Isolated limb perfusion (ILP) provides an excellent tool in the treatment of locally
advanced tumours. During ILP, high local drug concentrations are possible due to minimal
leakage into the systemic circulation, and the effect on vital organs is limited,
allowing high dosages to be used. We and others have demonstrated that addition of
tumour necrosis factor (TNF) to an ILP with melphalan increased the tumour response
dramatically as compared to melphalan alone (Lienard et al, 1992; Eggermont et al,
1996).
Melphalan is used most commonly in ILP, but other agents have been applied with varying
success in limb or organ perfusion (Rossi et al, 1992; Weksler et al, 1994; Abolhoda
et al, 1997). We observed in ILP that local toxicity was dose-limiting at suboptimal
doxorubicin concentrations (Van Der Veen et al, 2000).
The formulation of doxorubicin in long-circulating liposomes (Stealth liposomal doxorubicin,
Doxil®) prolongs circulation time, decreases toxicity and augments localisation in
tumour tissue (Gabizon et al, 2003). We hypothesised that the use of Doxil® in ILP
may reduce local toxicity while augmenting tumour accumulation and improving tumour
response. In this study, we examined the efficacy of Doxil® in a TNF-based ILP in
sarcoma-bearing rats.
MATERIALS AND METHODS
Chemicals
Human recombinant TNF-α was kindly provided by Dr G Adolf (Bender Wien GmbH, Wien,
Austria). Pegylated liposomal doxorubicin (Doxil®, Caelyx™) was kindly provided by
Dr Working (ALZA Corporation, Mountain View, CA, USA). Doxorubicin hydrochloride (adriblastina)
was purchased from Pharmacia (Brussels, Belgium).
Animals and tumour model
Male inbred BN rats (soft-tissue sarcoma model, BN175) and WAG/RIJ rats (osteosarcoma
model, ROS-1) were obtained from Harlan-CPB (Austerlitz, the Netherlands). Small fragments
(3 mm) of tumour were implanted subcutaneously in the right hindleg, as previously
described (de Wilt et al, 1999). Tumour growth was recorded by calliper measurements,
and tumour volume was calculated using the formula 0.4(A
2
B) (where B represents the largest diameter and A the diameter perpendicular to B).
All animal studies were done in accordance with protocols approved by the Animal Care
Committee of the Erasmus University Rotterdam, the Netherlands (Workman et al, 1998).
A tumour response indicates either a partial remission (PR, decrease of tumour volume
between –25 and 90%) or a complete remission (CR, tumour volume less than 10% of initial
volume).
Isolated limb perfusion protocol
Rat limbs were perfused as previously described (de Wilt et al, 1999). Tumour necrosis
factor (50 μg), Doxil® or doxorubicin (400 μg BN175 and 200 μg ROS-1) were added as
boluses to the oxygenation reservoir. Control rats were perfused with Haemaccel or
placebo liposomes alone. The concentration of TNF was adapted from previous animal
studies, and doxorubicin concentrations that yielded no local toxicity were used.
All animal studies were approved as stated above (Workman et al, 1998).
Assessment of doxorubicin accumulation in solid tumour during ILP
Accumulation of doxorubicin in tumour and muscle was determined directly after ILP,
as previously described (Mayer et al, 1989; Van Der Veen et al, 2000). As the ILP
included a thorough washout, there was no intravascular doxorubicin present. All animal
studies were approved as stated above (Workman et al, 1998).
Statistical analysis
The results were evaluated for statistical significance using the Mann–Whitney U-test
with SPSS for Windows. P-values below 0.05 were considered statistically significant.
RESULTS
Tumour response to Doxil® in TNF-based ILP
Perfusion with Doxil®, TNF or buffer alone resulted in progressive disease in all
soft-tissue sarcoma-bearing rats (Figure 1A
Figure 1
(A) Tumour volumes of subcutaneous implanted soft-tissue sarcoma BN175 after isolated
limb perfusion with perfusate alone (n=6), 400 μg Doxil® (n=4), 50 μg TNF (n=6), Doxil®
plus 50 μg TNF (n=8), 400 μg free doxorubicin (DXR) (n=7), or a combination of TNF
and free DXR (n=6). (B) Tumour volumes of subcutaneous implanted osteosarcoma ROS-1
after isolated limb perfusion with perfusate alone (n=6), 200 μg Doxil® (n=6), 50 μg
TNF (n=8), 200 μg Doxil® plus 50 μg TNF (n=7), 200 μg free doxorubicin (DXR) (n=6),
or a combination of TNF and DXR (n=6). The mean tumour volumes are shown±s.e.
