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.

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.

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.

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.

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.

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.

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.

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.

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.

[Nuclear magnetic resonance study of the interaction of zinc with thymulin].

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.

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.

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.

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.

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.

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.

[Enhancement of the activity of hepatitis B virus vaccine by association with 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.

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.

[Role of zinc and other metals in the biological activity of the serum thymic factor (thymulin)].

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.

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.