Muramyl peptides. Variation of somnogenic activity with structure.

Sleep-promoting activities of muramyl dipeptide (MDP) (NAc-Mur-L-ala-D-isogln) and the naturally occurring muramyl peptide(s), factor S, have recently been demonstrated. We now have amplified our understanding of structural requirements for somnogenic activity. The effects of several analogs of MDP on rabbit slow-wave sleep are presented and these results are compared to the dose-response relationship for MDP. Some tentative conclusions as to structural requirements for somnogenic activity are presented; most notably, amidation of the free gamma-carboxyl of MDP and several of its analogs resulted in the loss of somnogenic activity. MDP also can induce febrile and immunostimulatory responses. In the present paper, we show that some analogs possess immunostimulatory and pyrogenic activity but not somnogenic activity, thus suggesting that these biological activities of muramyl peptides may, in part, be mediated by separate mechanisms.

Activation of alveolar macrophage tumoricidal activity and eradication of experimental metastases by freeze-dried liposomes containing a new lipophilic muramyl dipeptide derivative.

The ability of a member of a new class of lipophilic muramyl dipeptide (MDP) derivative, muramyl dipeptide-glyceryldipalmitate (MDP-GDP), to induce alveolar macrophage cytotoxic activity in vitro towards B16 melanoma cells when incorporated into two types of liposome was studied. MDP-GDP incorporated into conventionally prepared liposomes formulated from distearoylphosphatidylcholine and phosphatidylserine (7:3 molar ratio) was 10-fold more effective than liposomes containing MDP, and 7000-fold more effective than free MDP in inducing macrophage cytotoxic activity. MDP-GDP incorporated into freeze-dried liposomes was 50,000- to 100,000-fold more effective than free MDP in inducing such activity. Freeze-dried liposomes containing MDP-GDP were efficiently localized in the lungs of normal mice, and induced cytotoxic activity in the alveolar macrophages. Such liposomes were able to significantly reduce the pulmonary metastatic burden of mice carrying the B16 melanoma. These data provide evidence that this class of lipophilic MDP derivative, when incorporated into freeze-dried liposomes, is a potent inducer of macrophage cytotoxic activity in vitro and in situ, and has antitumor activity in vivo. In addition, the use of a freeze-drying procedure allows the preparation and long-term storage of reproducible liposome formulations.

Decrease in pyrogenicity of muramyl dipeptide after coupling with luteinizing hormone-releasing hormone.

Muramyl dipeptide (MDP) and its adjuvant active derivative lysine-MDP (Lys-MDP) have been demonstrated to be pyrogenic and to induce endogenous pyrogen (EP) production in vivo and in vitro. It has recently been shown that immunologic castration can be achieved in mice by immunization with luteinizing hormone-releasing hormone (LHRH) directly conjugated by carbodiimide to Lys-MDP, termed LHRH-Lys-MDP (cdi), or with a linear monomeric MDP-linked molecule obtained by total synthesis, termed LHRH-Lys-MDP (s). These preparations were tested in the rabbit for their capacity to induce fever and were found to be devoid of pyrogenicity at dosage levels of Lys-MDP that induced fever. This decrease of pyrogenicity of Lys-MDP after coupling to LHRH seems to be related to the structure of the conjugate because the derivative LHRH-LysNH2-MDP exhibited the same pyrogenic activity as the free glycopeptide. Surprisingly, nonpyrogenic LHRH-Lys-MDP induced production of EP and interleukin-1 (IL-1) in vitro and increased in vivo modifications of metal levels attributed to the action of IL-1. Moreover, LHRH-Lys-MDP reduced the pyrogenic effect of an exogenous dose of EP.

Effect of a collagen derived octapeptide on different steps of the platelet/collagen interaction.

The interaction of platelets with collagen involves short aminoacid sequences which recur along the fibres. Platelet aggregation by collagen and serotonin release is inhibited by a synthetic octapeptide LYS-PRO-GLY-GLU- PRO-GLY-PRO-LYS- derived from type III collagen. In contrast, this octapeptide inhibits only weakly the retention of platelets labelled with 111Indium to collagen, suggesting that it has a limited effect on platelet adhesion. Preincubation of the octapeptide with platelets inhibits the rise of cAMP level caused by activating adenylate cyclase by various concentrations of PGI2. The octapeptide at 5 mM reverses the inhibition by PGI2 of the adhesion of platelets to collagen. These results suggest that the octapeptide affects the intrinsic activity (manifested as platelet aggregation and secretion) more than the recognition of collagen by its receptor (manifested by adhesion).

