Abstract:
The invention relates to peptide conjugates comprising a sequence of at least 3 amino acids derived from a thymic hormone selected amongst thymuline and thymopoietine, the amino acids being independently in the form D, L or DL, said sequence being chemically or physically conjugated with at least one compound selected amongst monocarboxylic acids having the general formula (I): HOOC—R, as well as alcohol, aldehyde or amide derivatives, the dicarboxylic acids having the general formula (II): HOOC—R 1 —COOH. The invention also relates to the use of such conjugates as medicaments, and pharmaceutical or cosmetological compositions containing them.

Description:
CROSS-REFERENCES TO RELATED APPLICATIONS 
     This application is a National Stage application of International Application No. PCT/FR96/01812 filed Nov. 15, 1996. 
    
    
     BACKGROUND OF THE INVENTION 
     The invention relates to novel synthetic derivatives of two thymic hormones, i.e. thymulin and thymosin, and to their uses as a medicament or in the field of cosmetology. 
     The large number of studies which have been carried out on the functioning of the immune system have demonstrated the cardinal role played by the thymus and the thymic hormones in stimulating the immune defences of the organism. 
     The thymic hormones, in particular thymulin, “J. F. Bach et al.”, thymopoietin “Grandstein” and thymosin “Dayne” have been identified as being of a polypeptide or peptide nature. 
     These hormones, principally thymulin and thymopoietin, possess the property of inducing maturation of the T lymphocytes and of stimulating the immune response of the organism. 
     It has been scientifically demonstrated that a close relationship exists between the decrease in thymic functions with age and cutaneous ageing under normal physiological circumstances. 
     At a pathological level, the loss of thymic activity, i.e. “involution”, finds expression in the appearance of physiological disorders: “autoimmune diseases, alopecias (abnormal hair loss), etc.”. 
     It has also been shown that a relationship exists between the physiological properties of the thymus and certain cells of the epidermis. 
     Thus, a structural similarity exists between certain cells of the thymus and keratinocytes. It has also been shown that, under certain conditions, the keratinocytes also secrete a hormone of the thymopoietin type which is identical to that secreted by the thymus and are able to induce post-thymic lymphocyte maturation and in this way to take over from the thymus. 
     This relationship between the physiological activities of the cells of the thymus and the keratinocytes has recently been confirmed by demonstrating that keratin and keratohyalin, which have identical structures to those of the keratin and keratohyalin which are secreted by the epidermal keratinocytes, are present in the cells of the thymus. 
     Furthermore, the scientific demonstration that keratinocytes which are in the differentiation phase secrete a substance, i.e. ETAF, which activates thymocyte growth, confirms the physiological relationships which exist between the thymus and the keratinocytes. (ETAF: epidermal thymocyte activating factor). 
     A parallel exists between the “involution” of the thymus and the decrease in the synthesis of ETAF by the keratinocytes as a function of age, with, however, a functional time-lag in favour of the keratinocytes (post-thymic lymphocyte maturation). 
     The thymic hormones, in particular thymulin and thymopoietin, have been used therapeutically in a large number of pharmacological and clinical studies with the aim in view of stimulating and regulating the immune defenses of the organism. 
     When used in the form of extracts or of molecules obtained by chemical synthesis, these products have yielded variable and disappointing results as a result of their lack of stability and a ubiquitous effect linked to the absence of dose-effect relationships. 
     SUMMARY OF THE INVENTION 
     The present invention relates to using chemical synthesis to obtain peptide derivatives of the thymic hormones, the structure of which derivatives has been adjusted so as to provide the derivatives with a high degree of physicochemical stability and a precisely defined pharmacological activity. 
     Using all the knowledge which our research groups have recently acquired and developed concerning the essential role of the epidermis in immune defense and its relationships with the thymus (International Society of Development and Comparative Immunology), the present invention relates to the creation of synthetic peptide derivatives whose physiological and therapeutic activity is directed toward stimulating and modulating the immune defenses of the corium and the epidermis in preference to any systemic action. 
     In one aspect, the invention preferably relates to using chemical synthesis to create metallopeptides whose physiological properties are specifically directed toward activating the cutaneous immune system and stimulating the germinative functions of the basal cells of the epidermis to the exclusion of any secondary effect on systemic (general) immunity. 
     The present invention relates to the creation of peptide molecules which are homologs or derivatives of the active peptide sequences of the principal thymic hormones, i.e. thymulin and thymopoietin. 
     The large number of clinical tests which have been carried out using these hormonal mediators have failed to demonstrate that these mediators have any therapeutic relevance. 
     Thus, when these hormones have been used in the form of injectable preparations for treating immune deficiencies and autoimmune disorders, the results obtained have all been negative because of the ubiquitous activity of the hormones in terms of the doses administered. 
     It appears that these ubiquitous effects are linked to the structure of these compounds which results from synthesis and which does not enable them to bind normally to globulins and cell receptors, taking into account the fact that they are administered parenterally. 
     For this reason, one aspect of the invention relates to creating compounds in the form of metallopeptides which are obtained by synthesis and whose physiological activities are directed toward modulating the immune system and activating the germinative cells of the epidermis, by means of local topical applications in accordance with the pharmacological principle of “structure-activity relationships” (Hänch&#39;s principle). 
     More specifically, the present invention relates to a pharmaceutical or cosmetological composition which contains at least one peptide conjugate which comprises a sequence of at least three amino acids which are derived from the total sequence of thymulin or of thymopoietin, with it being possible for the amino acids to be in the D, L or DL form, the said sequence being conjugated chemically or physically with at least one compound which is selected from: 
     monocarboxylic acids of the general formula 
     
       
         HOOC—R  (I) 
       
     
      in which R represents a straight-chain or branched, optionally substituted C 1 -C 20  aliphatic radical which can contain one or more unsaturations, as well as the alcohol or aldehyde or amide derivatives of the acids of the formula I; 
     dicarboxylic acids of the general formula 
     
       
         HOOC—R 1 —COOH  (II) 
       
     
      in which R 1  represents a straight-chain or branched divalent aliphatic radical which comprises at least 3 carbon atoms, preferably from 3 to 10 carbon atoms, in particular an alkyl, alkylene, alkenylene or alkynylene radical, and which can contain one or more unsaturations and can optionally be substituted, in particular by one or more amino, hydroxyl or oxo groups or by a C 1 -C 3  alkyl radical, with it also being possible for R 1  to form a cycle with one of the acid functions. 
     The conjugates according to the invention are low-molecular-weight derivatives which are obtained, in particular, in the form of salts, esters or amides of the compounds of the formula I or II. 
     The compounds of the formula I which are particularly preferred are carboxylic acids which have a metabolic activity which is essential for the Krebs tricarboxylic cycle and which act in the mitochondria as coenzymes of oxidative decarboxylation, and whose properties are to bind, preferably covalently, to the ε amino functions of certain peptides, in the form of lipoamides, thereby facilitating presentation of the peptide conjugates, which are obtained in accordance with the invention, to the G class β receptor of the epidermal cells. 
     In the case of these acids, and of others which are suitable for implementing the invention, R can represent a substituted or unsubstituted, straight-chain or branched C 1 -C 20 , in particular C 1 -C 4 , aliphatic radical, in particular an alkyl, alkenyl or alkynyl radical, which can contain one or more unsaturations and which can be substituted by one or more radicals which are selected from the group comprising: NH 2 , OH, oxo or thiol or a nonaromatic cyclic radical which contains from 5 to 6 atoms in the cycle, 1 or 2 of which can be different from carbon, in particular S, O or N, with it being possible for the said cycles to be substituted by C 1  to C 3  alkyl radicals, in particular methyl. 
     In particular, when R represents a C 1 -C 20  aliphatic chain, it can be substituted by a cycle which is selected from                           
     Other compounds of the formula I which are suitable for implementing the invention are those in which, in the general formula I, R can represent a monounsaturated radical, having the cis or trans configuration, of the general formula 
     
