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Sleep-promoting materials isolated from human urine and rabbit brain are muramyl peptides (MPs). The most active component of the urinary material is N-acetylglucosaminyl-1,6-anhydro-N-acetylmuramyl-Ala-Glu-diaminopimel yl-Ala; 1 pmol, infused into a lateral cerebral ventricle of rabbits, induced excess slow-wave sleep (SWS) for several hours. MP-induced sleep is normal in that it is similar to the deep sleep that follows sleep deprivation. Other biological actions of MPs (e.g., pyrogenicity and immunomodulatory activity) could be dissociated, but only in part, from somnogenic actions. Interleukin 1, a substance thought to mediate many MP activities, is somnogenic, and thus may be involved in MP-induced sleep. That MPs and other immunologically active substances can greatly enhance SWS suggests that some immunological mechanisms integrate sleep in their actions.

A synthetic peptide whose sequence was derived from the circumsporozoite protein of Plasmodium knowlesi coupled to bovine gamma globulin has been shown to be immunogenic when administered with Freund complete adjuvant. The present experiments were designed to test the immunogenicity of the peptide when attached to a tetanus toxoid carrier and administered with alum or murabutide, both acceptable clinical adjuvants. In both cases, the use of an adjuvant increased the levels of circulating anti-peptide antibodies over those observed when no adjuvant was used. However, when the antisera were tested for reactivity with the native protein, animals of the group receiving the conjugate associated with murabutide always had titers greatly exceeding those observed in animals that received the conjugate with alum. Moreover, the sera of the murabutide-treated group were shown to be more active in eliciting shedding of the circumsporozoite protein than were sera of animals of the Freund complete adjuvant-treated group. The use of tetanus toxoid in secondary immunizations could be eliminated when the mice primed with peptide-tetanus toxoid and murabutide were boosted with a polymer of the peptide. The results indicate that the synthetic malarial peptide-tetanus toxoid conjugate is capable of stimulating high levels of biologically active antibodies only when administered with murabutide.

The efficacy of N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP), 6-O-stearoyl-MDP (L18-MDP), N alpha-acetylmuramyl-L-alanyl-D-isoglutaminyl-N epsilon-stearoyl-L-lysine (MDP-Lys-L18) and N-stearoylmuramyl-L-alanyl-D-isoglutamine (2N-L18-MDP) for augmenting host-resistance to viral infection was examined in Sendai virus infected mice. L18-MDP and MDP-Lys-L18 augmented the non-specific host-resistance to infection with Sendai virus. MDP showed a slight enhancement of host-resistance to this infection but 2N-L18-MDP was ineffective. The protective effect of MDP-Lys-L18 was seen only when the drug was administered a few days before the virus challenge. The intranasal administration of MDP-Lys-L18 was effective at 1 microgram but only slight activity was observed in mice treated intravenously or intraperitoneally even at the 100 microgram dose level. MDP-Lys-L18 treatment preceding infection augmented interferon production in the lung of the mice but MDP-Lys-L18 treatment alone induced no interferon.

Multiple intralesional injections of quinonylmuramyl dipeptide-66, a new synthetic immunoadjuvant, with a vehicle oil such as squalene or squalane resulted in the regression of line-10 hepatocarcinoma in strain-2 guinea pigs. This chemically synthesized adjuvant should be a useful immunotherapeutic agent for cancer.

The purpose of these studies was to determine whether the combination of a low-dose local thoracic irradiation (LTI) followed by systemic activation of macrophages with liposomes containing muramyl tripeptide phosphatidylethanolamine (MTP-PE) would significantly decrease established experimental fibrosarcoma lung metastases. Male C3Hf/Kam mice were given i.v. injections of 1 X 10(5) fibrosarcoma cells. Five days later, groups of mice were treated with saline, with 8 Gy LTI, or with liposomes containing MTP-PE or first with 8 Gy LTI and followed by multiple i.v. injections of liposomes containing MTP-PE. Most of the mice in the groups treated with liposomes died by day 42 of the experiment. In contrast, 60% of the mice treated with the combination of LTI and liposomes containing MTP-PE were alive by day 140 of the study. These mice were killed and were found to be free of tumors. Control studies demonstrated that liposomes administered i.v. to mice given LTI were trapped in the capillary bed of the lungs and activated the tumoricidal properties of lung macrophages. We conclude that, in this combination, low-dose LTI, which can lead to both tumor cell death and inflammatory changes in the lung capillaries, could precede i.v. administration of liposomes containing MTP-PE. This combination of treatments can lead to destruction of tumor foci in the lung that cannot be achieved with either treatment alone.

