GB1572368A

GB1572368A - Pharmaceutical compositions containing glycolipids

Info

Publication number: GB1572368A
Application number: GB2661577A
Authority: GB
Original assignee: Individual
Current assignee: Individual
Priority date: 1976-06-25
Filing date: 1977-06-24
Publication date: 1980-07-30
Also published as: FR2355506A1; FR2355506B1; JPS533514A

Description

(54) PtIARMACEUTICAL COMPOSITIONS CONTAINING GLYCOLIPIDS (71) We, ICHIRO AZUMA, of 1-2, Aoyamadai, Suitas-hi, Japan and YUICHI YAMAMURA, of 1-9-22, Nikawa-takadai, Takarazuka-shi, Japan, both Japanese subjects, 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 present invention relates to pharmaceutical compositions and particularly to pharmaceutical compositions for use as an adjuvant.

In the immunotherapy of various diseases, a substance which is capable of enhancing the immunity of an antigen when introduced into the body of a host together with this antigen, is known as an adjuvant. Recently, various attempts have been made to use microorganisms or compounds produced by the fermentation of microorganisms as adjuvants in the immunotherapy of tumours.

For example, a fatty acid ester of trehalose produced by using a bacterium of the genus Mycobacterium and designated as "cord factor" has been reported to exhibit the activity of an adjuvant but possesses a high toxicity [Saikin-nai Dokuso (homma et al) 495-499 (1973) published by Kodansha Tokyo, Japan and H. Noll, Advanc.

Tuberc. Res. 7, 149 (1956)].

The present invention is based upon the discovery that glycolipids in which the sugar moiety is selected from fructose, sucrose and glucose possess good adjuvant activity and low toxicity and are of potential interest in enhancing the activity of protein antigens and tumour antigens. The glycolipids for use according to the present invention are thus fatty acid esters of fructose, sucrose and glucose and may, for example, be prepared by fermentation of microorganisms.

According to one feature of the present invention there is provided a pharmaceutical composition comprising as active ingredient at least one glycolipid, the sugar moiety of which is selected from fructose, sucrose and glucose, in association with a sterile pharmaceutical carrier or excipient.

The term "pharmaceutical composition" as used herein does not include within its scope a composition which is merely a solution of at least one glycolipid, the sugar moiety of which is selected from fructose, sucrose and glucose, in solely non-sterile water or a common solvent.

The pharmaceutical composition is conveniently in a form suitable for subcutaneous, intracutaneous, intramuscular, intravenous or intradermal administration. Preferably the pharmaceutical composition is contained in ampoules or vials, each ampoule or vial preferably containing from 2 y to 10 mg, especially from 4 to 300 P of the said active ingredient. If desired the pharmaceutical composition may additionally contain a protein antigen such as an amylase, egg white albumin or phytohaemaglutinin and/or a tumour antigen, for example, mastocytoma P815.

According to a further feature of the present invention there is provided a pharmaceutical composition comprising as active ingredients (1) at least one glycolipid (the sugar moiety of which is selected from fructose, sucrose and glucose) and (2) a protein antigen and/or a tumour antigen, in association with a pharmaceutical carrier or excipient.

As stated above the protein antigen may, for example, comprise a-amylase, egg white albumin or phytohaemoglutinin. The pharmaceutical composition may, for example, be in dosage unit form (as hereinafter defined) e.g. in the form of tablets, pills, capsules or vials or ampoules. Each dosage unit form preferably contains from 2 y to 10 mg, especially 4 to 300 y, of the said active ingredient (i.e.

the glycolipid).

According to a still further feature of the present invention there is provided a pharmaceutical composition in dosage unit form (as hereinafter defined) comprising as active ingredient at least one glycolipid, the sugar moiety of which is selected from fructose, sucrose and glucose, in association with a pharmaceutical carrier or excipient.

