Enhancement by muramyl peptides of the protective response of interferon-alpha/beta against encephalomyocarditis virus infection.

The use of interferon-alpha (IFN-alpha) in the treatment of infectious diseases has shown limited efficacy and dose-limiting toxicity. We have selected safe immunomodulators of the muramyl peptide family with the potential of enhancing the efficacy of IFN-alpha without resulting in increased toxicity. One of these synthetic muramyl dipeptide (MDP) derivatives, namely murabutide which is in a clinical stage of development, has been recently found to synergize with IFN-alpha 2a in the selective induction of anti-inflammatory mediators and to enhance the biological activities of the therapeutic cytokine. The present study was performed to assess the antiviral activity of such muramyl peptides and a possible potentiation of the antiviral activity of IFN-alpha/beta by associated therapy using the classical assay of Encephalomyocarditis virus (EMCV) infection. In vitro, pretreatment of Moloney Sarcoma virus (MSV)-transformed cell line with MDP derivatives followed by treatment with IFN-alpha/beta showed a synergistic protection against the cytopathogenic effect of a subsequent EMCV infection. None of the MSV cultures could be protected by stimulation with muramyl peptides alone. In vivo, all of the muramyl peptide derivatives tested were found to be more potent than the parent molecule MDP in inducing protection against death or in the prolongation of the mean survival time of infected mice. Sequential administration of suboptimal doses of exogenous IFN-alpha/beta and muramyl peptides established a strong antiviral state and considerably improved the protective effect of the cytokine, frequently leading to an abortive infection. Our findings suggest that combination therapy with safe muramyl peptides and IFN-alpha/beta could constitute a highly effective and new regimen for the treatment of viral infections in humans.

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.

NMR study of a lymphocyte differentiating thymic factor. An investigation of the Zn(II)-nonapeptide complexes (thymulin).

Detailed investigations of a serum peptide (less than Glu1-Ala2-Lys3-Ser4-Gln5-Gly6-Gly7-Ser8-++ +Asn9) were carried out by 1H and 13C NMR spectroscopy to elucidate the structure of the complex formed with Zn(II), thymulin, which has been found to be active in vivo. These experiments were performed in dimethyl sulfoxide-d6 solution at different metal:peptide ratios. The results suggest the following conclusions. (i) The Zn(II) complexation corresponds to a fast exchange on the NMR time scale. (ii) The evolution of 1H and 13C NMR chemical shifts indicates the existence of two types of complexes: a 1:2 species associating two peptide molecules and one Zn(II) ion and a complex with 1:1 stoichiometry. The former is predominant for metal:peptide ratios below unity. (iii) In the 1:2 complex, Zn(II) is coordinated by the Ser4-O gamma H and Asn9-CO2- sites, while in the 1:1 complex, Ser8-O gamma H is the third ligand to the Zn(II) ion. The results are compared with those for the [Ala4] and [Ala8] analogues, and those for the complexes of thymulin with other metal ions (Cu2+ and Al3+) in terms of its biological activity. These comparative studies suggested that the 1:1 complex is the only conformation recognized by the antibodies.

Structural requirements for the induction of "immunological castration" by linear monomeric LHRH-lys-MDP administered in saline.

It was previously shown that "castration" could be obtained in male mice by immunizing them in saline with a conjugate referred to as LHRH-Lys-MDP and containing the decapeptide hypothalamic hormone LHRH covalently linked to the adjuvant glycopeptide MDP-Lys. Since coupling was made using carbodiimide, it could have produced oligomers or isomers as well as monomers. In the present investigation male and female mice were immunized in saline with a linear monomeric MDP linked LHRH molecule obtained by total synthesis. Histological studies showed gonadal alterations in both male and female mice. The study of analogs provided a correlation between the "castrative" activity of LHRH-Lys-MDP and its chemical and antigenic structures. However, because LHRH antibody levels were not very high, mechanisms other than antibody response are discussed. Such totally synthesized molecules including a safe adjuvant could make a clinical use of LHRH immunization possible in endocrine-dependent cancers.

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.

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.

Synthesis of conjugates between luteinizing hormone releasing hormone (LH-RH) and N-acetyl-muramyl-L-alanyl-D-isoglutamine (MDP) models of totally synthetic vaccines.

Two glycopeptides associating the aminoacid sequence of LH-RH with MDP were prepared, using a Lys residue as a linker. These conjugates, N alpha MDP N epsilon (LH-RH)-Lys and N alpha MDP N epsilon (LH-RH)-Lys-NH2 obtained by condensation of fragments were synthesized by liquid as well as solid phase methods. Both compounds were able to induce anti LH-RH antibodies and immunological castration. They retained the immuno-adjuvant activity of MDP. Such antigen-adjuvant constructs, devoid of carrier and obtained by chemically defined and reproducible synthetic methods could offer suitable tools for structure-activity relationship studies aiming at defining synthetic vaccines.

