EP0693082A1

EP0693082A1 - Immuno modulated peptide derivatives of leukokinin fragments

Info

Publication number: EP0693082A1
Application number: EP94913125A
Authority: EP
Inventors: René-Pierre BÜNTER
Original assignee: Borella Fabio
Current assignee: Borella Fabio
Priority date: 1993-04-09
Filing date: 1994-04-08
Publication date: 1996-01-24
Also published as: ITMI930724A1; WO1994024154A2; WO1994024154A3; IT1264129B1; ITMI930724A0

Abstract

The present invention relates to oligopeptide derivatives of leukokinin fragments, containing at least one residue deriving from 1-amino-adamantane, having immunostimulating, immunoadjuvant and antiviral activity, a process for their preparation, and therapeutical compositions containing them as the active principles.

Description

IMMUNO MODULATING PEPTIDE DERIVATIVES OF LEUKOKININ FRAGMENTS.

FIELD OF THE INVENTION

The present invention relates to oligopeptide derivatives of leukokinin fragments, having immunomodulating activity, a process for their preparation and pharmaceutical compositions containing them.

PRIOR ART DISCLOSURE

Immunomodulating agents are substances able to act on the organism natural defence, either by stimulating the immune system in case this is depressed, or by inhibiting same if it is hyperexcited.

Some of these compounds found a therapeutical application, for example in the immunotherapy of tumours and in the treatment of recurrent infections and allergic diseases. In particular these substances are defined as immunostimulants when showing a prevailingly stimulating action and as immunoadjuvants when they are able to enhance antigens immunostimulating action.

Immunoadjuvants can be advantageously used in the field of vaccines, if administered in association with the antigens which form the same vaccines.

As the result of an intensive research carried out during the course of the last twenty years, several compounds have been identified, characterized by showing immunomodulating activity, comprising in their molecular structure, peptides, glycopeptides or lipopeptides.

Most of these compounds have not been clinically tested, whereas other compounds did not prove sufficiently active when clinically experimented. Some of them gave serious side-effects, other compounds showed too short half-lives or a very wide non specific activity pattern, that it resulted very difficult to use them for long time periods. Particularly, among the compounds having immunomodulating activity we mention muramyl dipeptide (MDP) or N-acetylmuramyl-L- alanyl-D-isoglutamine, a product of bacterial origin which shows pyrogenic activity, and its correlated compounds, called muramyl or desmuramyl peptides because of the presence or the absence in their structure of muramic acid residue, respectively. In particular it was found that some derivatives free from this residue maintain immunoadjuvant and immunomodulating activity, and that the insertion of lipophylic residues both in muramyl and in desmuramyl peptides brought to a biologic activity increase. (Adam A., Synthetic adjuvants, Modern Concepts in Immunology. Ed. C.A. Bona, J. Wiley and Sons, Inc. N.Y. pp 10-17, 1 85).

In addition, it is known that the effect of several immunostimulants having peptidic origin may be increased when they are administered in combination with mineral oils or if they are incorporated in liposome type structures (Masek K. et al., Experientia, 3**, 1363. 1978). On the base of the preceding research with muramyl peptides having bacterial origin, more than ten years ago Masek and co- workers were able to identify an other compound having immunostimulating and immunoadjuvant activity, where the muramyl dipeptide essential molecule, namely L-alanine-D-isoglutamine , was linked to 1 - amino- admantane.

The resulting compound, known as adamantyl dipeptide (AdDp) , proved to possess also rather considerable antiviral activity (Masek K. et al . , The immunomodulatory property of a novel synthetic compound, adamantyl amide-dipetide, Meth. Find. Exptl.

Clin. Pharmacol. 6, 667, 1984; Masek K. , Immunopharmacological Properties of Synthetic Muramyl Peptides and Their Analogs, in: Advances in the Bioscience 68, Immunomodulators and Nonspecific Host Defence Mechanism, against Microbial Infections, Eds. K.N. Masihi and W. Lange, Pergamon Press, pages 11-19, 1988).

It is known that tetrapeptides derived from fragments 289- 92 of long chain leukokinins increase the macrophages phagocytosis activity as well as the cytolytic activity of granulocytes and T cells, stimulating through these and probably other activities both the humoral and the cell-mediated immunity (V.A. Na jar and K. Nishioka, Nature 228,672, 1970; V.A. Najjar et al.. The Chemistry and Biology of Tuftsin in: Lymphokines Reports , Vol.l, PP 157-179. academic Press, N.Y., I98O ; K. Nishioka et al., Biochim. Biophys. Acta 310, 230, 1973). The presence of adamantane residue in oligopeptides imparts these compounds advantageous pharmacokinetic properties (Walder P.et al., Pharmacokinetic profile of the immunomodulating compound adamantylamide dipeptide (AdDP) , a muramyl dipeptide derivative in mice. Immunopharm, and Immunotoxicol . 13, PP 101-119, 1991) and in particular it improves the transfer of oligopeptides to various organs and immuno competent cells (Tsuzuki N. et al., Adamantane as a brain directed drug carrier for poorly adsorbed drug : antinociceptive effects of (D-ala ) Leu-Enkephalin derivatives conjugated with 1 -adamantane moiety, Biochem. Pharmacol.41, R5-R8, 1991 ) •

The need is felt to find new and very active compounds, which are well tolerated by organism and have a high immunostimulating and immunoadjuvant activity. DETAILED DESCRIPTION The Applicant has now unexpectedly found a new type of immunomodulating and immunoad uvant compounds, where the hydrophobic and metabolically stable structure of adamantane is linked to endogenous leukokinin fragments. In particular, the derivatives of the present invention are oligopeptide derivatives of endogenous leukokinin fragments, containing at least one residue L-Glu(NH-Ad) , and their pharmaceutically acceptable salts.

As the salts we mean those obtained by salification with acids or with bases both of organic or inorganic type. The residue L-Glu(NHAd) is L-glutamic acid gamma- (1- adamantylamide) ; Ad indicates the hydrocarbon residue present in

1- amino- adamantane (Ad-NI^) •

In the present description the peptides nomenclature used was that recommended in "Nomenclature and Symbols for aminoacids and peptides" , Eur. J . Biochem. 138, 9 (1984) .

The symbols relating to aminoacids denote, where not differently indicated, the L configuration of the carbon atom bringing the α- aminic group .

Endogenous leukokinin are peptides of natural origin present in eukaryotic cells , for example of mammals .

In particular, as the oligopeptide derivatives of leukokinin fragments we mean in the instant description oligopeptides consisting of aminoacids present in endogenous leukokinin fragments . More in particular , the present derivatives consist of oligopeptides having aminoacids in common with endogenous leukokinin fragments , in which one or more , preferably from 1 to

3 , L-Glu(NHAd) residues , replace or alternate the aminoacids residues of these fragments . Examples of leukokinin fragments are 282-292 residue , also known as tuftsin (Thr-Lys-Pro-Arg) , 245-248 residue (Pro-Lys-Pro-Lys ) and 341-344 residue (Gly-Gln-Pro-Arg) .

The aminoacid residues number in the present oligopeptides ranges from 2 to 10, preferably from 4 to 5 - The oligopeptides of the invention are completely different from MDP and AdDP immunomodulators, because they contain a peptidic portion of animal (mammal in particular) , rather than bacterial origin.

