Current Cancer Drug Targets, 2006, 6, 123-133123
Liposomal Muramyl Tripeptide Phosphatidylethanolamine: Targeting and
Activating Macrophages for Adjuvant Treatment of Osteosarcoma
A. Nardin*, M.-L. Lefebvre, K. Labroquère, O. Faure and J.-P. Abastado
IDM, Immuno-Designed Molecules, 172 rue de Charonne, 75011 Paris, France
Abstract: About one third of osteosarcoma patients develop lung metastasis refractory to chemotherapy.
Recent studies indicate that biological response modifiers activating the patient’s immune system may help
controlling minimal residual disease via pathways distinct from those used by cytotoxic drugs, and therefore
prove effective against tumor resistance. Muramyl tripeptide phosphatidylethanolamine (MTP-PE) is a
synthetic lipophilic glycopeptide capable of activating monocytes and macrophages to a tumoricidal state.
When intercalated in multilamellar liposomes (L-MTP-PE) and injected intravenously, it targets lung, liver,
and spleen macrophages. Therapeutic activity of L-MTP-PE was demonstrated in several preclinical models of
experimental lung metastasis and in clinical trials in dogs with osteosarcoma. Although macrophage
activation was shown to be directly involved in the in vivo anti-metastatic activity of this molecule, cytokine
and chemokine secretion by activated macrophages could induce recruitment and stimulation of other immune
cells, which may in turn indirectly contribute to the anti-tumor effect. L-MTP-PE has undergone
development in humans. In early trials, most side effects of L-MTP-PE were minimal. L-MTP-PE showed signs
of efficacy in treatment of patients with recurrent osteosarcoma and the encouraging results from phase II
studies led to a phase III trial conducted by the Children’s Oncology Group in patients with newly diagnosed
high-grade osteosarcoma. Patients were treated with or without L-MTP-PE in combination with multi-drug
chemotherapy in adjuvant setting; significantly higher overall survival and disease-free survival were
observed in the group receiving L-MTP-PE.
Keywords: Osteosarcoma, macrophages, innate immunity, muramyl peptides, liposomes, chemotherapy.
Osteosarcoma is the most common form of bone cancer
[1-3]. It primarily occurs in children and adolescents. The
incidence is very low: there are approximately 900 new cases
of osteosarcoma per year in the United States and around the
same number in Europe. The standard treatment consists in
surgical resection of the primary tumor, associated with
chemotherapy in neoadjuvant and adjuvant settings. With
this treatment, long-term survival is achieved in about 70%
of patients. The remaining patients usually develop
pulmonary metastasis, and represent an unmet therapeutic
In recent years, results from animal models and early
clinical trials have indicated that biological response
modifiers activating the patient’s immune system may help
controlling minimal residual disease via pathways either
complementary or synergistic to those used by cytotoxic
drugs, and may therefore prove effective in preventing the
development of tumor resistance. Toll-like receptor (TLR)
ligands as CpG oligonucleotides or lipid A derivatives
activate adaptive and/or innate immune responses against
tumors [1-3]. Muramyl tripeptide phosphatidylethanolamine
(MTP-PE), a synthetic molecule derived from muramyl
dipeptide (MDP), is also a stimulator of innate immunity.
Formulated in multilamellar liposomes, it has been
developed for the treatment of osteosarcoma.
In this review we will present an update of the preclinical
and clinical studies conducted with liposomal MTP-PE (L-
MTP-PE), and we will discuss the mechanisms of action in
THE MOLECULE AND ITS FORMULATION
MTP-PE is a synthetic molecule derived from MDP, the
minimal peptidoglycan motif common to both Gram-
positive and Gram-negative bacteria. The covalent addition
of an alanine and of dipalmitoylphosphatidylethanolamine to
MDP results in MTP-PE, a more lipophilic molecule due to
the presence of two lipid acid tails. The chemical structure is
shown in Fig. (1A).
