Hyperthermia and smart drug delivery systems for solid tumor therapy.

Chemotherapy is a cornerstone of cancer therapy. Irrespective of the administered drug, it is crucial that adequate drug amounts reach all cancer cells. To achieve this, drugs first need to be absorbed, then enter the blood circulation, diffuse into the tumor interstitial space and finally reach the tumor cells. Next to chemoresistance, one of the most important factors for effective chemotherapy is adequate tumor drug uptake and penetration. Unfortunately, most chemotherapeutic agents do not have favorable properties. These compounds are cleared rapidly, distribute throughout all tissues in the body, with only low tumor drug uptake that is heterogeneously distributed within the tumor. Moreover, the typical microenvironment of solid cancers provides additional hurdles for drug delivery, such as heterogeneous vascular density and perfusion, high interstitial fluid pressure, and abundant stroma. The hope was that nanotechnology will solve most, if not all, of these drug delivery barriers. However, in spite of advances and decades of nanoparticle development, results are unsatisfactory. One promising recent development are nanoparticles which can be steered, and release content triggered by internal or external signals. Here we discuss these so-called smart drug delivery systems in cancer therapy with emphasis on mild hyperthermia as a trigger signal for drug delivery.

Expression and prognostic significance of thymidylate synthase (TS) in pancreatic head and periampullary cancer.

BACKGROUND: Pancreatic cancer has a dismal prognosis. Attempts have been made to improve outcome by several 5-FU based adjuvant treatment regimens. However, the results are conflicting. There seems to be a continental divide with respect to the use of 5-FU based chemoradiotherapy (CRT). Furthermore, evidence has been presented showing a different response of pancreatic head and periampullary cancer to 5-FU based CRT. Expression of thymidylate synthase (TS) has been associated with improved outcome following 5-FU based adjuvant treatment in gastrointestinal cancer. This prompted us to determine the differential expression and prognostic value of TS in pancreatic head and periampullary cancer. PATIENTS AND METHODS: TS protein expression was studied by immunohistochemistry on original paraffin embedded tissue from 212 patients following microscopic radical resection (R0) of pancreatic head (n = 98) or periampullary cancer (n = 114). Expression was investigated for associations with recurrence free (RFS), cancer specific (CSS) and overall survival (OS), and conventional prognostic factors. RESULTS: High cytosolic TS expression was present in 26% of pancreatic head tumours and 37% of periampullary tumours (p = .11). Furthermore, TS was an independent factor predicting favourable outcome following curative resection of pancreatic head cancer (p = .003, .001 and .001 for RFS, CSS and OS, respectively). In contrast, in periampullary cancer, TS was not associated with outcome (all p > .10). CONCLUSION: TS, was found to be poorly expressed in both pancreatic head and periampullary cancer and identified as an independent prognostic factor following curative resection of pancreatic head cancer.

Cytokines and vascular permeability: an in vitro study on human endothelial cells in relation to tumor necrosis factor-alpha-primed peripheral blood mononuclear cells.

Tumor response is strongly enhanced by addition of tumor necrosis factor (TNF)-alpha to chemotherapy in local-regional perfusion. TNF primarily targets the endothelial lining of the tumor-associated vasculature, thereby improving permeability of the vascular bed. This augments uptake of the coadministered chemotherapeutic drug in the tumor. In vitro, however the high dose of TNF did not directly affect endothelial cells, indicating that other factors, most likely TNF-induced, are involved in the antivascular activities observed in vivo. This is supported by in vivo studies in our laboratory in which depletion of leukocytes resulted in loss of the antivascular activity of TNF. The present study examined the role of peripheral blood mononuclear cells (PBMCs) on endothelial cells by exposing them to TNF, interferon (IFN)-gamma, and PBMCs. We observed morphological changes of the endothelial cells when exposed to TNF in combination with IFN. Endothelial cells became elongated. and gaps between the cells formed. Addition of PBMCs enhanced these alterations. The endothelial layer became disrupted with highly irregular-shaped cells displaying large gap formations. PBMCs also contributed to an increased permeability of the endothelial layer without augmenting apoptosis. Replacing PBMC by interleukin (IL)-1beta produced similar effect with regard to inhibition of cell growth, morphological changes, and induction of apoptosis. Blocking IL-1beta with a neutralizing antibody diminished the effects inflicted of PBMCs. These observations indicate that endogenously produced IL-1beta by primed PBMCs plays an important role in the antivascular effect of TNF.

Isolated limb perfusion with actinomycin D and TNF-alpha results in improved tumour response in soft-tissue sarcoma-bearing rats but is accompanied by severe local toxicity.