       
         R 2 —CH═CH— 
       
     
     in which R 2  can represent a straight-chain or branched alkyl radical which comprises at least 6 carbon atoms, preferably from 6 to 16 carbon atoms, and which is optionally substituted by an amino, hydroxyl or oxo group. 
     Advantageously, in the formula II, R 1  represents an optionally substituted C 4 -C 8  alkylene radical. 
     Peptide conjugates according to the invention are, in particular, those in which the acid of the general formula I is selected from acetic acid, pyroglutamic acid, DL-lipoic acid, dihydrolipoic acid, N-lipoyllysine, hydroxydecenoic and decenoilic acids, retinoic acid and its derivatives, retinal and retinol, myristic acid and its derivatives, palmitic acid, in the form of salts, esters or amides, or else the acid of the general formula II is selected from adipic acid, α-aminoadipic acid, pimelic acid or sebacic acid, and their derivatives. 
     The acids of the general formulae I or II are preferably selected from acetic acid and its derivatives, pyroglutamic acid, α-DL-lipoic acid and its derivatives, dihydrolipoic acid, N-lipoyllysine, adipic acid, α-aminoadipic acid, pimelic acid, sebacic acid and its derivatives, trans-10-hydroxy-Δ2-decenoic acid and trans-9-oxo-2-decenoic acid, retinoic acid and its derivatives, retinol and retinal, myristic acid and palmitic acid. 
     The amino acid sequence of the conjugates according to the invention will contain one of the following 3 sequences: 
     Gln-Gly-Gly 
     Arg-Lys-Asp 
     Lys-Asp-Val 
     Thus, more specifically, the present invention relates to the creation of two series of peptide molecules, whose activities are complementary, for use in the fields of human medicine and dermocosmetology. 
     The present invention preferably relates to peptide conjugates which are derived from circulating thymic hormone, i.e. thymulin, and whose physiological activities are directed toward effecting maturation of the immune system and of the different mediators of the cutaneous immune system. 
     Thus, these peptide conjugates are particularly directed toward treating autoimmune diseases of the skin, and in particular preventing and treating alopecias whether they be of physiological (aging), traumatic or pathological origin. 
     The present invention also relates to creating peptide conjugates which are derived from cellular thymic hormone, i.e. thymopoietin, and whose physiological activities are also directed toward effecting maturation of the immune system of the skin while possessing a preferential activity for stimulating the metabolism of the cells of the epidermis, in particular of the germinative keratinocytes, in which latter they stimulate both secretion formation and the intracellular synthesis of the “low-molecular-weight keratin and keratohyalin” structural molecules. 
     Thus, this series of peptide conjugates is particularly indicated for the treatment of disorders of the skin which are linked to immune deficiencies and, in particular, for the prophylactic or therapeutic treatment of cutaneous ageing which is of a physiological (age) or pathological nature. 
     Preferably, the amino acid sequence of the peptide conjugates which are derived from thymulin corresponds to the following general formula: 
     
       
         X-Gln-Gly-Gly-Y (SEQ ID NO:1) 
       
     
     in which Gln represents glutamine or a derivative of glutamine, Gly represents glycine or a derivative of glycine; the derivatives include, in particular, the halogenated derivatives of the amino acids in question. The amino acids can be in a natural or unnatural form. 
     According to one of the aspects of the invention, the peptide conjugates will therefore have the following formula: 
     
       
         A-X-Gln-Gly-Gly-Y (SEQ ID NO:1)  (III) 
       
     
     in which A is a compound of the general formula I or II, as previously defined, in particular DL-lipoic acid, dihydrolipoic acid or N-lipoyllysine. 
     X is: 
     Ser, Lys-Ser, Ala-Lys-Ser, Pyr-Ala-Lys-Ser (SEQ ID NO:2), a bond 
     bond or Glx-Ala-Lys-Ser (SEQ ID NO:3) 
     in which Glx is: Pyr-Glu, Glu or Gly and its derivatives 
     Y is: 
     Ser-Asn-OH 
     Ser-Asn-NH2 
     Ser-OH 
     Ser-NH2 
     with the amino acids being in the L, D or DL form. 
     In the formulae which follow, Pyr represents pyroglutamic acid. 
     The peptide conjugates of the formula III preferably include a sequence of at least 4 amino acids, in particular of from 4 to 9 amino acids, with this sequence advantageously comprising the sequence Gln-Gly-Gly-Ser. 
     The present invention very specifically relates to the following conjugates: 
     
       
         
               
               
             
               
               
             
               
               
             
               
               
             
               
               
             
               
               
             
               
               
             
               
               
             
               
               
             
               
               
             
               
               
               
             
               
               
             
               
               
             
           
               
                   
                 (SEQ ID NO:5) 
               
             
          
           
               
                 I 
                 A-Pyr-Ala-Lys-Ser-Gln-Gly-Gly-Ser-Asn-NH 2   
               
               
                   
               
             
          
           
               
                   
                 (SEQ ID NO:6) 
               
             
          
           
               
                 II 
                 A-Pyr-Ala-Lys-Ser-Gln-Gly-Gly-Ser-NH 2   
               
               
                   
               
             
          
           
               
                   
                 (SEQ ID NO:7) 
               
             
          
           
               
                   III 
                 A-Ala-Lys-Ser-Gln-Gly-Gly-Ser-Asn-NH 2   
               
               
                   
               
             
          
           
               
                   
                 (SEQ ID NO:8) 
               
             
          
           
               
                   IV 
                 A-Ala-Lys-Ser-Gln-Gly-Gly-Ser-NH 2   
               
               
                   
               
             
          
           
               
                   
                 (SEQ ID NO:9) 
               
             
          
           
               
                 V 
                 A-Lys-Ser-Gln-Gly-Gly-Ser-Asn-NH 2   
               
               
                   
               
             
          
           
               
                 VI 
                 A-Lys-Ser-Gln-Gly-Gly-Ser-NH 2   
                 (SEQ ID NO:10) 
               
               
                   
               
               
                 VII 
                 A-Ser-Gln-Gly-Gly-Ser-Asn-NH 2   
                 (SEQ ID NO:11) 
               
               
                   
               
               
                 VIII 
                 A-Ser-Gln-Gly-Gly-Ser-NH 2   
                 (SEQ ID NO:10) 
               
               
                   
               
               
                 IX 
                 A-Gln-Gly-Gly-Ser-Asn-NH 2   
                 (SEQ ID NO:10) 
               
               
                   
               
               
                 X 
                 A-Gln-Gly-Gly-Ser-NH 2   
                 (SEQ ID NO:10) 
               
               
                   
               
             
          
           
               
                   
                 (SEQ ID NO:26) 
               
             
          
           
               
                 XXIII 
                 A-Ala-Lys-Ser-Gln-Gly-Gly-Ser-Asn-OH 
               
             
          
         
       
     
     with A having been defined previously, as well as to the derivatives of these molecules in the form of salts, esters or amides. 
     The abovementioned amino acid sequences can be obtained in the terminal NH 2  form and in the terminal OH form. 
     Similarly, it is possible, in some cases, for some of these amino acids to possess functions, for example glycosylations. 
     According to another aspect, the invention relates to peptide conjugates which are derived from thymopoietin and which correspond to the following general formula: 
     
       
         A-W-Lys-Asp-Z (SEQ ID NO:15)  (IV) 
       
     
     in which A is a compound of the general formula I or II, as previously defined, or the corresponding radical, in particular acetic acid and its derivatives, DL-α-lipoic acid and its derivatives, dihydrolipoic acid, N-lipoyllysing, adipic acid, α-aminoadipic acid, pimelic acid, sebacic acid and its derivatives, trans-9-oxo-2-decenoic acid or trans-10-hydroxy-2-decenoic acid. 
     