Like MDP, MTP-PE activates in vitro monocytes and
macrophages. This activation is measured by increased
tumoricidal activity and secretion of cytokines and pro-
inflammatory factors, including TNF-α, IL-6, IL-8, IL-1β,
nitric oxide, PGE2 [4-9]. Fogler and Fidler demonstrated a
superiority of MTP-PE over MDP in the activation of
human monocytes . This was ascribed to the lipophilic
properties of MTP-PE, resulting in higher cell uptake via
passive transfer through the cytoplasmic membrane, with
increased availability for an intracellular receptor. In
addition, the lipophilic MTP-PE could be efficiently
incorporated in the lipid bilayer of liposomal structures.
Fidler and colleagues developed a liposomal formulation
that, when injected intravenously, distributes primarily in
the liver, spleen, and lungs, where it is phagocytosed by
monocytes and macrophages [10-12]. These liposomes are
concentric multilamellar vesicles (MLV) of approximately 2-
3 µm. They are constituted by the two synthetic
*Address correspondence to this author at the IDM Research laboratory,
IFR58, 15 rue de l’Ecole de Médecine, 75006 Paris, France; Tel: +33 1 53
10 17 75; Fax: +33 1 53 10 17 80; E-mail: email@example.com
1568-0096/06 $50.00+.00 © 2006 Bentham Science Publishers Ltd.
124 Current Cancer Drug Targets, 2006, Vol. 6, No. 2Nardin et al.
phospholipids phosphatidylcholine and phosphatidylserine
at a 7:3 w/w ratio. MTP-PE is intercalated within the
vesicles at a 1:250 w/w ratio. A freeze-fracture electron
microscopy photo of a multilamellar liposome is shown in
Fig. (1B). Formulation
phospholipids vesicules improves activation of macrophage
and monocyte tumoricidal properties in vitro, and prolongs
its presence in the lungs [10, 12, 13]. Compared to the free
lipopeptide, the MLV formulation undergoes very rapid
clearance from the circulation, due to drug targeting to the
phagocytic cells of the reticuloendothelial system [12, 14].
Pharmacokinetics studies in dogs showed that, 5 minutes
of MTP-PE into these
after i.v. injection of L-MTP-PE, only 0.5% was found in
plasma, compared to 93% when administrated as free
lipopeptide . Possibly because of this fast clearance,
MTP-PE formulated in liposomes showed a no-adverse-
event level ten times higher than free MTP-PE in rabbit and
PRECLINICAL STUDIES AND ANIMAL TRIALS
In vitro, both free and liposomal MTP-PE are potent
activators of monocytes and macrophages. Increased
tumoricidal activity on melanoma and sarcoma cell lines has
3rd peptide Ala
Fig. (1). Liposomal MTP-PE. A. Molecular structure of MTP-PE. B. Electron microscopy picture of a multilamellar liposome. The
lipophilic molecule MTP-PE is intercalated in the lipid bilayers at a MTP-PE:phospholipid w/w ratio of 1:250.
Liposomal Muramyl Tripeptide PhosphatidylethanolamineCurrent Cancer Drug Targets, 2006, Vol. 6, No. 2 125
been measured with murine alveolar macrophages (AM),
Kupffer cells, or peritoneal macrophages after treatment with
this molecule [6, 15, 16]. Interestingly, tumor cells
surviving six sequential exposures to activated peritoneal
macrophages did not show any reduction in susceptibility to
macrophage killing compared to the parent cell line: thus,
tumor cell destruction by activated macrophages was
nonselective and did not lead to the development of resistant
cells . Tumor killing by L-MTP-PE-treated rat liver
macrophages was reduced by about 75% with anti-TNF-α
antibodies, indicating that TNF-α may be involved in anti-
tumor activity .
In vitro-treated human monocytes killed allogeneic and
autologous tumors (including melanoma, ovarian, colon,
and renal carcinoma [7, 18-20]), but had no by-stander
toxicity towards normal cells, even when mixed with the
tumor targets [4, 21]. Importantly, several of these studies
showed that monocytes from cancer patients could be
activated to similar levels of tumoricidal activity as cells
from healthy donors [7, 18-20].
Increased tumoricidal activity by isolated murine Kupffer
cells and AM [15, 22, 23], dog monocytes and AM [5, 24]
was also measured ex vivo, that is after treatment of the
animals with L-MTP-PE and isolation of cells. Dog AM
cytotoxicity was even greater if the animals were treated with
both doxorubicin and L-MTP-PE .
