Synthetic Muramyl Dipeptide Derivative Research Articles

Intrapleural therapy with MDP-Lys (L18), a synthetic derivative of muramyl dipeptide, against malignant pleurisy associated with lung cancer

N 2-[( N-acetylmuramoyl)- l-alanyl- d-isoglutaminyl]- N 6-stearoyl- l-lysine (MDP-Lys (L18), romurtide) is a synthetic muramyl dipeptide derivative, and has immunomodulating activities including activation of cells of monocyte-macrophage lineage. We examined the effect of intrapleural instillation of MDP-Lys (L18) against malignant pleurisy associated with lung cancer. Six patients with cytologically-positive malignant pleural effusion (four with adenocarcinoma, one with small cell carcinoma and one with large cell carcinoma) were treated with single intrapleural instillation of MDP-Lys (L18) of 200 μg. Clinically, no reaccumulation of pleural effusion for at least 4 weeks was observed in four patients. No major side effects were observed. Total cell number elevated remarkably 4 h after instillation, and main increased population was that of neutrophils. Levels of chemotactic cytokines, such as interleukin (IL)-8, and monocyte chemotactic protein (MCP)-1 and levels of pro-inflammatory cytokines, such as tumor necrosis factor (TNF)-α, interleukin (IL)-1β and IL-6, elevated in pleural effusion, and peak IL-1β and IL-6 levels tended to be higher in clinical responders than non-responders. These results suggest MDP-Lys (L18) instilled by intrapleural route had a potential local immunomodulatory activity. Further study is warranted to further determine the critical factors which correlate with the clinical response.

Study of the adjuvant activity of new MDP derivatives and purified saponins and their influence on HIV-1 replication in vitro

Muramyl dipeptide (MDP), eight new lipophilic MDP derivatives (MDPs) and three purified saponins were evaluated for their ability to induce immune responses in mice immunized with HIV-1 envelope protein rgp160 and for their ability to influence the HIV-1 replication in vitro. Three of nine new synthetic MDP derivatives (β-butyl-MDP, MTPO-26 and β-cholesteryl-MDP) and one saponin (Taurosid I) have been shown to induce strong humoral immune responses to HIV-1 envelope glycoproteins rgp160 and rgp120. Three substances (β-butyl-MDP, MDP-cholyl and β-G27-MDP) induced high levels of T-cell stimulation to HIV-1 rgp160. β-butyl-MDP induced the strongest B- and T-cell responses to HIV-1 glycoproteins. Two substances (β-butyl-MDP and Taurosid I) did not induce an enhancement of HIV-1 replication in vitro and can be considered as promising adjuvants.

Suppression of in vivo IgE and tissue IL-4 mRNA induction by SDZ 280.636, a synthetic muramyl dipeptide derivative

Modulation of IgE isotype expression on B cells is one of the numerous effects of muramyl peptides on the regulation of the immune system. A non toxic diacyl glycerol derivative of muramyl dipeptide (MDP), in which the l-alanine is replaced by l-threonine (MDP-Threo-GDP; SDZ 280.636), is currently under investigation as lead compound for the development of an anti-allergic drug. In this report, the modulatory effect of orally administered SDZ 280.636 in a murine model on polyclonally induced IgE levels is described. In this model, mice are injected i.v. with goat anti mouse IgD (GAMD) and challenged three to four weeks later with goat IgG (GIG). Both the primary and secondary immune responses lead to an increase of serum IgE levels. We demonstrate the efficacy of this muramyl dipeptide derivative in selectively inhibiting a polyclonal IgE response in GAMD-primed, GIG challenged mice without affecting the levels of other immunoglobulin classes. It is further shown that the induction of interleukin 4 (IL-4) gene transcript levels in lymphoid organs, which is observed as a consequence of boosting GAMD pretreated mice with GIG, is selectively suppressed in gut associated lymphoid tissues (GALT) and mesenteric lymph nodes but not in spleen. In contrast, interleukin 13 (IL-13) mRNA levels are not affected by SDZ 280.636. The findings that SDZ 280.636 inhibits polyclonal IgE responses and suppresses IL-4, but not IL-13 mRNA expression point towards differences in the regulatory pathways of IL-4 and IL-13 gene transcription in lymphoid organs. Thus the mechanism of action appears to involve a specific suppression of IL-4 gene transcription in cells occurring in Peyer's patches and mesenteric lymph nodes which are among the first constituents of the immune system encountered by an orally administered drug.