The term "dosage unit" as used herein means a pharmaceutical composition of the present invention in a form adapted to provide a single or unitary dosage of the said active ingredient. Thus, for example, tablets, pills and capsules may be in a form adapted to provide a single or unitary dose of the said active ingredient and thus such tablets, pills and capsules may be considered to be in dosage unit form.

As stated above each dosage unit preferably contains from 2 y to 10 mg, especially 4 to 30q y, of the said active ingredient and may, or course, additionally contain a protein antigen and/or a tumour antigen as described above.

According to a yet still further feature of the present invention there is provided a pharmaceutical composition in the form of granules, drops, syrups, emulsions or spray compositions comprising as active ingredients at least one glycolipid the sugar moiety of which is selected from fructose, sucrose and glucose.

Where the composition is in the form ofdrops, syrups, emulsions or spray compositions, such compositions preferably contain from 10 y to 10 mg/ml, especially from 100 y to 2 mg/ml of the said active ingredient.

The pharmaceutical compositions of the present invention preferably contain as active ingredient a glycolipid of the formula:

[I] Fructolipid [II] Sucrolipid [111] ' Giucolipid wherein R represents a mycolic acid residue, a nocardomycolic acid residue or a corynomycolic acid residue. Preferred active ingredients according to the present invention include glycolipids wherein the fatty acid residue is of the formula:

wherein R1and R2, which may be same or different, each represents an alkyl group.

With respect to the number of carbon atoms in the various above-mentioned acids, the following table illustrates the carbon atom content of each acid:- Acid R, R2 Mycolic acid various alkyl groups various alkyl groups in the range C2 > 24, of 2-8 types (C4167) mainly C22 Nocardomycolic various alkyl groups various alkyl groups acid in the range C10~18, of 2-8 types (C21~41) mainly C12-14 Corynomycolic various alkyl groups various alkyl groups acid in the range C10~18, of 2-8 types (C1123) mainly C2-14 Main peaks for the number of carbon atoms in R1 and R2 for glycolipids derived from certain microorganisms are exemplified as follows: Acid R1 R2 Origin Mycolid acid 20 & 22 57, 59, 61 & 63 Mycobacterium avium 20 & 22 55, 57, 59 & 61 M. paraffinicum Nocardomycolic acid 12 & 14 33, 37 & 39 Nocardia rubra acid l2 & 14 33, 35 & 37 N. parnffinica Corynomycolic acid acid 12 17, 19 & 21 Corynebacterium hydrocarboclastus 12 & 14 19, 21 & 23 Arthrobacter paraffineus The production and recovery of glycolipids for use in the pharmaceutical compositions of the present invention may be effected according to the methods described in Japanese Patent Publication No. 7349/72 and Japanese Patent Application No. 48186/75 (laid open to public inspection), each corresponding to U.S. Patents Nos. 3,637,461 and 3,909,356 respectively.

The glycolipids for use in the pharmaceutical compositions of the present invention may, for example, be prepared by culturing a microorganism of the genus Arthrobacter, Corynebacterium, Nocardia or Mycobacterium which is capable of producing a glycolipid by metabolizing a sugar. The cultivation is effected in a medium containing the sugar as main carbon source under aerobic conditions to accumulate a glycolipid corresponding to the sugar in the microorganism or medium, from which the glycolipid is recovered. Certain microorganisms which may be used with advantage for the purpose of the present invention are exemplified as follows.

Arthrobacter paraffineus ATCC 15591 Arthrobacter hydrocarboglutamicus ATCC 15583 Arthrobacter roseoparaffineus ATCC 15584 Arthrobacter simplex ATCC 15799 Corynebacterium hydrocarboclastus ATCC 15592 Corynebacterium hydrocarboclastus ATCC 21628 Corynebacterium pseudodiphtheriticum ATCC 10701 Nocardia paraffinica ATCC 21198 Nocardia globerula ATCC 13130 Nocardia rubra IFM 18;NRRL 11094 Mycobacterium rubrum ATCC 14346 Mycobacterium paraffinicum ATCC 12670 Mvcobacterium smegmatis ATCC 21293 Mvcobacterium smegmads ATCC 12297 Mvcobacterium avium IFO 3153; NRRL B-11095 The method of culturing these microorganisms is known per se, and a large amount of glycolipid may, for example, be obtained when the fermentation is effected at 25400C and at a pH of 4--9 (preferably 6-8) for 1--7 days. The method of recovering the glycolipid is also known per se.