Cell activation was associated with production of
cytokines and other inflammatory factors, including TNF-α,
IL-6, IL-8, IL-1, nitric oxide, PGE2 [4-9, 17, 25]. Fig. (2A)
shows that human macrophages stimulated in vitro with
MTP-PE secrete TNF-α, IL-1β, IL-6 and increased IL-8. No
IL-12p70 and IL-10 were however detected. Asano and
colleagues reported that the inflammatory chemokine CCL2
was also induced . On human monocytes, L-MTP-PE
stimulated up-regulation of the adhesion molecules LFA-1
and ICAM-1, which could be important for interaction with
tumor cells; indeed, anti-LFA-1 antibody inhibited killing
of melanoma cells .
Several studies demonstrated that IFN-γ synergizes with
MTP-PE to increase tumoricidal activity and to induce
secretion of cytokines [20, 23, 28-31]. We found that MTP-
PE synergizes with IFN-γ in inducing tumor killing by
human macrophages, and augmenting secretion of TNF-α
and IL-1β (Fig. (2)). The mechanism for this potentiation is
not known, but a 2-fold increase in liposome phagocytosis
was observed after treatment of human monocytes with IFN-
Fig. (2). MTP-PE, in synergy with IFN-γ, activates tumoricidal properties and secretion of TNF-α, IL-1β, IL-6, IL-8 in human
macrophages. Macrophages were differentiated from monocytes by a 6-day incubation of total apheresis in the presence of GM-CSF
. The cells were then treated for 18 h with 1 µg/ml MTP-PE +/- 166 U/ml of IFN-γ. A. Cytokine concentration (mean and SD from 4
independent experiments) was measured in culture supernatants harvested 18 h after cell activation. No IL-12p70 or IL-10 were
detected. B. Activated and non-activated macrophages were purified and their tumoricidal activity against the human breast cancer
cell line SK-BR-3 was evaluated in a 48-h cytotoxicity assay, at effector:target ratio of 20:1. Data from 4 independent experiments are
shown. * p<0.05 by paired one-tailed Student’s t test. ND, not determined.
126 Current Cancer Drug Targets, 2006, Vol. 6, No. 2Nardin et al.
Consistent with the in vitro findings, intravenous
injection of L-MTP-PE showed therapeutic activity in
several models of experimental metastasis or neoplasia. In
the B16 melanoma model developed by Fidler and
colleagues, spontaneous lung and lymph node metastasis
occur subsequent to the growth of the primary tumor
implanted in the footpad of mice. Injection of L-MTP-PE
after surgical removal of the primary tumor increased
survival from 10% (control animals receiving placebo
liposomes) to 70% . Regression of established
metastasis was associated with tumoricidal macrophages
residing within the metastasis. Efficacy was dependent on
the regimen and the tumor burden at the moment of
treatment: best results were obtained starting the treatment 3
days after tumor removal, twice weekly for 4 weeks .
When the beginning of the treatment was delayed, the
efficacy decreased, probably due to large metastatic lesions.
Importantly, lung cells from surviving mice injected s.c.
into syngeneic hosts did not develop tumors, indicating
complete eradication by the treatment. Synergy with IFN-γ
was also observed in this in vivo model, provided that IFN-
γ was present in the same liposomes as MTP-PE .
In addition, i.v. L-MTP-PE delayed tumor growth and
increased animal survival in mice wih UV-induced skin
tumors [34, 35], and in rats with liver metastasis [36, 37].