       
         
               
               
             
           
               
                 W represents: 
                 Glu-Gln-Arg, Gln-Arg, Arg, 
               
               
                   
                 Arg-Lys-, Arg-Lys-Asp or a bond 
               
               
                 Z represents: 
                 Val-Tyr-NH 2 , Val-Tyr-OH 
               
               
                   
                 Val-NH 2 , Val-OH, Tyr-OH, Tyr-NH 2 , 
               
               
                   
                 OH or NH 2   
               
             
          
         
       
     
     with W and Z being selected such that at least one of the sequences Arg-Lys-Asp or Lys-Asp-Val is present in the compounds of the formula IV. 
     Peptide conjugates of the formula IV which include the sequence Arg-Lys-Asp-Val (SEQ ID NO:16) yield excellent results. 
     The amino acids can be in the D, L or DL form and can, where appropriate, by glycosylated. 
     The following conjugates of the formula IV are peptide conjugates which are particularly suitable and whose biological activities are complementary to those of the peptide conjugates of the formula III: 
     
       
         
               
               
             
               
               
             
               
               
             
               
               
             
               
               
             
               
               
             
               
               
               
             
           
               
                   
                 (SEQ ID NO:17) 
               
             
          
           
               
                  XI 
                     A-Glu-Gln-Arg-Lys-Asp-Val-Tyr-NH 2   
               
               
                   
               
             
          
           
               
                   
                 (SEQ ID NO:17) 
               
             
          
           
               
                   XII 
                  A-Glu-Gln-Arg-Lys-Asp-Val-Tyr-OH 
               
               
                   
               
             
          
           
               
                   
                 (SEQ ID NO:18) 
               
             
          
           
               
                   XIII 
                 A-Gln-Arg-Lys-Asp-Val-Tyr-NH 2   
               
               
                   
               
             
          
           
               
                   XIV 
                  A-Gln-Arg-Lys-Asp-Val-Tyr-OH 
                 (SEQ ID NO:18) 
               
               
                   
               
               
                 XV 
                 A-Arg-Lys-Asp-Val-Tyr-NH 2   
                 (SEQ ID NO:19) 
               
               
                   
               
               
                 XVI 
                 A-Arg-Lys-Asp-Val-Tyr-OH 
                 (SEQ ID NO:19) 
               
               
                   
               
               
                 XVII 
                 A-Lys-Asp-Val-Tyr-NH 2   
                 (SEQ ID NO:20) 
               
               
                   
               
               
                 XVIII 
                 A-Lys-Asp-Val-Tyr-OH 
                 (SEQ ID NO:20) 
               
               
                   
               
               
                 XIX 
                 A-Arg-Lys-Asp-Val-NH 2   
                 (SEQ ID NO:21) 
               
               
                   
               
               
                 XX 
                 A-Arg-Lys-Asp-Val-OH 
                 (SEQ ID NO:21) 
               
               
                   
               
               
                 XXI 
                 A-Arg-Lys-Asp-NH 2   
               
               
                   
               
               
                 XXII 
                 A-Arg-Lys-Asp-OH 
               
             
          
         
       
     
     with “A” being as previously defined, as are the derivatives of these molecules in the form of salts, esters or amides. 
     The abovementioned amino acid sequences can be sequences of natural or unnatural amino acids. 
     It is hereby pointed out that the abovementioned peptide conjugates can be obtained in the NH 2 -terminal or OH-terminal form. 
     The peptide conjugates according to the invention can also be present in the form of molecular complexes with a metal, in particular zinc. 
     The conjugates according to the invention can be obtained by synthesis, using techniques known to the skilled person. 
     The free peptides can be synthesized by the MERRIFIELD techniques, using two methods. 
     In the first method, a glutamine derivative is used whose γ amide function is protected with “Mbh”, i.e. 4,4-dimethoxybenzhydryl. 
     In the second method, the synthesis is carried out without protecting the γ amide of the glutamine residues. 
     When the first procedure and the second procedure are used, the protected C-terminal part of the dipeptide is respectively linked to the protected tetrapeptide Ser-Gln-Gly and to the protected hexapeptide Ala-Lys-Gln-Gly-Gly (SEQ ID NO:22). 
     Thus, the polypeptides of the sequence 
      X-Gln-Gly-Gly-Y (SEQ ID NO:23), 
     in which Y represents Ser-Asn and X represents Ser or Ala-Lys-Ser, are prepared by linking the protected dipeptide Ser-Asn to the protected peptide X-Gln-Gly-Gly and possibly consecutively eliminating the protecting groups. 
     Therefore, the octapeptide Ala-Lys-Ser-Gln-Gly-Gly-Ser-Asn (SEQ ID NO:14) is obtained when the second procedure is used while the same octapeptide is prepared from the hexapeptide Ser-Gln-Gly-Gly-Ser-Asn (SEQ ID NO:11), by way of the heptapeptide Lys-Ser-Gln-Gly-Gly-Ser-Asn (SEQ ID NO:9), when the first procedure is used. 
     The octapeptide Ala-Lys-Ser-Gln-Gly-Gly-Ser-Asn (SEQ ID NO:14) then enables the two forms to be prepared by adding on the first residue, i.e. PyroGlu or Gln. 
     The process employed for adding the PyroGlu residue onto the sequence Ala-Lys-Ser-Gln-Gly-Gly-Y (SEQ ID NO:24) is the same whatever the possible meanings of Y, namely Ser and Ser-Asn, and the possible meanings which are derived therefrom by altering an amino acid, for example in the case of Ser-Asp or Ala-Asn (SEQ ID NO:24). 
     This addition to the sequence Ala-Lys-Ser-Gln-Gly-GLy-Y is advantageously obtained using the derivative PyrGlu-OTcp, with it being possible for the side chain functions of certain amino acids in the sequence to be protected. 
     The peptide derivatives to which the present invention relates, and which have been described previously, can be used in their peptide form or in the form of conjugates with the previously described carboxylic acids which correspond to the general formula I. 
     The same peptide conjugates can be used in the form of molecular complexes with a metal. 
     The metal employed for preparing the metallopeptide complexes of the present invention is, for example, zinc, in the form of ZnCl 2 , which can be coupled, in salt form, to a carboxylic group of the terminal amino acid, or in the form of “Zn 2 -peptide” complexes in the following proportions of 0.5 to 5%, preferably 1%, of ZnCl 2  per peptide molecule. 
     These proportions are variable depending on the molecular weight of the peptide conjugate. 
     The metallopeptide complex can, in particular, be obtained in a 10% ZnCl 2  solution, for one peptide molecule, after heating at 20° C. for 10 minutes. 
     The complex is then purified, for example on a P-2 biogel column, and eluted with distilled water, after which it is lyophilized. 
     A stable colyophilizate is obtained which contains 1% zinc bound by affinity to the peptide molecule. 
     All of the compounds according to the present invention can be used as medicaments. 
     In particular, the conjugates according to the present invention can be used for preparing a medicament which is intended for correcting deficiencies of the immune system. 
     The pharmaceutical compositions can comprise at least one conjugate according to the invention and excipients which are suitable for administration by the parenteral route or by the external topical route. 
     These compositions can be used in the fields of both human medicine and veterinary medicine; while they can be administered by the oral route or the parenteral route, they are preferably in a form which can be administered by the external topical route. 
     Compositions which are particularly suitable for implementing the invention are those which comprise one of the following conjugates: 
     acetic acid-Arg-Lys-Asp-Val-Tyr-NH 2  (SEQ ID NO:19) 
     Pyr-Ala-Lys-Ser-Gln-Gly-Gly-Ser-Asn-OH (SEQ ID NO:25) 
     where appropriate in the form of a complex with a metal. 
     The peptide conjugates to which the invention relates, their derivatives and their pharmaceutical compositions can be in the form of creams, milks, lotions appropriate, other active principles. 
     They can be used alone or in the form of pharmaceutical combinations or compositions for treating autoimmune disorders in the different fields of dermatology. 
     The invention also relates to the use of the peptide conjugates in dermocosmetology and in cosmetology. 
     The present invention therefore relates to galenic, pharmaceutical and cosmetological compositions which comprise at least one of the previously described peptide conjugates. 
     The peptide conjugates of the invention, their derivatives and their combinations with known active principles can be used in the form of cosmetological and capillary preparations, in particular for rejuvenating and renewing the superficial layers of the skin and, in particular, for the cosmetocapillary treatment of hair loss. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     FIG.  1 : effect of ETF on DNA synthesis. 
     FIG.  2 : effect of ETF, administered by the topical route, on NK cells (target cells: YAC1). The percentage lysis for the different effector cell/target cell ratios is shown for days 1, 3, 6, 9, 15, 18 and 21 of the injection. 
     FIG.  3 : effect of ETF, when administered by the topical route, on NK cells (target cells P815). 
     FIG.  4 : effect of ETF on NK cells in vitro. The figure shows the percentage lysis as a function of the effector cell/target cell ratio for different doses of ETF. 
     FIG.  5 : effect of ETF, when administered by injection, on NK cells. Percentage lysis as a function of the effector cell/target cell ratio is shown both after stimulation with 15 μg of ETF and in the case of control animals. 
     FIG.  6 : role of interferon in stimulation of NK cells by ETF when administered by injection. The percentage lysis is given as a function of the effector cell/target cell ratio in the presence or absence of antiinterferon. 
     FIG.  7 : effect of ETF, when administered by injection, on NK cells: kinetics of the response at 1, 3 and 8 days after administering 15 μg of ETF by the intraperitoneal route. 
    