In this manner, glycolipids represented by the formulae [I], [II] and [III] as hereinbefore defined may be produced. For example, when Arthrobacter paraffineus (ATCC 15591) is cultured by using sucrose as the main carbon source, it is possible to obtain sucrolipids represented by the following formulae [IV] and [V]:

in which R represents an alkyl group with 1018 carbon atoms and R2 represents an alkyl group with 11-23 carbon atoms.

When fructose is used as main carbon source for culturing the same microorganism, fructolipids represented by the following formulae [VI] and [VII] are obtained:

in which R, represents an alkyl group with 10--18 carbon atoms and R2 represents an alkyl group with 11-23 carbon atoms.

When Corynebacterium hydrocarboclastus (ATCC 21628) is cultured by using sucrose as the main carbon source, it is possible to produce glycolipids of the formulae [IV] and [V] as hereinbefore defined, and by using fructose as main carbon source the glycolipids of formulae [VI] and [VII] as hereinbefore defined may be produced.

The pharmaceutical compositions of the present invention may be formulated according to conventional methods known per se, for example, by adding the glycolipid of the present invention to a mineral oil, surfactant, physiological salt solution, or Freund's incomplete adjuvant (an emulsion of the water-in-oil type).

The composition thus obtained may, if desired, by used as a complete adjuvant.

The liquid pharmaceutical compositions of the present invention are preferably in a concentration of from 10 y/ml to 10 mg/ml (preferably 100 y/ml to 2 mg/ml). The amount to be administered in one week is conveniently 50 y/man to 10 mg/man (usually 100 to 300 y/man) for the average patient, the weight of such a patient being approximatley 60 Kg. The entire weekly dose may be administered at one time. The total dosage to be administered and the interval between doses may depend upon the response of the patient.

The composition may, for example, be administered subcutaneously, intracutaneously, intramuscularly, intravenously or intradermally. The composition comprising the glycolipid as active ingredient may be administered alone or in combination with an appropriate antigen, or the composition may itself contain the appropriate antigen.

The pharmaceutical compositions according to the present invention serve to enhance the immunity effected by protein antigens such as for example a-amylase, egg white albumin and phytohaemaglutinin, as well as the immunity of various tumour antigens, with low toxicity. Thus the pharmaceutical compositions of the present invention are of potential interest in human and/or veterinary medicine.

The activity of the pharmaceutical compositions according to the present invention was determined by using guinea pigs and mice as test animals. The results are shown in the following Examples, in which the activity is in general indicated by the enhancing activity upon the delayed-type hypersensitive reaction or upon the formation of the body liquid-type antibody. In the Examples, a known trehalose glycolipid is described in Journal of The National Cancer Institute, 52 95101 from Mycobacterium tubrcuThsis Aoyama B. The method of producing this glycolipid is described in Journal of The National Cancer Institute, 52, 95-101 (1974), and the glycolipids of the present invention used in the Examples were produced in a similar manner to that described in Japanese Patent Publication No.

7349/72 and Japanese Patent Application No. 48186/75 (laid open to public inspection).

Sucrolipids (corynomycolic acid) used in the following Examples may be produced in the following manner.

Arthrobacter paraffneus (ATCC 15591) was cultured with shaking in a medium containing sucrose (3 g/dl), meat extract (1.0 g/dl), peptone (1.0 g/dl) and sodium chloride (0.3 g/dl) and having a pH of 7.2 before sterilization, at a temperature of 30"C for 24 hours, and was then inoculated into another medium (3.0 litre) put in a 5 litre jar fermentor at a ratio of 10% by volume. This medium had the following composition: NaHPO4. 12H2O (0.2 g/dl); MgSO4. 7H2O (0.1 g/dl); FeSO4. 7H2O (0.05 g/dl); ZnSO4. 7H2O (0.001 g/dl); corn steep liquor (0.3 g/dl); KH2PO4 (0.2 g/dl); (NH4)2SO4 (0.5 g/dl); MnSO4 . 4H2O (0.002 g/dl); CaCI2 . 2H2O (one mg/I) and sucrose (10 g/dl).