Clinical trials were conducted at the University of
Wisconsin in dogs and cats with various malignancies:
osteosarcoma, splenic hemangiosarcoma, melanoma or
mammary adenocarcinoma. Results from these studies are
particularly relevant because spontaneous tumors in these
animals have similar characteristics and undergo similar
treatment interventions as in humans. L-MTP-PE was
administrated as adjuvant treatment after resection of primary
tumor, at a dose of 2 mg/m2 twice weekly for 8 weeks,
combined or not with chemotherapy (doxorubicin and
cyclophosphamide, or cisplatin). MacEwen and colleagues
observed significantly longer disease-free survival (DFS) and
overall survival (OS) in dogs with osteosarcoma, splenic
hemangiosarcoma or early-stage melanoma treated with L-
MTP-PE [38-42]. Some of the data from these trials are
summarized in Table 1. In contrast, L-MTP-PE had no effect
in cats and dogs with mammary tumors after mastectomy
[43, 44]. This lack of efficacy might have been due to a
suboptimal activation of local macrophages: while some
pulmonary recurrences were seen in the placebo group,
recurrences in the treated group occurred only locally,
possibly as a consequence of the selective activation of lung
Routes of administration other than i.v were tested in
some pre-clinical models. Given orally to mice, free or
liposomal MTP-PE (phosphatidylcholine
activated both alveolar and peritoneal macrophages, and
inhibition of lung and lymph node metastasis was observed
[45, 46]. Intranasal or intratracheal MTP-PE stimulated
macrophages and granulocytes in the lungs of rat, and
increased mice survival in the B16 lung metastasis model
[47, 48]. Intraperitoneal treatment enhanced survival of mice
with i.p. syngeneic fibrosarcoma or human ovarian
xenografts [49, 50]. Intravesical treatment with L-MTP-PE
reduced tumor incidence in nude mice grafted orthotopically
with human bladder carcinoma; activated macrophages were
found in the bladder of treated mice . Thus, MTP-PE
may be active in several indications, provided that the drug
is efficiently delivered to local macrophages.
INSIGHTS ON THE MECHANISMS OF ACTION
The outcome of L-MTP-PE administration is likely to be
the result of
1- the cellular targets of the liposomes
2- the cellular receptor expression
3- additional indirect effects secondary to activation of
the primary cellular targets.
I.v.-injected liposomes are taken-up by macrophages
adjacent to sinusoids in liver and spleen. They also
accumulate in lung capillaries, from which they cannot
however spontaneously extravasate . Circulating
monocytes, rapidly activated by L-MTP-PE in the lung
capillaries, can however cross the endothelium, be recruited
to the alveoli as activated AM, and act at the level of the
micrometastasis. One day after L-MTP-PE administration to
mice, 15% of AM presented internalized liposomes .
Although both MTP-PE and lipopolysaccharide, a
common monocyte/macrophage activator binding to TLR4,
have been shown to activate the MAP kinases ERK-1/2 and
the transcription factors NF-κB and AP-1, the signaling
pathways of the two molecules are clearly distinct [9, 17].
Fogler and Fidler found that MTP-PE is taken up by
monocytes in a non-saturable, receptor-independent fashion,
and therefore suggested that its receptor may be intracellular
Table 1. Randomized Clinical Trials in Dogs: Median Survival Times after Treatment with L-MTP-PE or Placebo Liposomes in
Indication DesignMedian survival timep-valueRef.
Osteosarcoma L-MTP-PE or placebo immediately after surgeryL-MTP-PE: 222 d
Placebo: 77 d
Osteosarcoma After cisplatin (non-metastatic dogs)L-MTP-PE: 14.4 mo
Placebo: 9.8 mo
Osteosarcoma Together with cisplatinL-MTP-PE: 10.3 mo
Placebo: 7.6 mo
Hemangiosarcoma Together with doxorubicin and cyclophosphamideL-MTP-PE: 277 d
Placebo: 143 d
Liposomal Muramyl Tripeptide PhosphatidylethanolamineCurrent Cancer Drug Targets, 2006, Vol. 6, No. 2 127
Fig. (3). Direct and indirect cellular targets of intravenous administration of L-MTP-PE. Tumoricidal macrophages are elicited in
lungs, liver and spleen. They secrete cytokines and chemokines that may directly affect the micrometastasis, and increase the
recruitment and activation of other cells, including dendritic cells, granulocytes, NK cells.
