg of antigen constituted by the albumin of bovine serum (BSA) and 0.1 mg of the tested substance in an isotonic saline solution. This high dose of antigen, since it is situated at the limit of the paralysing dose with respect to the immunising response, therefore carries with it a weak response or no response to the antigen alone in the tests; it then constitutes a severe criterion for showing the activity of an adjuvant substance. Thirty days later, the mice receive a further dose containing 0.1 mg of the same antigen.

The strength of the antibody is determined by passive hemagglutination by using the red blood corpuscles of sheep treated with formalin and recovered from the antigen studied by the method described by A. A. Hirata and M. W. Brandiss (J. Immunol., 100, 641-648, 1968). The taking of the blood occurred 14, 28, 34 and 36 days after the first injection.

By way of comparison, the mice receive, instead of the Mur - NAc - L - Ser D - isoGln - OH, either lipopolysaccharides (LPS) (extract of S. Enteritidis by the water-phenol method), or the adjuvant denoted by the name "WSA" and described by Adam et al. [Infect. Immun. (1973)7,855-861]. The control mice only received the antigen.

The results of these tests are given in the following Table.

TABLE 1 Strength of antibody

14th 28th -34th 36th day day day day Control BSA < 3 < 3 < 3 < 3 BSA + LPS (100 g) < 3 6 50 1310 BSA + WSA (30011g) < 3 < 3 < 3 6 BSA + Mur-NAc-L-Ser-D-isoGlnH 12 25 1600 1600 (100;1g) These tests were carried out again with limitation of the dose of antigen in the first injection to 0.05 mg, the other conditions being the same. This weaker dose of antigen gives by itself a positive response with the controls which have received the antigen without adjuvant. It thus allows to be shown a substance which inhibits the response of immunising the disorder. Moreover, an adjuvant substance, under these conditions, always increases the strength of antibody with respect to that of the controls.

In these tests, the determination of the strength of antibody has been effected simultaneously by passive hemagglutination (PA) and by the method of fixation of the antigen by the serum (ABC), such as described by P. Minden and S. Parr (Handbook of Exp. Immunol., P. 463, D. M. Weir ed. Blackwell Scient. Pub.

Oxford and Edinburgh, 1967).

The results of these tests are shown in the following Table.

TABLE 2 Strength of antibody

42nd 14th day 28th day 34th day 36th day day PA ABC PA ABC PA ABC PA ABC PA BSA (control) < 3 < 20 < 3 < 20 6 < 20 50 25 25 BSA + LPS < 3 < 20 25 < 20 400 300 3200 500 800 BSA + WSA (100gig) < 3 < 20 < 3 < 20 6 < 20 3 < 20 3 < 20 3 < 20 3 BSA + WSA (300/lug) < 3 < 20 < 3 < 20 < 3 < 20 < 3 < 20 < 3 BSA + Mur-NAc-L-Ser-D- isoGln-OH (1001lug) < 3 < 20 3 < 20 50 < 20 800 230 260 These results show that the Mur - NAc - L - Ser - D - isoGln - OH, administered in saline isotonic solution, causes a considerable increase in the strength of antibody formed, although, under the same conditions, the WSA is practically inactive.

In the second series of tests, for which the dose of antigen, in the first injection, is weak, the presence of WSA even seems to inhibit the formation of antibody, although the Mur - NAc - L - Ser - D - isoGln - OH, on the contrary, favours the appearance of the antibodies.

The Mur - NAc - L - Ser - D - isoGln - OH may be considered in a general way as comparable to the LPS for its capacity to stimulate the formation of the antibodies, but, contrary to the LPS, it is without toxicity.

There are thus obtained adjuvant compositions without toxicity and which may be used to increase the efficiency of vaccines of bacterial or viral origin, especially when these are slightly immunising.

They may be used especially for promoting the immunisation of the host (human or animal patients) with regard to infections of bacterial or viral origin, or antigens of tumours. They are also effective for the production of serum containing active antibodies with regard to these antigens.

Claims (11)

WHAT WE CLAIM IS:

1. Compounds of the general formula:

wherein R1 is OH, NH,, NHCH3, N(CH3)2, SOCH2, OC2Hs or OC3H7 and R2 is OH, NHCH3, N(CH3)2, OCH3, OCH2Hs or OC3H,.

2. 2 - (2 - acetamido - 2 - deoxy- 3 - O- D - glucopyranosyl) - D propionyl - L - seryl - D - isoglutamine, which corresponds to the formula

3. A process for the preparation of a compound of formula (I) in which the peptide chain L-Ser-D-isoGln, L-Ser-D-Glu or an ester or amide derivative thereof as defined in claim 1 is attached to N-acetyl muramic acid whereby the reactive groups of the peptide chain other than the amino group carried by the L-seryl moiety and the reactive groups of the N-acetyl-muramic acid, other than its propionyl group have been previously blocked, and then the protected functions are freed.

4. A medicinal composition containing a product according to claim 1 and a pharmaceutically acceptable vehicle.

5. A medicinal composition which is constituted of an injectable solution of a product according to claim 1.

6. A medicinal composition constituted by an isotonic aqueous saline solution in which a product according to claim 1 is dissolved.

7. A medicinal composition constituted by a water-in-oil emulsion in which a product according to claim 1 is dissolved.

8. A medicinal composition according to claim 4 in a form for oral administration, or in a form for application on mucous membranes.

9. A medicinal composition according to any one of claims 4 to 8, in which the product according to claim I is associated with an immunising agent.

10. A medicinal composition according to claim 4 in which the product according to claim 1 is associated with an immunising agent and the composition is in the lyophilised state.

11. A pharmaceutical composition for the extemporaneous preparation of injectable solutions, comprising a dose of about 2 mg of a product according to claim 1 in a lyophilised state, in association with about 10 mg of lactose.