Chemistry of immunomodulators.

A large number of synthetic derivatives, inorganic compounds or naturally occurring substances are able to depress, regulate or enhance the immune response. Immunomodulators, among which some are chemically well defined and others are complex preparations, exhibit a great variety of chemical structures which are briefly reviewed, without details on their immunopharmacological properties. These molecules allow access to a new type of therapy which aims at acting on the host defense mechanisms.

A comparative 1H-n.m.r. study of MurNAc-L-Ala-D-iGln (MDP) and its analogue murabutide: evidence for a structure involving two successive beta-turns in MDP.

The active principle, MurNAc-L-Ala-D-iGln (MDP), of complete Freund's adjuvant and its analogue, MurNAc-L-Ala-D-Gln-OnBu (murabutide), which express immunomodulatory as well as other biological properties, have been studied by 2D-1H-n.m.r. spectroscopy at 500 MHz. The results suggest the presence in MDP of two successive turns involving the MurNAc-L-Ala and L-Ala-D-iGln moieties, respectively, whereas only the former turn persists in murabutide. This turn mimics the type II beta-turn found in L-D depsipeptides, whereas the other is a typical type II beta-turn for L-D peptides.

Colony-stimulating activity induced by synthetic muramyl peptides: variation with chemical structure and association with anti-infectious activity.

The in vivo induction of colony-stimulating activity (CSA) by N-acetylmuramyl-L-alanine-D-isoglutamine has been demonstrated recently. In this study we increased our understanding of this property by testing muramyl peptides of several structures and activities for their capacity to induce CSA in vivo. A comparison of the anti-infectious and adjuvant activities of these molecules revealed no correlation between the capacities of these compounds to be adjuvant active and to induce CSA: all adjuvant-inactive compounds induced CSA, and certain adjuvant-active molecules did not induce CSA. In contrast, all anti-infectious compounds induced CSA, but the reverse was not true; some compounds devoid of anti-infection activity were able to induce CSA only if they were adjuvant active.

Synthesis of N-acetyl-muramyl-L-alanyl-D-isoglutamine, an adjuvant of the immune response, and of some n-acetyl-muramyl-peptide analogs.

A relatively easy synthetic method is reported for the production of the immunoadjuvant glycopeptide, N-acetyl-muramy-L-alanyl-D-isoglutamine (MDP). Most of the details in this method were also applied to the preparation of some analogs, that were synthesized in order to study structure-activity relationships.

Macrophage stimulation in vitro by an inactive muramyl dipeptide derivative after conjugation to a multi-poly(DL-alanyl)-poly(L-lysine) carrier.

It has been previously reported that N-acetyl-muramyl-L-alanyl-D-isoglutamine (MDP), which represents the minimal structure that can substitute for mycobacteria in Freund complete adjuvant, activated macrophages in vitro and in vivo. In the present study we show that, in contrast to MDP, the nonadjuvant MDP(DD) stereoisomer has no effect on cytostatic activity of thioglycolate-induced macrophages as measured by uptake of [3H]thymidine. However, surprisingly, after conjugation to an inert carrier, multi-poly(DL-alanyl)-poly(L-lysine), this compound activates macrophages in vitro and becomes at least as effective as MDP. It has also been shown in other studies that after conjugation MDP(DD) remained devoid of antigenicity and of adjuvant activity although such a conjugate could increase resistance to infection. It, therefore, appears that there exists no correlation between the structure required for adjuvant activity and the structure required for macrophage activation or for enhancement of nonspecific immunity.

Synthesis of N-acetyl-muramyl-L-alanyl-D-glutamic-alpha-amide(MDP) or -alpha-methyl ester derivatives, bearing a lipophilic group at the C-terminal peptide end.

We report the synthesis of nine lipophilic derivatives of N-acetyl-muramyl-L-alanyl-D-glutamic-alpha-amide (MDP) or -alpha-methyl ester in which the gamma-carboxyl function of the D-glutamyl residue is either esterified by a medium chain alcohol or substituted by an L-alanyl residue esterified by a medium or long chain alcohol. A new method is described which easily allows one to obtain derivatives of MDP, bearing a free or substituted amino-acyl or peptidyl residue on the gamma-carboxyl function.

[Nuclear magnetic resonance study of the interaction of zinc with thymulin]. / Etude par résonance magnétique nucléaire de l'interaction du zinc avec la thymuline.