. Bacterial-derived structures are often recognized by
members of the Toll-like receptor family. In particular, the
TLR2/TLR6 heterodimer is known to bind biacetylated
lipopeptides like MALP-2. As MTP-PE
biacetylated, and TLR2 can be found intracellularly, in the
phagosomal compartment , an interaction between the
two molecules was conceivable. On the other hand, recent
evidence indicates that MDP, the natural precursor of MTP-
PE, signals through Nod2, an intracellular protein that
induces NF-κB activation and is implicated in innate
immune defense against
predominantly expressed in cells of the myeloid lineage,
including monocyte/macrophages, myeloid dendritic cells,
and granulocytes . D. Philpott and colleagues recently
found that MTP-PE is a Nod2, but not TLR2, agonist (J.
Fritz et al. personal communication).
Thus, the cellular targets of the liposomes seem to be the
same cells that also express the receptor for MTP-PE. In
vivo, phagocytic cells of the myeloid lineage take up the
liposomes containing MTP-PE and slowly degrade the
multilamellar vesicles in the endo-lysosomal compartment
. MTP-PE is released into the cytosol where it interacts
with Nod2, and the cells are consequently activated.
Although the anti-tumoral activity of activated macrophages
is far from being fully understood, direct tumor killing
through TNF-α or other cytokines is a possible mechanism
 (Fig. (3)).
There is direct evidence that the anti-metastatic activity
of L-MTP-PE is due to macrophages. In the murine model
bacteria . Nod2 is
of melanoma lung metastasis, Fidler and colleagues showed
that when MDP liposomes were not retained in the lung
vessels (a particular type of liposomes entirely composed of
phosphatidylcholine), they failed to activate AM or inhibit
tumor growth . More importantly, treatment with agents
selectively impairing macrophage
carrageenan, hyperchlorinated drinking water) also resulted in
lack of metastasis eradication by liposomal MDP, while
depletion of NK or T cells had no effect. Finally, the authors
demonstrated reduction of lung metastasis after adoptive
transfer of syngeneic macrophages activated in vitro.
Despite this compelling experimental evidence for the
role of macrophages, it seems difficult to exclude that effects
secondary to their activation may contribute to L-MTP-PE
anti-tumor activity in vivo. Talmadge and co-workers
isolated activated NK cells from lung and liver of mice
treated with L-MTP-PE. They also demonstrated, by NK
cell depletion, that the anti-metastatic activity of L-MTP-PE
in a prophylactic model was completely resting on NK cells
. As NK cells were not activated in vitro by L-MTP-PE,
it is likely that their activation was induced by factors
produced in vivo by macrophages. Dendritic cells (DC)
maturation could also be stimulated during L-MTP-PE
treatment, either directly (particularly for myeloid DC,
which express the Nod2 receptor) , or indirectly,
monocyte/macrophages. If matured, pulmonary DC that had
taken up tumor antigens could migrate to the draining
lymph nodes and stimulate a T cell response to the tumor.
Finally, granulocytes may take up the liposomes and be
cytokine production by
128 Current Cancer Drug Targets, 2006, Vol. 6, No. 2Nardin et al.
directly stimulated by L-MTP-PE, while their recruitment to
the lung could be enhanced by IL-8 produced by activated
macrophages (Fig. (3)). The precise role of these other cell
types in the in vivo anti-tumor response triggered by L-
MTP-PE awaits further investigations.
inflammatory cytokines by activated monocytes [62, 63].
Biological activity was consistently observed during the
phase I and II trials, and included: increased monocyte
tumoricidal activity and secretion of IL-1, as well as
increased TNF-α, IL-6, C-reactive protein, neopterin in
plasma (but no IFN-γ or IL-1) [57, 59, 61-65]. Chronic
treatment resulted in monocyte tachyphylaxis for cytokine
secretion but not tumor cytotoxicity, which remained
In later trials, including the phase III study, the dose of
L-MTP-PE was escalated in each patient until occurrence of
fever, chills or increase in the C-reactive protein: from a
starting dose of 2 mg/m2 to a maximal dose of 2 mg/m2 + 2
mg . In the phase III study, where L-MTP-PE was
associated to chemotherapy (see below), there was no
difference among the treatment arms for most of the
toxicities reported .