    
     DETAILED DESCRIPTION 
     The examples which follow are intended to illustrate the invention, in particular the immunostimulatory activity of the synthesized thymic peptides when administered by the topical route, without in any way limiting its scope. 
     EXAMPLE NO. 1 
     Preparation of Conjugate No. II 
     A-Pyr-Ala-Lys-Ser-Gln-Gly-Gly-Ser-NH 2  (SEQ ID NO: 6) 
     A=DL-lipoic acid 
     The conjugate is synthesized in accordance with the following steps using described general methodology: 
     1—Boc-Gln-Gly-Gly-OMe 
     2—Trifluoroacetate-Gln-Gly-Gly-OMe 
     3—Z-Ser-NH-NH 2 (But)→Bop→DIEA→DMF 
     4—Z-Ser-Glu-Gly-Gly-Ser-NH 2  (SEQ ID NO:12) 
     5—Lipoic acid→BOP→DIEA→DMF 
     6—Lipoyl-Pyr-Ala-Lys-Ser-Gln-Gly-Gly-Ser-NH 2  (SEQ ID NO:6) 
     HPLC analysis of the amino acids 
     Asp-1.02; Ser-1.77; Glu-1.0; Gly-2.00; Ala-0.91. 
     EXAMPLE NO. 2 
     Preparation of Conjugate No. III 
     A-Ala-Lys-Ser-Gln-Gly-Gly-Ser-Asn-NH 2  (SEQ ID NO:26) 
     A=adipic acid 
     The conjugate is synthesized in accordance with the following steps using general methodology: 
     1—Z-D-Ala-Lys-(Boc)-Ser(But)→DMF 
     2—Lys-(Boc)-Ser(But)-Gln(Mbh)→DMF 
     3—Gly-Gly-Ser(But)-Asn-O-But (SEQ NO:27) 
     4—Me.OH→CH 3 COOH→D-Ala-Lys(Boc)-Ser(But)-Gln(Mbh) (SEQ ID NO:28)→Gly-Gly-Ser-Asn(But) (SEQ ID NO:29) 
     5—Adipic acid→BOP→DIEA 
     6—Adipoyl-Ala-Lys-Ser-Gln-Gly-Gly-Ser-Asn-NH 2  (SEQ ID NO:26) 
     HPLC analysis of the amino acids 
     Asp-1.02; Ser-1.77; Glu-1.0; Gly-2.00; Ala-0.91. 
     EXAMPLE NO. 3 
     Preparation of Conjugate No. I 
     A-Pyr-Ala-Lys-Ser-Gln-Gly-Gly-Ser-Asn-NH 2  (SEQ ID NO:5) 
     A=acetic acid 
     The methodology described in Example 1 is used to obtain this conjugate from the derivative: Ala-Lys(Boc)-Ser(But)-Gln-(Mbh)-Gly-Gly-Ser(But)-Asn(But) (SEQ ID NO:30) as previously described. 
     A homogenous product is obtained by chromatography at pH 2.3 in solvents G and H. 
     HPLC analysis of the amino acids. 
     Asp-0.97; Ser-1.67; Glu-1.84; Gly-2.00; Ala-0.98. 
     EXAMPLE 4 
     Measurement of Activity on IA+Langerhans Cells and THY.1.2+ Thymocytes of the Epidermis of C57-BL/6 Mice 
     I Summary 
     Keratinocytes of “C57-BL/6” mice which are stimulated with the peptide derivatives of the thymic hormones release mediators (cell messengers), such as ETAF, IL1 and IL6, which induce activation of the immune response of the immunocompetent cells of the epidermis, i.e. the Ia+Langerhans cells and the Thy 1.2+ thymocytes. 
     The measurement of this activation using immunofluorescence techniques is characteristic for the immunomodulatory activity of the thymic peptides. 
     II Materials and Methods 
     I—Experimental Samples—Code: ETF 
     In solution in propylene glycol at a concentration of 10 −5  M/ml 
     ETF—peptide—No. I 
     ETF—peptide—No. III 
     ETF—peptide—No. XV 
     ETF—peptide—No. XIX 
     control=propylene glycol 
     2—Methodology 
     5 to 7-week-old male C 57-BL/6 mice, which are divided into groups of 4 per cage, and which have free access to water and food and which are subjected to a photoperiod of 12 h of light per 24 hours, are given a topical application of the peptide in propylene glycol (50 μl vol) on the dorsal surface of the ear daily for 5 consecutive days. 
     The animals are sacrificed on day 6. The ears are removed, with the dorsal and ventral surfaces being separated. 
     The tissues of the dorsal surface are immersed in Na EDTA (0.020 M) at 37° C. for 2 h. 
     After incubation, the epidermis is removed in the form of an intact sheet, fixed with acetone and rehydrated in PBS (phosphate buffer). 
     The Thy 1-2+ and Ia+ EDC (epidermal dendritic cells) are identified by indirect immunofluorescence double labeling. 
     In the case of each epidermal sheet, the number of cells per mm 2  is determined using the peripheral regions of the same surface. 
     The EDC (epidermal dendritic cells) carrying the Ia+ marker were identified using an anti-Ia+ monoclonal antibody. 
     The epidermal sheets are fixed with acetone and then incubated, at 37° C. for 1 h, in the presence of the anti-Ia+ monoclonal antibody, which has been labeled by the indirect immunoperoxidase method (Kit). 
     After having been incubated at 37° C. for 10 minutes with a solution of aminoethylcarbazole, the preparation is washed with PBS. 
     The Thy 1.2+ EDC were identified by indirect immunofluorescence double labeling. The epidermal sheets were fixed with acetone and then rehydrated in PBS. 
     The tissues were labeled simultaneously, at 4° C. for 16 h, with mouse anti-Thy 1.2+ antibody which was diluted 1:100. The tissue samples were washed three times in PBS for 60 min and then incubated, at 37° C. for 60 min, either with goat antimouse antibody which was coupled to rhodamine and diluted 1:20, or with goat antirabbit antibody which was coupled to fluorescein (fluorescine and diluted 1:20 in PBS. The tissues were then washed in PBS before being mounted as previously described. 
     The controls comprised epidermal sheets which were not labeled with primary antibody and which were incubated only in the presence of the secondary reagents in order to demonstrate any possible crossreactions with the antibodies which were conjugated to rhodamine and to fluorescein. 
     In the case of each epidermal sheet, the number of Ia+ Langerhans cells or Thy 1.2+ EDCs per square millimeter was determined by means of immunofluorescence, using a confocal Zeiss microscope which was equipped for this purpose, in four peripheral areas of the same surface. At least four animals were studied in each of the groups. The density of Ia+ Langerhans cells and Thy 1.2+ EDCs is shown for each measurement. 
     3—Results 
     The results summarized in Table I show that, when administered to C 57 BL/6 mice by the topical route, ETF and its homologs increase the activity of the resident cells of the cutaneous immune system in a highly significant manner. 
     In particular, a significant increase is seen, at doses of from 0.1 to 1 nanogram, in the phenotypes of the Ia+—(HLADR) Langerhans epidermal dendritic cells. 
     In parallel, a significant increase is also observed, at identical doses, in the activity of the epidermal thymocytes of the Thy 1.2+ phenotype, i.e. ETAF-dependent thymocytes. 
     The cutaneous immune response is dependent on the activity of the nucleated keratinocytes which secrete ETAF and the mediators of immunity, that is the 2nd signal without which there is no normal physiological immune function (L.D.H. immunodermatology). ETF and its homologs can be regarded as reestablishing the immune functions of the epidermis by activating the keratinocytes of the basal layers in a physiological manner, as opposed to exogenous antigenic stimulation. 
     