Many adjuvant-active analogs of muramyl dipeptide (MDP) have been described. Unfortunately, most have been found to induce pyrexia or other adverse side effects in several species. In the guinea pig, these side effects include the guinea pig distress syndrome, which lasts 24 to 48 h, and enhancement of endotoxin shock. A dose-related response to MDP or N-acetylmuramyl-L-alpha-aminobutyryl-D-isoglutamine [( Abu1]-MDP) was observed. However, we found several adjuvant-active analogs which were completely innocuous in guinea pigs. 2-(2-Acetamido-2-deoxy-D-glucose-3-O-yl)-D-hexanoyl-L-alanyl-D-isoglutam ine (3'-n-propyl-MDP) was one such compound. To understand the differences between the toxic and nontoxic compounds, macrophage stimulation in vitro was studied. Both compounds stimulated guinea pig peritoneal macrophages to produce lymphocyte-activating factor in vitro. However, the supernatant from 3'-n-propyl-MDP-stimulated macrophages had no observable effects on guinea pigs, whereas the dialyzed supernatant from [Abu1]-MDP-stimulated cells readily induced the guinea pig distress syndrome. Sephadex G-150 chromatography showed that both analogs stimulated production of a major (60,000) and minor (11,000) peak of lymphocyte-activating factor activity. The factor causing the guinea pig distress syndrome was found only in the low-molecular-weight fraction of [Abu1]-MDP-treated cell supernatants. Thus, one difference between the nontoxic and toxic compounds is their ability to produce lymphocyte-activating factor in the absence of the toxic factor. The lack of toxicity of 3'-n-propyl-MDP makes it an attractive compound for use in vaccines.

The effects on adriamycin cardiotoxicity of an immunoadjuvant, 2-[2-acetamido-2-deoxy-6-0-[10-(2,3-dimethoxy-5-methyl-1, 4-benzo-quinon-6-yl) decanoyl]-D-glucopyranos-3-0-yl]-D-propionyl-L-valyl D-isoglutamine methyl ester (QMDP-66), and a reference compound, ubiquinone-10, were studied in Wistar Kyoto (WKY) rats. Adriamycin, 1 mg/kg/day intra-peritoneally (i.p.) for 23 days, elicited cardiotoxicity, as judged by widening of the QRS complexes in ECGs. Electron microscopic examination of myocytes from the treated rats revealed many cytoplasmic vacuoles possibly originating from deranged endoplasmic reticula or mitochondria. In addition, the treatment significantly inhibited body weight gain, and decreased ventricular weight. QMDP-66 alone, 1 mg/kg/day i.p. for 23 days, had no effect on the parameters described above. When QMDP-66 (1 mg/kg/day, i.p.) or ubiquinone-10 (3 mg/kg/day, i.p.) was administered together with adriamycin, the widening of the QRS complexes was significantly depressed, and cytoplasmic vacuoles in myocytes were rarely observed. The QMDP-66 or ubiquinone-10 treatment, however, did not alleviate the decrease in body weight gain or ventricular weight due to adriamycin. Heart rate was not significantly changed by any of the treatments. These findings suggest that QMDP-66 is an effective antidote against adriamycin cardiotoxicity.

The antifilarial activity of combination of diethylcarbamazine (DEC) and an immunomodulator, N-Palmitoylmuramyl-L-alanyl-D-isoglutamine (NP-MDP) was evaluated against Litomosoides carinii in cotton rat (Sigmodon hispidus) and Mastomys natalensis. DEC was used at 6 mg/kg in cotton rat whereas it was 75 mg/kg x 5 days in mastomys. The immunomodulator was administered at 62.5 to 500 micrograms/animal x 2 days. Combination therapy with optimum dose of immunomodulator resulted in prolonged and significant suppression of microfilaraemia in comparison to infected animals treated only with DEC. The effective doses of immunomodulator alone or in combination with DEC also caused enhanced antibody titre in treated animals. Though combination therapy resulted in prolonged suppression of microfilaraemia, the effect disappeared slowly and caused no damage to adult worms.

The restoration of resistance by N alpha-acetylmuramyl-L-alanyl-D-isoglutaminyl-N epsilon-stearoyl-L-lysine [MDP-Lys(L18)] on herpes simplex virus (HSV) type-1 infection was examined in cyclophosphamide (CY)-treated mice. MDP-Lys(L18) was shown to restore the resistance to HSV infection in CY-treated mice when it was injected either subcutaneously, intravenously, or intraperitoneally before infection. Treatment with MDP-Lys(L18) in CY-treated mice restored impaired activity for inhibiting HSV growth in the liver.

