Systemic activation of tumoricidal properties in mouse macrophages and inhibition of melanoma metastases by the oral administration of MTP-PE, a lipophilic muramyl dipeptide

The purpose of these studies was to determine whether the oral administration of a lipophilic analog of muramyl dipeptide, MTP-PE, can produce in situ activation of tumoricidal properties in mouse macrophages. MTP-PE was dissolved in a phosphate-buffered saline to produce micelles. Single or multiple oral administrations of MTP-PE produced tumoricidal activation in both lung and peritoneal macrophages. This was in direct contrast to the i.v. or i.p. administrations of MTP-PE incorporated in liposomes, which produced activation in only lung or only peritoneal macrophages, respectively. The distribution and fate of [3H]-labeled MTP-PE subsequent to oral administration revealed that MTP-PE was found in various organs independent of reticuloendothelial activity. Finally, the repeated twice-weekly oral administrations of MTP-PE inhibited lung and lymph node metastasis in C57BL/6 mice by syngeneic B16 melanoma cells. The oral administration of MTP-PE, however, was not effective in eradicating well-established melanoma metastases. We conclude that the oral administration of a lipophilic muramyl dipeptide produces systemic activation of macrophages. The feasibility of enhancing host defense against infections and cancer by the oral administration of an immunomodulator has obvious clinical advantages.     

Stimulant effect of the immunomodulator MTP-PE on proliferation of monocytic cells in the guinea pig

The effect of a single subcutaneous injection of various doses of the lipophilic muramyl tripeptide MTP-PE on cell proliferation was investigated in autoradiographs of histological sections of various organs of the guinea pig. The animals either received first MTP-PE in saline and then one 3H-thymidine pulse 1 h prior to sacrifice, or they were prelabeled with 3H-thymidine and then received MTP-PE. The number of proliferating cells increased up to between two and fivefold (marginally after 0.3 mg/kg and maximally after 30 mg/kg MTP-PE), but differed in the various organs. In addition, the time of the maximal increase varied between 5 h and 72 h after MTP-PE treatment and also depended on the organ. The majority of proliferating cells were of the monocyte lineage seen in conjunction with the vascular system. They were apparently promonocytes still capable of proliferation. Evidence for this conclusion is derived from (i) the distribution of 1 h-pulse-labeled cells in the various organ compartments in relation to the stimulated proliferation of the bone-marrow cells, and (ii) the distribution of the prelabeled, mainly bone-marrow derived cells, to the various organs. The augmented proliferation of the monocyte lineage is preceded by a dose-dependent, short-lasting increase in the proliferation of some epithelia and also by an increase in body temperature and a transient change in plasma proteins. These effects are part of a limited inflammatory reaction and may contribute to the immunostimulation.     

Inhibitory effect of cholesterol on the uptake of liposomes by liver and spleen

The effect of cholesterol content of small unilamellar (SUV) and reverse phase (REV) liposomes on blood clearance and tissue distribution has been studied. [¹⁴C]Inulin has been used as an aqueous marker of liposomes to represent the uptake of intact liposomes in tissues. The blood clearance of the intravenously-injected SUV and REV liposomes depends on the cholesterol content of liposomes. The cholesterol-free (0 mol%) liposomes are cleared more readily from the circulation than the cholesterol-poor liposomes (20 mol%) and the cholesterol-poor are cleared more rapidly than the cholesterol-rich (46.6 mol%) liposomes. This clearance pattern of liposomes from the circulation is not attributed to the change of size of liposomes due to the increase in cholesterol content of liposomes. However, poor stability of cholesterol-free or cholesterol-poor liposomes in the circulation is partly responsible, but the predominant factor responsible for the observed blood clearance pattern is the inhibitory effect of cholesterol on the uptake of liposomes by reticuloendothelial-rich tissues liver and spleen. Uptake of liposomes by these organs is decreased with increasing cholesterol content of vesicles. It is suggested that to produce liposome preparations with a long circulating half life in vivo it is necessary to inhibit their uptake by liver and spleen.     

