Synthesis and immunostimulating properties of lipophilic ester and ether muramyl peptide derivatives.

Macrophages can become cytotoxic toward tumor cells when activated by immunomodulators. Three different muramyl peptides were synthesized: one hydrolyzable lipophilic ester derivative (MTP-Chol) and two nonhydrolyzable lipophilic ether derivatives (MTP-octadecane and MTP-heptadecafluorooctadecane). Activation of the RAW 264.7 cell line was studied by measuring nitrite production as an indication of NO-synthase activity. The lipophilic ester derivative, incorporated within nanocapsules, was as active as free muramyl dipeptide, whereas the lipophilic ether derivatives were unable to activate macrophages. MTP-octadecane in micellar form was not capable of inducing macrophage cytotoxicity either. These results indicate that lipophilic muramyl peptides need to be hydrolyzed to yield a hydrosoluble metabolite in order to activate macrophages.

MDP and LPS act synergistically to induce arginine-dependent cytostatic activity in rat alveolar macrophages.

Rat alveolar macrophages can be activated in vitro for cytostatic activity against tumor cells by MDP and LPS acting in synergy. MDP can be substituted for by analogs active as adjuvants. Macrophage activation correlates with an increased production of nitrite and citrulline. NG-monomethyl-L-arginine, a specific inhibitor of the L-arginine metabolism having nitrite and citrulline as end products, abolishes the cytostatic activity. We therefore conclude that, in this model, the main effector mechanism of the cytostatic activity is mediated by molecules derived from L-arginine through the newly described NOo-generating pathway.

Anti-metastatic activity in vivo of MDP-L-alanyl-cholesterol (MTP-Chol) entrapped in nanocapsules.

A lipophilic muramylpeptide (MTP-Chol), capable of rendering macrophages cytostatic towards tumour cells, was encapsulated within polyisobutylcyanoacrylate nanocapsules and administered to mice carrying an experimental model of liver metastasis. Treatment by intravenous injections twice a week beginning before the establishment of metastases significantly reduced the number of liver colonies. Treatment started later was less effective. The dose of MTP-Chol in each injection, and the tumour burden in the mice did not change the percentage inhibition of metastases significantly. Anti-metastatic activity was also observed after administering nanocapsules containing MTP-Chol by the oral route.

Second antibody for improvement of antibody imaging: liposome-entrapped and free preparations in animal and human studies.

When anti-tumour antibodies are given systematically for tumour imaging or therapy, second antibody directed against the first (anti-tumour) antibody can be used to accelerate clearance of first antibody, thus improving discrimination between tumour and normal tissues. Liposome-entrapped, and free second antibodies (LESA and FSA, respectively) have been compared in an animal tumour model system and in patients with cancer. Nude mice bearing xenografts of human colon carcinoma were given goat antibody to carcino-embryonic antigen (CEA) as first antibody and horse anti-goat second antibody. Patients with gastrointestinal carcinomas received i.v. 131I-labelled goat anti-CEA or mouse monoclonal 17-1A first antibody and unlabelled horse angi-goat or rabbit anti-mouse second antibody, respectively. Antibody distribution was studied by serial gamma camera imaging. The effectiveness of LESA and FSA depended on dose. Tumour-to-blood ratios were increased up to eight-fold by either method in animals. Tumour imaging was enhanced among 15 patients with gastrointestinal cancer and tumour was correctly identified at five sites where it was not seen by a background subtraction method. No significant toxicity occurred in patients nor in rabbits studied for evidence of immune complex mediated disease. LESA and FSA appear to be equally effective.

Use of mannosylated liposomes for in vivo targeting of a macrophage activator and control of artificial pulmonary metastases.

From a mannosylated mycobacterial phospholipid, we prepared an original type of liposome which was taken up by macrophages by means of the mannose/fucose receptor. When a lipophilic immunomodulator, MDP-L-alanyl-cholesterol (MTP-CHOL) was included in such liposomes, they were able to activate WAG rat alveolar macrophages for cytotoxicity against syngeneic tumour cells in vitro. The presence of suboptimal levels of endotoxin was essential for this activation. Cytotoxic macrophages could also be induced in vivo by injecting immunomodulator-loaded liposomes intravenously 24 h before harvesting macrophages. A decrease in experimental pulmonary colonies arising from i.v. injected tumour cells was observed following repeated administration of such liposomes.