As fever and chills, the major side effects following L-
MTP-PE infusion, could be prevented by administration of
anti-inflammatory drugs, a series of studies were conducted
to determine whether COX inhibitors interfere with L-MTP-
PE action. In vitro, only high doses of ibuprofen suppressed
the generation of tumoricidal phenotype, as well as IL-1 and
TNF-α production, in monocytes treated with L-MTP-PE
. Therefore, for the management of L-MTP-PE side
CLINICAL DEVELOPMENT: FROM PHASE I TO
Safety and Biological Activity
Exploratory phase I clinical studies were conducted in
approximately 150 patients with various advanced
malignancies (colorectal, melanoma, renal cell carcinoma,
lung, breast, stomach and salivary gland cancers as well as
sarcoma) to determine L-MTP-PE maximum tolerated dose
(MTD) and optimal biological dose (OBD) (Table 2) [57-
60]. MTD was found to be 4-6 mg/m2 whereas the OBD,
based on monocyte activation, was 0.5-2.0 mg/m2 .
Most side effects of L-MTP-PE were minimal and included
fever and rigors in the majority of patients, some tachycardia
and nausea in approximately half the patients, as well as
chills, myalgias, malaise and fatigue. Side effects decreased
in intensity and frequency during the course of treatment
[58, 59, 61]. They were believed to result directly from L-
MTP-PE biological activity, particularly the secretion of
Table 2.Clinical Trials with L-MTP-PE
Various metastaticI 28 patients
Dose escalation, PK
[57, 58, 62]
Advanced malignanciesI 37 patients
Dose escalation, biological activity
Advanced malignanciesI 27 patients
Dose escalation, biological activity
Metastatic cancersI 14 patients
PK, biological activity
Advanced melanomaI 14 patients
Stage III and IV melanomaI/II 18 patients
Adjuvant or neo-adjuvant
Stage III and IV melanoma I/II18 patients 
Relapsed OsteosarcomaII 28 patients, historical ctr
Randomized; Dose escalation
[64, 71, 73]
Soft tissue sarcomas II20 patients 
IIb 9 patients
Adjuvant or neo-adjuvant
Combination with ifosfamide
Osteosarcoma less than 30 days after
III 677 patients adjuvant
Combination with chemotherapy; Randomized to chemotherapy alone
Liposomal Muramyl Tripeptide PhosphatidylethanolamineCurrent Cancer Drug Targets, 2006, Vol. 6, No. 2 129
effects, the authors suggested not to exceed blood ibuprofen
levels of 10 µg/ml. On the other hand, the anti-tumor
activity of diclofenac and L-MTP-PE in a murine
fibrosarcoma model were lost when the two agents were used
in combination , indicating that COX inhibitors can
attenuate the anti-tumor effect of L-MTP-PE in vivo.
. More recently, Meyers and colleagues showed that
addition of ifosfamide to a three-drug regimen had no
significant effect on DFS and OS .
Before testing the efficacy of combining chemotherapy
and L-MTP-PE, it was first necessary to define whether
cytotoxic agents interfere with monocyte activation by L-
MTP-PE. Kleinerman and co-workers measured the
tumoricidal activity of blood monocytes from osteosarcoma
patients treated with cisplatin, high-dose methotrexate,
cyclophosphamide or doxorubicin, following in vitro
activation with L-MTP-PE. They found no difference with
monocytes from normal
cyclophosphamide was used together with doxorubicin, a
combination that inhibited monocyte activation .
Combination therapy was therefore evaluated during a
phase IIb study (Table 2). Nine patients with pulmonary
lesions that had developed during chemotherapy, or were
present at diagnosis, persisted despite chemotherapy and
recurred after surgical excision, were treated with ifosfamide
and L-MTP-PE in either adjuvant or neo-adjuvant setting.
No increase in ifosfamide toxicity was observed upon
combination with L-MTP-PE. Serum cytokines and
monocyte-mediated tumoricidal activity were not different
from what had been reported following treatment with L-
MTP-PE alone. Signs of tumor fibrosis and infiltration by
inflammatory cells were observed in surgical specimens from
patients in the neo-adjuvant group, strengthening the notion
that chemotherapy does not mitigate L-MTP-PE activity and
supporting the rationale for combination .
donors, except when
Phase II studies were performed in various indications
including melanoma, non-small cell lung carcinoma,
colorectal cancer and osteosarcoma (Table 2).