Augmentation of NK cell activity in tissue specific sites by liposomes incorporating MTP-PE

Liposomes incorporating a variety of immunomodulators have been shown to activate macrophages and monocytes for tumoricidal activity both in vivo and in vitro. We report that in addition to the activation of macrophages, the i.v. injection of liposomes (multilamellar vesicles) that have encapsulated muramyl tripeptide-phosphatidylethanolamine (MTP-PE) could also augment interstitial natural killer (NK) cell activity in the lung and the liver. In contrast, liposomes incorporating MTP-PE were unable to augment NK cell activity in the spleen, peripheral blood, or peritoneal cavity (after i.p. injection). In addition, liposomes did not augment splenic NK cell activity in vitro. This suggests that the augmentation of NK cell activity in the lungs and liver was not due to direct effects of the liposomes but may have been a secondary effect mediated by a monokine. The augmentation of pulmonary NK cell activity was paralleled by the nonspecific immunoprophylaxis of experimental pulmonary metastases. The augmented NK cell activity, as well as the enhanced nonspecific immunoprophylactic activity, was reduced by pretreatment of the mice with anti-asialo GM1 antiserum. Thus, the augmentation of organ-associated NK cell activity by liposomes incorporating MTP/PE plays a major role in the host's increased resistance to the formation of experimental metastases.     

Systemic Activation of Macrophages by Liposomes Containing Muramyltripeptide Phosphatidylethanolamine for therapy of Cancer Metastasis

Abstract

The heterogeneous response of metastases to conventional therapy is a major cause of failure in cancer treatment. Evidence that activated macrophages can recognize and destroy neoplastic cells in vitro without regard to their phenotypic diversity has stimulated efforts to develop effective approaches to the activation of macrophages in situ. Systemic administration of liposomes containing immunomodulators activates macrophages in situ and augments host destruction of spontaneous metastases.

Liposomes are a useful carrier system for the transport of agents to phagocytic cells in vivo. Once in the circulation, liposomes are cleared by phagocytic cells, and this passive localization provides an effective mechanism for targeting liposome-entrapped materials, such as muramyltripeptide phosphatidylethanolamine (MTP-PE), to macrophages.

Macrophage destruction of metastases in vivo is significant, provided that the total tumor burden at start of treatment is minimal. For this reason, we advocate using chemotherapy or radiotherapy first to reduce the tumor burden in patients with metastases. Tumoricidal macrophages that can differentiate neoplastic from bystander nonneoplastic cells are then used to destroy the few tumor cells that escape destruction by conventional therapeutic methods.     

Differential distribution of liposome-entrapped [H]methotrexate and labelled lipids after intravenous injection in a primate

Positive liposomes consisting of phosphatidylcholine, cholesterol and stearylamine and negatively charged liposomes consisting of phosphatidylcholine, cholesterol and phosphatidylserine, were double labelled with either ³H-labelled dipalmitoyl phosphatidylcholine and [¹⁴C]cholesterol or with [¹⁴C]cholesterol and [³H]methotrexate entrapped in the aqueous phase. The plasma levels and urinary excretion of radioactivity from sonicated and non-sonicated liposomes were then compared with the levels of radioactivity from free [³H]methotrexate during a 4 h experimental period after an initial intravenous injection in cynomolgous monkeys. Tissue uptake at the completion of the 4 h experimental period was also measured.It was found that plasma radioactivity from [³H]methotrexate and [¹⁴C]cholesterol in sonicated positive liposomes was cleared more slowly than from comparable non-sonicated liposomes, and considerably slower than from free [³H]methotrexate. Radioactivity from sonicated negative liposomes was cleared more rapidly than from positive sonicated liposomes. Positive liposomes captured considerably more [³H]methotrexate than negative liposomes and showed very low permeability to [³H]methotrexate in in vitro studies, even in the presence of high concentrations of serum.[¹⁴C]Cholesterol radioactivity was cleared more rapidly from plasma than ³H-radioactivity from liposome-entrapped [³H]methotrexate for double-labelled sonicated liposomes and generally showed greater uptake into tissues and red blood cells. ³H-labelled dipalmitoyl phosphatidylcholine in sonicated positive liposomes was cleared faster than [¹⁴C]cholesterol during the first 3 h. The more rapid disappearance of [¹⁴C]cholesterol from the plasma was complemented by greater uptake into a number of tissues, and positive non-sonicated liposomes were taken up to a greater extent by the spleen than equivalent sonicated liposomes.Renal excretion of ³H from liposome-entrapped [³H]methotrexate was considerably less than that of ³H from free [³H]methotrexate. There was insignificant excretion, however, of ¹⁴C from cholesterol in the urine.Entrapment in liposomes completely prevented the otherwise considerable breakdown of free methotrexate to ³H-containing products in plasma and partially prevented its breakdown in tissues.These studies indicate marked differences in the distribution of liposomes in vivo due to surface charge and size, and some degree of exchange of the lipid components of the liposome bilayer independent of the distribution of the entrapped species. They also show that entrapment in liposomes can reduce metabolic degradation of a drug, maintain high plasma levels and reduce its renal excretion.     