The fermentation was effected at a temperature of 30"C for 45 hours with shaking (600 rpm) and aeration of sterilized air (one litre/litre/minute). The pH of the medium was automatically adjusted to 6.8-7.2 with ammonia. After completion of the fermentation, the cultured liquor (2.8 litre) was centrifuged to give microbial bodies (250 g by weight in wet state) which were extracted with a mixture of chloroform and methanol (1:1) 3 times. The extracted solution (3 litre) was then concentrated by flash evaporation and the residue was extracted with chloroform (100 ml). The solvent was evaporated off andthe residue was dissolved in n-hexane (21 ml). By centrifuging of the solvent, there was obatined an n-hexane solution (20 ml) containing a mixture of each two types of sucrose esters and of phospholipids.

The solution was transferred to a column (inner diameter 4 cm; height 20 cm) packed with silica gel (commercially available from Mallinckrodt Chemical Works, USA). Chloroform (1200 ml) was passed through the column to elute pigments and liberated fatty acids, and a mixture of chloroform (99-93) and methanol (1--7) (1500 ml) was passed through the column to elute fractions (each 50 ml). Fraction Nos. 15-27 were collected and combined, from which the solvent was removed.

The residue was dissolved in warm acetone (15 ml) and allowed to stand while cooling with ice to give precipitates which were then washed with cold acetone (20 ml). 'After drying there were obtained white powders (1.1 g) which represented sucrolipids (corynomycolic acid) used in the following examples, viz. sucrolipids of the formula [Il] as hereinbefore defined, in which R being a reside of corynomycolic acid, in which R being various alkyl groups having carbon atoms of 1018 (main peaks at 12 and 14) and R2 being various alkyl groups having carbon atoms of 11-23 (main peak at 23).

Glucolipids (mycolic acid) used in the examples may be produced in a similar manner to that described above with the exception that glucose is used instead of sucrose and that Mycobacterium avium (IFO 3153; NRRL B-11095) is used instead of Arthrobacter paraffineus (ATCC 15591). The thus-produced glucolipids correspond to glucolipids (mycolic acid) of the formula [III] as herein before defined, in which R being a residue of mycolic acid, in which R, being various alkyl groups having carbon atoms of 20--24 (main peak at 22) and R2 being various alkyl groups having carbon atoms of 4167 (main peaks at 57, 59, 61 and 63).

Fructolipids (mycolic acid) used in the examples may be produced in a similar manner to that described in the abovementioned reference with the exceptions that Mycobacterium avium (IFO 3153; NRRL B-1 1095) is cultured instead of Arthobacter paraffineus (ATCC 15591) and fructose is used instead of sucrose. The thusproduced fructolipids correspond to fructolipids of the formula [I] as hereinbefore defined, in which R being a residue of mycolic acid, in which R being various alkyl groups having carbon atoms of 20--24 (main peaks at 20 and 22) and R2 being various alkyl groups having carbon atoms of 41-67 (main peaks at 61 and 63).

Fructolipids (corynomycolid acid) used in the examples may be produced in a similar manner to that described in the above-mentioned reference with the exception that fructose is used instead of sucrose. The thus-produced fructolipids correspond to the fructolipids of the formula [I] as hereinbefore defined, in which R being a residue of corynomycolic acid, in which R1 being various alkyl groups having carbon atoms of 1018 (main peaks at 12 and 14) and R2 being various alkyl groups having carbon atoms of 11-23 (main peaks at 19, 21 and 23).