N2-[(N-Acetylmuramoyl)-L-alanyl-D-isoglutaminyl]-N6-stearoyl-L-lysine (MDP-Lys(L18), muroctasin), a muramyl dipeptide derivative, has been reported to enhance host resistance to infections, as well as displaying adjuvant activities on the humoral and cellular immune responses. This paper reviews briefly its immunopotentiating activities, which were found to be attributed to its augmenting effect on interleukin 1 production.

Cell walls isolated from 29 strains of 24 gram-positive bacterial species, whose peptidoglycans belong to the group A type of Schleifer and Kandler's classification, with one exception (Arthrobacter sp.), were shown to activate the complement cascade in pooled fresh human serum mainly through the alternative pathway and partly through the classical one. The complement-activating effect of cell walls (5 species) possessing group B type peptidoglycan, except those of Corynebacterium insidiosum, was weaker than that of the walls with group A type peptidoglycan. Preparations of peptidoglycan isolated from cell walls of Staphylococcus aureus, Streptococcus pyogenes, and Lactobacillus plantarum also activated the alternative pathway of the complement cascade, but less effectively than the respective parent cell walls. A water-soluble "polymer" of peptidoglycan subunits (SEPS), which was prepared from Staphylococcus epidermidis peptidoglycans by treatment with a cross-bridge degrading endopeptidase, retained most of the complement-activating ability of the parent cell walls. A peptidoglycan "monomer," SEPS-M, which was obtained by hydrolysis of the glycan chain of SEPS with endo-N-acetylmuramidase to disaccharide units did not activate complement. In conformity with this finding, neither synthetic N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP) nor MDP-L-Lys-D-Ala activated the complement cascade. Among several lipophilic derivatives of MDP, 6-O-(3-hydroxy-3-docosylhexacosanoyl)-MDP-L-Lys-D-Ala (BH48-MDP-L-Lys-D-Ala) and 6-O-(2-tetradecylhexadecanoyl)-MDP (B30-MDP) were shown to activate complement through the alternative as well as the classical pathway and exclusively through the classical pathway, respectively. The finding that a D-isoasparagine analog of B30-MDP caused the same effect as the parent molecule strongly suggests that the activation of complement by B30-MDP is different from that caused by cell wall peptidoglycans and a water-soluble "polymer" of peptidoglycan subunits.

Decomposition of N2-[(N-acetylmuramoyl)-L-alanyl-D-isoglutaminyl]-N6-stearoyl-L-lysine (MDP-Lys(L18), muroctasin) under extreme conditions was investigated. MDP-Lys(L18) in an aqueous solution was heated under reflux for 4 h to give the decomposition products D-1, D-2, D-3 and D-4. Reaction of MDP-Lys(L18) with 0.1N NaOH at room temperature for 5 h gave D-1, and that with 1 N HCl under reflux for 1 h afforded D-4. MDP-Lys(L18) was found to be stable to light and heat. MDP-Lys(L18) in a powder form was stored at room temperature or at 25 degrees C and 75% relative humidity for 3 days to 18 months. Quantitative analysis, thin-layer chromatography, and tests for appearance and color change, etc., were performed in the stored samples. MDP-Lys(L18) was found to be stable, but hygroscopic.

Muramyl dipeptide (MDP) and the butyl ester derivative, N-acetylmuramyl-L-alanyl-D-glutamine-alpha-n-butyl ester (MDP[Gln]OnBu), were shown to induce the in vitro proliferation of oil-induced guinea pig peritoneal exudate cells (PEC). Both agents induced 10-20 fold increases in tritiated thymidine incorporation in PEC cultures. The maximal effects occurred in 72 h cultures stimulated with either 0.1 microgram MDP or 10 micrograms MDP[Gln]OnBu. The mitogenic effects of MDP appeared to be mediated by a macrophage product detected in the supernatants of MDP-stimulated cultures. Supernatants of MDP- or MDP[Gln]OnBu-stimulated PEC cultures were also inhibitory to normal fibroblast growth and cytotoxic to L929 tumor cells. These results indicated that these agents may stimulate macrophages by modulating secretory functions. In addition, either peptidoglycan was capable of activating bactericidal activity in in vitro macrophage cultures. Initial studies of possible mechanisms of action revealed an early increase in the level of cyclic GMP. The possible role of cyclic GMP in mediating the stimulation of macrophage secretory processes is discussed.