[3H]cyclo(Histidyl-Proline) in rat tissues: Distribution,clearance and binding

Cyclo(Histidyl-Proline), a metabolite of TRH, has been demonstrated to have a number of biological activities. The clearance, distribution and binding of the peptide in the rat was studied. Cyclo(His-Pro) was cleared from the circulation biphasically ($\text{tl}\text{2}\text{= 1.25 and 33 min}$). Unmetabolized cyclo(His-Pro) appeared rapidly in urine. Accumulation of [³H]cyclo(His-Pro) in adrenal, liver and kidney was demonstrated. Membrane preparations from adrenal and liver, but not from kidney, brain, pituitary, and other tissues were shown to bind cyclo(His-Pro) specifically.     

The lipophilic muramyltripeptide MTP-PE, a biological response modifier, is an activator of protein kinase C

The lipophilic immunomodulator MTP-PE is able to activate purified protein kinase C (PKC) by substituting phosphatidyl-serine (PS) or the synthetic diacylglycerol, DiC8, in the assay system. In addition, MTP-PE inhibited [3H]-phorbol-12, 13-dibutyrate ([3H]-PDBu) binding to PKC in a reconstituted receptor system as well as on intact cells (MCF-7). Furthermore, MTP-PE was also able to reduced the epidermal growth factor binding of MCF-7 cells to an extent similar to that found with DiC8 or PDBu. These data indicate that MTP-PE is able to compete for the phorbol ester binding site on PKC both in vivo and in vitro. The components of the MTP-PE molecule, MTP (muramyl-tripeptide) and PE (phosphatidylethanolamine) exerted only marginal effects on PKC activity, did not affect the phorbol ester binding of PKC and the EGF binding of intact MCF-7 cells. Our results suggest that only the complete molecule of the immunomodulator MTP-PE is able to interact with PKC.     

Liposomal delivery of biological response modifiers to macrophages

Macrophages activated to the tumoricidal state can recognize and destroy neoplastic cells and leave normal cells unharmed. Systemic activation of macrophages can be achieved by the intravenous administration of liposomes containing various immunomodulators. Much like any particle, liposomes are cleared from the circulation by phagocytic cells. This passive but specific targeting of immunomodulators to macrophages results in their activation forin vitro andin vivo lysis of tumor cells that can be resistant to conventional therapies.     

The effect of elimination of macrophages on the tissue distribution of liposomes containing [H]methotrexate

In the present study the tissue distribution of [³H]methotrexate was studied after intravenous injection of [³H]methotrexate-containing liposomes in normal and macrophage-depleted mice. Elimination of macrophages was performed by treatment with dichloromethylene diphosphonate- (DMDP)-containing liposomes. After thorough elimination of the macrophages from spleen and liver, by two intravenous injections of DMDP liposomes 6 and 4 days before tissue distribution studies, we found dramatic changes in the localization pattern of [³H]methotrexate liposomes in the blood, due to a decreased uptake of [³H]methotrexate liposomes by the DMDP liposome-treated liver. Because of the absence of these macrophages that are able to clear the blood of liposomes, and because of the resulting higher blood level of liposomes, we found an enhanced uptake of [³H]methotrexate liposomes by the spleen. It may be concluded that, in the spleen, apart from uptake of liposomes by macrophages, at least one other mechanism is responsible for the clearance of liposomes from the circulation. When comparing cholesterol-rich with cholesterol-poor liposomes, we found basically the same results, although uptake of cholesterol-rich liposomes by macrophages was smaller than that of cholesterol-poor liposomes, as found in several other studies. We suggest that pretreatment with DMDP liposomes can help to maintain a high level of intravenous-injected liposome-entrapped material in the blood, which otherwise would be removed by macrophages.     