       
         
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
               
               
             
           
               
                   
               
               
                 Results expressed on 4 batches of 4 C 57 BL/6 mice 
               
               
                 Number of Thy 1.2+ and Ia+ cells per mm 2  of epidermis 
               
             
          
           
               
                   
                 Doses 
                 Thy 1.2+ cells 
                   
                 % 
                 Ia+ cells 
                   
               
             
          
           
               
                 Samples 
                 Nanog. 
                 1 
                 2 
                 3 
                 4 
                 Mean 
                 T 
                 1 
                 2 
                 3 
                 4 
                 Mean 
                 % 
               
               
                   
               
               
                 PG controls 
                   
                  80 
                  76 
                  87 
                 110 
                  88.25 
                   
                  98 
                 102 
                 100 
                 110 
                 102.50 
                   
               
               
                 ETF-I 
                 0.10 
                 197 
                 180 
                 192 
                 207 
                 194.00 
                 120 
                 368 
                 388 
                 378 
                 395 
                 382.25 
                 273 
               
               
                   
                 0.50 
                 200 
                 208 
                 199 
                 219 
                 206.50 
                 134 
                 360 
                 392 
                 386 
                 399 
                 384.25 
                 275 
               
               
                   
                 1.00 
                 214 
                 210 
                 208 
                 226 
                 214.50 
                 143 
                 372 
                 410 
                 390 
                 412 
                 396.00 
                 286 
               
               
                 ETF-III 
                 0.10 
                 168 
                 165 
                 180 
                 176 
                 172.25 
                  95 
                 376 
                 382 
                 387 
                 399 
                 386.00 
                 277 
               
               
                   
                 0.50 
                 190 
                 190 
                 205 
                 200 
                 196.25 
                 122 
                 380 
                 388 
                 299 
                 388 
                 363.75 
                 255 
               
               
                   
                 1.00 
                 200 
                 220 
                 216 
                 210 
                 211.50 
                 140 
                 305 
                 400 
                 398 
                 410 
                 378.25 
                 269 
               
               
                 ETF-XV 
                 0.10 
                 160 
                 158 
                 160 
                 170 
                 162.00 
                  84 
                 315 
                 326 
                 320 
                 330 
                 322.75 
                 215 
               
               
                   
                 0.50 
                 176 
                 168 
                 170 
                 186 
                 175.00 
                  98 
                 305 
                 345 
                 330 
                 345 
                 331.25 
                 223 
               
               
                   
                 1.00 
                 198 
                 180 
                 188 
                 199 
                 191.25 
                 117 
                 348 
                 360 
                 348 
                 350 
                 351.50 
                 243 
               
               
                 ETF-XIX 
                 0.10 
                 216 
                 210 
                 219 
                 212 
                 214.25 
                 143 
                 360 
                 390 
                 378 
                 320 
                 362.00 
                 253 
               
               
                   
                 0.50 
                 220 
                 215 
                 228 
                 216 
                 219.75 
                 149 
                 388 
                 398 
                 386 
                 400 
                 393.00 
                 283 
               
               
                   
                 1.00 
                 227 
                 238 
                 230 
                 225 
                 230.00 
                 161 
                 400 
                 410 
                 438 
                 415 
                 415.75 
                 306 
               
               
                   
               
             
          
         
       
     
     EXAMPLE 5 
     Measurement of ETF No. IX Activity on Human Skin Keratinocytes Derived from Primary Culture 
     I Materials and Methods 
     1—Cell Model 
     Culture of normal human keratinocytes 
     
       
         
               
               
               
             
           
               
                   
                   
               
             
             
               
                   
                 Sources: 
                 Mammaplasty 
               
               
                   
                 Primary culture: 
                 The tissues, which have been freshly 
               
               
                   
                   
                 biopsied under conditions of total 
               
               
                   
                   
                 asepsis, are digested in 0.25% tryp- 
               
               
                   
                   
                 sin, at +4° C., in order to separate 
               
               
                   
                   
                 the epidermis from the corium. 
               
               
                   
                   
               
             
          
         
       
     
     The epidermis is then dissociated under gentle mechanical action, or enzymically, in order to obtain the individual cells. Only the basal cells are capable of multiplying in vitro. 
     The culture is carried out at 37° C. in a controlled moist atmosphere (95% o 2 —5% CO 2 ). 
     In monolayer culture, the calcium concentration is between 0.05 and 0.1 mM. Under these conditions, the keratinocytes proliferate rapidly but do not form strata. The synthesis of the keratinocytes is maintained. The intercellular spaces are wide: cell connections are maintained by microvillosities and not by tight junctions. While the tonofilaments of the perinuclear space can be seen, they are still not attached to the anchoring plaques. 
     Stratification can be induced by increasing the final calcium concentration in the culture medium to 1.2 mM. The formation of desmosomes then appears after 2 hours. 
     The level of intracellular potassium and sodium increases from the 12th hour onward; furthermore, it is not blocked by the usual Ca 2+ /Na+ flux inhibitors. 
     Vertical stratification can be seen after 2 days, and cornified envelope cells complete their terminal differentiation. 
     2—Samples Analyzed 
     
       
         
               
               
               
             
           
               
                   
                   
               
             
             
               
                   
                 I 
                 ETF derivative No. IX, which has been lyophilized 
               
               
                   
                   
                 in Dextran (40 g/l) and which is at a concen- 
               
               
                   
                   
                 tration of 10 −6  M in the final culture medium. 
               
               
                   
                 II 
                 Crist. SIGMA thymopoietin - at a concentration of 
               
               
                   
                   
                 10 −6  M in the final culture medium. 
               
               
                   
                 III 
                 Control: 15,000 D dextran - (40 g/l). 
               
               
                   
                 IV 
                 Cell proteins - 50 micrograms per ml cell 
               
               
                   
                   
                 homogenate reference sol. 
               
               
                   
                   
               
             
          
         
       
     
     3—Methodology 
     Human keratinocytes are cultured as described by Henning et al. (1983) or Fuchs et al. (1981). Air/liquid cultures are also carried out using the following methodology. 
     Collagen Lattices on Grids 
     Type I or type IV collagen is incorporated into the culture medium. 
     3T3 fibroblasts are added to this solution at a concentration of 1.5×10 5  /ml and seeded on 35 mm Petri dishes which are then placed at 37° C. 
     After the gel has been formed, medium is added so as to cover the lattices completely. The keratinocytes are seeded and maintained in submerged culture in this way for 7 days. 
     At this stage, the lattices are transferred onto stainless steel grids. These grids are placed in medium-containing dishes for from 7 to 24 days. 
     Artificial Basal Membrane 
     Bovine corneal endothelial cells are cultured on collagen-covered filters. After having been stimulated with a growth factor of the FGF type, these cells secrete collagen IV and laminin, and other molecules, which are deposited on the collagen, thereby reconstituting a basal pseudomembrane. 
     After the bovine endothelial cells have been removed, the keratinocytes are seeded on this support. 
     Artificial Corium 
     Fibroblasts are incorporated into a collagen gel. After retraction of the gel, the keratinocytes are seeded on this lattice. 
     Basal Membrane of the Corium 
     Fragments of human skin are de-epidermalized by being immersed at 37° C. for 5 days in PBS. After such a treatment, the epidermis detaches completely above the basal membrane. The fibroblasts are then killed by successive freezings and thawings. This tissue, which comprises the corium and the basal membrane, is then used as a nutrient layer for the keratinocytes. 
     