A synthetic muramyl dipeptide derivative N2-[(N-acetylmuramoyl)-L-alanyl-D-isoglutaminyl]-N6-stearoyl-L-lysine (MDP-Lys(L18), muroctasin) was studied for mutagenicity using the Ames method, in vitro cytogenetics and micronucleus test. MDP-Lys(L18) had no mutagenic effect on S. typhimurium (TA1535, TA1537, TA1538, TA98 and TA100) or E. coli (WP2 uvrA) in the reverse mutation assay. In the cytogenetic study, MDP-Lys(L18) had no effect on the chromosomes of the Chinese hamster cells at cytotoxic doses. Single subcutaneous treatment of MDP-Lys(L18) at dose levels of 3.5, 35 or 350 mg/kg in the mouse micronucleus test did not increase the incidence of micronucleated polychromatic erythrocytes. These results show that MDP-Lys(L18) has no demonstrable mutagenic potential.

A previous paper has demonstrated that enhanced tumor-specific immunity could be induced by priming mice with Bacillus Calmette Guerin (BCG) and subsequently immunizing them with syngeneic tumor cells modified with BCG-cross-reactive muramyl dipeptide (MDP) hapten. The present study establishes a tumor-specific immunotherapy protocol for a murine chronic leukemia based on the above T-T cell collaboration between antitumor effector T cells and anti-MDP hapten helper T cells induced by BCG priming. BALB/c mice which had been primed to BCG were injected intravenously (i.v.) with viable, syngeneic BCL1 leukemia cells. One week later, these mice were immunized intraperitoneally (i.p.) with unmodified or MDP hapten-modified, 10,000 R X-irradiated BCL1 cells, followed by 4 booster immunizations at 5-day intervals. The administration of unmodified BCL1 tumor cells into BCG-primed mice failed to prevent them from tumor death due to the persistent growth of preinjected BCL1 cells. In contrast, the immunization of BCG-primed, BCL1 leukemia-cell-bearing mice with MDP-modified BCL1 cells resulted in a high growth inhibition of leukemia cells and protection of these mice from death by leukemia. It was also revealed that potent tumor-specific, T-cell-mediated immunity was generated in mice which survived in this immunotherapy model. Thus, these results indicate that administration of MDP hapten-modified, syngeneic leukemia cells into leukemia-bearing mice which have been primed with BCG results in potent tumor-specific, T-cell-mediated immunity attributable to preventing the growth of disseminated leukemic cells.

Muramyl dipeptide (MDP) and adjuvant-active derivatives were confirmed as unable directly to induce differentiation of mouse myeloid leukemia M1 cells. They were, however, found effective in stimulating either rabbit macrophages or human blood monocytes to produce differentiation-stimulating activity (D factor). The various conditioned media (CM) thus obtained were able to induce differentiation of the myeloblastic M1 cell line as indicated by the appearance of Fc receptors and inhibition of cell proliferation. Among the synthetic glycopeptides inducing the production of D factor, murabutide (MDP[Gln]-OnBu) was as effective as MDP, although it did not stimulate monocytes to simultaneously release endogenous pyrogen. The absence of pyrogenicity in murabutide CM was attested by IV or intracerebroventricular administration to rabbits. However, in the same CM, LAF(IL1) activity estimated by potentiation of the in vitro proliferative response to phytohemagglutinin of mouse thymocytes was usually higher than that induced by MDP.

The potentiation of endotoxin-induced necrosis and regression of solid syngeneic Meth A tumors in mice previously observed following administration of N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP) was investigated further by use of various muramyl peptide analogues and two unrelated synthetic adjuvants, viz. the pluronic polyol L121 and dimethyldioctadecylammonium bromide (DDA) instead of MDP. All agents were administered in aqueous solution by the IV route. None of the muramyl peptide analogues nor L121 or DDA had any strong antitumor action of their own. Two 6-O-acylated muramyl peptides (L2-MDP and B30-MDP) and muramyl dipeptide stearoyllysine [MDP-Lys (L18)] clearly potentiated endotoxin-induced necrosis and regression. In contrast, MDP with L- instead of D-isoglutamine was completely inactive. Optimal activity of B30-MDP and MDP-Lys (L18) was only achieved by adding of suitable amounts of a nonionic surfactant. L121 and DDA could not replace muramyl peptides as potentiating agent. The combination of endotoxin, MDP, and L121 caused complete tumor regression in all mice, but was highly toxic. On the basis of the data in the literature on the biological response-modifying activities of the agents used it is concluded that the potentiating activity of muramyl peptides cannot yet be related to their immunoadjuvant action or their capacity to activate macrophages or to enhance nonspecific bacterial resistance.