Previously we demonstrated that addition of Tumour Necrosis Factor-alpha to melphalan or doxorubicin in a so-called isolated limb perfusion results in synergistic antitumour responses of sarcomas in both animal models and patients. Yet, 20 to 30% of the treated tumours do not respond. Therefore agents that synergise with tumour necrosis factor alpha must be investigated. Actinomycin D is used in combination with melphalan in isolated limb perfusion in the treatment of patients with melanoma in-transit metastases and is well known to augment tumour cell sensitivity towards tumour necrosis factor alpha in vitro. Both agents are very toxic, which limits their systemic use. Their applicability may therefore be tested in the isolated limb perfusion setting, by which the tumours can be exposed to high concentrations in the absence of systemic exposure. To study the beneficial effect of the combination in vivo, BN-175 soft tissue sarcoma-bearing rats were perfused with various concentrations of actinomycin D and tumour necrosis factor alpha. When used alone the drugs had only little effect on the tumour. Only when actinomycin D and tumour necrosis factor alpha were combined a tumour response was achieved. However, these responses were accompanied by severe, dose limiting, local toxicity such as destruction of the muscle tissue and massive oedema. Our results show that isolated limb perfusion with actinomycin D in combination with tumour necrosis factor alpha leads to a synergistic anti-tumour response but also to idiosyncratic locoregional toxicity to the normal tissues. Actinomycin D, in combination with tumour necrosis factor alpha, should not be explored in the clinical setting because of this. The standard approach in the clinic remains isolated limb perfusion with tumour necrosis factor alpha in combination with melphalan.

Liposome-encapsulated tumor necrosis factor-alpha enhances the effects of radiation against human colon tumor xenografts.

Recent reports have shown that tumor necrosis factor-alpha (TNF-alpha) can augment the effects of radiation against certain tumor types. However, the high concentrations of intravenous infusion of TNF-alpha needed to cause tumor regression can induce many systemic side effects. The aims of this study were to determine if TNF-alpha encapsulated in sterically stabilized (Stealth, ALZA Corporation, Mountain View, CA), PEGylated liposomes (SL) augments the antitumor effects of radiation and to compare its efficacy and possible toxicity with free TNF-alpha in the LS174T human colon tumor xenograft model. Nude mice were injected subcutaneously (s.c.) with LS174T cells and treated intravenously (i.v.) with Stealth-liposomal TNF-alpha (SL-TNF-alpha) with and without radiation or TNF-alpha with or without radiation when tumor size was approximately 200 mm(3). In phase 1, a significant decrease (p = 0.047) in tumor growth was observed with radiation at day 21 but not with SL-TNF-alpha or free TNF-alpha alone. By the end of phase 1 (day 27) with continued treatments, the SL-TNF-alpha plus radiation group had significantly smaller tumors (p = 0.044) than those in the free TNF-alpha plus radiation group. In phase 2, where a similar tumor growth reduction pattern was observed, the addition of TNF-alpha to radiation, either as free protein or within SL, increased lymphocyte activation and natural killer (NK) cell numbers in both blood and spleen. The effect was generally more pronounced with SL-TNF-alpha. Systemic toxicity, based on hematologic analyses and body weight, was absent or minimal. Collectively, the data show that pretreatment with SL-TNF-alpha can enhance more effectively, and possibly more safely, the effects of radiation against human colon tumor xenografts than can free TNF-alpha and that the increased antitumor action may involve upregulation of lymphocytes.

Nitric oxide synthase inhibition results in synergistic anti-tumour activity with melphalan and tumour necrosis factor alpha-based isolated limb perfusions.

Nitric oxide (NO) is an important molecule in regulating tumour blood flow and stimulating tumour angiogenesis. Inhibition of NO synthase by L-NAME might induce an anti-tumour effect by limiting nutrients and oxygen to reach tumour tissue or affecting vascular growth. The anti-tumour effect of L-NAME after systemic administration was studied in a renal subcapsular CC531 adenocarcinoma model in rats. Moreover, regional administration of L-NAME, in combination with TNF and melphalan, was studied in an isolated limb perfusion (ILP) model using BN175 soft-tissue sarcomas. Systemic treatment with L-NAME inhibited growth of adenocarcinoma significantly but was accompanied by impaired renal function. In ILP, reduced tumour growth was observed when L-NAME was used alone. In combination with TNF or melphalan, L-NAME increased response rates significantly compared to perfusions without L-NAME (0-64% and 0-63% respectively). An additional anti-tumour effect was demonstrated when L-NAME was added to the synergistic combination of melphalan and TNF (responses increased from 70 to 100%). Inhibition of NO synthase reduces tumour growth both after systemic and regional (ILP) treatment. A synergistic anti-tumour effect of L-NAME is observed in combination with melphalan and/or TNF using ILP. These results indicate a possible role of L-NAME for the treatment of solid tumours in a systemic or regional setting.