       
         
               
               
               
               
             
               
               
               
               
               
             
           
               
                   
                   
               
               
                   
                   
                 Kerat- 
                 67 kD 
               
               
                   
                 Culture on: 
                 ohyalin 
                 keratin 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 1. Plastic + immersion 
                   
                 − 
                 − 
               
               
                   
                 2. Filter covered with coll. I 
                 S 
                 − 
                 − 
               
               
                   
                 or IV 
                 E 
                 − 
                 − 
               
               
                   
                 3. Filter covered with an 
                 S 
                 − 
                 − 
               
               
                   
                 artificial basal membrane 
                 E 
                 − 
                 − 
               
               
                   
                 4. Artificial corium 
                 S 
                 ± 
                 − 
               
               
                   
                   
                 E 
                 + 
                 ++ 
               
               
                   
                 5. Natural corium which has been 
                 S 
                 ± 
                 − 
               
               
                   
                 de-epidermalized and killed 
                 E 
                 ++ 
                 ++ 
               
               
                   
                   
               
               
                   
                 S = submerged  
               
               
                   
                 E = immersed  
               
             
          
         
       
     
     Differentiation of the epidermis is monitored by electron microscopy (Prunieras, 1988), by biochemical analysis (Tinois et al., 1993) and by immunological analysis (Woodcock et al., 1982, Prunieras, 1988). This latter analysis can make use of antiinvolucrin, antiepidermal transglutaminase, antifilaggrin and antikeratin (AE1, AE2 and AE3) antibodies. 
     The 48 Kd keratins are the markers of proliferation. 
     The 50 and 58 Kd keratins are the permanent keratin markers. 
     The 56.5 and 65/67 Kd keratins are the markers of differentiation. 
     The cell markers which are selected are the low-molecular-weight 46/47 KD keratins, which are specific for the growth and cell differentiation of the germinative cells. 
     The filaggrins are markers of keratohyalin. 
     Indirect fluorescent labeling by the sandwich technique—48 hours after adding calcium to the culture medium (stratification). 
     I—“SIGMA” Primary Antibodies 
     rat antihuman 46-47 KD keratin antibody 
     human antihuman 56.5/68 KD keratin antibody 
     human antikeratohyalin (filaggrin) antibody 
     II—“SIGMA” Secondary Antibodies 
     FITC-labeled mouse antirat Ig G2 antibody. 
     FITC-labeled goat antihuman Ig G antibody. 
     Microscopic assays 
     “confocal laser scanner Zeiss microscope” 
     epifluorescent LSM 310 
     Use of immunofluorescence, with graphic recording, for measuring cell markers. 
     Spectrofluorometric assays 
     “Perkin Elmer LS-50 B spectrofluorimeter”. 
     on cell homogenate supernatants. 
     Assays of cell proteins 
     LOWRY technique using a Perkin Elmer UV spectrophotometer (cell homogenate supernatants). 
     II Results 
     Tables Nos. II and III summarize the effect of “ETF” peptide No. IX on the growth of the germinative cells of the basal and suprabasal layers of the epidermis—i.e. “young keratinocytes”. 
     The fluorescence intensity of the cells treated with ETF No. IX is assessed in comparison with the fluorescence intensity of the controls. 
     A significant increase in the germinative prekeratinocyte cells, and their metabolic activity, is observed. In particular, structures resembling those of the thymus medulla are seen in relation to syntheses of low-molecular-weight keratin and “filaggrin” keratohyalins. 
     
       
         
               
             
               
               
               
               
               
               
             
           
               
                 TABLE II 
               
             
             
               
                   
               
               
                 “Summary” 
               
               
                 Effect of ETF on the synthesis by keratinocytes of 46-47 
               
               
                 KD keratin and of cell proteins, as compared with a 
               
               
                 control (proteins expressed in micrograms/ml). 
               
             
          
           
               
                   
                   
                   
                   
                 Keratin/ 
                   
               
               
                   
                   
                 Concen- 
                 Keratin/ 
                 protein 
                 Stimu- 
               
               
                   
                   
                 tration 
                 protein 
                 standard 
                 lation 
               
               
                   
                 Product 
                 M 
                 mean 
                 deviation 
                 % 
               
               
                   
                   
               
               
                   
                 Thymo- 
                 10 −6   
                  94.8 
                  3.4 
                 19 
               
               
                   
                 poietin 
               
               
                   
                 ETF 
                 10 −6   
                 151.5 
                 14.9 
                 90 
               
               
                   
                 No. IX 
                 10 −8   
                 156.0 
                 13.2 
                 95 
               
               
                   
                   
                 10 −10   
                 148.9 
                 22.9 
                 86 
               
               
                   
                   
                 10 −12   
                 113.0 
                  5.6 
                 41 
               
               
                   
                   
               
             
          
         
       
     
     
       
         
               
             
               
               
               
               
               
               
             
           
               
                 TABLE III 
               
             
             
               
                   
               
               
                 “Summary” 
               
               
                 Effect of ETF on the synthesis by keratinocytes of 
               
               
                 keratohyalin and of proteins as compared with a control 
               
               
                 (proteins expressed in micrograms/ml). 
               
             
          
           
               
                   
                   
                   
                   
                 Keratin/ 
                   
               
               
                   
                   
                 Concen- 
                 Keratin/ 
                 protein 
                   
               
               
                   
                   
                 tration 
                 protein 
                 standard 
                 Stimula- 
               
               
                   
                 Product 
                 M 
                 mean 
                 deviation 
                 tion % 
               
               
                   
                   
               
               
                   
                 Thymo- 
                 10 −6   
                 4.99 
                 0.54 
                 35 
               
               
                   
                 poietin 
               
               
                   
                 ETF 
                 10 −6   
                 5.97 
                 0.12 
                 62 
               
               
                   
                 No. IX 
                 10 −8   
                 6.75 
                 0.66 
                 83 
               
               
                   
                   
                 10 −10   
                 5.13 
                 0.25 
                 39 
               
               
                   
                   
                 10 −12   
                 5.11 
                 0.39 
                 38 
               
               
                   
                   
               
             
          
         
       
     
     EXAMPLE NO. 6 
     Effect of ETF on “Mononuclear Phagocyte System” Granulocytes 
     I Introduction 
     The immune response of the epidermis is dependent on the keratinocytes and the Langerhans cells, in association with other “granulocyte-thymocyte” cell lines, etc. 
     The Langerhans cells are considerably less capable of carrying out phagocytosis than are the keratinocytes, a fact which formally differentiates them from macrophages. 
     Phenotypically and functionally, they resemble the veiled cells of the afferent lymph and the interdigitated cells of the lymphatic nodes. 
     The action of the Langerhans cells results from cellular cooperation, in particular with the “phagocyte” granulocytic cells under the dominance of the keratinocytes. 
     It was therefore of interest to assess the effect of ETF on the phagocytic activity of the polymorphonuclear granulocytes and related cell systems, in particular the NK cells (ENKAF, epidermal cell-derived natural killer activating factor). 
     II—Effect of ETF on Blood Granulocytes 
     1) Protocol 
     Human peripheral blood polymorphonuclear cells (neutrophil granulocytes), which are at a concentration of 10 6  cells/ml, are preincubated at 37° C. for 20 mm before adding luminol and serum-opsonized zymosan particles. 
     The chemiluminescence is measured every 3 minutes after the latter reagent has been added. The results for each point represent the mean of 5 independent measurements. (Standard deviation less than 5% on average). Two systems are employed: 
     A. The inducing agent is ETF peptide No. I, which is present during the whole of the test. 
     Normal human peripheral blood granulocytes, which are enriched by being sedimented in dextran, and which are freed of erythrocytes by hypotonic lysis. The ETF is tested at concentrations of 10, 1 and 0.1 μg/ml. 
     B. The ETF inducing agent is removed by washing following preincubation and prior to adding chemiluminescent reagents. 
     Normal human peripheral blood granulocytes, which are purified by settling through dextran and by hypotonic lysis of the erythrocytes. ETF is incubated with the granulocytes at 37° C. for 20 min and then removed by washing before opsonized zymosan and luminol are added. 
     2—Results: (the values represent the mean of 5 measurements) 
     System A 
     
       
         
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                   
               
               
                 Treatment of 
                 Chemiluminescence values at: (mm) 
               
             
          
           
               
                 the cells 
                 3 
                 6 
                 9 
                 12 
                 15 
                 18 
                 21 
               
