the conjugate according to the invention can be present either as a racemate or as optically pure substances . the bisacyloxypropylcysteine conjugate according to the invention is preferably a s -[ 2 , 3 - bis ( acyloxy )-( 2s )- propyl ]- l - cysteinylcarboxypolyethylene glycol ( bap - cys - peg ) in which the polyethylene glycol is bonded on by way of the carboxyl group of the cysteine and the amino group of the cysteine remains free . in another embodiment , the bisacyloxypropylcysteine conjugate according to the invention is preferably a s -[ 2 , 3 - bis ( acyloxy )-( 2r )- propyl ]- l - cysteinylcarboxy - polyethylene glycol ( bap - cys - peg ) in which the polyethylene glycol is bonded on by way of the carboxyl group of the cysteine and the amino group of the cysteine remains free . customary modifications and substitutions , which are known to the chemist , can be performed on the molecule while retaining its function or , in this case , its physiological effect . the radicals r 1 , 2 of the bisacyloxypropylcysteine conjugate according to the invention can be identical or different . those which are preferred at present are c 7 - 25 -, preferably c 8 - 22 - alkyl , - alkenyl or - alkynyl groups , with the unsaturated positions preferably being present in the cis configuration . the alkyl , alkenyl and alkynyl radicals can be branched or unbranched , cyclic or cycloalkyl - substituted radicals . suitable radicals r 1 and r 2 are sufficiently well known from fatty acid chemistry . with regard to the group y , it is simply a matter of establishing a stable linkage to the conjugate radical r 3 so as to ensure that the bisacyloxypropylcysteine is adequately bonded to r 3 and thereby remains water - soluble . the radical r 3 is preferably a polyethylene glycol radical . however , in a general manner , this radical is a covalently , ionically or associatively bonded , physiologically tolerated conjugate radical which is suitable for converting the bisacyloxypropylcysteine into an active water - soluble form . covalently bonded polymers are presently preferred . however , as an alternative , the conjugate radical can also be a dextran , a sugar , a polyvinylpyrrolidone , an alginate , a pectin or a collagen . in particular , dextran is used as a blood expander and is unobjectionable in view of the fact that it is physiologically tolerated . particularly in the case of highly polymerized conjugate radicals , it is also possible for several bap - cys units to be bonded to one conjugate radical r 3 . the molecular weight of the water - soluble polymer radical is preferably selected such that it amounts to from 100 to 30 000 daltons per bisacyloxypropylcysteine molecule . in the case of polyethylene glycol , a chain length m of from 5 to 700 , preferably of from 100 to 500 , is preferred but not imperative . in one preferred embodiment , the s -[ 2 , 3 - bis ( acyloxy )-( 2s )- propyl ]- l - cysteinylcarboxypolyethylene glycol is a s -[ 2 , 3 - bis ( palmitoyloxy )-( 2s )- propyl ]- l - cysteinyl - carboxypolyethylene glycol . in another preferred embodiment , the s -[ 2 , 3 - bis ( acyloxy )-( 2r )- propyl ]- l - cysteinylcarboxypolyethylene glycol is a s -[ 2 , 3 - bis ( palmitoyloxy )-( 2r )- propyl ]- l - cysteinylcarboxy - polyethylene glycol . the bisacyloxypropylcysteine conjugate according to this invention exhibits the advantage , as compared with previously known macrophage activators , that it combines good solubility in water with a macrophage activity which is comparatively very high ( see below ). it is not immunogenic , which means that no antibodies develop against the preparation when it is used in humans or animals . the invention furthermore encompasses pharmaceutical compositions which comprise the bisacyloxypropyl - cysteine conjugates according to the invention . these compositions include , inter alia , solutions of the bisacyloxypropylcysteine conjugate according to the invention . additional pharmaceutical additives and auxiliary substances can be present in these compositions . in particular , the bisacyloxypropyl - cysteine conjugate can be bonded , or adsorbed , to a pharmaceutically acceptable excipient . suitable excipients are known to the skilled person and are also available commercially . the pharmaceutical composition is preferably present in the form of a formulation which is suitable for injection , for inhalation or for intranasal or topical administration , with excipient - bonded administration forms being included . ointments , creams , tinctures , solutions and the like , inter alia , are provided for local administration , as known to the skilled person for this purpose . the bisacyloxypropylcysteine conjugates according to the invention , or the appurtenant pharmaceutical compositions , can be employed , inter alia , for stimulating macrophages , for stimulating antibody synthesis , for defense against infection , for stimulating immunity , particularly in regard to tumors , for preventing and treating septic shock , for wound healing and as an adjuvant for vaccines . substances which are administered together with the actual antigen ( i . e . the substance which provokes the desired immune