Romurtide, a synthetic muramyl dipeptide derivative, promotes megakaryocytopoiesis through stimulation of cytokine production in nonhuman primates with myelosuppression

The response of megakaryocytes and cytokines to the administration of romurtide, a synthetic muramyl dipeptidederiivative, was investigated in monkeys with myelosuppression by carboplatin-treatment. Romurtide increased the number of megakaryocytes and promoted the shift of megakaryocytes towards high ploidy class indicative of the promotion of the proliferation and maturation of megakaryocytes. The serum levels of interleukin-6, stem cell factor, and erythropoietin elevated significantly before the enhanced response of megakaryocytes induced by romurtide was observed. Romurtide also enhanced production of colony-stimulating factors (CSFs), such as granulocyte-CSF, macrophage-CSF, and granulocyte-macrophage CSF by monkey mononuclear cells. The stimulating effect of romurtide on the production of those cytokines and CSFs is likely to be responsible for the subsequent promotion of the proliferation and maturation of marrow megakaryocytes.

Romurtide, a synthetic muramyl dipeptide derivative, accelerates peripheral platelet recovery in nonhuman primate chemotherapy model

We investigated the therapeutic effects of romurtide, a synthetic muramyl dipeptide derivative, on experimental thrombocytopenia induced by carboplatin in cynomolgus monkeys. A prolonged thrombocytopenia due to a severe myelosuppression was induced by carboplatin. Romurtide given subcutaneously elevated significantly the peripheral platelet counts during both early initiation and later recovery phase of thrombocytopenia, thereby shortening the time required for recovery to a normal platelet level and the duration of thrombocytopenia. An oral administration of romurtide was also found to have a similar therapeutic efficacy to subcutaneous administration. These results demonstrated a possible therapeutic potential of romurtide in the management of thrombocytopenia associated with myelosuppression.

Oral application of romurtide, a synthetic muramyl dipeptide derivative, stimulates nonspecific resistance to microbial infections and hematopoiesis in mice

Romurtide given orally enhanced the nonspecific resistance against microbial infections and hematopoiesis up to the levels achieved by subcutaneous (s.c.) injection of the compound in mice. Oral romurtide conferred protection and, in consequence, enhanced therapeutic efficacy of antibiotics against systemic infections in mice. The leukocytosis followed by the elevations of colony stimulating activity in serum and the colony forming unit of granulocyte-macrophage (c.f.u.-GM) in femoral bone marrow was observed as successive event in mice treated orally with romurtide. To obtain a comparable potency to s.c. injection of the compound at a dose of 0.1 mg per mouse, oral application required doses of 3 and 10 mg per mouse for stimulating the nonspecific resistance to infection and hematopoiesis, respectively.

Enhancement of platelet recovery in X-irradiated guinea pigs by romurtide, a synthetic muramyl dipeptide derivative

The response of megakaryocytes and platelets to the administration of romurtide, a synthetic muramyl dipeptide derivative, was investigated in normal and irradiated guinea pigs. Romurtide was administered subcutaneously in a single dose or daily doses at levels of 1 to 100 micrograms/animal/day to normal animals to assess the dose response. Subsequently, dosage at 100 micrograms/animal/d for 8 consecutive days was initiated in separate groups of animals immediately after 1 Gy total body x-irradiation. In normal animals, a significant dose- dependent increase in the platelet count was noted, and a prolonged thrombocytopoiesis was detectable from 7 through 15 days after the initiation of romurtide administered for 8 days at a dose of 100 micrograms/animal/d. A significant increase in the white blood cell (WBC) count was also observed during days 1 through 11 after beginning romurtide treatment. In the irradiated animals, the treatment with romurtide increased platelet counts during the recovery phase of thrombocytopenia, thus apparently decreasing the time required for recovery to a normal platelet level. Before the rapid recovery of platelet counts by romurtide treatment, a marked increase in the number of megakaryocytes was noted as early as 7 days after irradiation. This increase was accompanied by an accelerated shift of the size distribution of megakaryocytes toward larger size class. Thus, the mean megakaryocyte size was significantly greater in guinea pigs receiving romurtide than in controls. Preceding the increase in the number of megakaryocytes, the serum interleukin-6 levels were found to be approximately 5 times greater than those in control animals. Treatment with romurtide diminished the WBC count nadir, resulting in significantly higher WBC count levels than in controls. Elevation of the plasma fibrinogen level was observed in the treated animals, and normalized gradually after discontinuation of romurtide treatment. These results indicate a possible therapeutic potential of romurtide in the management of thrombocytopenia associated with myelosuppression.