               
                   
               
               
                 PBS 
                 22,966 
                  77,598 
                 140,423 
                 178,951 
                 198,063 
                 191,781 
                 188,531 
               
               
                 ETF 10 μg/ml 
                 79,358 
                 140,100 
                 170,947 
                 183,578 
                 174,710 
                 160,718 
                 137,798 
               
               
                      1 μg/ml 
                 59,867 
                 138,846 
                 183,578 
                 207,713 
                 203,449 
                 188,901 
                 168,813 
               
               
                   0.1 μg/ml 
                 29,205 
                  86,761 
                 143,463 
                 176,924 
                 193,589 
                 188,573 
                 179,449 
               
               
                   
               
             
          
         
       
     
     System B 
     
       
         
               
               
             
               
               
               
               
               
               
             
           
               
                   
               
               
                 Treatment of 
                 Chemiluminescence values at: (mm) 
               
             
          
           
               
                 the cells 
                 6 
                 9 
                 12 
                 15 
                 18 
               
               
                   
               
               
                 PBS 
                  69,924 
                 114,751 
                 144,838 
                 162,594 
                 164,750 
               
               
                 ETF 10 μg/ml 
                 103,105 
                 119,677 
                 125,307 
                 119,952 
                 112,499 
               
               
                   0.1 μg/ml 
                 100,532 
                 124,819 
                 134,788 
                 129,950 
                 133,707 
               
               
                   
               
             
          
         
       
     
     3—Conclusions 
     A. At concentrations of 10 and 1 μg/ml, ETF has a considerable impact in potentiating the chemiluminescence right from the start. 
     B. Even after washing, ETF, at concentrations of 10 and 0.1 μg/ml, induces significant activation of the granulocytes. 
     EXAMPLE 7 
     Effect of ETF on DNA Synthesis 
     I—Protocol—Sample: ETF Peptide No. VII 
     A pool of normal mouse spleen cells is obtained and incubated with ETF alone, at increasing doses, in microtiter plates. The DNA is assayed on D1, D3 and D5. 
     The results are presented in FIG.  1 . 
     II Conclusions 
     The ETF shows a very clear response, which is proportional to the dose. The strongest dose employed (10 μg/ml) gives the greatest DNA synthesis on all the days of the control, with a maximum on day 3. It should be noted, however, that since the base values are higher on day 3, the specific response is perhaps in actual fact earlier. 
     EXAMPLE 8 
     Effect of ETF on Lysis Plaque-Forming Cells (PFC) 
     I Protocol 
     50 Balb/C mice which are being given 5 μg of ETF by the s.c. route. 10 8  SRBC (sheep red blood corpuscles) in 0.1 ml of PBS are injected i.v. 20, 10 and 3 days after the ETF or 2 days before it. PFC technique of Cunningham and Szenberg. 
     II Results 
     Number of PFC per 10 6  spleen cells. 
     
       
         
               
               
               
             
               
               
               
               
               
               
             
           
               
                   
                   
               
               
                   
                 Date of immuno- 
                 Number of PFC at 
               
             
          
           
               
                 Treatment 
                 stimulation 
                 D + 3 
                 D + 4 
                 D + 5 
                 D + 6 
               
               
                   
               
               
                 SRBC Control 
                 0 
                  86 
                 509 
                 374 
                 119 
               
               
                 5 μg of ETF s.c. 
                 D − 20 
                  69 
                 438 
                 318 
                  84 
               
               
                   
                 D − 10 
                 
                   323 
                 
                 
                   823 
                 
                 337 
                 104 
               
               
                   
                 D − 3  
                  57 
                 399 
                 353 
                 129 
               
               
                   
                 D + 2  
                 119 
                 410 
                 336 
                 135 
               
               
                   
               
             
          
         
       
     
     III Conclusions 
     A response is observed which exhibits a maximum at D+4, with a very significant, substantial increase in the number of PFC in the animals which were given the ETF 10 days before the antigen. 
     EXAMPLE 9 
     Effect of ETF on NK Cells 
     I Effect of ETF on NK Cells—Topical Route 
     1—General Protocol for the Topical Route 
     CBA/H mice (90 mice) target cells: 
     YAC-1 cells which are sensitive to NK cells 
     P 815 cells which are not sensitive to NK cells 
     ETF No. VII 
     Each day for 14 consecutive days, the mice are given 15 μg of ETF, in the form of a 10 −5  M/ml solution in propylene glycol, by the topical route. An identical booster dose is administered on day 18. 
     Lytic activity is measured every 3 days by counting the  51 Cr which is released during 4 hours of incubation for effector cell:target cell ratios of 200:1, 100:1, 50:1 and 25:1. 
     The results on NK cells, when the ETF is administered by the topical route, are presented in FIGS. 2 and 3. 
     The results are extremely clear. A highly significant increase in NK activity is observed, as a function of time, after beginning the treatment with ETF. This activity, which is discernible from day 3 onward, becomes very substantial on days 6 and 9 and is maintained at a very high level for the whole duration of the experiment (21 days). 
     2—Conclusions 
     Non-specific cytotoxicity on the P 815 cells is not detectable at any time during the experiment. This excludes the possibility that administration of the ETF might lead to the induction of non-specifically cytolytic cells or to the polyclonal activation of cytolytic T cells. 
     II Effect of ETF on NK Cells in vitro 
     1—“In-vitro” Assay Protocol 
     Normal CBA mouse spleen cells. Incubator at 37° C.+CO2. 10 7  cells per ml of RPMI 1640 medium+5% fetal calf serum. Incubation with varying quantities of the substance to be studied. YAC-1 target cells. 
     Lysis is measured by counting the  51 Cr which is released during 4 hours of incubation for effector cell/target cell ratios of 200:1, 100:1, 50:1 and 25:1. 
     The results are presented in FIG.  4 . 
     2—Conclusions 
     ETF very strongly stimulates the activity of the NK cells in vitro in a manner which is proportional to the dose. 
     At a concentration of 0.1 μg/ml, the activity of ETF is still much greater than that of 25 μg of poly I:C/ml. 
     (Activity is at least 100 times greater than that of the reference product poly I:C, which is an interferon inducer). 
     III Effect of ETF on NK Cells—Injectable Route 
     1—General Protocol for an Injectable Route 
     4 to 5-month-old CBA/H mice. (140 mice) Target cells: 
     YAC-1 cells which are sensitive to NK cells (lymphoma induced/moloney virus) 
     P 815 cells which are insensitive to NK cells (DBA/2 mastocytoma) 
     The substance to be tested (ETF No. VII) is injected in 0.2 ml of PBS by the i.p. route 24 hours before the test. Lysis is measured by counting the 51 Cr which is released during 4 hours of incubation for effector cell/target cell ratios of 200:1, 100:1, 50:1 and 25:1. 
     2—NK Results/Injectable Route 
     The results on the effect of the ETF in stimulating the NK cells are given in FIG.  5 . They show strong activation, which is very significant (P&lt;0.01), of the NK cells in the animals which were given the ETF (15 mg/i.p.) as compared with the controls. No non-specific cytotoxicity on the P815 cells was observed. 
     It can be seen from FIG. 6 that activation of the NK cells by ETF is due to an interferon induction effect. A drastic reduction in the NK cells is observed when antiinterferon (α-IF) is administered simultaneously. 
     The kinetics of the response of the NK cells on days 1, 3 and 8 of the injection is shown, in comparison with a PBS control, in FIG.  7 . When administration is by an injectable route, stimulation of the NK cells by ETF is of short duration. 
     At doses of tilorone of 1 mg per animal, ETF stimulates both NK cells and pre-NK cells identically in young animals (from 7 to 9 months). 
     Figure Legends 
     FIG.  1 : 
     ETF. DNA synthesis 
     FIG.  2 : 
     ETF. NK cells/topical route                           
     FIG.  3 : 
     ETF. NK cells/topical route                           
     FIG.  4 : 
     ETF: NK cells ‘in vitro’ 
     Comparison of 0.1→100 μg of ETF/ml with 25 μg of poly I:C/ml                           
     FIG.  5 : 
     ETF. NK cells, injectable route 
     FIG.  6 : 
     ETF. NK cells, injectable route 
     Role of interferon in stimulating NK cells with ETF 
     FIG.  7 : 
     ETF. NK cells, injectable route 
     Kinetics of the NK response to ETF 
     NK test on days 1, 3 and 8 following 15 μg of ETF by the i.p. route 
     BIBLIOGRAPHY 
     E. TINOIS, H. DUMAS, Q. GAETANI, D. DUPONT, X. POURADIER DUTEIL, A. ROUGIER Nouv. Dermatol. 12 No. 7, 510, 1993. 
     H. HENNING, K. A. HOLBROOK, S. H. YUSPA The J. of Invest. Dermatol. 81 50-55, 1983. 
     E. FUCHS, H. GREEN CELL, 25 617-625, 1981. 
     M. PRUNIERAS In-vitro methods in pharmacotoxicology, INSERM Symposium 170, 67-76, 1988. 
     J. WOODCOCK-MITCHELL, R. EICHNER, W. G. NELSON, T. SUN The J. of Cell Biol., 95 580-588, 1992. 
     