reaction ) in connection with an immunization in order to augment the humoral and / or cell - mediated immune response are termed adjuvants . the use of optimal adjuvants is of crucial importance when employing substances for immunization . antigens only rarely mediate an adequate immune response when they are administered without adjuvants . in addition to this , it is not only a matter of the strength of the elicited immune response but also a matter of its quality . stimulating an incorrect immunization pattern can lead to immunopathologic reactions and a deterioration of the symptoms of the infection . in this connection , the adjuvant can help to support the desired immune reaction . adjuvants can be combined , to form vaccines , with a very wide variety of antigens . the antigens which are selected can , in particular , be target antigens for the prophylactic treatment of infectious diseases , tumors , autoimmune diseases , allergies and chronic or acute inflammatory diseases . a vaccination is also understood as being a treatment with antigens for monitoring fertility in human or animal populations . these uses encompass activating macrophages / monocytes or other cells which carry the receptor combination toll - like receptors 2 and 6 , with all the indirect consequences due to mediators , in animals or humans . this implies the use , as adjuvant ( that is as an adjunct for vaccines ), for tumor therapy in the widest sense , including the in - vitro priming against tumor antigens of autologous cells which are to be reimplanted , or by means of direct therapy , which can be effected locally or systemically , for generating crosstolerance against endotoxin or other corresponding microbial components , which protects against sepsis , and for accelerating wound healing . 1 . synthesizing a bisacyloxypropylcysteine according to the invention where r 1 and r 2 = palmitoyl , y = nh and r 3 = peg a . ( i ) is synthesized in accordance with a described method ( metzger , j ., wiesmüller , k .- h ., schaud , r ., bessler , w . g ., and jung , g ., 1991 , “ synthesis and novel immunologically active tripalmitoyl - s - glycerylcysteinyl bisacyloxypropylcysteines as useful intermediates for immunogen preparations ”. int . j . pept . protein res . 37 : 46 - 57 ; metzger , j . w ., wiesmüller , k .- h ., and jung , g ., 1991 , “ synthesis of n - fmoc protected derivatives of s -( 2 , 3 - dihydroxypropyl )- cysteine and their application in peptide synthesis ”, int . j . pept . protein res . 38 : 545 - 554 ). in particular where : r 1 , 2 = c 15 h 31 —( palmitoyl -) and r 3 =—( ch 2 — ch 2 — o ) x — ch 2 — ch 2 — nh 2 b . the carboxyl group is subsequently linked , using known methods ( carbodiimide synthesis ), to , for example , polymers which contain water - soluble nh 2 groups , e . g . diamino - peg . 34 mg ( 38 μmol ) of ( i ) are dissolved in 1 ml of dry dimethylformamide / dichloromethane 2 : 1 after which 6 μl ( 38 μmol ) of diisopropylcarbodiimide and 6 mg ( 38 μmol ) of 1 - hydroxybenzotriazole are added consecutively . 76 mg ( 38 μmol ) of diamino - peg 2000 are added to this mixture . after 24 hours at room temperature , the mixture is concentrated down to dryness and the fmoc protective group is eliminated using 20 % piperidine in dimethylformamide . the compound ( ii ) is purified by means of silica gel chromatography . it is characterized by means of nmr and mass spectroscopy . it is further purified by means of hplc on a c18 column at 40 ° c ., buffer 1 : 0 . 1 % tfa in water ; buffer 2 : 0 . 1 % tfa in 2 - propanol . the substance elutes at approx . 80 % v / v of buffer 2 . content analysis is effected by means of fatty acid determination using internally admixed c14 standard , after hydrolysis and glc , in accordance with standard methods , or by means of amino group determination using fluorescamine . in principle , the activation of macrophages and monocytes can be measured by a large number of parameters , for example by means of the release of cytokines , chemokines or arachidonic acid metabolites in cultures of human monocytes or murine peritoneal macrophages . the test which is used here is based on the simultaneous stimulation of mouse peritoneal macrophages with interferon - γ and macrophage activator , e . g . bap - cys - peg , so as to release nitrogen monoxide . ( reference : muhlradt , p . f ., and frisch , m ., 1994 , “ purification and partial biochemical characterization of a mycoplasma fermentans - derived substance that activates macrophages to release nitric oxide , tumor necrosis factor , and interleukin - 6 ”, infect . immun . 62 : 3801 - 3807 ). in brief , peritoneal macrophages from c3h / hej mice were used as the macrophage source . they were cultured in 96 - well microtiter plates and stimulated simultaneously with rifn - γ and a serial dilution of macrophage activator . insofar as necessary , the macrophage activators were dissolved in 25 mm octylglucoside in the first dilution step and then diluted further with medium . after an incubating time of 45 - 48 hours , the nitrate was reduced with nitrate reductase and the starting substance nitrogen monoxide was determined , as the sum of nitrate and nitrite , using griess &# 39 ; reagent . 