EXAMPLE 1 Enhancing activity upon the delayed-type hypersensitive reaction (skin reaction test) Glycolipids shown in Table I were used as test samples.

TABLE 1 Skin Reaction Test (mm/mm) Guinea After 4 After 48 pig hours hours Trehalolipid** 1 16x16 17x1d (mycolid acid)* 2 15x15 16x14 3 17x17 15x12 4 15x15 14xl0 Sucrolipid 1 22x22 20x 16 (corynomycolic 2 20x20 19x18 acid)* 3 18x18 15x1l 4 17x17 13x13 Glucolipid 1 20x20 20x 14 (mycolic acid)* 2 20x20 19x 14 3 18x18 15x15 4 16x16 14x13 Fructolipid 1 18x18 20x20 (mycolic acid)* 2 16x16 19x18 3 14x14 19x17 4 14x14 16x13 Fructolipid 1 16x16 16x14 (corynomycolic 2 15x15 14x12 acid)* 3 15x15 13x10 4 15x15 10x8 No additive 1 20x20 4x4 (control) 2 18x18 7x7 3 18x18 0x0 **Reference adjuvant.

*Fatty acid moiety.

A mixture of a glycolipid (300 yg) and a bacterial a-amylase (300 g; prepared from Bacillus subtilis) which was available commercially from Seikagaku Kogyo K, K., Tokyo was homogenized after addition of a smallest possible amount of Drakeol 6VR (a mineral oil available commercially from Pennsylvania Refining Co., USA). After this, the mixture was added with a physiological solution of sodium chloride containing Tween 80 [a surfactant available commercially from Atlas Chemical Industries Inc., USA (the work 'Tween' is a registered Trade Mark)] in such an amount that gave a final concentration of 0.2 /" and was further homogenized to give an oil-in-water type mixture.

Some groups of guinea pigs, each consisting of 4 guinea pigs (weight 250300 g),. were used as test animals, each of which was immunized by intramuscular injection of the oil-in-water type mixture (0.2 ml in total) in four footpads.

After 4 weeks a physiological solution of sodium chloride (0.1 ml) containing the said a-amylase (100 yg) was intradermally administered to each of the test animals at its back to investigate the skin reaction. The results are shown in Table 1 wherein the skin reaction is evaluated by the sizes of the erythemata after 4 hours and the induration after 48 hours from the injection of the skin antigen to be determined.

A known trehalolipid and the antigen without addition of the glycolipid were used for reference and control purposes respectively. In Table 1 and the following tables, the fatty acid in each of the glycolipid is indicated in the parenthesis.

EXAMPLE 2 Glycolipids shown in Table 2 were used in the test which was carried out in a similar manner to that described in a report by Okada et al [J. Biochem., 54, 477483 (1963)].

A smallest possible amount of Drakeol 6VR was added to a mixture of a glycolipid (300 elg) and a bacterial a-amylase prepared from Bacillus subtilis (300 g). The mixture was homogenized and was added with a physiological solution of sodium chloride which contain Tween 80 in such an amount that gave a final concentration of 0.2%. The mixture was further homogenized to give an oil-inwater type mixture.

Some groups of guinea pigs, each consisting of 4 guinea pigs (weight 2(X300 g.),. were used as test animals, each of which was immunized by intramuscular injection of the oil-in-water type mixture (0.2 ml in total) into four footpads. A blood sample (25 cm1) was collected and centrifuged to give a serum sample (13,us) which was then diluted [x20, x 100 and x1000] with physiological solution of sodium chloride. The diluted serum was mixed with the bacterial a-amylase (0.6 clog) dissolved in physiological solution of sodium chloride.

The activity of -amylase in the serum was determined, from which there was calculated a ratio of the dilution of the serum required for neutralizing a given amount of a-amylase. The calculative value was indicated by the value of the antibody against a-amylase, viz. one unit of the antibody against a-amylase corresponds to the ratio of the dilution of the serum which is able to neutralize just 10 units of a-amylase activity. The results are shown in Table 2 where a known trehalolipid and a system without addition of the glycolipid are used for reference and control purposes respectively.