The Applicant has found that the oligopeptides of the invention are highly active "in vivo", in particular in stimulating the cellular and humoral immune reaction, and have a strong antiviral activity.

Moreover, the present oligopeptides have a low toxicity, rendering them well tolerated by the organism, and in particular they are not pyrogen.

The oligopeptides of the invention are useful in the human therapy, both alone and in combination with other therapeutically active substances; for example, because of their remarkable immunomodulating, in particular immunostimulating and immunoadjuvant properties, they find application in the treatment and in the prophylaxis of diseases associated with a deficiency of the immuno response, as for example tumors and infections of various origin, for example of bacterial or viral origin, and in recurrent infections. The association with other therapeutically active substances concerns any kind of therapeutically suitable association, but preferably the association with substances whose activity is increased by the present oligopeptides, for example with substances having antigenic properties. The compounds of the present invention are therefore useful adjuvants in vaccines. Among the preferred derivatives of the present invention, we mention peptides characterized by containing in the aminoacid sequence from 1 to 2 L-Glu(NHAd) residues, at least a Lys residue and at least a residue selected from the group consisting of Arg, Pro and Pro-Arg.

More preferred derivatives are the following: H-Lys-Glu(NH-Ad)-Pro-Arg-Glu(NH-Ad)-OH (A) H-Lys-Glu(NH-Ad)-Arg-Glu(NH-Ad)-OH (B) H-Lys-Glu(NH-Ad)-Pro-Glu(NH-Ad)-OH (C) H-Glu(NH-Ad)-Lys-Pro-Arg-NH₂ (D)

The presence of a hydrophobic residue derived from 1- aminoadamantane not only improves the oligopeptides transfer through the biological membranes and affects oligopeptides flexibility and conformation , but it also improves the bond between immunocompetent cells and cellular membranes , bringing, as a result, to a favourable pharmacokinetic profile. If compared to the known compounds , the present oligopeptides exhibit a more specific activity on the humoral immunity (see for example the compound B ) or on the cellular one ( see the compounds C and D) .

Further, the compounds of the invention have the advantage to be apyrogenic , more stable at the metabolism, and to exhibit a favourable pharmacokinetic profile . The Applicant has furthermore found that the oligopeptides of the present invention having an arginine residue at the C-terminal end (the one bearing an α-carboxygroup not involved in the peptide bound) , such as compound D, show a considerable activity on the enzymatic synthesis of Nitrogen oxide (NO) , by activating macrophages towards its production. The activity on NO synthesis is important for antimicrobic activity of phagocytes, and furthermore for other biologic activity promoted by NO, such as smooth muscle relaxation, immune system regulation, neurotransmission, platelet inhibition and penis erection ("Biochemistry of Nitric oxide and its Redox - Activated forms", J.S. Stamler et al., Science, Vol.258, 18 December 1992) .

The compounds of the invention having a residue of arginine at the C-terminal end, such as compound D, find therapeutic applications in the treatment of diseases of the nervous system and immune system, more specifically in tumor immunotherapy, autoimmune diseases, diseases linked with neurotransmission disturbances and vascular diseases.

The oligopeptides of the invention are synthesized according to the solid phase method, utilizing a semiautomatic synthesizer or manually by using a syringe.

The solid phase method comprises condensing an aminoacid on a suitably functionalized insoluble polymer, followed by assembling the remaining aminoacids in the successive steps. All the aminoacids are utilized after suitably protecting the α- a inogroup. The other functional groups optionally present in the side chain of the amino acid are suitably protected with acetyl (Ac) , benzyl (Bz), formyl (For), terbutyloxycarbonyl (Boc) , Tosyl (Tos) or 4-methyloxy-2,3,6 trimethylbenzenesulfonyl (Mtr) groups. The condensation of the first amino acid on to the polymer occurs by reacting its carboxy group with the polymer functional groups. After removing the protecting group of the α-amino group of the aminoacid supported on the polymer, the other aminoacids are assembled by forming an α-amide bond between their α-carboxy group and the α-amino group of the aminoacid linked to the polymer.

The formation of the α-amide bond may be accomplished in different ways, for example the carboxy group of the aminoacid to be assembled can be activated by forming a mixed anhydride or an activated ester, according to methods commonly known in the field of peptide synthesis.

At the end of the synthesis, the protecting groups are removed by using suitable techniques. The present invention further relates to a process for preparing the present derivatives, comprising the following steps : a) condensing on a resin the first aminoacid protected at the α- amino group with P^, wherein P-^ is a protecting group of the α- amino group selected from the group consisting of terbutoxycarbonyl (Boc) and fluorenylmethyloxycarbonyl (Fmoc) , by treating a resin with chloromethyl groups with the Cesium salt of the above mentioned protected first aminoacid, in a polar aprotic solvent at a temperature comprised between +40 and +90°C, preferably between +60° and +70°C; or by treating a resin with p- methylbenzhydrylamino groups with the above first aminoacid protected at the α-amino group, after treating it with a compound P₂0H known for the activation of the aminoacid α-carboxy group, in the presence of a dialkylcarbodiimide, at room temperature (about +20°C - +30°C) ; b) removing the above mentioned protecting group Pi from the amino acid supported on the resin obtained in a) , by treatment, when P^ is Boc, with trifluoroacetic acid, at a temperature comprised between 0 and +50°C, preferably between +20^βC and +30°C, followed by neutralization with a base (an alyphatic tertiary amine substituted with C^-Cg alkyl groups) , and when P^ is Fmoc, with a solution of a saturated heterocyclic amine in an organic solvent; c) assembling onto the resin the next aminoacid protected at the alpha-aminogroup with P^ , where P-^ is above defined, in an organic solvent at a temperature comprised between 0 and +50°C, preferably between +20°C and +30°C, after treating said successive aminoacid with P2OH, where P2OH is a hydroxy compound known for the activation of the aminoacid carboxy group, in the presence of a condensing agent, at a temperature comprised between 0 and +50°C, preferably between +20°C and +30°C; d) removing as in step b) the protecting group P^ and the other Boc or Fmoc groups possibly present in the lateral chain of the peptide supported on the resin obtained in step c) ; e) cleaving the final oligopeptide from the polymeric support by means of treatment with liquid HF, at a temperature between -20° and 0°C, in the presence of a "scavenger", followed by the recovery of the thus obtained oligopeptide.

Under preferred conditions from 1 to 1.2 equivalents of cesium salt, 2 equivalents of the successive aminoacid, 2 equivalents of P2OH and 2 equivalents of condensing agent are used in respect of the amount of resin used in step a) .

In the present description the equivalents refer to the amount of resin initially used in step (a).