       
         
           
             30 
           
           
             1 
             9 
             PRT 
             Artificial Sequence 
             
               Description of Artificial Sequence synthetic
      peptide derived from thymic hormones 
             
           
            1
Xaa Xaa Xaa Xaa Gln Gly Gly Xaa Xaa
  1               5 
           
             2 
             4 
             PRT 
             Artificial Sequence 
             
               Description of Artificial Sequence synthetic
      peptide derived from thymic hormones 
             
           
            2
Xaa Ala Lys Ser
  1 
           
             3 
             4 
             PRT 
             Artificial Sequence 
             
               Description of Artificial Sequence synthetic
      peptide derived from thymic hormones 
             
           
            3
Xaa Ala Lys Ser
  1 
           
             4 
             4 
             PRT 
             Artificial Sequence 
             
               Description of Artificial Sequence synthetic
      peptide derived from thymic hormones 
             
           
            4
Gln Gly Gly Ser
  1 
           
             5 
             9 
             PRT 
             Artificial Sequence 
             
               Description of Artificial Sequence synthetic
      peptide derived from thymic hormones 
             
           
            5
Xaa Ala Lys Ser Gln Gly Gly Ser Asn
  1               5 
           
             6 
             8 
             PRT 
             Artificial Sequence 
             
               Description of Artificial Sequence synthetic
      peptide derived from thymic hormones 
             
           
            6
Xaa Ala Lys Ser Gln Gly Gly Ser
  1               5 
           
             7 
             8 
             PRT 
             Artificial Sequence 
             
               Description of Artificial Sequence synthetic
      peptide derived from thymic hormones 
             
           
            7
Ala Lys Ser Gln Gly Gly Ser Asn
  1               5 
           
             8 
             7 
             PRT 
             Artificial Sequence 
             
               Description of Artificial Sequence synthetic
      peptide derived from thymic hormones 
             
           
            8
Ala Lys Ser Gln Gly Gly Ser
  1               5 
           
             9 
             7 
             PRT 
             Artificial Sequence 
             
               Description of Artificial Sequence synthetic
      peptide derived from thymic hormones 
             
           
            9
Lys Ser Gln Gly Gly Ser Asn
  1               5 
           
             10 
             6 
             PRT 
             Artificial Sequence 
             
               Description of Artificial Sequence synthetic
      peptide derived from thymic hormones 
             
           
            10
Lys Ser Gln Gly Gly Ser
  1               5 
           
             11 
             6 
             PRT 
             Artificial Sequence 
             
               Description of Artificial Sequence synthetic
      peptide derived from thymic hormones 
             
           
            11
Ser Gln Gly Gly Ser Asn
  1               5 
           
             12 
             5 
             PRT 
             Artificial Sequence 
             
               Description of Artificial Sequence synthetic
      peptide derived from thymic hormones 
             
           
            12
Ser Gln Gly Gly Ser
  1               5 
           
             13 
             5 
             PRT 
             Artificial Sequence 
             
               Description of Artificial Sequence synthetic
      peptide derived from thymic hormones 
             
           
            13
Gln Gly Gly Ser Asn
  1               5 
           
             14 
             8 
             PRT 
             Artificial Sequence 
             
               Description of Artificial Sequence synthetic
      peptide derived from thymic hormones 
             
           
            14
Ala Lys Ser Gln Gly Gly Ser Asn
  1               5 
           
             15 
             7 
             PRT 
             Artificial Sequence 
             
               Description of Artificial Sequence synthetic
      peptide derived from thymic hormones 
             
           
            15
Xaa Xaa Xaa Lys Asp Xaa Xaa
  1               5 
           
             16 
             4 
             PRT 
             Artificial Sequence 
             
               Description of Artificial Sequence synthetic
      peptide derived from thymic hormones 
             
           
            16
Arg Lys Asp Val
  1 
           
             17 
             7 
             PRT 
             Artificial Sequence 
             
               Description of Artificial Sequence synthetic
      peptide derived from thymic hormones 
             
           
            17
Glu Gln Arg Lys Asp Val Tyr
  1               5 
           
             18 
             6 
             PRT 
             Artificial Sequence 
             
               Description of Artificial Sequence synthetic
      peptide derived from thymic hormones 
             
           
            18
Gln Arg Lys Asp Val Tyr
  1               5 
           
             19 
             5 
             PRT 
             Artificial Sequence 
             
               Description of Artificial Sequence synthetic
      peptide derived from thymic hormones 
             
           
            19
Arg Lys Asp Val Tyr
  1               5 
           
             20 
             4 
             PRT 
             Artificial Sequence 
             
               Description of Artificial Sequence synthetic
      peptide derived from thymic hormones 
             
           
            20
Lys Asp Val Tyr
  1 
           
             21 
             4 
             PRT 
             Artificial Sequence 
             
               Description of Artificial Sequence synthetic
      peptide derived from thymic hormones 
             
           
            21
Arg Lys Asp Val
  1 
           
             22 
             5 
             PRT 
             Artificial Sequence 
             
               Description of Artificial Sequence synthetic
      peptide derived from thymic hormones 
             
           
            22
Ala Lys Gln Gly Gly
  1               5 
           
             23 
             8 
             PRT 
             Artificial Sequence 
             
               Description of Artificial Sequence synthetic
      peptide derived from thymic hormones 
             
           
            23
Xaa Xaa Xaa Gln Gly Gly Ser Asn
  1               5 
           
             24 
             8 
             PRT 
             Artificial Sequence 
             
               Description of Artificial Sequence synthetic
      peptide derived from thymic hormones 
             
           
            24
Ala Lys Ser Gln Gly Gly Xaa Xaa
  1               5 
           
             25 
             9 
             PRT 
             Artificial Sequence 
             
               Description of Artificial Sequence synthetic
      peptide derived from thymic hormones 
             
           
            25
Xaa Ala Lys Ser Gln Gly Gly Ser Asn
  1               5 
           
             26 
             8 
             PRT 
             Artificial Sequence 
             
               Description of Artificial Sequence synthetic
      peptide derived from thymic hormones 
             
           
            26
Ala Lys Ser Gln Gly Gly Ser Asn
  1               5 
           
             27 
             4 
             PRT 
             Artificial Sequence 
             
               Description of Artificial Sequence synthetic
      peptide derived from thymic hormones 
             
           
            27
Gly Gly Xaa Xaa
  1 
           
             28 
             4 
             PRT 
             Artificial Sequence 
             
               Description of Artificial Sequence synthetic
      peptide derived from thymic hormones 
             
           
            28
Ala Xaa Xaa Xaa
  1 
           
             29 
             4 
             PRT 
             Artificial Sequence 
             
               Description of Artificial Sequence synthetic
      peptide derived from thymic hormones 
             
           
            29
Gly Gly Ser Xaa
  1 
           
             30 
             8 
             PRT 
             Artificial Sequence 
             
               Description of Artificial Sequence synthetic
      peptide derived from thymic hormones 
             
           
            30
Ala Xaa Xaa Xaa Gly Gly Xaa Xaa
  1               5