1 unit ( u )/ ml is defined as the dilution at which half - maximal stimulation takes place . the results of the macrophage activation test are shown in fig1 . it can be seen from the figure that bispalmitoyloxy - propylcysteine - peg ( bpp - cys - peg ), i . e . a macrophage activator according to this invention , has a markedly higher potential for activating macrophages than has the known pam 3 - cys - peg ( in this present case designated tpp - cys - peg ). the figure shows that bpp - cys - peg already achieves the same degree of macrophage activation at a concentration which is approx . forty times lower than that of tpp - cys - peg . the figure furthermore shows that this outstanding and unexpected activation effect in the case of bpp - cys - peg is not noticeably improved by adding a solubilizer , in this case octylglucoside , whereas such an addition is required for the effect of pam 3 - cys - peg to be displayed optimally . the novel bisacyloxypropylcysteine conjugate according to this invention does not , therefore , require any additional , and possibly physiologically disadvantageous , solubilization by means of an organic solvent or detergent . another advantage of bap - cys - peg as compared with pam3 - cys - peg is the greater cell specificity , which can be attributed to the fact that this substance requires the cooperation of toll - like receptors 2 and 6 whereas the simultaneous presence of toll - like receptors 1 and 2 on the cell to be stimulated is sufficient for stimulation by pam 3 - cys - peg . the expression of toll - like receptor 6 is restricted to specific cells whereas toll - like receptor 1 is expressed ubiquitously by almost all the cells in the body . as in the case of diabetic patients , wound healing is also disturbed in diabetic mice and proceeds more slowly than in wild - type mice . the diabetic mouse is therefore an established animal model for wound healing . an area of approx . 4 × 4 cm in size was shaved on the backs of 20 diabetic mice ( 32 c57bkls / bom - db ) and this area was depilated with veet two days later , after which the cream was carefully removed . after a further two days , the depilated backs of the mice were disinfected with braunol . after anaesthesia had been initiated with isoflurane / air , an additional local anaesthesia was performed by means of the i . c . injection of 2 % xylocaine directly into the site envisaged for the excision , and the skin was defatted with trichloroethylene . after that , scissors were used to cut out a circular skin wound of 1 . 3 cm in diameter in each of the mice . the wounds were closed with a transparent plaster ( hydrofilm , f . hartmann ) and dressing adhesive . an additional , larger plaster , having an aperture corresponding to the wound , making possible continued observation of the wound , was adhered on top of the first plaster . 3 × 200 kunits of bpp - cys - peg , in methylcellulose and 5 % mouse serum ( as carrier ), were administered , per animal , through the transparent plaster on days 0 , 2 and 5 . control mice were only given the carrier mixture . the mice were observed for a period of 29 - 30 days . the plasters were changed at what were normally intervals of 5 days . administering bpp - cys - peg ( 3 × 200 ku ) accelerated the wound healing significantly ( see fig2 ). 3 . using malp - 2 derivatives or bpp - cys - peg as a mucosal adjuvant for eliciting an effective humoral response at both the systemic and mucosal levels . experimental procedure : 6 - 8 - week - old female balb / c ( h - 2d ) mice ( harlan winkelmann gmbh , borchen , germany ) were used for the experiments . groups of in each case 5 mice were administered 50 μg of β - galactosidase ( β - gal ) ( boehringer , mannheim , germany ), either on its own or together with 0 . 5 μg of synthetic r - malp - 2 , s - malp - 2 or bpp - cys - peg , as adjuvant , by the nasal route ( 25 μl ) on days 1 , 14 and 21 . serum samples were withdrawn on day 31 and stored at − 20 ° c . until the β - gal - specific antibodies were determined . nunc - immuno maxisorp test plates containing 96 wells ( nunc , roskilde , denmark ) were coated with 100 μl of β - gal ( boehringer , mannheim , germany ), containing 5 μg / ml in 0 . 05 mol carbonate buffer ( ph 8 . 2 ), per well . serial double dilutions of the sera or washings in pbs containing 1 % bsa and 0 . 05 % tween 20 were added ( 100 μl / well ) and the plates were incubated at 37 ° c . for 2 hours . after the plates had been washed , biotinylated γ - chain - specific goat anti - mouse igg ( sigma chemie , deisenhofen , germany ) was added and the plates were incubated at 37 ° c . for a further hour . after the plates had been washed four times , 100 μl of peroxidase - conjugated streptavidin ( pharmingen ) were added to the wells and the plates were incubated at 37 ° c . for 30 minutes . after the plates had been washed four times , the reactions were developed with abts in 0 . 1 mol citrate phosphate buffer ( ph 4 . 35 ) containing 0 . 01 % h 2 o 2 . the titers at the end point were given as the reciprocal log 2 of the last dilution which , after a 30 - minute incubation , gave an optimal density at 405 nm of 0 . 