The invention relates to substituted glyceroi derivatives. It concerns 3-0-[N- acetylmuramyl-L-lysyl-D-isoglutaminyl]-1 ,2-di-O-palmitoyl-sn-glycerol of formula I

in free form or in salt form, hereinafter referred to briefly as "the compound of the invention".

Compounds with a similar structure and related activity are known from e.g. ANVA EP 165123 and SANDOZ EP 406175. The compound of the invention is, however, nowhere specifically disclosed nor suggested in the above art. It possesses vastly more beneficial properties than the state of the art compounds. Especially, all the molecules of the MDP-GDP family which have been produced previously were found not to be soluble in water. This posed significant problems at the level of galenics since excipients used to dissolve or to incorporate such molecules, e.g. pluronic alcohols or liposomes were either interfering with the activity or rendering upscaling difficult. The compound of the invention is the first member of this family which can be dissolved in water and solubilization of at least 10 mg/ml is complete after 5 to15 minutes of sonication.

The present invention further concerns a process for the preparation of the compound of the invention which comprises deprotecting a corresponding compound of formula II

wherein R, to R ₄ independently are protecting groups, and recovering the resultant compound of formula I in free form or salt form.

The process of the invention may be effected in conventional manner for splitting off protecting groups. The starting material may be the α- or β-glycoside anomer or a mixture thereof. Deprotection may be effected in one or in several reaction steps. The compound of formula I is normally obtained as a mixture of both anomeric forms. The process may e.g. be effected reductively, preferably with hydrogen in a palladium catalyst on charcoal, using e.g. acetic acid as solvent, whereby any conventional protecting group susceptible of hydrogenation may be used, e.g. benzyloxycarbonyl (Z) or benzyl. R_, and R ₂ may also form together a common protecting group such as benzylidene. Deprotection may also be effected e.g. under acidic conditions. A preferred protecting group for deprotection under acidic conditions is tert.butoxycarbonyl (BOC).

The compound of formula I can exist in free form or in salt, especially acid addition salt form, e.g. the acetate or hydrochloride salt form. Salts can be prepared from the free form in conventional manner and vice-versa.

The starting material may also be prepared in conventional manner, e.g. according to the following reaction scheme:

Compounds of formula II

In the above scheme R, to R ₄ are as defined above, R' is an amino protecting group, X is an activated carboxylic acid group, BOP is a group benztriazol-1 -yloxytris- (dimethylamino)phosphonium hexafluorophosphate and BOC is tert.butoxycarbonyl.

Splitting off of R' preferably is effected under hydrogenolytic conditions. R' preferably is benzyloxycarbonyl. X preferably is -C(=0)-Oalkyl, such as -C(=0)-OCH ₂ CH(CH ₃ ) ₂ .

The following Example illustrates the invention. All temperatures are in degrees Centigrade.

The abbreviations used have the following meaning:

BOC = tert.butoxycarbonyl

Bz = benzyl

Z-D-iGIn = carbobenzoxy-D-isoglutamine

Pd/C = palladium on charcoal tBDMS = tert.butyldimethylsilyl

Z = benzyloxycarbonyl

iNOS: inducible nitric oxide synthase

OVA: ovalbumin, chickenegg albumin

Substance A: compound of the invention in acetate salt form

BPO-KLH: benzylpenicilloyl-bovine serum albumin

BSA: bovine serum albumin

ELISA: enzyme-linked immunosorbent assay

IL-1 (IL-1 B): interleukin-1 (interleukin-1 β)

LPS: lipopolysaccharide

MDP: muramyi dipeptide

MDP-GDP: 3-0-[N-acetylmuramyl-L-alanyl-D-isoglutaminyl]-1 ,2-di-0- palmitoyl-sn-glycerol (compound of Example 1 in ANVAR EP 165123)

TNF-α: tumor necrosis factor α

INF-γ ^* . interferon γ

PBS: phosphate-buffered saline

FIA: Freund's incomplete adjuvant

PCA: passive cutaneous anaphylaxis

Møs: murine peritoneal macrophages

Example: 3-0-[N-Acetylmuramyl-L-lysyl-D-isoglutaminyl]-1 ,2-di-0-palmitoyl-sn- glycerol