). Perfusion with Doxil® plus TNF resulted in a short growth delay followed by rapid
outgrowth of the tumour, and all rats showed progressive disease. Application of free
conventional doxorubicin resulted only in a slight inhibition of the tumour growth,
and no rats showed a tumour response. Isolated limb perfusion with conventional doxorubicin
combined with 50 μg TNF increased the antitumour activity with a response rate of
83% (PR and CR combined) (P<0.01 compared with doxorubicin alone).
Isolated limb perfusion in osteosarcoma-bearing rats with buffer or conventional doxorubicin
alone had no significant effect on tumour growth (Figure 1B). Isolated limb perfusion
with TNF alone resulted in a response rate of 25%. Isolated limb perfusion with conventional
doxorubicin combined with TNF further increased the tumour response to 83% (P<0.05
compared with TNF alone or doxorubicin alone). Isolated limb perfusion with Doxil®
only, induced slight tumour growth delay comparable to free conventional doxorubicin.
Strikingly, ILP with Doxil® plus TNF diminished the tumour response, and none of the
rats showed a tumour response.
Accumulation of doxorubicin in solid tumour after ILP
We observed that addition of TNF did not significantly augment the accumulation of
Doxil® in soft-tissue sarcoma or osteosarcoma when compared to ILP with Doxil® alone
(data not shown). Levels of doxorubicin were significantly increased when TNF was
added to ILP with free doxorubicin (Van Der Veen et al, 2000).
DISCUSSION
In the present study, we demonstrated that ILP treatment with Doxil® combined with
TNF in sarcoma-bearing rats does not provide a useful alternative to free conventional
doxorubicin. The lack of efficacy of Doxil® is not due to failure of the drug to be
active at the tumour site, as dramatic synergy between Doxil® and TNF after systemic
treatment has been shown (ten Hagen et al, 2000). Rather, we speculate that the liposomes
are unable to extravasate into the tumours during the relatively short ILP interval.
In spite of the indicated usefulness of doxorubicin in ILP for the treatment of sarcoma,
we and others observed dose-limiting local toxicity after a TNF-based ILP with conventional
doxorubicin (Di Filippo et al, 1999; Van Der Veen et al, 2000). Biodistribution and
pharmacokinetic studies with Doxil® demonstrated a favourable profile of the liposomal
formulations over the free drugs, that is, circulation time was extended, toxicity
reduced and tumour localisation was increased (Gabizon et al, 2003). Therefore, we
envisioned that Doxil® could be a good alternative to free conventional doxorubicin
in ILP. However, Doxil® failed to induce any response in sarcoma-bearing rats, even
when applied in combination with TNF. Minimal accumulation of Doxil® in tumour after
ILP with or without TNF was observed, whereas considerably higher levels of doxorubicin
were found in tumour after ILP with free doxorubicin plus TNF. A possible explanation
for the difference in accumulation between free conventional doxorubicin and liposomal
doxorubicin is the particle size. Whereas distribution of a small molecule like doxorubicin
is diffusion dependent, the transport of particulate matter is convection dependent
(Jain et al, 1990; Hobbs et al, 1998). This would indicate that, during ILP, drug
distribution is mostly diffusion dependent and not convection dependent.
The increased tumour localisation of Doxil seen after systemic administration is reportedly
due to the ability of the pegylated liposomes to avoid accumulation in the liver and
spleen and other parts of the mononuclear phagocytic system (MPS), which results in
a long circulation time and extravasation through the leaky vasculature of tumours
(Gabizon et al, 2003). The volume of distribution of Doxil® is markedly smaller than
that of doxorubicin given systemically, reflecting the broad tissue distribution of
the latter. The short 30-min circulation time imposed by the ILP procedure is likely
inadequate for the circulation time advantage of Doxil® to have an effect on its distribution,
and, of course, the restriction of circulation to the isolated limb obviates the value
of avoiding the MPS. Thus, the rapid distribution properties of the small doxorubicin
molecule makes it a better choice for ILP procedures, particularly when it is used
in combination with TNF.