1 units above the values of the negative controls after a 30 minute incubation . vaginal and lung washings were obtained by rinsing the organs with 1 mm pbs which contained 50 mm edta , 0 . 1 % bsa and 10 mm pmsf . the washings were then centrifuged in order to remove tissue debris ( 10 min at 3000 × g ) and the supernatants were stored at − 20 ° c . in order to determine the concentrations of total iga in the lung and vagina washings , serial dilutions of the corresponding samples were incubated in microtiter plates with these plates having been previously coated with goat anti - mouse iga ( sigma chemie , deisenhofen , germany ) as the capturing antibody ( 100 μl / well ). serial dilutions of purified mouse iga ( sigma chemie , deisenhofen , germany ) were used for obtaining a standard curve . in order to investigate the capacity of r - malp - 2 , s - malp - 2 and bpp - cys - peg for stimulating effective humoral immune responses , the serum titers of β - gal - specific antibodies were determined in the inoculated mice . as fig3 shows , administering β - gal in the presence of bpp - cys - peg leads to the elicitation of antigen - specific igg titers which rise more rapidly than those obtained using r - malp - 2 . in order to determine the ability of the above derivatives to stimulate mucosal responses to antigens which were together intranasally ( i . n . ), the production of β - gal - specific iga was investigated in lung and vaginal washings from immunized animals . whereas no β - gal - specific iga was produced in the lung washings following i . n . inoculation with pure β - gal or mixtures of β - gal and the s - malp - 2 derivative , a significant increase in antigen - specific iga antibodies was observed in the case of the animals which were immunized with β - gal and , in addition , either r - malp - 2 or bpp - cys - peg ( fig4 ). the simultaneous administration of r - malp - 2 , or bpp - cys - peg , and antigen resulted in an effective iga production being stimulated even at distant mucosae , as was demonstrated by the presence of significant concentrations of β - gal - specific iga in vaginal washings ( fig5 ). 4 . using bpp - cys - peg as a mucosal adjuvant leads to the elicitation of an effective t cell - mediated proliferation response . the spleens were removed and taken together for analyzing the cell immune responses . the cells were propagated in rpmi 1640 , additionally containing 10 % calf serum , 100 u of penicillin / ml , 15 μg of streptomycin / ml , 5 × 10 − 5 m 2 - mercaptoethanol and 1 mm l - glutamine ( gibco brl , karlsruhe , germany ), and stored at 37 ° c . in a moist atmosphere containing 5 % co 2 . the cell suspensions were adjusted to 5 × 10 6 cells / ml in complete medium and sown , at the rate of 100 μl per well , in a 96 - well flat - bottomed microtiter plate ( nunc ); the plates were then incubated for 4 days in the presence of different concentrations of soluble β - gal . each concentration was tested in triplicate . 1 μci of [ 3 h ]- thymidine ( amersham international , freiburg , germany ) was added to each well for the last 8 hours of culture . these cells were then harvested on filter paper ( filtermat a ; wallac , freiburg , germany ) using a cell harvester ( inotech , wohlen , switzerland ) and the quantity of [ 3 h ] thymidine taken up into the dna of proliferating cells was determined using a scintillation counter ( wallac 1450 , micro - trilux ). the results were recorded as the ratios between the values ( mean values , determined in triplicate ) of stimulated and unstimulated samples ( stimulation index ). whereas the i . n . administration of β - gal on its own did not elicit any induction of detectable cell proliferation , the simultaneous administration of r - malp - 2 or bpp - cys - peg together with antigen led to an effective proliferation response ( fig6 ). it is noted that the strongest t cell proliferation response was observed in the spleen cells of mice which were immunized with bpp - cys - peg and β - gal . using s - malp - 2 gave a markedly weaker stimulation index and only led to restimulation when using the highest dose of β - galactosidase . the results which were obtained clearly show that bpp - cys - peg is at least as effective as , or even more effective than , r - malp - 2 as a mucosal adjuvant . effective humoral and cellular immune responses were obtained , both at the systemic level and the mucosal level , against antigens which were administered simultaneously with bpp - cys - peg .
the invention provides lipopeptide conjugates in which a cysteine that is double - substituted by a fatty acid is bonded by means of the carboxyl group to a highly soluble , physiologically compatible and non - immunogenic , polymeric conjugate group . the conjugates exhibit an excellent macrophage stimulant action and do not require addition solutizing . they can be used in a wide range of applications , in particular for stimulating macrophages , for stimulating antibody synthesis , for combating infection , as an immunostimulant , in particular in relation to tumors , for preventing and treating septicaemic shock , for would healing and as an adjuvant for vaccines .