120 mg 3-0-[1 -α-0-benzyl-4,6-0-benzyliden-N-acetylmuramyl-(N _ε -Z-L-lysyl-)-D- isoglutaminyl]-1 ,2-di-0-palmitoyl-sn-glycerol are dissolved at 4° in 20 ml of 100 % acetic acid and reacted with prehydrogenated catalyst [90 mg 10 % Pd/C, 15 mg palladium chloride (PdCI ₂ ) in 20 ml of acetic acid 100 %, hydrogenated for 45 minutes with hydrogen]. The mixture is stirred for 2 hours under hydrogen, the catalyst is filtered off, the solution concentrated and evaporated to dryness thrice with toluene. The residue is chromatographed over siiicagel using dichloromethane/methanol/diisopropylether/ water/acetic acid (80/20/6/2/1 ) as the eluant. The resultant product is lyophylised and the title product is obtained as salt of the acetic acid.

¹ H-NMR: 0.88 (t, J=7Hz, 6H); 1 .27 (m, 48H); 1 .41 (d, J=7Hz, 3H); 1 .52 (m, 1 H); 1 .62 (m, 4H); 1 .70 (m, 2H); 1.80 (m, 1 H); 1.86 (m, 1 H); 1 .98 (s, 3H); 2.23 (m, 1 H); 2.33 (m, 4H); 2.47 (m, 2H); 2.93 (m, 2H); 3.45-3.90 (m, 6H); 4.18 (m, 2H); 4.23-4.40 (m, 5H); 5.20 (d, J=3Hz, 1 H); 5.30 (m, 1 H).

The starting material is obtained as follows:

a) 3-0-fBenzyloxycarbonyl-D-isoglutaminyll-1 .2-di-Q-palmitoyl-sn-glvcerol

740 mg Z-D-iGIn are dissolved in 6 ml of a mixture of dry dimethylformamide/ tetrahydrofuran (1/1 ) and reacted in darkness with 1 .46 g of benztriazol-1 -yloxytris-

(dimethylamino)phosphonium hexafluorophosphate (= BOP) and 365 μl of

N-methylmorpholine. After 30 minutes 1 .14 g of 1 ,2-di-O-palmitoyl-sn-glycerol and 270 mg of imidazole are added and the reaction mixture is stirred further in darkness for 4 days. After evaporation of the solvent mixture the residue is chromatographed over siiicagel using dichloromethane/methanol (20/1 ) as the eluant.

¹ H-NMR: 0.89 (t, J=7Hz, 6H); 1 .28 (s, 48H); 1 .62 (m, 4H); 1 .95 (m, 1 H); 2.15 (m, 1 H); 2.34 (m, 4H); 2.46 (m, 2H); 4.25 (m, 5H); 5.12 (s, 2H); 5.27 (m, 1 H); 7.35 (s, 5H).

b) 3-0-[tert.Butyloxycarbonyl-(N _c -Z-L-iysyπ-D-isoglutaminyl1-1 .2-di-Q-palmitoyl-sn- glycerol

400 mg of the compound obtained under a) are dissolved in 20 ml of 100 % acetic acid and reacted with 40 mg Pd/C 10%. Stirring is pursued for 2 hours under a hydrogen atmosphere, the catalyst is filtered off and the residue evaporated thrice to dryness with toluene. The residue is dissolved in 8 ml of dichloromethane under addition

of 60 μl of of N-methylmorpholine (= solution A). 152 mg of N _α -BOC-N _c -Z-lysine are dissolved in 8 ml of dichloromethane together with 233 μl of N-methylmorpholine and 73 μl of chloroformic acid isobutylester and the mixture is stirred at room temperature for 45 minutes (= solution B). Solution B is cooled to +4° and solution A is added thereto. The mixture is stirred for 18 hours at room temperature, the solvent is evaporated and purification effected by chromatography over siiicagel using a dichloromethane/methanol

(100/1 → 100/3) as the eluant.

¹ H-NMR: 0.88 (t, J=7Hz, 6H); 1 .28 (m, 48H); 1.40 (s, 9H); 1.60 (m, 4H); 1 .80 (m, 4H); 2.34 (m, 4H); 2.41 (m, 2H); 3.18 (m, 2H); 3.64 (m, 1 H); 4.00 (m, 1 H); 4.14 (m, 2H); 4.27 (m, 2H); 4.45 (m, 1 H); 5.10 (m, 2H); 5.34 (m, 1 H); 7.35 (m, 5H).

c) 3-0-[1-α-0-Benzyl-4.6-Q-benzy ιden-N-acetylmuramyl-(N _c -Z-L-lysyπ-D- isoglutaminyl]-1.2-di-Q-palmitoyl-sn-glvcerol