TABLE 2 Antibody value against a-amylase (unit) in the serum Antibody value (unit) Guinea pig Average value Trehalolipid 1 1141 1250+91 (mycolic acid) 2 1498 (44.6) (reference) 3 1097 4 1266 Sucrolipid 1 313 176+47 (corynomycolic 2 162 (6.3) acid) 3 116 4 111 Glucolipid 1 133 99+21 (mycolic acid) 2 72 (3.5) 3 69 4 v 55 Fructolipid 1 347 264+50 (mycolic acid) 2 314 (9.5) 3 235 4 162 Fructolipid 1 265 176+61 (corynomycolic 2 175 (6.3) acid) 3 87 4 not measured Without addition 1 43 28+8 of glycolipid 2 26 (1.0) (control) 3 17 EXAMPLE 3 Glycolipids shown in Table 3 were used for the test. A glycolipid (100 yg), phytohemagglutinin (500 yg; commercially available from Difco Laboratories, USA) and Freund's incomplete adjuvant (0.05 ml) were mixed together and the mixture was administered to each test animal by subcutaneous injection into the right hind paw. Some groups of mice, each consisting of 10 mice (weight 20.5 g in average) were used as test animals and immunized in this manner. After 12 days from the injection, phytohemagglutinin (20 frog) was subcutaneously injected into the left hind paw. After 24 hours, the thickness of the left paw was measured and compared with the thickness before injection. The difference was used to determine the strength of the delayed-type hypersensitive reaction. In Table 3 showing the results, a known trehelolipid was used for reference purpose and also a system without addition of the glycolipid was used as control.

TAUTr z Delayed-type hypersensitive reaction (1/10 mm) Trehalolipid (mycolic acid) 8.2 Sucrolipid (corynomycolic acid) 3.5 Glucolipid (mycolic acid) 4.9 Fructolipid (mycolic acid) 7.8 Fructolipid (corynomycolic acid) 4.0 Control (without addition of glycolipid) 2.1 EXAMPLE 4 Test on the lysis of target cells Glycolipids shown in Table 4 were used for the test. Tumor cells (2x 104/ml) of mastocytoma P 815 were mixed with an oil-in-water type mixture prepared in a similar manner to that described in Example 2 except that a-amylase was not used.

The mixture was administered to each of the test animals by intraperitoneal injection. Some groups of mice, each consisting of 4 mice (weight 30 g in average), were used for this purpose. After 14 days the spleen cells of the test animals were excised. The spleen cells (one ml containing 1x107 cells) were mixed with mastocytoma P815 (one ml containing lx105 cells) labelled with radioactive chrome (51chrome) and were incubated at 370C for 20 hours. The evolved amount of 5'Cr was counted by a well-type scintillation counter. The degree of the lysis of the target cells was indicated by a ratio of the amount of the evolved radioactive chrome to the radioactivity in total. In Table 4 showing the results, a known trehalolipid and a mixture which does not contain the glycolipid are used for reference and control purposes respectively.

TABLE 4 Ratio of the lysis of target cells (%) Trehalolipid (mycolic acid)** 68 Sucrolipid (corynomycolic acid) 30 Glucolipid (mycolic acid) 65 Fructolipid (mycolic acid) 55 Fructolipid (corynomycolic acid) 38 Control (without addition of glycolipid) 8 **Reference.

EXAMPLE 5 The actue toxicities of the glycolipids according to the present invention were investigated in the following manner. Glycolipids shown in Table 5 were used in the test. As test animals, some group of mice, each consisting of 5 male mice (weight 20.2 g in average), were used. A mixture of Tween 80 (a surfactant; 0.2 ml), physiological solution of sodium chloride (0.5 ml) and a glycolipid was administered to each of the test animals by intraperitoneal injection. The animals were observed for 14 days. The amount of the glycolipid in the mixture was changed from 150 mg/kg to 500 mg/kg through 250 mg/kg, and the results are shown in Table 5, from which it is apparent the acute toxicities of all of the glycolipids according to the present invention being more than 250 mg/kg.