Clearance and distribution of acid-stable trypsin inhibitor (ASTI)

The clearance, organ distribution and metabolic pathway of the acid-stable trypsin inhibitor (ASTI) were studied in mice using 125I-labeled urinary trypsin inhibitor (UTI), the most typical ASTI in the urine. Following intravenous injection of 125I-UTI, the radioactivity disappeared rapidly from the circulation with a half-life of 4 min for the initial part of the curve. Gel filtration of plasma samples revealed that the rapid disappearance of the radioactivity was due to elimination of free inhibitor from the plasma. 125I-UTI was cleared primarily in the kidney. Gel filtration of urine samples showed that part of the radioactivity in the urine appeared at the same elution volume as 125I-UTI in the plasma, indicating that the origin of UTI was ASTI in the plasma.     

Pharmacokinetics of the SPECT benzodiazepine receptor radioligand [123I]iomazenil in human and non-human primates

The pharmacokinetics of [¹²³I]iomazenil (Ro 16-0154) in 5 healthy human volunteers were compared to those in 2 hypothermic and 3 normothermic anesthetized monkeys. Following intravenous injection in humans and monkeys, [¹²³I]iomazenil rapidly diffused outside the vascular bed and was cleared from the arterial plasma triexponentially. The clearance half-times in hypothermic animals were protracted to values closer to those of the human. [¹²³I]lomazenil was metabolized mainly to a polar radiometabolite (not extracted by ethyl acetate) in the human whereas an additional lipophilic radiometabolite was detected in the monkey. In vitro and in vivo studies showed that [¹²³I]Iomazenil established equal concentrations in association with the cellular and plasma component of the blood, indicating that the plasma clearance of [¹²³I]iomazenil mirrors that of the blood. Analysis of organs from a monkey given [¹²³I]iomazenil showed that the parent compound was actively taken up by peripheral organs; the polar radiometabolite accumulated mainly in the bile and the kidneys whereas the non-polar radiometabolite accumulated in the urine and kidneys. Greater than 90% of the radioactivity in the different regions of the brain was unchanged parent [¹²³I]iomazenil.     

Differential distribution of liposome-entrapped [3H]methotrexate and labelled lipids after intravenous injection in a primate

Positive liposomes consisting of phosphatidylcholine, cholesterol and stearylamine and negatively charged liposomes consisting of phosphatidylcholine, cholesterol and phosphatidylserine, were double labelled with either ³H-labelled dipalmitoyl phosphatidylcholine and [¹⁴C]cholesterol or with [¹⁴C]cholesterol and [³H]methotrexate entrapped in the aqueous phase. The plasma levels and urinary excretion of radioactivity from sonicated and non-sonicated liposomes were then compared with the levels of radioactivity from free [³H]methotrexate during a 4 h experimental period after an initial intravenous injection in cynomolgous monkeys. Tissue uptake at the completion of the 4 h experimental period was also measured.It was found that plasma radioactivity from [³H]methotrexate and [¹⁴C]cholesterol in sonicated positive liposomes was cleared more slowly than from comparable non-sonicated liposomes, and considerably slower than from free [³H]methotrexate. Radioactivity from sonicated negative liposomes was cleared more rapidly than from positive sonicated liposomes. Positive liposomes captured considerably more [³H]methotrexate than negative liposomes and showed very low permeability to [³H]methotrexate in in vitro studies, even in the presence of high concentrations of serum.[¹⁴C]Cholesterol radioactivity was cleared more rapidly from plasma than ³H-radioactivity from liposome-entrapped [³H]methotrexate for double-labelled sonicated liposomes and generally showed greater uptake into tissues and red blood cells. ³H-labelled dipalmitoyl phosphatidylcholine in sonicated positive liposomes was cleared faster than [¹⁴C]cholesterol during the first 3 h. The more rapid disappearance of [¹⁴C]cholesterol from the plasma was complemented by greater uptake into a number of tissues, and positive non-sonicated liposomes were taken up to a greater extent by the spleen than equivalent sonicated liposomes.Renal excretion of ³H from liposome-entrapped [³H]methotrexate was considerably less than that of ³H from free [³H]methotrexate. There was insignificant excretion, however, of ¹⁴C from cholesterol in the urine.Entrapment in liposomes completely prevented the otherwise considerable breakdown of free methotrexate to ³H-containing products in plasma and partially prevented its breakdown in tissues.These studies indicate marked differences in the distribution of liposomes in vivo due to surface charge and size, and some degree of exchange of the lipid components of the liposome bilayer independent of the distribution of the entrapped species. They also show that entrapment in liposomes can reduce metabolic degradation of a drug, maintain high plasma levels and reduce its renal excretion.     

