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Orally administered muramyl dipeptide (MDP) was found to prime induction of endogenous tumor necrosis factor (TNF) in mice. This priming effect was observed after oral administration of MDP of more than 100 micrograms/mouse; the maximal time interval between oral administration of MDP and i.v. injection of OK-432, a triggering agent for induction of endogenous TNF, was extended over 3-10 h and then decreased after 24 h. Antitumor effect against Meth-A, MH134, and MM46 tumor cells in mice was observed after oral administration of MDP followed by i.v. injection of OK-432. These findings suggest that orally administered MDP can be used as a priming agent for inducing endogenous TNF in cancer patients, and that MDP, a component of enteric bacteria, must have an important role in maintaining homeostasis through activation of macrophages.

A phase I clinical trial of the macrophage activator, muramyl tripeptide-phosphatidylethanolamine has been carried out in 37 patients (47 courses) at doses of 0.01-6.0 mg/m2 intravenously twice weekly for 4 weeks. Activation of peripheral blood monocytes and drug toxicity were used as the parameters to monitor the trial. Toxicity was acute systemic responses of fever, chills, and hypertension without a clear dose response. No major organ-related toxicity was seen. A dose of 4.0 mg/m2 biweekly produced activation of blood monocytes; a dose of 6.0 mg/m2 produced inhibition. There was one complete response of 3 months duration in a patient with renal cell carcinoma with pulmonary metastases. The optimum dose for phase II studies is in the range of 1-4 mg/m2 twice weekly for 4 weeks, a dose that is well tolerated.

Interleukin-6 (IL-6) was measured in sera from 26 patients with advanced malignancies before and after an I.V. infusion of muramyltripeptide-phosphatidylethanolamine (MPT-PE) in liposomes. Significantly elevated IL-6 could be measured 2 and 4 h after medium (0.25-0.5 mg/m2) and high (1.0-6.0 mg/m2) doses of the drug accompanied by a rise in body temperature. The biological activity of IL-6 in sera could be inhibited in vitro by monoclonal antibodies against IL-6. Other biological effects of MTP-PE in vivo such as leukocytosis and elevated acute phase reactants are discussed in view of increased IL-6 levels. It is concluded that MTP-PE in liposomes generates increased amounts of IL-6 measurable in serum several hours after administration. IL-6 could therefore play an important role in the biological response modification induced by the drug.

Some biological functions of macrophages can be stimulated by muramyl dipeptide (MDP) in vitro. Such stimulation is more efficient when MDP is bound to macromolecule carriers. The macrophage stimulation by MDP bound to glycosylated serum albumin (BSA) or bound to gluconoylated and glycosylated poly-L-lysine (PLK) is investigated. These two types of MDP conjugates are more efficient than free MDP in rendering mouse peritoneal and rat alveolar macrophages cytostatic against various tumor cells. However, the release of mitogenic factor or cytotoxic factor (CF) by activated macrophages varies according to the nature of the carrier (BSA or PLK) and to the nature and content of sugar residues bound to the macromolecule carrier (mannose or 6-phosphomannose). Macrophages activated by MDP bound to glycosylated BSA release mitogenic factor and CF into the medium; anti-recombinant tumor necrosis factor (rTNF) totally inhibits the cytotoxicity of the supernatant. On the contrary, MDP bound to glycosylated PLK induces no secretion of mitogenic factor and a very small amount of CF in the culture medium. The role of CF in the cytostatic activity of activated macrophages is discussed. The released CF is not involved in the cytostatic activity, but TNF-like molecules, expressed at the membrane level, could be implied because anti-rTNF abrogates 40% of the cytostatic activity of the macrophages.

The antiviral activity of a novel biological response modifier (murabutide MDP derivative) has been investigated in 3-week-old OF1 mice infected with influenza (A/Texas/1/77) virus. In each experimental and control group, 10 mice were infected intranasally with a viral dose producing 50% mortality in 5 days and received murabutide via the subcutaneous or intranasal route at various doses either in a simple or in daily repeated administration. All experiments were done in triplicate. Significant prophylactic or therapeutic effects were observed when murabutide was administered the same day as virus, 4 days or 2 days before virus, and 2 days later. These effects varied with the route of administration and the doses of the compound.

Muramyl dipeptide and muramyl tripeptide in free form or encapsulated within multilamellar liposomes were injected intravenously into (C57BL/6 X C3H)F1 mice and strain 2 guinea pigs to evaluate their potential acute, subacute, or chronic toxicity. Animals received either single or multiple (X 3, X 4, X 6, X 8) injections. A definite species variation was established with regard to the toxic nature of muramyl peptides. Mice failed to exhibit any changes of a clinical, biochemical, functional, or morphological nature in response to repeated intravenous administrations of high doses of muramyl peptide (free or in liposomes). In contrast, strain 2 guinea pigs were very sensitive to muramyl peptide, regardless of the dose or number of injections, or whether it was given in liposomes or as micelles (free form). Most guinea pigs exhibited vascular pathology reminiscent of the Schwartzman phenomenon. Further toxicity studies of muramyl peptides administered at relevant biological doses are recommended, as well as studies aimed at elucidating the reasons for the species variations.

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.