TABLE 5 500 250 150 (mg/kg) Sucrolipid (corynomycolic acid) 1/5 0/5 0/5 Glucolipid (mycolic acid) 3/5 0/5 0/5 Fructolipid (mycolic acid) 0/5 0/5 0.5 Fructolipid (corynomycolic acid) 1/5 0/5 0.5 WHAT WE CLAIM IS: 1. A pharmaceutical composition (as herein defined) comprising as active ingredient at least one glycolipid, the sugar moiety of which is selected from fructose, sucrose and glucose, in association with a sterile pharmaceutical carrier or excipient.

2. A composition as claimed in Claim 1 contained in an ampoule or vial.

3. A composition as claimed in Claim 2 wherein each ampoule or vial contains from n }; to 10 mg of the said active ingredient.

4. A composition as claimed in Claim 3 wherein each ampoule or vial contains from 4 to 300 y of the said active ingredient.

5. A composition as claimed in any one of the preceding claims which further contains a protein antigen and/or a tumour antigen.

6. A composition as claimed in Claim 5 wherein the protein antigen comprises c-amylase, egg white albumin and/or phytohaemaglutinin.

7. A pharmaceutical composition comprising as active ingredients (1) at least one glycolipid (the sugar moiety of which is selected from fructose, sucrose and glucose) and (2) a protein antigen and/or a tumour antigen, in association with a pharmaceutical carrier or excipient.

8. A composition as claimed in Claim 7 wherein the protein antigen comprises a-amylase, egg white albumin and/or phytohaemaglutinin.

9. A composition as claimed in Claim 7 or Claim 8 in the form of dosage units (as herein defined).

10. A composition as claimed in Claim 9 wherein each dosage unit contains from 2 y to 10 mg of the said active ingredient.

**WARNING** end of DESC field may overlap start of CLMS **.

Claims

**WARNING** start of CLMS field may overlap end of DESC **. were used for this purpose. After 14 days the spleen cells of the test animals were excised. The spleen cells (one ml containing 1x107 cells) were mixed with mastocytoma P815 (one ml containing lx105 cells) labelled with radioactive chrome (51chrome) and were incubated at 370C for 20 hours. The evolved amount of 5'Cr was counted by a well-type scintillation counter. The degree of the lysis of the target cells was indicated by a ratio of the amount of the evolved radioactive chrome to the radioactivity in total. In Table 4 showing the results, a known trehalolipid and a mixture which does not contain the glycolipid are used for reference and control purposes respectively. TABLE 4 Ratio of the lysis of target cells (%) Trehalolipid (mycolic acid)** 68 Sucrolipid (corynomycolic acid) 30 Glucolipid (mycolic acid) 65 Fructolipid (mycolic acid) 55 Fructolipid (corynomycolic acid) 38 Control (without addition of glycolipid) 8 **Reference. EXAMPLE 5 The actue toxicities of the glycolipids according to the present invention were investigated in the following manner. Glycolipids shown in Table 5 were used in the test. As test animals, some group of mice, each consisting of 5 male mice (weight 20.2 g in average), were used. A mixture of Tween 80 (a surfactant; 0.2 ml), physiological solution of sodium chloride (0.5 ml) and a glycolipid was administered to each of the test animals by intraperitoneal injection. The animals were observed for 14 days. The amount of the glycolipid in the mixture was changed from 150 mg/kg to 500 mg/kg through 250 mg/kg, and the results are shown in Table 5, from which it is apparent the acute toxicities of all of the glycolipids according to the present invention being more than 250 mg/kg. TABLE 5 500 250 150 (mg/kg) Sucrolipid (corynomycolic acid) 1/5 0/5 0/5 Glucolipid (mycolic acid) 3/5 0/5 0/5 Fructolipid (mycolic acid) 0/5 0/5 0.5 Fructolipid (corynomycolic acid) 1/5 0/5 0.5 WHAT WE CLAIM IS:

1. A pharmaceutical composition (as herein defined) comprising as active ingredient at least one glycolipid, the sugar moiety of which is selected from fructose, sucrose and glucose, in association with a sterile pharmaceutical carrier or excipient.