Stimulation of cell proliferation in rabbits by MTP-PE, a lipophilic muramyl peptide

The in vivo effect of the lipophilic muramyl peptide MTP-PE on the proliferation of blood cells and various tissues of the rabbit was studied by means of 3H-thymidine. Animals were killed up to 120 h after one or two i.v. injections of MTP-PE (10 mg/kg). MTP-PE caused a drastic effect on white blood cells: (1) neutropenia and lymphocytopenia occurring within 5 h was followed by leukocytosis of neutrophils and their juvenile forms by 24 h and thereafter, (2) within 24 h the number of prelabelled, i.e. recently regenerated, mononuclear cells in the bone marrow and the vascular system of various tissues increased approximately threefold, and (3) within 48 h the concentration of proliferating monocytic cells (1-h pulse labelling) rose to maximum levels of up to 20-fold in the lumina of blood vessels, particularly in capillaries of many organs. The number of proliferating cells also increased in the adventitia of medium and small arteries with a maximum at 48 h, whereas this occurred only later in the media and hardly at all in the intima. Thus, these proliferating, apparently monocytic cells are blood derived, and migrate into the tissue within 24 h after MTP-PE administration. In addition, proliferation in the epithelium of the bile ducts and oesophagus was also stimulated with a maximum at 24 h after MTP-PE. In contrast, enhanced proliferation occurred more slowly and to a lesser extent in hepatocytes, hepatic interstitial cells, and renal epithelial cells, consistent with a regenerative process after an inflammatory or toxic event.     

Studies of the pathogenesis of Gaucher's disease: tissue distribution and biliary excretion of [14C]L-glucosylceramide in rats

The time course of the clearance from the blood and the tissue localization of [14C]L-glucosylceramide, a nonmetabolizable enantiomorph of D-glucosylceramide that accumulates in Gaucher's disease, has been determined. 14C-labeled L-glucosylceramide injected intravenously in the form of micelles or liposomes is rapidly removed from the circulation. Most of this lipid is taken up by the liver where it is found in both hepatocytes and nonparenchymal cells. This sphingolipid analog is promptly cleared from hepatocytes and a significant portion is recovered in the bile. The clearance of [14C]L-glucosylceramide from Kupffer cells is greatly prolonged in comparison with its brief residence in hepatocytes. These findings have significant implications regarding the pathogenesis and treatment of Gaucher's disease.     

The effect of elimination of macrophages on the tissue distribution of liposomes containing [3H]methotrexate

In the present study the tissue distribution of [³H]methotrexate was studied after intravenous injection of [³H]methotrexate-containing liposomes in normal and macrophage-depleted mice. Elimination of macrophages was performed by treatment with dichloromethylene diphosphonate- (DMDP)-containing liposomes. After thorough elimination of the macrophages from spleen and liver, by two intravenous injections of DMDP liposomes 6 and 4 days before tissue distribution studies, we found dramatic changes in the localization pattern of [³H]methotrexate liposomes in the blood, due to a decreased uptake of [³H]methotrexate liposomes by the DMDP liposome-treated liver. Because of the absence of these macrophages that are able to clear the blood of liposomes, and because of the resulting higher blood level of liposomes, we found an enhanced uptake of [³H]methotrexate liposomes by the spleen. It may be concluded that, in the spleen, apart from uptake of liposomes by macrophages, at least one other mechanism is responsible for the clearance of liposomes from the circulation. When comparing cholesterol-rich with cholesterol-poor liposomes, we found basically the same results, although uptake of cholesterol-rich liposomes by macrophages was smaller than that of cholesterol-poor liposomes, as found in several other studies. We suggest that pretreatment with DMDP liposomes can help to maintain a high level of intravenous-injected liposome-entrapped material in the blood, which otherwise would be removed by macrophages.     