Low-dose tumor necrosis factor-alpha augments antitumor activity of stealth liposomal doxorubicin (DOXIL) in soft tissue sarcoma-bearing rats.

It has previously been demonstrated in the setting of an isolated limb perfusion that application of high-dose TNF-alpha 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-alpha augments the antitumor activity of a liposomal formulation of doxorubicin (DOXIL(R)). Addition of TNF-alpha to a DOXIL(R) 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-alpha had been co-administered. Administration of TNF-alpha augmented DOXIL(R) accumulation only after repeated injections, whereas accumulation of free doxorubicin was not affected by TNF-alpha. Drug levels in the tumor interstitium appeared crucial as intracellular levels of free or liposome-associated doxorubicin were not increased by TNF-alpha. Therefore, we hypothesize that low-dose TNF-alpha 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-alpha, these findings may be important for enhanced tumor targeting of various liposomal drug formulations.

TNF-alpha augments intratumoural concentrations of doxorubicin in TNF-alpha-based isolated limb perfusion in rat sarcoma models and enhances anti-tumour effects.

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-alpha). This is in line with observations in patients. Here we show that ILP with doxorubicin in combination with TNF-alpha has comparable effects in two different rat sarcoma tumour models. The addition of TNF-alpha 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-alpha on the activity of cytotoxic agents in ILP is still unclear. We investigated possible modes by which TNF-alpha 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-alpha 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-alpha fails to augment drug uptake in tumour cells or to increase cytotoxicity of the drug. These findings make it unlikely that TNF-alpha directly modulates the activity of doxorubicin in vivo. As TNF-alpha 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.

In vivo isolated kidney perfusion with tumour necrosis factor alpha (TNF-alpha) in tumour-bearing rats.

Isolated perfusion of the extremities with high-dose tumour necrosis factor alpha (TNF-alpha) plus melphalan leads to dramatic tumour response in patients with irresectable soft tissue sarcoma or multiple melanoma in transit metastases. We developed in vivo isolated organ perfusion models to determine whether similar tumour responses in solid organ tumours can be obtained with this regimen. Here, we describe the technique of isolated kidney perfusion. We studied the feasibility of a perfusion with TNF-alpha and assessed its anti-tumour effects in tumour models differing in tumour vasculature. The maximal tolerated dose (MTD) proved to be only 1 microg TNF-alpha. Higher doses appeared to induce renal failure and a secondary cytokine release with fatal respiratory and septic shock-like symptoms. In vitro, the combination of TNF-alpha and melphalan did not result in a synergistic growth-inhibiting effect on CC 531 colon adenocarcinoma cells, whereas an additive effect was observed on osteosarcoma ROS-1 cells. In vivo isolated kidney perfusion, with TNF-alpha alone or in combination with melphalan, did not result in a significant anti-tumour response in either tumour model in a subrenal capsule assay. We conclude that, because of the susceptibility of the kidney to perfusion with TNF-alpha, the minimal threshold concentration of TNF-alpha to exert its anti-tumour effects was not reached. The applicability of TNF-alpha in isolated kidney perfusion for human tumours seems, therefore, questionable.

Biodistribution and tumor localization of stealth liposomal tumor necrosis factor-alpha in soft tissue sarcoma bearing rats.

The blood residence half-life and organ distribution of recombinant human tumor necrosis factor-alpha (TNF-alpha) encapsulated in sterically stabilized liposomes, were investigated in rats bearing a soft tissue sarcoma in the hind leg. We studied the decay in blood concentration of "empty" liposomes using the aqueous marker 67gallium-desferal, as well as the blood concentration of soluble TNF-alpha and liposome encapsulated TNF-alpha using l25I. Encapsulation efficacy of TNF-alpha was 24%. The pharmacokinetics of TNF-alpha were markedly altered after encapsulation in liposomes, with a 33-fold increase in mean residence time of TNF-alpha in the blood, and a concomitant 14-fold increase in the area under the plasma concentration vs. time curve for liposomal TNF-alpha. Although the liposomes exhibit Stealth characteristics, uptake by mononuclear phagocyte-rich organs (e.g., liver and spleen) was noticeable, especially at later time points. Encapsulation of TNF-alpha in sterically stabilized liposomes resulted in a marked increase in localization of the cytokine in tumor measured as total uptake over time. However, peak TNF-alpha concentration levels in tumor were not significantly enhanced compared with free TNF-alpha. Besides the augmented localization of TNF-alpha after encapsulation in sterically stabilized liposomes, a diminished toxicity was observed.