580 mg of the compound obtained under b) are reacted at +4° with 20 ml of trifluoroacetic acid and the mixture is stirred for 30 minutes. The solution is concentrated and evaporated to dryness twice with toluene. The residue is reacted with 10 ml of dichloromethane and 65 μl of N-methylmorpholine (= solution A). 278 mg α-O-benzyl-

4,6-benzylidene-N-acetylmuramic acid are dissolved in 10 ml of dichloromethane together with 259 μl of N-methylmorpholine and 81 μl of chloroformic acid isobutylester and the mixture is stirred for 35 minutes at room temperature (= solution B). Solution A is added dropwise to solution B at +4° and the mixture is allowed to stand for 2 days at room temperature. After evaporation of the solvent the residue is chromatographed over siiicagel using dichloromethane/methanol (100/1 → 10/1 ) as the eluant.

¹ H-NMR: 0.88 (t, J=7Hz, 6H); 1.25 (m, 48H); 1.37 (d, J=7Hz, 3H); 1.62 (m, 4H); 1.95 (m, 1 H); 1.98 (s, 3H); 2.12 (m, 1 H); 2.34 (m, 4H); 2.45 (m, 2H); 3.10 (t, J=7Hz, 2H); 3.65 (m, 4H); 4.00-4.40 (m, 1 1 H); 4.58 (dd, J=13 u. 40Hz); 4.97 (d, J=3Hz, 1 H); 5.25 (m, 1 H); 5.53 (s, 1 H); 7.30 (m, 15H).

Explanation of the Figures:

Figure 1 : Mean values of net induced total IgE (μg/ml) during the tertiary response.

Figure 2: Mean values of the ovalbumin specific IgE (Units/ml) during the tertiary response.

Figure 3: Mean values of total IgE (μg/ml) in the groups of mice that are treated with water or substance A during the secondary and tertiary responses.

Figure 4: Mean values of the ovalbumin specific IgE (Units/ml) in the groups of mice that are treated with water or substance A during the secondary and tertiary responses.

Figure 5: Passive cutaneous anaphylaxis reaction to the murine serum obtained on day 5 of tertiary response.

Figure 6: Cytokine gene expression in the murine elicited peritoneal macrophages 6 hours after in-vitro stimulation with water, LPS, IFN-γ or substance A in water or their combinations: Line 1. water 100 μl/ml

2. LPS 10 ng/ml

3. IFN-γ 10 U/ml

4. substance A 1 μg/ml

5. substance A 10 μg/ml

6. substance A 100 μg/ml 7 LPS (10 ng/ml) + substance A 1 μg/ml

8. LPS (10 ng/ml) + substance A 10 μg/ml

9. LPS (10 ng/ml) + substance A 100 μg/ml

10. IFN-γ (10 U/ml) + substance A 1 μg/ml

1 1. IFN-γ (10 U/ml) + substance A 10 μg/ml

12. IFN-γ (10 U/ml) + substance A 100 μg/ml

The compound of formula I in free form or pharmaceutically acceptable salt form, hereinafter briefly referred to as "the agent of the invention" or, when in acetate salt form, as substance A, possesses pharmacological activity. It is therefore indicated for use as a pharmaceutical. In particular it has been found to have a pronounced immunomodulating, antiallergic, antiviral, antiinflammatory and adjuvant activity. These activities can be demonstrated using various test methods explained in more detail hereinafter. Further, it has been found that the agent of the invention is characterized by much less side effects as compared to structurally similar compounds such as MDP- GDP; it is therefore better tolerated.

Test methods and results:

1. Pyrogenicity in the rabbit:

The test method is as described in the literature, e.g. in the US Pharmacopeia. The results obtained in this assay are shown in table 1 and demonstrate a marked improvement in side effects:

Table 1 Pyrogenicity in the rabbit

Substance Highest non-pyrogenic Lowest pyrogenic dose (μg/kg i.v.) dose (μg/kg i.v.)