NMR study of thymulin, a lymphocyte differentiating thymic nonapeptide. Conformational states of free peptide in solution.

The nonapeptide less than Glu-Ala-Lys-Ser-Gln-Gly-Gly-Ser-Asn (formerly called serum thymic factor) is a factor produced by the thymic epithelium, which needs a zinc ion to express its immunoregulatory properties. We report here on 1H and 13C NMR investigation of the conformational properties of the free peptide in aqueous medium and in dimethyl sulfoxide-d6 solution by a combination of homo- and heteronuclear one- and two-dimensional experiments. The various resonances have been assigned in a straightforward manner on the basis of 1H,1H COSY spectroscopy for the recognition of the proton spin systems; two-dimensional NOESY spectra with the correlation peaks across amide bonds and for the amino acid sequence assignment; amide bonds and for the amino acid sequence assignment; 13C,1H COSY experiments using selective polarization transfer from 1H- to 13C-nucleus via the 13C,1H long-range couplings for the attribution of the carboxyl and carbonyl groups; and 13C,1H COSY experiments with selective polarization transfer via the 13C,1H direct couplings for the assignment of all the aliphatic carbons. Other experiments such as pH-dependent chemical shifts, combined use of multiple and selective proton-decoupled 1H and 13C NMR spectra, the temperature and the concentration dependence of the proton shifts of the amide resonances, the solvent dependences of peptide carbonyl carbon resonances, and comparison of the spectra with three different analogues were performed. In aqueous solution, the data are compatible with the assumption of a highly mobile dynamic equilibrium among different conformations, whereas in dimethyl sulfoxide-d6, a more rigid structure is found involving three internal hydrogen bonds. These observations provide an insight into the conformational tendencies of this peptidic hormone in two different media.

Activation of macrophage cytostatic and cytotoxic activity in vitro by liposomes containing a new lipophilic muramyl peptide derivative, MDP-L-alanyl-cholesterol (MTP-CHOL).

The ability of liposomes containing a new lipophilic muramyl peptide derivative, MDP-L-alanyl-cholesterol (MTP-CHOL), to induce peritoneal macrophage cytostatic activity and alveolar macrophage cytotoxic activity toward tumor cell targets in vitro was determined. MTP-CHOL was shown to be efficiently incorporated and subsequently retained in distearoylphosphatidylcholine/phosphatidylserine liposomes (DSPC/PS; 7:3 molar ratio), whereas hydrosoluble muramyl dipeptide (MDP) was rapidly lost due to leakage. Liposomes containing MTP-CHOL were able to stimulate mouse peritoneal macrophage cytostatic activity under conditions where free MDP was without effect. MTP-CHOL incorporated into liposomes was approximately eightfold more effective than liposomes containing entrapped MDP and 7,400-fold more effective than free MDP in inducing rat alveolar macrophage cytotoxic activity. These results provide evidence that the coupling of MDP to a lipophilic molecule, cholesterol, results in the formation of a viable liposome formulation that is a potent inducer of macrophage-mediated antitumor activity.

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.

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.

[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.

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.

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.

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).

[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.

Contribution of zinc and other metals to the biological activity of the serum thymic factor.

The serum thymic factor (FTS) utilized in its synthetic or natural form loses its biological activity in a rosette assay after treatment with a metal ion-chelating agent, Chelex 100. This activity is restored by the addition of Zn salts and, to a lesser extent, certain other metal salts. FTS activation is secondary to the binding of the metal to the peptide. The metal-to-peptide molar ratio of 1:1 provides the best activation. These data indicate the existence of two forms of FTS. The first one lacks Zn and is biologically inactive; the second one contains Zn and is biologically active, for which we propose the name of "thymulin" (FTS-Zn). The presence of Zn in synthetic FTS was confirmed by atomic absorption spectrometry. The interaction between Zn and FTS was further suggested by microanalysis demonstrating the presence of this metal in thymic reticuloepithelial cells.

Biological activity of a new synthetic muramyl peptide adjuvant devoid of pyrogenicity.

Immunostimulant activities of muramyl dipeptide (enhancement of specific immune responses and of nonspecific resistance to infection) were retained by its N-acetylmuramyl-L-alanyl-D-glutaminyl-n-butyl ester derivative, although very large amounts administered intravenously, or even by the very sensitive intracerebroventricular route, did not elicit fever in the rabbit. This analog also appeared to be devoid of other secondary effects which have been observed after administration of muramyl dipeptide.