After each step the functionalized resin is washed with organic solvents as encompassed in the solid phase technique. Steps b) and c) are repeated for the necessary number of times and aminoacids to complete the desired peptide sequence. During step d) , Boc and Fmoc groups, both in α-aminic position and, if any, in lateral chain, are removed before cleaving the peptide from the resin. in case of the oligopeptides A, B and C the first protected aminoacid is P₁-Glu(NHAd)-OH, and P-^ is preferably Boc. In case of oligopeptide D, the first aminoacid derivative is P^- Arg-(Mtr)-OH, wherein Mtr is 4-methoxy-2,3,6 trimethylbenzenesulfonyl and P-^ is preferably Fmoc. in the present process the functional groups in the side chain of arginine are protected for example with tosyl (Tos) or with 4- methoxy-2,3,6 trimethylbenzensulfonyl (Mtr); those of Lysine with terbutoxycarbonyl (Boc) or with fluorenylmethyloxycarbonyl

(Fmoc) . The protected aminoacid preferably used are:

Boc-Glu(NHAd)-OH (oligopeptides A, B and C)

Boc-Arg(Tos)-OH (oligopeptides A, B and C)

Fmoc-Lys(Fmoc)-OH (oligopeptides A, B and C)

Boc-Pro-OH (oligopeptides A, B and C) ; Fmoc-Glu(NHAd)-0H (oligopeptide D)

Fmoc-Arg(Mtr)-0H (oligopeptide D)

Fmoc-Lys(Boc)-0H (oligopeptide D)

Fmoc-Pro-OH (oligopeptide D) .

In the present process a resin in the chloromethyl form such as the Merrifield C—R)'resin is preferably used for the compounds A, B and C, while a p-methylbenzhydrylamino resin is preferably used for compound D.

The p-methylbenzhydrylamino resin is suitable for the preparation of peptides in the form of α-C0NH2 amides at the α-C terminal end (such as compound D) , resulting from the acidic cleavage.

For the preparation of compound D, the p-methylbenzhydrylamino resin, commercially available in HC1 form, is neutralized, before carrying out step (a). The organic solvents can be halohydrocarbons such as dichloromethane and chloroform, ethereal solvents, such as ethylether and tetrahydrofuran and the polar aprotic solvents such as dimethylformamide, dimethylsulfoxide, dioxane, or a mixture thereof.

In the preparation of compounds A, B, and C, dimethylformamide is preferably used as the solvent in step (a) and in step (c) the organic solvent preferably consists of a mixture dichloromethane:dimethylformamide 4:1 (volumetric ratio).

In the synthesis of derivative D, dimethylformamide is preferably used in steps a) and c) and to wash the functionalized resin.

The above mentioned cesium salt may be obtained by treating the first aminoacid, protected with P-^, with cesium carbonate, at room temperature and in a hydroalcoholic environment, preferably in a mixture water-methanol, evaporating then the solvent to dryness, preferably under vacuum.

The cleavage of the Boc groups is preferably accomplished with solutions of trifluoroacetic acid (TFA) in a halohydrocarbon solvent, such as dichloromethane, having a TFA concentration ranging from 40 to β0% (weight/volume or volume/volume) , at temperatures of +20°C - +30^°C.

The tertiary amine used for neutralizing trifluoroacetic acid is preferably ethyldiisopropylamine.

The saturated heterocyclic amine used for the Fmoc cleavage is preferably piperidine, in dimethylformamide, at +20° - +30°C.

The Tos and Mtr groups, possibly present on the guanidine group of the arginine residue, can be cleaved in strongly acidic conditions, for instance with trifluoromethanesulphonic acid, or with other known methods .

Referring to P2-OH compounds , they are selected among those normally used in the peptide synthesis for the formation of active esters , thus activating the α-carboxy group towards the formation of the peptide bond.

P2-OH can be for example N-idroxybenzotriazole ( OHBt ) , or a substituted phenol , as for example pentafluorophenol (PfpOH) , p- nitrophenol or 2 , 4-dinitrophenol , and preferably is N- hydroxybenzotriazole . The condensing agent can be a dialkylcarbodiimide , such as dicyclohexylcarbodiimide or diisopropylcarbodiimide , preferably disopopyl carbodimide .

The scavanger can be thioanisole or p-thiocresol or a mixture thereof , and preferably a mixture of these thioderivatives is used .

In the presence of this mixture the remotion of the oligopeptide from the polymeric matrix is accelerated and proceeds substantially in the absence of undesired side reactions. The oligopeptides obtained according to the present invention are recovered after HF cleavage by evaporation at laboratory temperature under reduced pressure (by means of a common water pump) , followed by extraction with an acetic acid aqueous solution. Once the oligopeptides are recovered, they can be purified by chromatography, utilizing for example a SephadexS^G 10 (dextrane by gel filtration) column and 0.1 M acetic acid as the eluant. Oligopeptide D is purified on a Bioge⁽lS^/P2 column. Oligopeptide D may be further purified by inverse phase preparative HPLC (by using for example a Vydacs- Clδ column) . The oligopeptides of the invention can be obtained in solid form by lyophilizing the solutions coming from step (e) or from chromatographic purification.

The lyophilization is carried out by following conventionally known techniques. The compound P₁~Glu(NHAd) -OH can be prepared starting from glutamic acid in the protected form as compound, wherein P^ has the above mentioned meaning and is preferably Boc, P2 is the residue of the above mentioned compound P2OH (for example P₂ = Pfp, namely the aromatic residue penta-fluorophenyl) and P is a protecting group of the carboxy group, for example the benzyl group. Pi-Glu(0P₂)0Po can be prepared condensing the diprotected aminoacid P^-Glu-OP , for instance Boc-Glu-OBz (that is N-t-Boc-L-Glutamic acid α-Benzyl ester) , with P2θH=pentafluorophenol in the presence of a diakylcarbodiimide then it is converted into the corresponding j^"7adamantylamide, by treatment with 1-aminoadamantane with methods known to the skilled in the art. Alternatively, P^-Glu-OP (Boc-Glu-OBz) can be reacted with 1-aminoadamantane after treatment with P 0H=N- hydroxybenzotriazole in the presence of a diakylcarbodiimide. P is then removed according to conventional techniques, such as, for the -OBz group, treatment with H /Pd.

The present invention further relates to therapeutical composition comprising as the active principle a therapeutically effective amount of at least one oligopeptide according to the present invention, or a pharmaceutically acceptable salt thereof, in combination with pharmaceutically acceptable excipients.

These compositions are useful in human therapy, in particular for the treatment of the above cited diseases.

Besides the above cited active principle and excipients, the compositions of the present invention may also contain other active principles having a complementary or in any case useful activity.

Particularly preferred therapeutical compositions are those containing , in association with the oligopeptides of the invention , one or more therapeutically active substances , whose activi ty is enhanced by them , and in particular antigenic substances .

In order to obtain the desired effects , the therapeutic compositions may be administered to patients by various routes , for example by the oral and the parenteral ones .

The compositions of the invention may be realized according to the known techniques , such as those described for example in

"Remington ' s pharmaceutical Science handbook" , Hack Publishing

Co . , USA . The compositions of the invention are administered on average once or twice a day, anyway they may be more frequently administered, at least in some cases, depending on the patient's conditions and the administration type. When orally administered, this active principle is formulated in the form of solid or liquid preparations, such as capsules, tablets, pills, powders, solutions, suspensions, emulsions, optionally in controlled release forms.

The unitary solid dose may be a soft or hard jelly capsule, containing lubricating agents and inert excipients such as lactose, saccharose and starch.