A 3000 >1000

MDP-GDP 10 20

2. Adjuvant activity:

The ability of substance A to increase antibody responses to an antigen has been evaluated in BALB/C mice. Control and experimental mice (3-4 per group) weighing 20 grams each receive 20 μg of bovine serum albumin in 0.2 ml of PBS. For experimen¬ tal groups, substance A or MDP is added to the antigen to give final dosages of 50, 500 or 5000 μg/kg. All mice are bled on day 14 (primary response), are boosted on day 15 with 20 μg of BSA alone and are bled again on day 25 (secondary response). Pooled sera from each group are tested for antibody levels against BSA using standard ELISA

and horse radish peroxidase-conjugated anti-mouse IgM and anti-mouse IgG. Results presented as means of ELISA titers of 2 separate experiments for IgM and 3 separate experiments for IgG are shown in table 2. A very strong adjuvant activity is noted for substance A which is comparable to the activity of the prototype molecule MDP:

Table 2 Adjuvant activity of substance A

IMMUNIZATION MEAN ANTI-BSA TITER (units/ml) ^* )

Primary Secondary response response

First Second IgM IgG IgG μg/kg μg/kg

BSA 1000 BSA 1000 61 ± 8 25 ± 4 1 17 ± 33

BSA + Subst. A 50 BSA 1000 98 ± 45 67 ± 26 1525 ± 520

BSA + Subst. A 500 BSA 1000 495 ± 340 515 ± 85 10757 ± 1553

BSA + Subst. A 5000 BSA 1000 768 ± 136 893 ± 141 12306 ± 1588

BSA + MDP 50 BSA 1000 167 ± 103 204 ± 18 4597 ± 1078

BSA + MDP 500 BSA 1000 428 ± 73 621 ± 266 10925 ± 886

BSA + MDP 5000 BSA 1000 1248 ± 362 1565 ± 171 14667 ± 576

^* ) Titers are assessed by standard ELISA and units/ml are calculated from a standard curve which is constructed from a positive standard anti-BSA serum containing 1000 units/ml.

3. Antiallergic activity: Down-regulation of serum antigen specific IgE:

The control of specific IgE production against defined allergens appears to be the key for the control of several allergic reactions and diseases. The only available animal models for in vivo studies in rats and BALB/c mice are based on the induction and downregulation of i) BPO-KLH specific IgE forming cells derived from organs tested (D.L. Auci et al., Immunopharmacol. 26 [1992] 157-169) or ii) serum IgE levels after treatment with anti mouse IgD antibodies as well as proinflammatory cytokines in the organs tested (F.Kricek et al., European Immunology Meeting, Barcelona, June 1994).

3.1. Animal model and treatment schedule:

The ability of substance A to down-regulate serum antigen-specific IgE is evaluated in BALB/c mice. Control and experimental mice (7 per group) weighing 20 grams each are immunized on day 0 with 400 ng of Pertussis toxin in 0.2 mi of PBS i.p. and 50 μg of OVA in Freund's incomplete adjuvant (FIA) s.c. All mice are boosted on days 21 (secondary response) and 42 (tertiary response) i.p. with 10 μg of OVA in aluminium hydroxide gel. The mice are fed by gavage during the secondary and tertiary response (treatment A) or only during the tertiary response (treatment B). Control groups receive excipient (water) and experimental groups receive substance A which is given in the final dose of 2, 10 or 50 mg/kg.

Treatment A (during the secondary and tertiary responses): Group 1 = water, 0.4 ml per mouse, days 23 and 43. Group 2 = substance A / 200 μg per mouse (10 mg/kg), days 23 and 44. Group 3 = substance A / 1000 μg per mouse (50 mg/kg), days 23 and 44. Group 4 = substance A / 40 μg per mouse (2 mg/kg), days 22, 23, 24, 25, 26, 43,

44, 45 and 46. Group 5 = substance A / 200 μg per mouse (10 mg/kg), days 22, 23, 24, 25, 26, 43,

44, 45 and 46. Treatment B (during the tertiary response): Group 1 = water, 4 x 0.4 ml per mouse, days 43, 44, 45 and 46. Group 2 = substance A / 200 μg per mouse (10 mg/kg), day 44. Group 3 = substance A / 1000 μg per mouse (50 mg/kg), day 44. Group 4 = substance A / 40 μg per mouse (2 mg/kg), days 43, 44, 45 and 46. Group 5 = substance A / 200 μg per mouse (10 mg/kg), days 43, 44, 45 and 46.