Optimization and limitations of systemic treatment of murine melanoma metastases with liposomes containing muramyl tripeptide phosphatidylethanolamine

Summary The purpose of these studies was to determine the optimal conditions and limitations for the eradication of spontaneous melanoma metastases by the systemic administration of liposomes containing MTP-PE. Mice whose primary melanoma had been excised were given i.v. injections of liposomes at various schedules. Optimal treatment was achieved by twice weekly administration for 4 weeks (eight i.v. injections). Bioassays failed to reveal the presence of melanoma cells in lungs of mice surviving to day 250 of the experiment. The success of liposome treatment of metastases diminished when the first i. v. injection of liposomes-MTP-PE commenced on day 10 after surgical excision of the local melanoma, as compared with day 3 or day 7 after surgery. We conclude that the major limitation for macrophage-mediated destruction of metastases is the number of tumor cells in the lesions. Because of this limitation, it is unlikely that the systemic activation of macrophages could be used as a single modality for treatment of advanced metastases.     

Tumor cytotoxicity and interleukin 1 production of blood monocytes of lung cancer patients

Summary The effects of lung cancer on the abilities of blood monocytes to produce interleukin-1 and to mediate antitumor activity were examined. The functional integrity of blood monocytes was determined by their capacity to respond in vitro to a variety of activating agents and become tumoricidal, as assessed by a radioactive release assay and ability to produce interleukin-1 in vitro. The results show that the presence of lung cancer significantly increased the number of harvested blood monocytes and that the spontaneous tumoricidal activity of these monocytes was slightly high as compared to monocytes obtained from healthy donors. The production of interleukin-1 by monocytes of healthy donors and lung cancer patients was similar. Blood monocytes obtained from lung cancer patients were less cytotoxic against allogeneic A375 melanoma cells as compared with those of healthy donors subsequent to incubation with a soluble muramyl dipeptide analog or lipopolysaccharide, but were as tumoricidal as those from healthy donors when activated with lipophilic muramyl tripeptide (MTP-PE) entrapped in multilamellar liposomes. The finding that monocytes of patients with lung cancer can respond to MTP-PE encapsulated in liposomes, recommends the use of these liposomes in therapy of human lung cancer.     

Ganglioside GM1 and Hydrophilic Polymers Increase Liposome Circulation Times by Inhibiting the Association of Blood Proteins

Abstract

Several agents have been shown to prolong the circulation lifetime of liposomes. These agents, such as ganglioside G_M1 or phosphatidylethanolamine-derivatives of monomethoxypolyethyleneglycols, provide insight into the mechanism(s) by which liposomes are cleared from the circulation. It is suggested here that the primary mechanism by which these molecules alter the biodistribution of liposomes in vivo involves an inhibition of the association of blood proteins to liposomes, resulting in a diminished rate of clearance of liposomes from the circulation.     

Systemic activation of macrophages by liposome-entrapped muramyl tripeptide in mice pretreated with the chemotherapeutic agent adriamycin

Summary The purpose of these studies was to determine whether macrophages of mice pretreated with the chemotherapeutic agent adriamycin (ADR) could be systemically activated by IV injection of liposomes containing muramyl tripeptide phosphatidylethanolamine (MTP-PE), a lipophilic derivative of muramyl dipeptide. Lower than normal levels of alveolar macrophages or peritoneal exudate macrophages were found in mice following IV injection of ADR. This decrease was dose-dependent and, in mice given <10 mg ADR/kg, it was transient (14 days). Peritoneal macrophages surviving the administration of 15 mg ADR/kg were tumoricidal.At various times after single or repeated administration of ADR, mice were given IV or IP injections of liposomes containing MTP-PE. One day thereafter, the cytotoxic activity of the in situ-activated macrophages (alveolar or peritoneal exudate) was assessed in culture against syngeneic melanoma cells. Our data demonstrate that under defined conditions the systemic administration of ADR does not interfere with the in situ activation of tumoricidal properties of murine macrophages after IV injection of liposomes containing a macrophage-activating agent.