The compounds of the present invention may be formulated as well in the form tablets utilizing the conventional excipients such as lactose, saccharose, starch, jelly, alginic acid , stearic acid, magnesium stearate. When parenterally administered the compounds of the present invention may be administered in injectable formulations, particularly they are dissolved or suspended in pharmacologically acceptable diluents, with a vehicle, being a sterile vehicle such as water or an oil, in the presence or in the absence of other components.

The oils to be used are of animal, vegetable, or synthetic origin, such as peanuts oil, soybean oil and mineral oil. Generally as the vehicle for the injectable solutions, water, mineral salts aqueous solutions, dextrose or other sugars aqueous solutions , ethanol , glycols such as propylen- or polyethylen- glycol, may be used.

Some examples of the present invention are hereinbelow reported for illustrative, but not limitative purpose.

PEPTIDES A.B.C AND D PREPARATION The following abbreviations are used:

Boc : tert-butyloxycarbonyl

Fmoc : fluorenylmethyloxycarbonyl

Tos : tosyl

Mtr : 4-methoxy-2,3,6-trimethylbenzensulfonyl NH-Ad : 1-adamantylamino

HOBt : N-hydroxybenzotriazole

TFA : trifluoroacetic acid

EDIA : ethyldiisopropylamine

MeOH : methanol DIC : diisopropylcarbodiimide

DCC : dicyclohexyl carbodiimide

DMF : dimethylformamide

Et₂0 : dimethylether

DCM : dichloromethane MBH : p-methylbenzhydrylamine

Materials and methods

General procedure for the peptide synthesis in solid phase are reported in the following publications :

J.M. Stewart, J.D. Young, Solid Phase Peptide Synthesis, Pierce Chem. Comp., 1984; E. Atherton and R.C. Sheppard, Solid Phase Peptide Synthesis-Practical Approach, RTL Oxford Press 1989.

Peptides A, B, C were prepared by using a semi automatic synthesizer built in laboratory, or manually in a polypropylene syringe on a chloromethyl Merrifield*^resin, 1% DVB crosslinking (substitution 1.03 mmoles Cl/g) .

A p-methylbenzhydrylaminic resin (MBH-resin) (substitution 0.5 mmoles/g) was used for the preparation of peptide D.

Analytical methods:

Thin layer chromatography (TLC) is carried out on TLC Kieselgel Merclr^-UV-254 plates in ethyl acetate : pyridine : acetic acid : water 5 : 5 : 1 : 3 (SI) system; the products revelation is accomplished with ninhydrin or by the chlorination/TMD method

(TMD = tetramethyl-4'-diaminodiphenylmethane) .

Mass spectra are carried out on a ZAB-EQ mass spectrometer. The FAB ionization technique is executed at 8 kV (Xe) in a thioglycerin : glycerin 3 : 1 matrix.

The aminoacids analysis is carried out by using a Durrum analyser. The analysis samples are hydrolyzed for 24 hours in M

HC1 in the presence of phenol. The following aminoacid derivatives are utilized for the synthesis:

Boc-Glu(NHAd)-OH (oligopeptides A, B and C)

Boc-Arg(Tos)-OH (oligopeptides A, B and C)

Fmoc-Lys(Fmoc)-0H (oligopeptides A, B and C) Boc-Pro-OH (oligopeptides A, B and C); Fmoc-Glu(NHAd)-OH (oligopeptide D)

Fmoc-Arg(Mtr)-OH (oligopeptide D)

Fmoc-Lys(Boc)-OH (oligopeptide D)

Fmoc-Pro-OH (oligopeptide D) .

All these derivatives, with the exception of Fmoc-Arg(Mtr)-OH

(Bachem^ commercial product) are prepared in our laboratory, using conventional methods known for introducing Boc, Fmoc and Tos groups. Experiments description in the case of A, B and C peptides the first esterification step is carried out following Gisin method (Gisin, B.F., Helv. Chem. Acta 56, 1476-1482, 1973).

600 mg Boc-Glu(NHAd)-OH are dissolved in a mixture of 10 ml water and 4 ml methanol. The carboxylic group titration is accomplished by using a 105. cesium carbonate solution (pH 7 Λ ) .

After evaporation, the Cesium salt of Boc-Glu(NHAd) -OH is obtained which, utilized in excess of 1.1 equivalents with respect to the chloromethyl resin Merrifielc , is dissolved in 10 ml DMF. 1.4 g of the above mentioned resin are added to the DMF solution and the so obtained suspension is maintained under stirring at 6 ^°C for 3 days. Then the polymer is removed by filtration and washed with DMF, water, methanol and ether. 1.64 g of esterified resin are obtained. The picric acid test reveals a substitution degree of 0.46 mmoles/g. A, B and C peptides assembly occurs on the Boc-Glu(NHAd)-resin, according to the general scheme hereinbelow reported:

1. Polymer washing in 10 ml DCM 3x2 min.;

2. Cleavage of Boc protecting group Boc remotion in 10 ml 0JU TFA for 30 min. ;

3. polymer washing in 10 ml DCM 3x2 min.;

4. neutralization with a 10 EDIA solution, 2x5 min.;

5. polymer washing in 10 ml DCM 3x min.;

6. condensing the next aminoacid with HOBt active esters prepared in situ, up to a negative response to the blu Bromophenol test

(V. Krchnak et. al., Coll. Czech. Chem. Commun. , 53, 25 3, 1988) and to Kaiser ninhydrin test.

HOBt active esters preparation in situ

The suitably protected aminoacid derivative, (two times in excess with respect to the resin) is dissolved in 10 ml of a DCM : DMF 4

: 1 mixture.

The calculated amounts of HOBt and DIC (2 equivalents) are added and the so obtained mixture is sucked in the syringe containing the polymer-deprotected peptide (prepared as above described) . The peptide-resin suspension is left in the coupling medium under rest or under magnetic stirring for 20/40 minutes.

The condensation reaction completion is evidenced by a negative response to the above mentioned colorimetric tests.

The condensation of the sterically hindered adamantylamide derivatives proceeds more slowly (1 hour) and generally is repeated twice.

In the last synthesis step both Boc and Fmoc groups are removed.

For removing Fmoc, a 20% piperidine solution in DMF (10 ml) for

20 minutes is used. The final washing is carried out with DMF 3x10 ml, DCM 3x10 ml,

MeOH 3x10 ml and ethyl ether 3 10 ml for about 2 min. for every washing, and in the end, the polymer-peptide is dryed in a dryer.

Peptide D is synthesized starting from Fmoc aminoacid derivatives, utilizing the continuous flux method. Fmoc-Arg(Mtr)-0H, as the C-terminal aminoacid is linked to the

MBH-polymer by means of the HOBt active ester method before described at +20°C - +30°C.

The first assembly step of the peptide in this special case is the neutralization of the commercially available resin in HC1 form.

The general preparation scheme for compound D is analogous to the one previously described for compounds A, B and C, with the exceptions herein below reported.

The only solvent utilized for washing and condensing is DMF. The cleavage of the Fmoc protecting group is carried out in a 20% piperidine solution in DMF for 20 min.

Obviously, in the synthesis of compound D the neutralization step required after the TFA cleavage of Boc protecting groups is omitted. The remotion of A, B, C and D peptides from the polymeric support is realized in 10 ml liquid HF for 1 hour at -10°C, in the presence of thioanisole (0.2 ml) and p-thiocresole (0.2 ml), utilized as scavengers.