2. A composition as claimed in Claim 1 contained in an ampoule or vial.

11. A composition as claimed in Claim 10 wherein each dosage unit contains

from 4 to 300 y of the said active ingredient.

12. A pharmaceutical composition in dosage unit form (as herein defined) comprising as active ingredient at least one glycolipid, the sugar moiety of which is selected from fructose, sucrose and glucose, in association with a pharmaceutical carrier or excipient.

13. A composition as claimed in Claim 12 in the form of tablets, capsules or pills.

14. A composition as claimed in Claim 12 or Claim 13 wherein each dosage unit contains from 2 y to 10 mg of the said active ingredient.

15. A composition as claimed in Claim 14 wherein each dosage unit contains from 4 to 300 y of the said active ingredient.

16. A pharmaceutical composition in the form of granules, drops, syrups, emulsions or spray compositions comprising as active ingredient at least one glycolipid the sugar moiety of which is selected from fructose, sucrose and glucose.

17. A composition as claimed in Claim 16 in the form of drops, syrups, emulsions or spray compositions which contain 10 y to 10 mv/ml of the said active ingredient.

18. A composition as claimed in Claim 17 which contains from 100 y to 2 mg/ml of the said active ingredient.

19. A composition as claimed in any one of the preceding claims wherein the glycolipid comprises a compound of the formula:

(wherein R represents a mycolic acid residue, a nocardomycolic acid residue or a corynomycolic acid residue).

20. A composition as claimed in any one of the preceding claims wherein the fatty acid residue of the glycolipid is of the formula:

wherein R, and R2, which may be the same or different, each represents an alkyl group.

21. A composition as claimed in Claim 20 wherein the fatty acid residue of the glycolipid is a mycolic acid residue of formula VIII in which R, represents an alkyl group with 20 to 24 carbon atoms and R2 represents an alkyl group with 41 to 67 carbon atoms.

22. A composition as claimed in Claim 20 wherein the fatty acid residue of the glycolipid is a nocardomycolic acid residue of formula VIII in which R, represents an alkyl group with 1018 carbon atoms and R2 represents an alkyl group with 21 to 41 carbon atoms.

23. A composition as claimed in Claim 20 wherein the fatty acid residue of the glycolipid is a corynomycolic acid residue of formula VIII in which R, represents an alkyl group with 10 to 18 carbon atoms and R2 represents an alkyl group with 11 to 23 carbon atoms.

24. A composition as claimed in any one of the preceding claims wherein the glycolipid is obtained by fermentation of a microorganism.

25. A composition as claimed in Claim 24 wherein the glycolipid is obtained by fermentation of a microorganism of the genus Arthrobacter, Corynebacterium, Nocardia, or Mycobacterium.

26. A composition as claimed in Claim 25 wherein a microorganism of the species A rthrobacter paraffineus, Arthrobacter roseoparaffineus, Arthrobacter hydrocarboclastus or Arthrobacter simplex is used.

27. A composition as claimed in Claim 25 wherein a microorganism of the species Corynebacterium hydrocarboclastus or Corynebacterium pseudodiphtheriticum is used.

28. A composition as claimed in Claim 25 wherein a microorganism of the species Nocardia paraffnica, Nocardia globerulla or Nocardia rubra is used.

29. A composition as claimed in Claim 25 wherein a microorganism of the species Mycobacterium avium, Mycobacterium rubrum, Mycobacterium paraffinicum or Mycobacterium smegmatis.

30. A composition as claimed in Claim 29 wherein the microorganism used is Mycobacterium avium (IFO 3153 NRRL B-11095).

31. A composition as claimed in Claim 26 wherein the microorganism used is Arthrobacter paraffineus (ATCC 15591).