Successful treatment of solid tumors with chemotherapeutics requires that adequate levels reach the tumor cells. Tumor vascular normalization has been proposed to enhance drug delivery and improve tumor response to chemotherapy.

Several possible mechanisms for the synergistic anti-tumour effects between tumour necrosis factor alpha (TNF-α) and melphalan after isolated limb perfusion (ILP) have been presented. We found a significant sixfold increase in melphalan tumour tissue concentration after ILP when TNF-α was added to the perfusate, which provides a straightforward explanation for the observed synergism between melphalan and TNF-α in ILP. © 2000 Cancer Research Campaign

We have shown previously that isolated limb perfusion (ILP) in sarcoma-bearing rats results in high response rates when melphalan is used in combination with tumour necrosis factor alpha (TNF-α). This is in line with observations in patients. Here we show that ILP with doxorubicin in combination with TNF-α has comparable effects in two different rat sarcoma tumour models. The addition of TNF-α exhibits a synergistic anti-tumour effect, resulting in regression of the tumour in 54% and 100% of the cases for the BN175-fibrosarcoma and the ROS-1 osteosarcoma respectively. The combination is shown to be mandatory for optimal tumour response. The effect of high dose TNF-α on the activity of cytotoxic agents in ILP is still unclear. We investigated possible modes by which TNF-α could modulate the activity of doxorubicin. In both tumour models increased accumulation of doxorubicin in tumour tissue was found: 3.1-fold in the BN175 and 1.8-fold in the ROS-1 sarcoma after ILP with doxorubicin combined with TNF-α in comparison with an ILP with doxorubicin alone. This increase in local drug concentration may explain the synergistic anti-tumour responses after ILP with the combination. In vitro TNF-α fails to augment drug uptake in tumour cells or to increase cytotoxicity of the drug. These findings make it unlikely that TNF-α directly modulates the activity of doxorubicin in vivo. As TNF-α by itself has no or only minimal effect on tumour growth, an increase in local concentrations of chemotherapeutic drugs might well be the main mechanism for the synergistic anti-tumour effects. © 2000 Cancer Research Campaign

MelphalanMelphalan (Alkeran, 50 mg per vial, Wellcome, Beckenham, UK) was diluted in 10 ml solvent. Further dilutions were made in 0.9% sodium chloride to give a concentration of 1 µg µl -1 . A volume of 40 µl (= 40 µg) was added to the perfusion circuit.Prerequisites for effective isolated limb perfusion using tumour necrosis factor alpha and melphalan in rats Summary An isolated limb perfusion (ILP) model using soft tissue sarcoma-bearing rats was used to study prerequisites for an effective ILP, such as oxygenation of the perfusate, temperature of the limb, duration of the perfusion and concentration of tumour necrosis factor (TNF). Combination of 50 µg TNF and 40 µg melphalan demonstrated synergistic activity leading to a partial and complete response rate of 71%. In comparison to oxygenated ILP, hypoxia was shown to enhance anti-tumour activity of melphalan alone and TNF alone but not of their combined use. Shorter perfusion times decreased anti-tumour responses. At a temperature of 24-26°C, anti-tumour effects were lost, whereas temperatures of 38-39°C or 42-43°C resulted in higher response rates. However, at 42-43°C, local toxicity impaired limb function dramatically. Synergy between TNF and melphalan was lost at a dose of TNF below 10 µg in 5 ml perfusate. We conclude that the combination of TNF and melphalan has strong synergistic anti-tumour effects in our model, just as in the clinical setting. Hypoxia enhanced activity of melphalan and TNF alone but not the efficacy of their combined use. For an optimal ILP, minimal perfusion time of 30 min and minimal temperature of 38°C was mandatory. Moreover, the dose of TNF could be lowered to 10 µg per 5 ml perfusate, which might allow the use of TNF in less leakage-free or less inert perfusion settings.

It has previously been demonstrated in the setting of an isolated limb perfusion that application of high‐dose TNF‐α in combination with chemotherapy (melphalan, doxorubicin) results in strong synergistic antitumor effects in both the clinical and preclinical settings. In this study, we demonstrate that systemic administration of low‐dose TNF‐α augments the antitumor activity of a liposomal formulation of doxorubicin (DOXIL®). Addition of TNF‐α to a DOXIL® regimen, which by itself induced some tumor growth delay, resulted in massive necrosis and regression of tumors. Furthermore, we could demonstrate a significant increase of liposomal drug in the tumor tissue when TNF‐α had been co‐administered. Administration of TNF‐α augmented DOXIL® accumulation only after repeated injections, whereas accumulation of free doxorubicin was not affected by TNF‐α. Drug levels in the tumor interstitium appeared crucial as intracellular levels of free or liposome‐associated doxorubicin were not increased by TNF‐α. Therefore, we hypothesize that low‐dose TNF‐α augments leakage of liposomal drug into the tumor interstitium, explaining the observed improved antitumor effects. Regarding the effects of systemic administration of low doses of TNF‐α, these findings may be important for enhanced tumor targeting of various liposomal drug formulations. Int. J. Cancer 87:829–837, 2000. © 2000 Wiley‐Liss, Inc.