After evaporating the reaction mixture (laboratory temperature, vacuum of a water aspirator) , the obtained solid material is washed with ethyl acetate, the peptides are extracted with a solution of 20% acetic acid in water and separated by lyophilization.

Raw A, B and C peptides are purified on a Sephadex*^G 10 column (50 x 1.5 cm) utilizing 0.1 M acetic acid as the eluant.

Peptide D is purified on a Biogel (^βR)P2 column (21 x 2.7 cm).

Purification progress is followed by TLC.

Pure fractions are collected and lyophilized. In addition peptide

D is purified by preparative HPLC. The amount of the utilized polymers and of the aminoacid derivatives, the analytical results and the yields are reported in the description for the individual compounds.

The assigned structures are confirmed by mass spectroscopy (see

Fig. 1-4) and by the aminoacid analyses. Peptide H-Lys-Glu(NH-Ad)-Pro-Arg-Glu(NH-Ad)-OH (A)

Polymer amount : 335 mg

Aminoacid derivatives amount : Boc-Glu(NHAd)-0H 117 mg

Boc-Pro-OH 66 mg

Boc-Arg(Tos)-0H 132 mg Fmoc-Lys(Fmoc)-0H 182 mg 106 mg of raw peptide are obtained from 347 mg of peptidyl-resin and after purification on Sephadex^G 10 column, 61 mg of peptide are obtained.

Mass spectra FAB⁺ [M⁺H]⁺ : 9 5-1. Aminoacids analysis : Glu (2) 2.08; Pro (1) 0-98; Lys (1) 0.92;

Arg (1) 1.02.

TLC in SI system.

Peptide H-Lys-Glu(NH-Ad)-Arg-Glu(NH-Ad)-OH (B)

Polymer amount : 335 mg Aminoacid derivatives amount : Boc-Glu(NHAd)-0H 117 mg

Boc-Arg(Tos)-0H 132 mg Fmoc-Lys(Fmoc)-0H 182 mg

The yield in peptidyl-resin is 365 mg; 80 mg of raw peptide are obtained; after purification on Sephadex^G 10 column, 31 mg of purified peptide are obtained.

Mass spectra FAB⁺ [M⁺H]⁺ : 827.5.

Aminoacids analysis : Glu (2) 2.19; Lys (1) 0.86; Arg (1) 0.94.

TLC in SI system.

Peptide H-Lys-Glu(NH-Ad)-Pro-Glu(NH-Ad)-OH (C) Polymer amount : 335 mg

Aminoacid derivatives amount : Boc-Glu(NHAd)-0H 117 mg

Fmoc-Lys(Fmoc)-0H 182 mg Boc-Pro-OH 66 mg

The yield in peptidyl-resin is 3 3 mg, 57 mg of raw peptide are obtained; after purification on Sephadex^ G 10 column, 37 mg of purified peptide are obtained.

Mass spectra FAB⁺ [M⁺H]⁺ : 769-0.

Aminoacids analysis : Glu (2) 1.97; Pro (1) 0.88; Lys (1) 1.14.

TLC in SI system.

Peptide H-Glu(NH-Ad)-Lys-Pro-Arg-NH₂(D)

MBH-polymer amount : 1600 mg, s=0.5 mmoles/g.

Aminoacid derivatives amount : Fmoc-Glu(NHAd)-OH 502 mg

Fmoc-Ar (Mtr)-OH 610 mg Fmoc-Lys(Boc)-0H 468 mg Fmoc-Pro-OH 337 mg

The yield in peptidyl-resin is 2.1 g; 280 mg of peptide are obtained after purification on a Sephadex G10 column. This product is subjected to a preparative HPLC on a Vydac&'Clδ column

(8x300 ml) (isocratic elution with mobile phase 12% MeOH/0.1% TFA in water; flux : 2.5 ml/min. ; UV detection at 230 nm) . After HPLC, 40 mg of peptide are obtained.

Mass spectra FAB⁺ [M⁺H]⁺ : 661.4.

Aminoacids analysis : Glu (1) 1.06; Lys (1) 1.02; Arg (1) 1.06;

Pro (1) 0.85.

TLC in SI system. PHARMACOLOGICAL TESTS

Immunoadjuvant and immunostimulating activity evaluation

The following abbreviations are used :

PBS : phosphate buffer saline solution

FBS : fetal calf serum RPMI -RPMI 1640 : Culture medium (Roswell Park Memorial

Institute)

TEST 1- Delayed hypersensitivity reaction to ovoalbumin in guinea pigs. 0.2 ml of a mixture composed of a physiological solution in an oily emulsion (Bayo±^: Arlace ^ : physiological solution 4 : 1 :

5) , containing 2 mg of crystalline ovoalbumin (FIA = Freund incomplete adjuvant) are injected into the pad of the left hind foot of albino female guinea pigs. The effect of oligopeptides A, B, C and D, which were added to the mixture in the amount of 100 μg, is compared with that of

Freund 's incomplete adjuvant (FIA) and with that of a known highly active bacterial adjuvant, Mycobacterium tubercolosis , contained in Freund' s complete adjuvant (FCA) . The cutaneous reaction is evaluated measuring the erythema diameter 14 days after the administration of 10 and 20 μg of ovoalbumin.

The so obtained erythema diameters, expressed in millimeters (mm), are reported in Table 1.

Table 1 Induction of delayed hypersensitivity to ovoalbumin in guinea pigs .

Ovoalbumine (dose) Ovoalbumine (dose)

Tested Dose 10 μg 20 μg

Substances

FIA - 1.4 ± 0.25 2.75 ± 0.47

A 100 μg 3.95 ± 0.40* 6.24 ± O.65*

B 100 μg 2.84 ± 0.34 4.80 ± O.56*

C 100 μg 4.22 ± 0.52* 7.55 ± 0.74*

D 100 μg 4.72 ± 0.62* 7.65 ± 0.82*

FCA 1 mg 3.25 ± 0.32* 6.12 ± O.56*

* P < 0.05

Results

As one can observe from Table 1, the administration of the oligopeptides A, B, C and D causes an increase of the delayed hypersensitivity reaction to ovoalbumine in guinea pigs and at the dose of 100 μg this effect is not only comparable to that of FCA but in certain cases (compound A, C and D) is even higher. TEST 2 GVHR (Graft-versus-host reaction)

Balb-c mice were divided in groups, each group of 4 animals, and utilized as splenocytes donors. Each group is treated by subcutaneous route with 100 μg of one compound selected from oligopeptides A, B or C or 1-aminoadamantane (AmAd) on days -2, -1 and 0 before the induction of the regional GVHR. A control group is not treated with any of the above mentioned substances. On day 0, a suspension of aggregated spleen cells obtained from the donors is prepared by passing the cut spleens in a RPMI 1640 medium through a fine sieve. After centrifugation (800 x g, 8 min.), the spherical mass obtained is resuspended in RPMI 1640 up to obtaining the final o density of 1 x 10 /ml (only nucleated cells).