A phase II study in 20 patients with metastatic soft
tissue sarcoma failed to show any clinical efficacy .
A pilot study in melanoma patients at high risk of
recurrence suggested a possible benefit in survival. Eighteen
patients with either resectable stage III or resectable stage IV
limited to lungs, lymph nodes and subcutaneous tissues
received L-MTP-PE before (for 4 weeks) and after (for 20
weeks) resection. Mean survival was 80.5 months (range 69
to more than 91 months) with 4 patients remaining free of
disease for more than 5 years after surgery .
The first signs of biological activity of L-MTP-PE at the
level of lung metastasis were obtained in osteosarcoma
patients who had recurrent lung nodules after treatment with
L-MTP-PE; in these nodules, peripheral tumor fibrosis was
frequently observed. This finding was clearly different from
the central necrosis generally observed after chemotherapy
and was therefore considered to result from a local effect of
L-MTP-PE . The incompleteness of the fibrosis
encouraged the investigators to extend treatment duration in
a second group of patients from 12 weeks to 24 weeks .
These phase II trials (described in more detail below) in
patients with osteosarcoma provided the basis for the phase
III trial design.
The choice of osteosarcoma as the indication was based
on the aforementioned signs of biological activity, the
availability of a clinically relevant model in dog, and the
prominent medical need. It is however worth mentioning
that Kleinerman et al.  suggested that other solid tumors
in which lung metastases pose a treatment problem could
benefit from further investigations with L-MTP-PE.
Another phase II trial (Table 2) was conducted in 28
osteosarcoma patients with lung metastases that developed
during adjuvant therapy or that were present at diagnosis,
persisted during therapy and recurred following surgical
excision. After surgical resection, 12 patients received L-
MTP-PE twice a week for 12 weeks (Group 1). Nodules that
recurred after L-MTP-PE therapy were excised in 6 patients
and compared with lesions that had been resected prior to
study entry. Based on the type of the histologic changes
observed in pulmonary lesions removed from patients in
Group 1 (see above), the therapy was altered for the next 16
patients, who were treated for 24 weeks instead of 12. An
historical control group (Group 3) included 21 patients who
had been treated post-operatively with chemotherapy in the
same institution. No significant
progression-free survival was observed in the first group of
patients compared to the historical controls. In the second
group of patients, receiving L-MTP-PE for 24 weeks,
progression-free survival was significantly longer than the
historical control group: 9 months versus 4.5 months,
respectively (p<0.03) .
From November 1993 through November 1997, the
Children's Cancer Group and the Pediatric Oncology Group
(now collectively known as Children’s Oncology Group)
conducted a phase III study to assess whether the addition of
L-MTP-PE and/or ifosfamide to a standard 3-drug
chemotherapeutic regimen (doxorubicin, cisplatin and high-
dose methotrexate) would increase DFS in newly diagnosed
patients with high-grade osteosarcoma. The trial design
Combination with Chemotherapy
The probability of 5-year disease-free survival (DFS) for
patients with osteosarcoma treated with surgery alone is less
than 20% . Incorporation of chemotherapy has
significantly improved both DFS and overall survival (OS):
patients presenting localized,
osteosarcoma and treated in a neo-adjuvant or adjuvant
setting have now a 5-year event-free survival (EFS) above
70% . Various chemotherapeutic agents and regimens
have been tested, none of them showing any obvious
superiority [75, 76]. For example, a study conducted by the
European Osteosarcoma Intergroup failed to reveal any
difference in survival of more than 400 patients randomized
to receive a two-drug regimen (doxorubicin and cisplatin) or
a complex multidrug regimen (vincristine, methotrexate,
doxorubicin, bleomycin, cyclophosphamide, dactinomycin)
130 Current Cancer Drug Targets, 2006, Vol. 6, No. 2Nardin et al.
Table 3. Phase III Clinical Trials with L-MTP-PE: Design and Results from Prospective Analysis. A Total of 664 Eligible
Patients with Non-Metastatic, Resectable Disease were Randomized to 4 Regimens
Regimen A: standard chemotherapy Regimen A + L-MTP-PE
Regimen B: standard chemotherapy + ifosfamide Regimen B + L-MTP-PE
# of patients
Relapses or deaths a
(A and B)
(A+ and B+)
33299 0.03061 0.039
a at the end of the study, corresponding to a median follow-up of 4.8 years.