Enhancement of Platelet Recovery in X-Irradiated Guinea Pigs by Romurtide, a Synthetic Muramyl Dipeptide Derivative

AbstractThe response of megakaryocytes and platelets to the administration of romurtide, a synthetic muramyl dipeptide derivative, was investigated in normal and irradiated guinea pigs. Romurtide was administered subcutaneously in a single dose or daily doses at levels of 1 to 100 µg/animal/day to normal animals to assess the dose response. Subsequently, dosage at 100 µg/animal/d for 8 consecutive days was initiated in separate groups of animals immediately after 1 Gy total body x-irradiation. In normal animals, a significant dose-dependent increase in the platelet count was noted, and a prolonged thrombocytopoiesis was detectable from 7 through 15 days after the initiation of romurtide administered for 8 days at a dose of 100 µg/animal/d. A significant increase in the white blood cell (WBC) count was also observed during days 1 through 11 after beginning romurtide treatment. In the irradiated animals, the treatment with romurtide increased platelet counts during the recovery phase of thrombocytopenia, thus apparently decreasing the time required for recovery to a normal platelet level. Before the rapid recovery of platelet counts by romurtide treatment, a marked increase in the number of megakaryocytes was noted as early as 7 days after irradiation. This increase was accompanied by an accelerated shift of the size distribution of megakaryocytes toward larger size class. Thus, the mean megakaryocyte size was significantly greater in guinea pigs receiving romurtide than in controls. Preceding the increase in the number of megakaryocytes, the serum interleukin-6 levels were found to be approximately 5 times greater than those in control animals. Treatment with romurtide diminished the WBC count nadir, resulting in significantly higher WBC count levels than in controls. Elevation of the plasma fibrinogen level was observed in the treated animals, and normalized gradually after discontinuation of romurtide treatment. These results indicate a possible therapeutic potential of romurtide in the management of thrombocytopenia associated with myelosuppression.

In vivo production of exotoxin A and its role in endogenous Pseudomonas aeruginosa septicemia in mice

We have examined the production of Pseudomonas aeruginosa exotoxin A (ETA) and its role in endogenous bacteremia in mice. Mice given P. aeruginosa D4 orally died of bacteremia between days 10 and 13 following cyclophosphamide-induced leukocytopenia. In this model, serum endotoxin was detected beginning on day 7 by the Limulus assay and P. aeruginosa was cultured from blood beginning on day 9. ETA and tumor necrosis factor alpha (TNF) were also detected in serum by enzyme-linked immunosorbent assay beginning on day 9. Purified ETA did not stimulate the production of TNF in normal mice primed with a synthetic derivative of muramyl dipeptide in the absence of endotoxin. However, ETA enhanced and primed endotoxin-induced TNF production in mice. The mortality rate of mice given ETA mutant PAO-PRI (5.0%) was significantly lower than that of mice given the parent strain (78.8%). These data indicate that ETA may be an important factor in the occurrence of P. aeruginosa bacteremia and/or the death of mice. Also, ETA may be responsible for enhancing the production of a lethal dose of TNF in the presence of endotoxin in P. aeruginosa bacteremia.