Serotonin at low micromolar concentrations inhibited binding of two [125I]-labeled muramyl peptides to resident mouse peritoneal cells and to a macrophage-derived cell line, PU5-1.8-F7. Binding of [3H]serotonin was inhibited in parallel fashion. Overnight incubation with serotonin or muramyl peptide enhanced the release of superoxide by both types of cells when later stimulated with phorbol myristate acetate. Serotonin antagonists decreased binding of muramyl peptide and serotonin and diminished the subsequent enhancement of superoxide release. A cell line variant lacking detectable binding sites for muramyl peptide was far less responsive (superoxide release) than the parent line, to either drug. The data are consistent with sharing of a common set of receptors on the macrophage by muramyl peptide and serotonin and with involvement of these receptors in enhancing superoxide release.

Thirteen 6-O-acyl-N-acetylmuramyl-L-alanyl-D-isoglutamines (6-O-acyl-MDPs), including four inactive D-isoasparagine and L-isoglutamine analogs, were tested for their pyrogenicity and immunopotentiating activity to stimulate primary humoral and cellular immune responses in guinea pigs to a model protein antigen, ovalbumin, when administered in various vehicles. Among them, derivatives whose muramic acid residue was substituted by alpha-branched (and beta-hydroxylated) higher fatty acids at the carbon-6 position, especially 6-O-(2-tetradecylhexadecanoyl)-MDP (B3O-MDP) and, to a lesser extent, 6-O-(3-hydroxy-2-docosylhexacosanoyl)-MDP (BH48-MDP) and its L-serine analog [BH48-MDP(L-Ser)], were found to exert strong adjuvant activity in both the induction of delayed-type hypersensitivity and the stimulation of circulating precipitating antibody levels when combined with nonirritating vehicles (liposomes, squalene-in-water emulsion, and phosphate-buffered saline). These vehicles did not efficiently support the adjuvant activity of MDP, the parent molecule of the above lipophilic derivatives. Pyrogenicity tests showed that introduction of alpha-branched higher fatty acid groups but not of straight, long-chain fatty acids at the 6-position of the muramic acid residue resulted in marked decrease of the pyrogenicity inherent to MDP via intravenous administration.

The effect of N-acyl derivatives of muramyl dipeptide (N-acetyl muramyl-L-alanyl-D-isoglutamine) on the activation of peritoneal adherent cells (PAC) in vivo and on the stimulation of nonspecific host resistance against Escherichia coli infection was examined in comparison with the effect of 6-O-stearoyl muramyl dipeptide. N-acyl muramyl dipeptide derivatives increased the release of hydrogen peroxide (H2O2) by PAC from mice treated 1 day before upon stimulation with phorbol myristate acetate, and their activities did not depend on the chain length or kinds of fatty acids introduced. The results obtained using N-stearoyl muramyl dipeptide analogs indicated that the acyl moiety combined to muramic acid played a more important role in the ability of PAC to release H2O2 than did the peptide moiety. PAC from mice treated with N-stearoyl muramyl dipeptide, N-(3-hydroxy-2-docosylhexacosanoyl) muramyl dipeptide, and 6-O-stearoyl muramyl dipeptide 1 day before, including 20 to 42% polymorphonuclear leukocytes, released large amount of H2O2, and most of the H2O2 released was due to the attribution of polymorphonuclear leukocytes. The cytostatic activity of PAC from mice treated with these three compounds reached a maximum on day 3 after injection, and the cytolytic activity of PAC was induced by N-stearoyl muramyl dipeptide on day 3 and by 6-O-stearoyl muramyl dipeptide on day 1 after injection. In contrast to the above results, N-acyl muramyl dipeptide derivatives did not stimulate nonspecific host resistance against E. coli infection in mice when compared to 6-O-stearoyl muramyl dipeptide.

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.