Improved radioimmunodetection of tumours using liposome-entrapped antibody.

The discrimination of radioimmunodetection of tumours is reduced by the presence of circulating radiolabelled antibody (primary antibody). We have prepared liposomes containing an antibody to the primary antibody (secondary antibody), with the intention of complexing and delivering to the liver primary antibody which is not associated with the tumour. In mice bearing xenografts of human tumours which secrete the marker carcinoembryonic antigen (CEA), liposomally entrapped secondary antibody was able to reduce the blood levels of 125I-labelled anti-CEA within 2 h, without reducing the amount of anti-CEA bound to the tumour. We therefore suggest that the use of liposomally entrapped secondary antibody would improve the diagnostic potential of radioimmunodetection of tumours and their metastases.

Clearance of injected radioactively labelled antibodies to tumour products by liposome-bound second antibodies.

Liposomes containing anti-goat immunoglobulin were injected 24 h after administration of 125I-labelled goat antibody against the carcinoembryonic antigen (anti-CEA) to groups of nude mice bearing human tumour xenografts, and normal mice. Controls in each group received radioactively labelled anti-CEA only. In liposome-treated mice, blood 125I levels were lower than those of controls 30 min to 24 h after liposome administration, with corresponding accumulation of 125I activity in the liver and spleen for the first 2 h after liposome injection. [14C]Cholesterol or 99mTc labels in the bilayer were eliminated rapidly from the blood, with uptake in the liver and spleen. In xenograft-bearing mice, 125I activity detected in the tumours up to 6 h after liposome injection was identical to that detected in the tumours of controls. However, 24 h after liposome injection a reduction in the tumour concentration of 125I-labelled anti-CEA was obtained, but the tumour/blood radioactivity was still increased. In two mice given 27 mumol lipid, the blood radioactivity count after 24 h was only 5% of that in the controls. In rabbits, 2 h after administration of liposomes containing anti-goat second antibody, the circulating 125I activity had dropped by 28-40%. The results suggest that administration of liposome-entrapped second antibody approximately 2 h prior to external scintigraphy may clear circulating radioactively labelled primary antibody by up to 50%.

Liposomally entrapped second antibody improves tumour imaging with radiolabelled (first) antitumour antibody.

The usefulness of antibodies directed against tumour products for localisation and therapy of cancer is limited by the large proportion of administered antibody which remains non-specifically in the circulation and extravascular space. Liposomally entrapped second antibody (LESA) directed against the first (antitumour) antibody has been used to accelerate clearance of non-tumour-bound first antibody without affecting its clearance from the tumour. Intravenously administered LESA binds the first antibody and LESA is cleared by the reticuloendothelial system. LESA accelerated clearance of radiolabelled antibody directed against carcinoembryonic antigen (CEA) from the circulation in four of five patients with gastrointestinal cancer, enhancing gamma-camera imaging of the tumour in three of them. These results suggest that LESA can improve the sensitivity and specificity of tumour imaging with radiolabelled antitumour antibody. This strategy may also have the potential to improve the therapeutic ratio of some toxic agents linked to antitumour antibody.

Activation of lysosomal enzymes and tumour regression caused by irradiation and steroid hormones.

The lysosomal enzyme activity and membrane permeability of mouse C3H mammary tumours has been studied using quantitative cytochemical methods following irradiation of the tumours with doses of 1500, 3500 or 6000 rad gamma rays. No change in the lysosomal enzyme activity was observed immediately after irradiation, but increased enzyme activity and increased membrane permeability were observed 24 hr after irradiation with doses of 3500 or 6000 rad. Twenty-four hours after injection of prednisolone there was a marked increase of lysosomal membrane permeability and enzyme activity, and injection of prednisolone soon after irradiation enhanced the effect of irradiation. After a dose of 6000 rad and prednisolone, the lysosomal membrane permeability increased to 191% of the control and the enzyme activity to 326% of the value of the controlled tumours. Measurement of tumour size after irradiation or after a combined treatment with irradiation and prednisolone showed that a close correlation exists between tumours regression and lysosomal enzyme activity. The experiments support the view that lysosomal enzymes play an important role in tumour regression following irradiation.