B30-MDP, a synthetic muramyl dipeptide derivative for tumour vaccination to enhance antitumour immunity and antimetastatic effect in mice

The effect of a muramyl dipeptide derivative (B30-MDP) on the augmentation of antitumour immunity against highly metastatic L5178Y-ML25 mouse lymphoma cells was examined in CDF1 (Balb/c × DBA/2) mice. Mice immunized with a mixture of X-irradiated tumour cells (103) and B30-MDP (100 μg) on 7 days prior to challenge by viable tumour cells displayed a significant decrease in metastasis towards the target organs, liver and spleen, compared with that of untreated mice. Immunization of mice with the mixture on day 5 or 7 after tumour challenge, when the level of glutamic-pyruvic transaminase (GPT) and glutamic-oxaloacetic transaminase (GOT) in sera of mice inoculated with viable tumour cells was observed to be normal, caused less metastasis than immunization with X-irradiated tumour cells alone. Sensitization with X-irradiated tumour cells admixed with B30-MDP induced almost two times higher cytotoxicity of spleen cells against L5178Y-ML25 lymphoma cells than sensitization with X-irradiated tumour cells without B30-MDP. In contrast, cytotoxic activity of spleen cells against another target, L1210 lymphoma cells derived from BDF1 mice, was not observed by immunization with X-irradiated L5178Y-ML25 cells with or without B30-MDP. Specific lysis by splenic cells of the immunized mice against L5178Y-ML25 cells decreased to the normal level when T cells were deleted from the immunized spleen cells by the treatment of rabbit anti-mouse Thy1.2 antibody and rabbit complement. These results indicate that B30-MDP is able to augment a specific tumour immunity due to the enhancement of cytotoxicity mediated by T lymphocytes, and is useful as an immunopotentiating agent for active immunization of inactivated tumour cells.

Augmentation by priming with interferon-gamma of the binding of a muramyl dipeptide derivative to macrophages resulting in synergistic macrophage activation.

Macrophage (MA) activation by recombinant murine interferon-gamma (rMuIFN-gamma) and a synthetic muramyl dipeptide derivative, N alpha-(N-acetylmuramyl-L-alanyl-D-isoglutaminyl)-N epsilon-stearoyl-L-lysine [MDP-Lys(L18)], was examined. The cytostatic activity of MA that had been primed with rMuIFN-gamma against Lewis lung adenocarcinoma cells was augmented extensively by exposure to MDP-Lys(L18) for a minimum of 15 min, though such treatment in the reverse sequence interfered with the effects of rMuIFN-gamma on MA. The binding assays revealed that rMuIFN-gamma bound to MA with an apparent dissociation constant (Kd) of 0.93 X 10(-9) M (16,000 binding molecules/cell), while MDP-Lys(L18) bound nonspecifically and was endocytosed by MA at 37 degrees. The amount of MDP-Lys(L18) bound to the rMuIFN-gamma-primed MA was significantly greater than that bound to the MA without rMuIFN-gamma priming. A small increase in the amount of MDP-Lys(L18) associated with MA was also observed at 4 degrees, but the amount was one order of magnitude less than that at 37 degrees. The binding of rMuIFN-gamma to MA was significantly suppressed by priming with MDP-Lys(L18). These results indicate that the binding of rMuIFN-gamma in preference to MDP-Lys(L18) to MA is of great importance in the mechanisms of MA activation.

Synergistic induction of cytotoxicity in macrophages by murine interferon-gamma and biological response modifiers derived from microorganisms.

The ability of recombinant murine interferon-gamma (rMuIFN-gamma) to activate murine macrophages with or without several biological response modifiers (BRM), including synthetic muramyl dipeptide derivatives (MDPs), was investigated. Mouse peritoneal macrophages were activated by rMuIFN-gamma alone to the cytostatic state, but not the cytolytic state. Other BRM as well as bacterial lipopolysaccharide (LPS), including a lyophilized preparation of an attenuated strain of Streptococcus hemolyticus, a cell wall skeleton of bacillus Calmette-Guerin and synthetic MDPs, were highly active in generating the synergism with rMuIFN-gamma. Macrophages were endowed with the cytolytic activities by combinations of rMuIFN-gamma and MDP-Lys(L18); the combination of 100 U/ml of rMuIFN-gamma with 10 ng/ml of MDP-Lys(L18) was sufficient to induce cytolytic activities in macrophages. The synergism was observed when the macrophages primed with rMuIFN-gamma were treated with LPS or MDP-Lys(L18), but not when the sequence of treatment was reversed. The cytotoxicity of macrophages induced by rMuIFN-gamma with MDP-Lys(L18) was suppressed by priming with MDP-Lys(L18). The suppressive effect was also observed by priming with LPS in combinations of rMUIFN-gamma and LPS. The reason for the suppression of macrophage activation by priming with LPS and MDP-Lys(L18) is at present unknown.