0.05 ml of the above mentioned suspension, containing 10 splenocytes are immediately injected into the pad of the left hind foot of hybrid F₁ (C3H x Balb) ( 10 + 12 animals per group). 8 days after the injection, the weight of the popliteal lymphonodus both in the legs subjected to the injection and in the contralateral legs are evaluated. Their difference is taken as GHRV quantitative measure. The obtained data are reported in Table 2.

TABLE 2 Test of the graft-versus-host reaction (GVHR)

Control A B C D Am Ad

Lympho- 1.03*0.17 1.50*0.21 1.20*0.17 1.65*0.17 1-99*0.24 0.60*0.14 nodus in mg

Am Ad = 1 - aminoadamantane Results

Compounds A, C and D resulted active, and among them the most active are compounds C and D.

TEST 3 - Hemoagglutination Sheep red blood cells (SRBC) are administered by intraperitoneal route to different mice groups in an amount of 5 x 10' in 0.50 ml

PBS for each animal and contemporaneously 100 μg oligopeptides A,

B, C or 1-aminoadamantane (AmAD) are administered by subcutaneous route. To the control group, no substance is administered. On the 5 day the mice are sacrificed by decapitation. Their serum is taken and heated to 56 °C for 30 min. in order to inactivate the complement.

Various serum dilution are prepared in PBS and an equal volume of

2% SRBC in PBS (volume/volume) is added. The so obtained samples are maintained at 4 °C for 18 hours before the final reading of the agglutination titre.

Titres are expressed as log₂ of the highest serum dilution still showing agglutination.

In Table 3 reported are the hemoagglutination titres obtained after 5 days from the immunization.

Table 3 Total hemoagglutination titre after primary immunization with sheep red blood cells (SRBC) .

5 days after immunization

Control A B C D Am Ad

Total 2.43*0.18 2.78*0.18 3-21*0.18* 2.77*0.18 N.D. 2.68*0.09 titre

#

P < 0.05

N, . D . = not determined

Results

Compound B is the most active, inducing a significant increase in total hemoagglutination titre.

TEST 4 - Response of plaque forming cells (PFC) 100 μg oligopeptides A, B and C and 1-amino adamantane (AmAD) are administered together with SRBC by subcutaneous route to different mice groups immunized with SRBC (5 x 10' per animal, administered by intraperitoneal route) . The control group has not received the substances to be tested. A single cell splenocytes suspension (2 x 10 /ml) is obtained on the 5 day after immunization.

0.2 ml of said suspension are mixed with an equal SRBC volume (1 x 10-Vml) and with guinea-pig complement (diluted 1 : 10).

Every component is prepared in the RPMI 1640 medium. The mixture is maintained in Cunningham chambers (0.1 ml) for 1 hour at 37 °C and at 100% relative humidity.

The number antibody-producing B-cells is evaluated as the PFC number per 10 splenocytes.

The obtained data are reported in Table 4.

Table 4 Plaque forming cells (PFC) number after primary immunization with sheep red blood cells (SRBC).

Control A B C D Am Ad

Number of 572*43 521±3δ 956*72 564*32 N.D. 657±27 PFC

** p < 0.01

N.D. = not determined

AmAd = 1-aminoadamantane

Results

As it can be seen from the obtained data, B is the only active compound in this test.

TEST - Splenocytes proliferation

According to Test 2, splenic cells are obtained from donors mice treated subcutaneously with 100 μg of the substances to be tested by passing their spleens cut in a RPMI 1640 medium through a fine sieve.

After centrifugation (800 x g, 8 min.) the obtained spheric mass is resuspended in RPMI 1640.

The obtained splenic cells (0.2 ml per well with a density of 2.5 x 10 /ml) are cultured for 72 hours in FB** 96-wells microplates in the complete RPMI 1640 medium (10% FBS, 0.005% gentamicine) , at 37°C, with 5% C0₂ and 100% relative humidity, both in the absence or in the presence of mitogens CON A (concanavalin A)(l μg/ml) and LPS (lipopolysaccharide) (LPS S.typhy murium, Sigma^ (20 μg/ml). During the last 6 hours of culture, cells are collected and their radioactivity is determined by using a scintillation beta- counter.

The obtained data are summarized in table 5, which reports the effects of splenocytes proliferation, measured as thymidine-^H incorporation.

TABLE 5 Splenocytes polyclonal activation in vitro in the absence or in the presence of mitogens.

p.p.m. Control A B C Am A

Without mitogens 965*117 1557*205 1392*169 1790*249 1350*142 CON A^b ( 1 μg/mol ) 794*187 1791*264 2240*317 1852*223 1 38*296 LPS^C (20 μg/mol ) 167 1*3874 22223*1797 31721*5104 21864*3957 21944*4921

^apulsation per minute ^clipopolysaccharide concanavalin A 1-aminoadamantane Results

All the tested oligopeptides are active in promoting splenocytes proliferation.

In the absence of mitogens, the most active compound is C. All compounds increase the mitogenic answer to CON A, nevertheless B results to be the most active compound.

Compound B is the most active also in increasing the mitogenic answer to LPS. Because LPS is considered to be a specific mitogen for B-cells and CON A for T-cells, the test shows an action specificity of the tested compounds versus B and T cells.

TEST 6- Pyrogenicity.

The pyrogenic effect of the above mentioned derivatives A, B, C and D is determined on rabbits having a weight of from 2 to 2.3 kg. The compounds are injected by intravenous route and the effect is compared with that obtained with muramyl dipeptide

(MDP), a very powerful pyrogenic adjuvant. Temperature is measured by a rectal thermistor thermometer every hour.

Results are shown in Table 6. The reported values are the average of values obtained from groups of 5 animals.

TABLE 6 Pyrogenic effect of the tested compounds Temperature (°C)

Tested Dose 1 h 2 h 3 h 4 h 5 h substances

Controls - + 0.2 - 0.2 - 0.1 + 0.2 0

A 1 mg + 0.3 + 0.4 - 0.2 + 0.3 + 0.2

B 1 mg - 0.2 0 + 0.3 - 0.2 + 0.1

C 1 mg + 0.1 0.3 + 0.4 + 0.4 + 0.2

D 1 mg - 0.2 0 + 0.2 + 0.1 - 0.2

MDP 100 μg + 0.9 1.6 1.2 1.2 0.7

Conclusions

The results obtained in these pharmacological tests highlight the quite different effects on the humoral and on the cell mediated immunity of the tested compounds.

For example, Compound B proves to be the most active in the stimulation of the humoral activity in both TESTS 3 and 4.

On the other side, all the tested compounds are active in stimulating cellular immunity. Among them, B compound has however the lowest activity, while C and D have a remarkable effect (as it can be seen from TESTS 1 and 2 which evaluate the cellular immunity) .

Moreover after the administration of the tested oligopeptides no pyrogenic effect and no toxicity sign are observed on animals. The pyrogenicity test is very significant because pyrogenicity is generally connected with I Interleukin release and therefore with a series of side effects determined by that substance. Test 7 ~ Effect of D compound in Lewis Lung Carcinoma (LLC) Materials and methods

C 57 Bl/6 (Charles River) female mice were used, weighing on average 20 g.

LLC (Lewis Lung Carcinoma) tumoral cells were obtained from animals affected by the transplanted tumor (16 passage). This tumor was subcutaneously transplanted into the axillary region. The total number of transplanted cells was 2X10-⁵ in 0.1 ml of Hanks solution.