Interaction of Zn2+ with thymulin was investigated by 1H NMR spectroscopy. In DMSO-d6 solution, Zn2+ forms a complex with a nonapeptide involving the C-terminal carboxylate and the hydroxyl groups of the two serine residues in position 4 and 8, in a tetrahedral structure.

Induction of murine macrophage tumoricidal activity and treatment of experimental pulmonary metastases by liposomes containing lipophilic muramyl dipeptide analogs.

The ability of three members of a new class of lipophilic muramyl dipeptide derivative to induce murine macrophage tumoricidal activity after liposomal incorporation was investigated. Liposomes containing the glycerol dipalmitate (GDP) derivatives of N-acetylmuramyl-L-alanyl-D-isoglutamine, N-acetylmuramyl-L-alanyl-D-glutamine-n-butyl ester, and N-acetylmuramyl-D-alanyl-D-isoglutamine were 5000, 2000, and greater than 10,000-fold more potent than the free muramyl dipeptides in inducing peritoneal macrophage tumoricidal activity in vitro. In situ activation of peritoneal macrophage tumoricidal activity showed that liposomal muramyl dipeptide-GDP derivatives were more potent than free hydrosoluble or sonicated muramyl dipeptide-GDP preparations. In situ induction of alveolar macrophage tumoricidal activity after i.v. treatment was observed with liposomes containing muramyl dipeptide-GDP derivatives, but not with hydrosoluble or sonicated lipophilic derivatives. Liposomes containing muramyl dipeptide-GDP derivatives were therapeutically active against experimentally induced pulmonary B16 melanoma tumors in C57BL/6 mice. These results demonstrate that when incorporated within liposomes this class of lipophilic muramyl dipeptide derivative is a potent inducer of macrophage tumoricidal activity both in vitro and in situ, and possesses antitumor activity in therapeutic treatment protocols.

Modulation of the immune response by a synthetic adjuvant and analogs.

N-Acetylmuramyl-L-alanyl-D-isoglutamine increases the humoral immune response of mice when given in aqueous media instead of the usual water-in-oil emulsions. Moreover, this compound is adjuvant active even by the oral route. In view of studying the relation between chemical structure and biological activity, several synthetic analogs were tested. The immune response could be modulated according to chemical modifications, and the synthetic analog with D- in place of L-alanine was shown to inhibit the immune response.

Relationship between chemical structure and adjuvant activity of some synthetic analogues of N-acetyl-muramyl-L-alanyl-D-isoglutamine (MDP).

Synthetic N-acetyl-muramyl-L-alanyl-D-isoglutamine, hereafter referred to as muramyl dipeptide or MDP, represents the minimal structure which can replace mycobacterial cells in Freund's complete adjuvant. This synthetic adjuvant can also enhance the humoral antibody response when administered to mice with an antigen in water by various routes including the oral route. The present paper reports some new results concerning the relationship between the structure and the adjuvant activity of MDP. Synthetic analogues of MDP were tested either in a water-in-oil emulsion in guinea-pigs or in saline in mice. The chemical modifications concerned essentially the D-glutamyl residue. The results obtained confirm the importance of maintaining the intact structure of the D-Glu residue and show that adjuvant activity can be abolished by certain modifications at the level of both carboxyl functions.

[Enhancement of the activity of hepatitis B virus vaccine by association with murabutide]. / Renforcement de l'activité d'un vaccin contre l'hépatite virale de type B par association avec le murabutide.

Murabutide (N-acetyl-muramyl-L-alanyl-D-glutamine-alpha-butylester), an MDP analogue, is a potential adjuvant for Human immunization. High levels of specific antibodies were obtained in Mouse and Guinea-Pig following administration with murabutide of low dosages of anti-hepatitis B viral vaccine containing the surface antigen (HBs). The effect of murabutide was enhanced without increasing the level of specific IgE by association with suboptimal dosages of aluminium hydroxide.

[Role of zinc and other metals in the biological activity of the serum thymic factor (thymulin)]. / Role du zinc et d'autres métaux dans l'activité biologique du FTS (thymuline).

The serum thymic factor (FTS) used in synthetic or natural form, loses its biological activity after passage on a chelating agent, Chelex 100. Such activity is recovered after addition of zinc and, to a lesser degree, of certain other metals. FTS activation is secondary to zinc binding to the peptide. These results indicate the existence of two FTS forms: the first one, deprived of zinc and biologically inactive, the second one containing zinc and biologically active, for which we purpose to coin the name of thymulin.