All mice are bled on days -1 , 20, 31 , 41 , 47, 49, 51 , 53 and 56. Serum from each mouse is tested for IgE levels against goat anti-mouse IgE (total IgE) or OVA (antigen specific IgE) using standard ELISA or glutaraldehyde modified ELISA and horseradish per-oxidase-conjugated anti-mouse IgE. Results presented as means of ELISA titers of 7 mice per tested group are shown in table 3 and figures 1 to 4. The values for IgE are given in μg/ml while specific IgE is given as units/ml. Significant downregulation of the serum IgE level is noted for substance A after multiple treatment in the secondary and/or tertiary response.

Table 3 Net induced total IgE (μg/ml) in a tertiary response

Group Net induced IgE on day Peak IgE response of mice.

No. 5 ^* 7 ^* 9 ^* between days 5 -14

1 41.6 ± 17.0 29.2 ± 18.8 14.3 ± 16.6 46.0 ± 15.7

2 40.7 ± 40.7 13.2 ± 16.6 -3.7 ± 14.7 42.0 ± 40.0 (NS) (NS) (NS) (NS)

3 33.7 ± 17.2 26.8 ± 22.9 13.6 ± 19.8 34.9 ± 19.0 (NS) (NS) (NS) (NS)

4 16.5 ± 8.2 16.7 ± 3.2 6.3 ± 5.3 20.6 ± 5.7 ( 0.0181 ) (NS) (NS) (0.0106)

5 15.4 ± 24 5.6 ± 8.0 -0.5 ± 5.5 18.5 ± 21 .5 ( 0.0476 ) ( 0.0127 ) (NS) (0.0350)

Statistical comparisons of net induced total IgE levels between groups performed using the

Mann Whitney U Rank test.

5 ^* = day 5 tertiary response corresponds to day 47

7 ^* = day 7 tertiary response corresponds to day 49

9 ^* = day 9 tertiary response corresponds to day 51

14 ^* = day 14 tertiary response corresponds to day 56.

3.2. Passive cutaneous anaphylaxis (PCA):

Serum from each control and experimental mouse is tested for anti OVA IgE levels using standard PCA reaction in SKH mice. Results are presented as mean diameters of the PCA lesions on the skin of SKH mice (figure 5). Treatment during the tertiary response is as described under 3.1.

There is evidence of downregulation of OVA-specific serum IgE obtained from the substance A treated animals at 24 hours after the last treatment.

4. Immunomodulatory activity; inhibition of macrophage production of proinflammatory cytokines and nitric oxide (NO):

There is evidence that high levels of nitric oxide (NO) produced by inflammatory cells on activation by immunological stimuli such as bacterial lipopolysaccharide (LPS), cytokines, and immunoglobulins play a critical mediator role in skin disorders such as immune complex diseases and psoriasis, as well as in toxic shock and sepsis.

In this test the effect of substance A is compared with that of MDP on the expression of inducible NO synthase (iNOS) and subsequent production of NO in activated thioglycollate-elicited murine peritoneal macrophages (Møs). Peritoneal macrophages are obtained by standard method and the cells are cultured for 24 hours with excipient, LPS (10 ng/ml), IFN-γ (10 U/ml), MDP (1-100 μg/ml) and substance A (1-100 μg/ml). Production of NO is measured by assaying nitrites, stable metabolic products of NO using Griess reagent. For the determination of the cytokine gene expression, the Møs are cultured for 4 and 6 hours with excipient, LPS (10 ng/ml), IFN-γ (10 U/ml), MDP (1-100 μg/ml) and substance A (1 -100 μg/ml). Total cellular RNA is extracted from the cells using RNA-now (Biogentex, TX, USA), according to the procedure given by supplier. Northern blotting and hybridization with cytokines-specific oligonucleotide probes reveals that messenger RNA transcripts for iNOS are not detected in excipient-stimulated Møs or cells incubated with MDP or substance A alone. There is also no detectable NO production measured as nitrite in such culture media. Incubation of the Møs with either LPS or IFN-γ for 4 and 6 hrs results in significant expression of mRNA transcripts for iNOS, TNF-α and accumulation of high levels of nitrites in cultures at 24 hours. Co-incubation of the Møs with LPS (10 ng/ml) and substance A (1 -100 μg/ml) produces a dose-dependent reduction in the levels of mRNA transcripts for iNOS, TNF-α and IL-1 B. Results are shown in figure 6. Similarly, the levels of nitrites in culture media increase dose-dependently when the Møs are incubated with LPS and MDP but decrease dose-dependently when LPS is combined with substance A, as shown in tables 4 and 5:

Table 4 Nitrites production in the murine elicited peritoneal macrophages 24 hours after stimulation with MDP, MDP(Thr)GDP, substance A, LPS, IFN-γ or their combination (nitrites concentration in μM/10 ⁶ cells)

Concent. + medium + IFN-γ + LPS μg/ml 10 U/ml 10 ng/ml stimulant alone 0 2 12

MDP 1 0 12 20

5 0 12 20

10 0 14 22

50 0 18 20

100 0 16 16

MDP(Thr)GDP 1 0 14 26

5 0 28 30

10 0 30 18

50 0 40 20

100 0 30 14

Substance A 1 0 2 1

5 0 6 2

10 0 8 2

50 0 21 0

100 0 40 0

Table 5 Effect of various concentrations of substance A on LPS induced nitrites production in the elicited peritoneal macrophage cultures (nitrites concentration in μM/10 ⁶ cells)

LPS ng/ml substance A (μg/ml)

0 1 5 10 50 100

0 0 0 0 0 0 0

0.1 2 2 0 0 0 0

1 4 2 2 3 0 0

10 24 20 15 5 0 0

100 30 24 22 8 0 0

ln contrast, co-incubation of the Møs with IFN-γ and substance A results in higher steady state levels of mRNA transcripts for iNOS, TNF-α (figure 6) and significant accumulation of nitrites in culture media at 24 hours as shown in table 6:

Table 6 Nitrites production of the elicited peritoneal macrophages after stimulation with various concentrations of substance A and INF-γ (nitrites concentration in μM/10 ⁶ cells)

subst. A IFN-γ units/ml μg/ml 0 1 5 10 50 100

0 0 0 . 0 0 0 3

0.1

0.5 0 0 0 0 1 4

1 0 0 0 0 3 4

5 0 0 0 0 4 3

10 0 0 0 0 11 15

50 0 0 0 0 14 14

100 0 0 5 12 21 23

0 0 4 12 19 22

In addition, substance A dose-dependently inhibits nitrites production in the Møs when: i) added at -1 hour before the LPS (10 ng/ml), ii) added simultaneously with LPS (10 ng/ml), iii) added at +1 hour after the LPS (10 ng/ml).

Substance A (1 - 100 μg/ml) synergizes with IFN-γ (10 U/ml) in nitrites production when added at -1 hr, simultaneously, or at +1 hr to the Møs culture. Results are presented as production of nitrites μM per 10 ⁶ cells after 24 hours of the culture and shown in table 7:

Table 7 Effect different treatment schedules of substance A on nitrites synthesis from the LPS (10 ng/ml), IFN-γ (10 U/ml) and LPS/IFN-γ stimulated elicited peritoneal macrophages (nitrites concentration in μM/10 ⁶ cells)

Treatment schedule Stimulant cone. μg/ml medium IFN-γ LPS IFN-γ/LPS

substance A, 0.1 0 0 0 0

- 1 hr pretreatment 1 0 0 0 0

10 0 0 0 1

50 0 12 0 11

100 0 7 0 10 medium only, 0.1 0 0 3 26

- 1 hr pretreatment 1 0 0 3 29

10 0 0 3 29

50 0 0 3 30

100 0 0 2 33 substance A, 0.1 0 0 0 0 simultaneous 1 0 0 0 0

10 0 0 0 0

50 0 16 0 13

100 0 18 0 16

medium only, simul¬ 0.1 0 0 1 26 taneous 1 0 0 1 26

10 0 0 7 31

50 0 0 6 32

100 0 0 5 37 substance A, 0.1 0 0 0 0

+1 hr post stimulation 1 0 0 0 1

10 0 3 0 1

50 0 17 0 12

100 0 19 0 13 medium only, 0.1 0 0 1 24

+1 hr post 1 0 0 3 25 stimulation 10 0 0 9 31

50 0 0 6 31

100 0 1 5 29

Moreover, LPS and IFN-γ induced nitrites production is dose-dependently downregulated when substance A is added at +1 hr, simultaneously, or -1 hr to the culture; this effect is LPS-specific and does not affect IFN-γ induced nitrites production.

The blocking mechanism of the LPS induced proinflammatory cytokine synthesis by substance A can be studied in the presence of cycloheximide as protein synthesis inhibitor. Co-incubation of cycloheximide with substance A and LPS for 4 hrs abolishes the down- regulatory effect of substance A. This result demonstrates the antiinflammatory properties of the agent of the invention by induction of de novo protein synthesis.