Type-specific opsonic antibodies evoked with a synthetic peptide of streptococcal M protein conjugated to polylysine without adjuvant.

A chemically synthesized copy (S-CB7) of a fragment (35 amino acid residues) of type 24 streptococcal M protein was covalently linked to polylysine with carbodiimide and injected subcutaneously into rabbits without adjuvant. Although the primary immune responses as measured by enzyme-linked immunosorbent assays at biweekly intervals were weak, the secondary responses as measured by both enzyme-linked immunosorbent assays and opsonophagocytic assays were as high as those obtained previously in rabbits immunized with the peptide conjugate emulsified in complete Freund adjuvant. Injection of murabutide, a synthetic muramyl dipeptide derivative of bacterial peptidoglycan, with the initial immunizing dose of peptide conjugate had no apparent effect on the secondary immune responses. These results indicate that protective immune responses may be raised against polylysine conjugates of chemically synthesized peptide copies of streptococcal M protein without adjuvant.

Polymorphonuclear leukocyte activation by a synthetic muramyl dipeptide analog.

N alpha-(N-Acetylmuramyl-L-alanyl-D-isoglutaminyl)-N epsilon -stearoyl-L-lysine, a synthetic derivative of muramyl dipeptide, stimulated the chemotactic mobility, phagocytic activity, and superoxide anion (O2-) productivity of peritoneal polymorphonuclear cells in mice. The chemotactic mobility of both cells preincubated with the adjuvant in vitro and those derived from the mice previously treated with the adjuvant was significantly enhanced. The phagocytic activity of cells preincubated in vitro with the adjuvant was also enhanced transiently, and that of the cells derived from the mice treated subcutaneously with the adjuvant 24 h before intraperitoneal inoculation with Escherichia coli was significantly greater than that of cells from the mice given phosphate-buffered saline instead of the adjuvant. The release of O2- from the cells derived from the adjuvant-treated mice was also greater than that from the cells of untreated control mice. However, the exposure of the cells derived from untreated mice to the adjuvant in vitro did not stimulate O2- generation by the cells.

Stimulation of resistance of immunocompromised mice by a muramyl dipeptide analog.

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.

Effect of L18-MDP(Ala), a synthetic derivative of muramyl dipeptide, on nonspecific resistance of mice to microbial infections.

By subcutaneous treatment with an aqueous solution of 6-O-stearoyl-N-acetylmuramyl-L-alanyl-D-isoglutamine [6-O-CH3-(CH2)16-CO-MurNAc-L-Ala-D-isoGln] [referred to here as L18-MDP(Ala)], an augmentation of the resistance of mice to Escherichia coli, Pseudomonas aeruginosa. Staphylococcus aureus, and Candida albicans infections was observed, but not to infections with Klebsiella pneumoniae and Listeria monocytogenes. Against E. coli infections, L18-MDP(Ala) was highly protective, irrespective of the administration route. Bacteremia occurring at an early phase of such infections was almost completely prevented by subcutaneous treatment 1 day before infection. Single or multiple doses were also effective against C. albicans infection. The phagocytosis of E. coli by mouse peritoneal polymorphonuclear cells was enhanced by treatment with the adjuvant, and the phagocytosis of K. pneumoniae was also enhanced, but only when the mice were treated either with rabbit normal serum or with a specific immune serum. The growth of the fungus in the kidneys was significantly inhibited, and growth was eliminated from the kidneys by treatment with the adjuvant once a day for 4 consecutive days, starting 1 day before infection. However, no growth suppression of L. monocytogenes in the livers or spleens of infected mice was observed when they were treated with a single dose of the adjuvant. This difference may be ascribed to the differences in the effector mechanisms of defense and to the different degree of augmentation of each defense mechanism by L18-MDP(Ala).