implied analysis in patients with non-metastatic disease that
was considered resectable at diagnosis, though patients with
metastatic or unresectable disease were also allowed to be
enrolled at selected sites. A total of 664 eligible patients
with non-metastatic disease amenable to resection were
enrolled and IDM evaluated them for disease-free and overall
survival. No significant improvement in DFS or OS was
observed by the addition of ifosfamide in the dose and
schedule used in this study (p=0.934 and 0.992
respectively). However, significant improvement in DFS and
OS were observed in patients randomized to receive L-MTP-
PE (p=0.030 and 0.039 respectively, Table 3). In this group
of patients, the risk of recurrence and death were decreased
by 25% and 30% respectively. The 6-year survival
probability went from 67.5% in the absence of L-MTP-PE
to 77.2% when it was added to the treatment.
Activated macrophages have been used in adoptive
therapies against various cancers, including bladder and
ovary cancers, mesothelioma and glioblastoma, and may be
able to eradicate microscopic but not macroscopic tumors
[80, 81]. The mechanism of in vivo anti-tumor activity of
activated macrophages is not fully understood, but it
probably implies pathways completely or partially distinct
from those used by chemotherapeutic agents. Despite many
attempts over the past decade to improve treatment of
osteosarcoma by chemotherapy, varying drugs and regimens,
it appears that about 30% of the patients present
chemotherapy-resistant tumor cells at diagnosis . MTP-
PE-activated monocytes and macrophages may eliminate
some metastases, provided they are small enough. It could
be speculated that the 10% increase in OS (from 67.5% to
77.2% at 6 years) observed in the phase III trial is due to a
L-MTP-PE beneficial effect on a subset of patients with
micrometastasis. The limited
macrophages on large metastatic lesions  would argue in
favor of L-MTP-PE addition as early as possible.
Combination therapy has always been a standard practice
in medical oncology. In the recent years, combination of
chemotherapy with immunotherapy is of increasing interest
in various types of cancer . The demonstration that L-
MTP-PE and chemotherapy can be used together effectively
in the treatment of osteosarcoma is encouraging and there is
great interest in attempting to optimize this combination to
take further advantage of any potential synergy.
efficacy of activated
This study was a factorial design with patients
randomized to one of four arms (3 drugs ± L-MTP-PE and 4
drugs ± L-MTP-PE) but with the intent to analyze based on
(i) 3 versus 4 drugs and (ii) ± L-MTP-PE. Because of a
suggested interaction between L-MTP-PE and ifosfamide
(which was however not significant in the prospectively
defined analysis), Meyers and colleagues performed an
analysis of individual arms . This analysis failed to
reveal any significant differences in DFS or OS, but the trial
was not powered for such comparison.
One hundred and thirteen patients entered the study with
metastatic or unresectable disease. We analyzed this
population separately. The trends in this group were the
same as in the larger group with non-metastatic resectable
disease, though the number was too small to observe any
significant differences. When all eligible patients (n=777)
were analyzed together, a highly significant improvement in
progression-free (p=0.011) and overall (p=0.015) survival
was observed in patients in the L-MTP-PE arms, with the
risk of recurrence/progression and death reduced by 26% and
In summary, although this study raised many additional
questions, including the best way to combine chemotherapy
and L-MTP-PE, it clearly demonstrated the clinical benefit
associated with L-MTP-PE in the treatment of osteosarcoma.
Liposomal Muramyl Tripeptide Phosphatidylethanolamine Current Cancer Drug Targets, 2006, Vol. 6, No. 2 131
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 Hofer, M.; Brezani,
Received: June 13, 2005 Revised: September 5, 2005Accepted: September 6, 2005