The agent of the invention is thus indicated for use as a modulator of unspecific antimicrobial resistance for systemic enhancement of immune response and unspecific immunity. It is indicated e.g. in the curative or supportive treatment (i.e. together with further specific or supportive forms of therapy) of conditions of decreased immune response, in particular conditions of decreased cellular and humoral immune response and conditions of decreased oversensitivity reactions of the delayed type, and further in the treatment of conditions generally in which a modulation of the immune response is desired. It is in particular indicated for use in the curative or supportive treatment of pathological conditions related to idiopathic immunodeficiencies or immunodeficiencies of the type encountered in geriatric patients or in patients with heavy burns or generalised infections.

For the above indications the dosage to be used will depend of course e.g. on the nature and severity of the disease to be treated and the mode of administration used. For the large subject a suitable parenteral dosage is from about 0.1 mg to about 70 mg, administered e.g. once for the achievement of an adjuvant effect, e.g. in supportive treatment, or daily. Repeated administration may conveniently be effected two to four times per day or in retard form. Indicated unit dosage forms include from about 0.025 mg to about 35 mg of agent of the invention in situations of repeated administration and up to about 70 mg when a single administration for adjuvant treatment is desired.

Its immunomodulating activity further makes the agent of the invention indicated for use as an adjuvant in vaccines. For this mode of utilization the indicated daily dosage is from about 0.1 mg to about 50 mg, preferably from about 0.5 mg to about 10 mg, especially about 7 mg, administered on the day of vaccination. Conveniently a second administration at the same dosage is effected 2 to 4 weeks thereafter.

Further, the agent of the invention is indicated for use as antiinflammatory agent and immunosuppressant and antiproliferative agent for topical and systemic use in the prevention and treatment of inflammatory and hyperproliferative conditions and of conditions requiring immunosuppression such as a) the treatment of inflammatory and hyperproliferative skin diseases, such as psoriasis, atopic dermatitis, contact dermatitis and further eczematous dermatoses, seborrhoeic dermatitis, Lichen planus, Pemphigus, bullous Pemphigoid, Epidermolysis bullosa, vasculitides, erythemas, cutaneous eosinophilias, Lupus erythematosus and acne; b) the prevention and treatment of allergic diseases such as extrinsic asthma, rhinitis, conjunctivitis, atopic eczema, urticaria/angioedema, food/drug allergy and anaphylaxis; c) the prevention and treatment of

- resistance in situations of organ or tissue transplantation, e.g. of heart, kidney, liver, bone marrow and skin,

- graft-versus-host diseases, such as following bone marrow grafts, and autoimmune diseases such as rheumatoid arthritis, systemic Lupus erythematosus, Hashimoto's thyroidis, multiple sclerosis, Myasthenia gravis, diabetes type I and uveitis,

- skin manifestations of immunologically-mediated disorders; d) the treatment of alopecia areata, toxic shock and sepsis.

The agent of the invention may be administered systemically or topically. For use in the above indications the appropriate dosage will, of course, vary depending upon, for example, the host, the mode of administration and the nature and severity of the condition being treated. However, in general, beneficial results are indicated to be obtained systemically at daily dosages of from about 1.5 μg/kg to about 100 μg/kg animal body weight. An indicated daily dosage in the larger mammal is in the range of from about 0.1 mg to about 70 mg, conveniently administered, for example, in divided doses up to four times a day or in retard form.

The agent of the invention also shows antiviral activity, especially against HIV-1 and is therefore useful in the curative or supportive treatment of viral infections such as disseminated Herpes and disseminated varicella infections, especially in the treatment of AIDS.

Pharmaceutical compositions containing the agent of the invention together with at least one pharmaceutically acceptable carrier or diluent are also a part of the present invention. They may be prepared in conventional manner, e.g. according to a process comprising mixing the agent of the invention with a pharmaceutically acceptable carrier or diluent. Further pharmaceutical compositions which are indicated are in the form of liposomes and of mixed micelles with e.g. lysophosphatidyl choline, n-octyl glucose or deoxycholate. Such sompositions may be prepared in conventional manner and be e.g. in the form of injectable solutions. They are also a part of the present invention. The invention further includes a method of treatment, curative or supportive, of conditions as described above comprising administering to a subject in need of such treatment a therapeutically effective amount of the agent of the invention. It further comprises the agent of the invention for use in the above indications, especially for use as an immunomodulator, as an antiviral agent and as an antiallergic agent.