The main metabolite of N2-[(N-acetylmuramoyl)-L-alanyl-D-isoglutaminyl]-N6-stearoyl-L-lysine MDP-Lys(L18), muroctasin), excreted to the urine and accounting for 15% of the dose, was identified to be R-D-lactic acid by the reverse isotope dilution method in which the metabolite was derivatized to p-bromophenacyl ester, recrystallized and separated into enantiomers by HPLC. The configuration of the lactate moiety in MDP-Lys(L18) was retained during the metabolism. Five metabolites were detected in the liver 5 hours after the administration and 4 of them were identified by TLC as des(GlcNAc)-MDP-Lys(L18), lactic acid, MDP-Lys(L18) and N-acetylmuramic acid in comparison with respective authentic compounds. N-Acetylmuramic acid was confirmed further by the reverse isotope dilution method in which it was converted to peracetylated methyl ester and separated by gas chromatography. It was the main metabolite and accounted for 40% of the radioactivity detected in the liver. MDP-Lys which was supposed to be the pharmacologically active metabolite was not detected. The metabolic pathway in which MDP-Lys(L18) was mainly metabolized to N-acetylmuramic acid, lactic acid and carbon dioxide, successively, was proposed. Some portion of the drug was converted to lactic acid via des(GlcNAc)-MDP-Lys(L18). A novel cleavage reaction of the ether linkage between the 3 position of sugar and lactic acid moiety in N-acetylmuramic acid was observed.

The efficacy of N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (nor-MDP) in controlling viral oncogenesis in mice was investigated. The tumors studied were blood cell malignancies induced by Friend leukemia virus in SJL/J mice, spontaneous mammary neoplasms in RIII/Imr and C3H/OuJ mice, and spontaneous lymphocytic leukemia in AKR/J mice. A transplantable tumor, Lewis lung carcinoma, in C57BL/6J mice was used as a nonvirally induced control model. The nor-MDP was dissolved in saline and made into an emulsion with an equal volume of squalene-Arlacel A and injected subcutaneously at 1- to 2-month intervals. Test and control mice were challenged with exogenous virus or tumor transplant 2 weeks after a second injection of nor-MDP. Administration was started at around 2 months of age in mice that develop spontaneous neoplasms. Electron microscopy studies were done on neoplastic tissues of selected test and control mice. This administration of nor-MDP prevented erythroleukemia in SJL/J mice caused by low doses of Friend leukemia virus (although erythroleukemia survivors were not protected from a late-developing lymphoma) and also delayed (possibly prevented) the development of a spontaneous mammary neoplasm in RIII/Imr mice. No antitumor effects were observed on the spontaneous neoplasms of C3H/OuJ and AKR/J mice or on the Lewis lung carcinoma implanted into C57BL/6J mice. Electron microscopic examinations of the various neoplastic tissues indicated that nor-MDP administration eliminated or reduced extracellular viruses. The results suggested that under the experimental conditions used nor-MDP appears to effect the viruses and not the malignant cells per se.

L18-MDP(Ala), a synthetic derivative of muramyl dipeptide, enhanced resistance against Escherichia coli and Candida albicans infections in aged mice and younger adult mice whose defense mechanism(s) had been depressed by X-ray irradiation or by treatment with cyclophosphamide.

Lipophilic derivatives of muramylpeptides, namely N alpha-(N-acetylmuramyl-L-alanyl-D-isoglutaminyl)-N epsilon-stearoyl-L-lysine [MDP-Lys (L18)] and 6-O-(2-tetradecylhexadecanoyl)-MDP (B30-MDP), were demonstrated to significantly enhance anti-bovine serum albumin (BSA) antibody production when they were incorporated in liposomes with BSA and administered by gastric intubation to BALB/c mice on days 0 and 1 (the primary immunization) and on days 27 and 28 (booster). N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP) itself showed negligible activity under the same experimental conditions. A stearoyl derivative of sodium beta-N-acetylglucosaminyl-(1-4)-N-acetylmuramyl- L-alanyl-D-isoglutaminyl-(L)-stearoyl(D)-meso-diaminopimelic acid-(D)-amide-D-alanine (GM-53) that was isolated by enzymatic degradation of L. plantarum cell wall peptidoglycans showed a powerful adjuvant effect by oral administration in liposomes with BSA. Similar or stronger adjuvant effects were observed by oral administration of LPS preparations, KO3 LPS isolated from K. pneumoniae (a noncapsulated mutant, LEN-1), Bacto lipopolysaccharide W derived from E. coli (O127:B8) and BIOSTIM F1 fraction derived from K. pneumoniae (O1:K2). Liposomes as a vehicle for oral administration were not always required for the manifestation of the adjuvant effects of MDP-Lys (L18) and BIOSTIM F1. These compounds, but not B30-MDP, showed a powerful adjuvant effect when orally administered with BSA in phosphate buffered saline.