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The systemic administration of doxorubicin (DXR) decreases the number of epithelial cells and leukocytes in the small intestine of mice. Oral administration of muramyl tripeptide phosphatidylethanolamine (MTP-PE) prevented both disruption of intestinal architecture, and a decrease in the number of macrophages, and it induced the expression of IL-6, G-CSF, GM-CSF, and TNF-alpha in the intestinal tissue. The data suggest that the oral administration of MTP-PE can prevent chemotherapy-induced toxicity to the intestinal mucosa and hence infections due to translocation of aerobic bacteria from the intestine to the blood.

The present study evaluated the in vivo biological activity of synthetic muramyl tripeptide, CGP 19835A, when encapsulated into phosphatidylcholine liposomes (POPC-19835A) and administered as an p.o. immunomodulator to BALB/c mice. Liposomes were rapidly absorbed in the intestine and reached the systemic circulation within 4 h. Alveolar macrophages harvested from the lungs of mice 24 h after a single p.o. feeding of POPC-19835A were tumoricidal toward syngeneic murine renal cell carcinoma target cells. Repeated daily feedings with POPC-19835A generated sustained activation of the alveolar macrophages. Activation of peritoneal macrophages to the tumoricidal state required at least three daily feedings of POPC-19835A. In vitro studies demonstrated the release of tumor necrosis factor alpha and interleukin 6 by macrophages activated by POPC-19835A in the presence of gamma-interferon. Interleukin 1 and nitric oxide were not induced in macrophages by this liposomal preparation. Daily administration of POPC-19835A after i.v. injection of renal cell carcinoma tumor in BALB/c mice inhibited the development of experimental lung metastasis and confirmed the potential role of long-term therapy with this new p.o. immunomodulator.

Subsequent to systemic administration of doxorubicin or whole-body X-irradiation, C57BL/6 mice exhibit a depletion in lymphoid cells (macrophages) lasting 2-3 weeks and returning to normal by 4 weeks after either treatment. We evaluated the ability of repeated oral administration of the synthetic macrophage activator muramyl tripeptide phosphatidylethanolamine (MTP-PE), either in free form or encapsulated into multilamellar liposomes, to reverse or prevent the decline in macrophages after cytoreductive therapies. The number of both resident peritoneal macrophages and peritoneal exudate cells elicited by thioglycollate-induced inflammation increased after the oral administration of MTP-PE (three times a week) for at least 3 weeks. The depletion of macrophage number from the peritoneal cavity that occurred after two IV injections of doxorubicin or X-irradiation with 1.5 or 3.0 Gy was prevented by repeated oral feedings of MTP-PE. Moreover pretreatment of mice with oral MTP-PE prevented depletion of macrophages resulting from IV injections of doxorubicin. Modest protection was also noted for blood leukocytes in mice receiving oral MTP-PE following systemic DXR. Similar effects were achieved when MTP-PE was encapsulated into phospholipid liposomes: this formulation also allowed macrophages to be readily activated to the tumoricidal state. The oral administration of MTP-PE offers an additional strategy for protection of host defense cells during cytoablative therapies.

The purpose of these experiments was to evaluate the possibility that the systemic administration of liposomes containing synthetic macrophage activation CGP 31362 or CGP 19835 in mice which were simultaneously receiving injections of gamma-interferon or interleukin 2 would lead to enhanced regression of spontaneous lung metastases produced by syngeneic renal adenocarcinoma. The kidneys of BALB/c mice were given injections of renal adenocarcinoma cells, and 10 days later the kidney with local tumor was surgically resected. These mice were then given injections i.v. with liposomes and with gamma-interferon (s.c.) or interleukin 2 (i.p.). Systemic administration of MLV-CGP 31362 and MLV-CGP 19835A significantly reduced the number of lung metastases in nephrectomized mice. Both lung tumor burden and regional recurrence were further reduced by the s.c. injection of gamma-interferon or i.p. injection of interleukin 2. Long-term survivors were observed only in the groups of animals treated with liposomes containing macrophage activators and with lymphokines. Evaluation of host responsiveness to this immunotherapy revealed in situ activation of alveolar macrophages by administration of MLV-CGP 31362 or MLV-CGP 19835A, which was enhanced in mice also treated with interleukin 2. Normal levels of natural killer cell activity were reduced in the spleens of tumor-bearing mice but were restored subsequent to treatment with MLV-CGP 31362. These results indicate the potential usefulness of treating metastatic renal cell carcinoma by systemic administration of liposomes containing synthetic macrophage activators in combination with parental injections of lymphokines.

Three syngeneic murine tumor models were used to determine potential interactions between chemotherapeutic drugs and the synthetic liposome-encapsulated macrophage activator, muramyl tripeptide phosphatidylethanolamine (MLV-19835). Experiments were designed to maximize any additive toxicity of the simultaneous administration of MLV-19835 on the known myelosuppressive effects of doxorubicin, ifosfamide, and cisplatin. Treatment with these drugs resulted in diminished blood leukocyte counts, altered leukocyte differentials, and decreased hematocrits, but the systemic administration of MLV-19835 produced no additional deleterious effects. Myelosuppression normally observed at 2 weeks following treatment of mice with doxorubicin was prevented by combination treatment with MLV-19835. In addition, there was no interference of the antitumor activity of ifosfamide or doxorubicin against subcutaneous, kidney, and spleen tumors. These studies and the recent demonstration of the biological activity of MLV-19835 in phase II trials of osteosarcoma recommend clinical testing of these combined modalities.