Compound D was dissolved in physiological solution and intraperitoneally (i.p.) administered at the dose of 15 mg/kg to groups of at least 10 mice, by following different protocols of treatment.

Described are hereinbelow the groups of treated animals, the day in which they received the specific treatment, and the received treatment protocol (the day 0 is that in which the tumoral cells transplant was conducted; the day -3 corresponds to 3 days before this transplant and +3 means 3 days after the same) .

Group 1 (control) day -3 : physiological solution day 0 : physiological solution + tumoral cells day +3 : physiological solution Group 2 (3x peptide D) day -3 peptide D day 0 peptide D + tumoral cells day +3 peptide D

Group 3 (2x peptide D) day -3 peptide D day 0 peptide D + tumoral cells day +3 physiological solution

Group 4 (2x peptide D) day -3 physiological solution day 0 peptide D + tumoral cells day +3 peptide D

Group 5 (lx peptide D) day -3 : physiological solution day 0 : peptide D + tumoral cells day +3 : physiological solution

On days 7,10 and 15, from tumoral cells inoculation, the tumoral growth was determined by palpation and evaluated by using the

"scoring" method, classifying the tumor as "large" or "small"

(see Table 7) .

On day 17 from tumoral cells inoculation, the animals were sacrificed, tumors were removed and weighed (see Table 8); lungs were examined by a histologist and metastases counted (see Table

9) Results

The results obtained in the Lewis Lung carcinoma model, show the effect of compound D both on the primary tumor and on the blood born metastases. The data reported in Table 7 show that when Compound D is administered three times (15 mg/kg) , respectively on days -3, 0 and 3, (Group 2), it significantly reduces the tumor growth as assessed through the scoring method on days 7, 10 and 1 . When compound D is administered however only twice (Group 3 and 4) or once (Group 5), no statistically significant results are obtained.

The data relating to the tumor weight after mice sacrifice (on day 17) , show that in the mice treated with Compound D under all the different protocols (Groups 2, 3. 4 and 5). the tumor average weight is always lower than in the control group (Group 1), though not reaching statistical significance.

Table 8 reports in particular the data on tumor weight, obtained by administering Compound D three times (3x) (Group 2), in comparison with the control group (Group 1). The results relating to lung metastases formation are the most important, since they show that Compound D is able to significantly reduce metastases incidence at lung level, some animals being free of metastases. The data of Table 9 concern in particular the metastases number observed in mice treated three times with Compound D (Group 2) in comparison with the control group (Group 1)

TABLE 7 Evaluation of the tumor by palpation, after 3 administrations (3x) of compound D according to Group 2 protocol. Tumor growth on day 7 from inoculation

Group 1 (control) Group 2 (3x compound D) mice No. mice % mice No. mice % large 10 83-3 3 27.3 small 2 16.7 8 72.7 chi-square 9-8 P < 0.01

Tumor growth on day 10 from inoculation

Group 1 (control) Group 2 (3x compound D) mice No. mice % mice No. mice % large 10 83-3 4 36.4 small 2 16.7 7 63.6 chi-square 3-8 P < 0.05

Tumor growth on day 15 from inoculation

Group 1 (control) Group 2 (3x compound D) mice No. mice % mice No. mice % large 12 100 9 81.8 small 0 0 2 18.2 chi-square 6.2 P < 0.05 Notes :

* = statistically significant datum. p = corresponds to the probability level for rejection of 0 hypothesis

TABLE 8 Tumor weight (grams) after administration of compound D (15 mg/kg; three times; on days: -3, 0,+3)

Group 1 Group 2

(control) (3x compound D) tumor weight (g) tumor weight (g)

Treatc 3d animals

1 (dead) 1.08

2 3-94 3-59

3 4.2 4.2

4 3-95 3-5

5 3.16 4.07

6 4-3 3.02

7 4.49 3-3

8 3-05 2.93

9 3-49 2.9

10 3-35 4.39

11 3.68 2.3

12 3-34 -

Sum 40.95 35-28

Average 3-72 3-21

SEM 0.15 0.28

Notes:

SEM = Standard error mean

Group 2 consists of 11 mice. TABLE 9 Number of lung metastases after compound D administration (15 mg/kg; three times; on days: -3, 0.+3)

Group 1 Group 2 (control) (3x compound D) No. of metastases No. of metastases Treated animals

1 (dead) 0

2 1 0

3 3 0

4 4 0

5 4 3

6 4 4

7 4 5

8 10 5

9 12 6

10 15 9

11 18 9

12 20

Sum 95 41

Average 8.64 3-73

SEM 2.1 1.05

% mice free from metastases 0 36.4 % mice with metastases 100 63-6 chi- square

Notes:

SEM = Standard error of mean

Group 2 consists of 11 mice

* = statistically significant datum

Test 8 - Macrophages Nitric Oxide (NO) production

Female mice of inbred strain Balb/c were used.

Compounds used and administration routes:

Compound D, sub-cutaneous injection

Physiological saline solution (saline) , sub-cutaneous injection

Lipopolysaccharide (LPS): S. typhy murium (Sigma), added to macrophages culture medium.

LPS is a substance known for its ability to activate N0- synthetase enzyme and therefore to activate NO production.

The control Group was made of 12 animals, divided in 2 sub-groups of 6 animals each.

Sub-group 1: was injected with saline, then macrophages culture.

Sub-group 2: was injected with saline, then macrophages culture in presence of LPS 5 U-

The Treated Group was made of 12 animals, divided in 2 sub-groups of 6 animals each.

Each animal got a single 200 μg sub-cutaneous injection of Compound D on day -1 prior to the macrophages cells isolation and assay of Nitric Oxide production.

Sub-group 3: Compound D 200 μg (-Id), then macrophages culture. Sub-group 4: Compound D 200 μg (-Id), then macrophages culture in presence of LPS 5 P- Macrophages culture conditions:

Peritoneal macrophages were isolated and cultivated in Nunc 96- well FB-microplates in RPMI-1640 medium (10% FBS, 0.005% Gentamycin, 0.05 μM 2-Mercaptoethanol) under the following conditions: 37°C, 5% C0₂, 100% relative humidity, 24 hours. Griess reagent was used to determine spectrophotometrically nitrite levels, carrying out the detection at 5 θ nm with an Uniscan II reader. The obtained results are reported in Table 10 here below:

TABLE 10

Cell Sub- Control Sub- Treated culture Group (saline) Group 1 x 200 μg (-Id)

- 1 AVG 0.014 3 AVG -0.008 NS SEM 0.01 SEM 0.003

LPS 2 AVG 0.031 4 AVG 0.251 ♦#*

5μg SEM 0.001 SEM 0.015

- *#* Notes :

- Nitrogen Oxide (NO) production is expressed as nitrites amount, which is in turn expressed as optical density at 540 nm (OD 540 nm) . - Average (AVG) is calculated on the results obtained on 6 animals.

- SEM = Standard Error of Mean. Results

These data show that Compound D is able, under certain conditions, to stimulate NO production by macrophages. On single dose of 200 μg/animal given 24 hours before assay markedly increased the LPS stimulatory effect on NO production.

Claims

CLAIMS 1. Oligopeptides derivatives endogenous leukokinin fragments containing at least one L-Glu(NH-Ad) residue and their pharmaceutically acceptable salts. 2. The oligopeptides as claimed in claim 1 containing from 1 to 3 L-Glu(NH-Ad) residues. 3- The oligopeptides as claimed in claim 2, containing from 1 to 3 L-Glu(NHAd) residues, at least a Lys residue and at least one residue selected from the group consisting of Arg, Pro and Pro- Ar - 4. The oligopeptides as claimed in claim 1, formed by a number of from 4 to 5 aminoacids. 5- The oligopeptides as claimed in claim 1, selected from the group consisting of: H-Lys-Glu(NH-Ad)-Pro-Arg-Glu(NH-A )-OH, H-Lys-Glu(NH-Ad) -Arg-Glu(NH-Ad) -OH , H-Lys-Glu(NH-Ad) -Pro-Glu(NH-Ad) -OH and H-Glu(NH-Ad) -Lys-Pro-Arg-NH₂ 6. A process for preparing oligopeptide, derivative of endogenous leukokinin fragments containing at least one L-Glu (NH-Ad ) residue, comprising the following steps : a) condensing on a resin the first aminoacid protected at the α- amino group with P^ , wherein P-^ is a protecting group of the α- amino group selected from the group consisting of terbutoxycarbonyl (Boc) and fluorenylmethyloxycarbonyl (Fmoc) , by 8 treating a resin with chloromethyl groups, with the Cesium salt

9 of the above mentioned protected first aminoacid, in a polar 20 aprotic solvent at a temperature comprised between +40 and +90°C,

11 preferably between +60° and +70 C; or by treating a resin with p-

12 methylbenzhydrylamino groups with the above first aminoacid

13 protected at the α-amino group, after treating it with a compound

14 P OH known for the activation of the aminoacid α-carboxy group;

15 in the presence of a dialkylcarbodiimide, at room temperature

16 (about +20%°C - +30%°C) ;

17 b) removing the above mentioned protecting group Pi from the

18 amino acid supported on the resin obtained in a), by treatment, ^⁹ when P^ is Boc, with trifluoroacetic acid, at a temperature

20 comprised between 0 and +50 C, followed by neutralization with a

²¹ base (an alyphatic tertiary amine substituted with C -Cg alkyl

22 groups), and when P^ is Fmoc, with a solution of a saturated

23 heterocyclic amine in an organic solvent when P-. is Fmoc;

24 c) assembling onto the resin the next aminoacid protected at the _2f- alpha-aminogroup with P-^, where P^ is above defined, in an „_ organic solvent at a temperature comprised between 0 and +50 °C, _„ after treating said successive aminoacid with P₂0H, where P₂0H is

28 ^a hydroxy compound known for the activation of the aminoacid

29 carboxy group, in the presence of a condensing agent, at a

30 temperature comprised between 0 and +50 °C, preferably between

31 +20 ^°C and +30 °C;

32 d) removing as in step b) the protecting group P^ and the other Boc or Fmoc groups possibly present in the lateral chain of the peptide supported on the resin obtained in step c) ; e) cleaving the final oligopeptide from the polymeric support by means of treatment with liquid HF, at a temperature between -20° and 0°C, in the presence of a "scavenger", followed by the recovery of the thus obtained oligopeptide. 7- A process as claimed in claim 6, wherein said oligopeptide derivative is selected from the group consisting of H-Lys-Glu(NHAd)-Pro-Arg-Glu(NHAd)-OH, H-Lys-Glu(NHAd)-Arg-Glu(NHAd)-OH and H-Lys-Glu(NHAd)-Pro-Glu(NHAd)-OH, the resin has chloromethyl groups, the first aminoacid protected at the α-amino group with P-^ is Boc-Glu(NHAd) , the cesium salt is obtained by reacting said first protected aminoacid with cesium carbonate, in a hydroalcoholic medium, at room temperature, then evaporating the solvent to dryness, the solvent in step a) is dimethylformamide, the temperature in step a) is +6θ°C - +70°C; in step c) , P 0H is N-hydroxybenzotriazole, the condensing agent is diisopropylcarbodiimide, the solvent of step c) is a dichlorometane/dimethylformamide mixture the temperature in step c) is +20°C - +30°C; in step e) the scavenger is a thioanisole-p- thiocresol mixture; the cleavage of Boc and Fmoc groups according to steps b) and d) is carried out in the case of Boc groups with 40%-60% solution of trifluoroacetic acid in an halogenated solvent, at a temperature of +20^°C - +30°C, followed by 20 neutralization with ethyldiisopropylamine; in the case of Fmoc

21 groups, piperidine in dimethylformammide at +20°C - +30°C is

22 used.

I 8. A process as claimed in claim 6, wherein said oligopeptide

2 derivative is

3 H-Glu(NHAd)-Lys-Pro-Arg-NH₂, the resin has p-methylbenzydrylamino groups, the first aminoacid

5 protected at the α-amino group with P_χ is Fmoc-Arg(Mtr)-OH, in

6 step a) and c), P 0H is N-hydroxybenzotriazole, the condensing

7 agent is diisopropylcarbodiimide, the solvent in step a) and c) is dimethylformamide, the temperature in step a) and c) is +20°C - +30°C; in step e) the scavenger is a thioanisole-p-thiocresol ° mixture; the cleavage of Boc and Fmoc groups according to steps b) and d) is carried out in the case of Boc groups with a 4θ%-6θ% 2 solution of trifluoroacetic acid in an halogenated solvent, at a 3 temperature of +20°C - +30^°C, followed by neutralization with 4 ethyldiisopropylamine; in the case of Fmoc groups, piperidine in 5 dimethylformammide at +20^°C - +30^°C is used. 9- Therapeutic compositions comprising as the active principle a therapeutically effective amount of at least one oligopeptide derived from fragments of endogenous leukokinin containing at least one L-Glu(NH-Ad) residue , or a pharmaceutically acceptable salt thereof, in combination with other excipients. 10. The therapeutic compositions as claimed in claim 9. useful in the human therapy for the treatment or the prophylaxis of diseases associated with a deficit in immune response. 11. The therapeutic compositions as claimed in claim 10, wherein these diseases are selected from the group consisting of tumours, and infections. 12. The therapeutic compositions as claimed in claim 9, comprising, besides this oligopeptide, one or more therapeutically active substances whose activity is enhanced by this oligopeptide. 13- The therapeutic compositions as claimed in claim 12, wherein these therapeutically active substances are provided with antigenic properties and are contained in vaccines. 14. The therapeutic compositions as claimed in claim 9, suitable to be orally administered. 15. The therapeutic compositions as claimed in claim 9, suitable to be parenterally administered. 16. The therapeutic composition as claimed in claim 9, wherein this oligopeptide has an arginine residue at the extreme C- terminal, said compositions being useful in the human therapy in the treatment of pathologies of the nervous system, of the immune system and in vascular diseases. 17. The therapeutic compositions as claimed in claim 16, wherein said pathologies are selected among tumors, autoimmune diseases, diseases linked with neurotransmission disturbances. 18. The therapeutical compositions as claimed in claim 16, wherein this oligopeptide is H-Glu(NH-Ad)-Lys-Pro-Arg-NH₂.