Parameters for Stable Water-in-Oil Lipiodol Emulsion for Liver Trans-Arterial Chemo-Eembolization.

PURPOSE: Water-in-oil type and stability are important properties for Lipiodol emulsions during conventional trans-arterial chemo-embolization. Our purpose is to evaluate the influence of 3 technical parameters on those properties. MATERIALS AND METHODS: The Lipiodol emulsions have been formulated by repetitive back-and-forth pumping of two 10-ml syringes through a 3-way stopcock. Three parameters were compared: Lipiodol/doxorubicin ratio (2/1 vs. 3/1), doxorubicin concentration (10 vs. 20 mg/ml) and speed of incorporation of doxorubicin in Lipiodol (bolus vs. incremental vs. continuous). The percentage of water-in-oil emulsion obtained and the duration until complete coalescence (stability) for water-in-oil emulsions were, respectively, evaluated with the drop-test and static light scattering technique (Turbiscan). RESULTS: Among the 48 emulsions formulated, 32 emulsions (67%) were water-in-oil. The percentage of water-in-oil emulsions obtained was significantly higher for incremental (94%) and for continuous (100%) injections compared to bolus injection (6%) of doxorubicin. Emulsion type was neither influenced by Lipiodol/doxorubicin ratio nor by doxorubicin concentration. The mean stability of water-in-oil emulsions was 215 ± 257 min. The emulsions stability was significantly longer when formulated using continuous compared to incremental injection (326 ± 309 vs. 96 ± 101 min, p = 0.018) and using 3/1 compared to 2/1 ratio of Lipiodol/doxorubicin (372 ± 276 vs. 47 ± 43 min, p = <0.0001). Stability was not influenced by the doxorubicin concentration. CONCLUSION: The continuous and incremental injections of doxorubicin in the Lipiodol result in highly predictable water-in-oil emulsion type. It also demonstrates a significant increase in stability compared to bolus injection. Higher ratio of Lipiodol/doxorubicin is a critical parameter for emulsion stability too.

Stabilization Improves Theranostic Properties of Lipiodol®-Based Emulsion During Liver Trans-arterial Chemo-embolization in a VX2 Rabbit Model.

PURPOSE: To demonstrate that stability is a crucial parameter for theranostic properties of Lipiodol®-based emulsions during liver trans-arterial chemo-embolization. MATERIALS AND METHODS: We compared the theranostic properties of two emulsions made of Lipiodol® and doxorubicin in two successive animal experiments (One VX2 tumour implanted in the left liver lobe of 30 rabbits). Emulsion-1 reproduced one of the most common way of preparation (ratio of oil/water: 1/1), and emulsion-2 was designed to obtain a water-in-oil emulsion with enhanced stability (ratio of oil/water: 3/1, plus an emulsifier). The first animal experiment compared the tumour selectivity of the two emulsions: seven rabbits received left hepatic arterial infusion (HAI) of emulsion-1 and eight received HAI of emulsion-2. 3D-CBCT acquisitions were acquired after HAI of every 0.1 mL to measure the densities' ratios between the tumours and the left liver lobes. The second animal experiment compared the plasmatic and tumour doxorubicin concentrations after HAI of 1.5 mg of doxorubicin administered either alone (n = 3) or in emulsion-1 (n = 6) or in emulsion-2 (n = 6). RESULTS: Emulsion-2 resulted in densities' ratios between the tumours and the left liver lobes that were significantly higher compared to emulsion-1 (up to 0.4 mL infused). Plasmatic doxorubicin concentrations (at 5 min) were significantly lower after HAI of emulsion-2 (19.0 µg/L) than emulsion-1 (275.3 µg/L, p < 0.01) and doxorubicin alone (412.0 µg/L, p < 0.001), and tumour doxorubicin concentration (day-1) was significantly higher after HAI of emulsion-2 (20,957 ng/g) than in emulsion-1 (8093 ng/g, p < 0.05) and doxorubicin alone (2221 ng/g, p < 0.01). CONCLUSION: Stabilization of doxorubicin in a water-in-oil Lipiodol®-based emulsion results in better theranostic properties.

Comparison of the effects of the repetition rate between microsecond and nanosecond pulses: electropermeabilization-induced electro-desensitization?

BACKGROUND: Applications of cell electropermeabilization are rapidly growing but basic concepts are still unclear. In particular, the impact of electric pulse repetition rate in the efficiency of permeabilization has not yet been understood. METHODS: The impact of electric pulse repetition rate in the efficiency of permeabilization was analyzed in experiments performed on potato tissue and partially transposed on mice liver. On potato tissue, pulses with durations of 100µs or 10ns are applied. The intensity of permeabilization was quantified by means of bioimpedance changes and electric current measurements and a new index was defined. RESULTS: For the two pulse durations tested, very low repetition rates (below 0.1Hz) are much more efficient to achieve cell permeabilization in potato tissue. In mice liver, using 100µs pulses, the influence of the repetition rate is more complex. Indeed, repetition rates of 1Hz and 10Hz are more efficient than 100Hz or 1kHz, but not the repetition rate of 0.1Hz for which there is an impact of the living mice organism response. CONCLUSIONS: We propose that the effects reported here might be caused by an electroporation-induced cell membrane 'electro-desensitization' which requires seconds to dissipate due to membrane resealing. GENERAL SIGNIFICANCE: This study not only reinforces previous observations, but moreover it sustains a new concept of 'electro-desensitization' which is the first unifying mechanism enabling to explain all the results obtained until now both in vitro and in vivo, with long and short pulses.

Electrochemotherapy: a new treatment of solid tumors.

Electrochemotherapy is a new local treatment of the solid tumors that can be defined as the local potentiation, by means of permeabilizing electric pulses, of the antitumor activity of non-permeant (e.g. bleomycin) or low-permeant (e.g. cisplatin) anticancer drugs. The electric pulses are delivered locally on the solid tumors, after the intravenous or intralesional injection of the chemotherapy agent. In this review, the basis of the electrochemotherapy are recalled. Then, after summarizing clinical data, we present some results of the European project Cliniporator, as well as the new pulse generator, the Cliniporator, that incorporates new features resulting from this research project, and that is fully conceived for a clinical use. Finally, future perspectives are discussed.

The effect of pulse repetition frequency on the uptake into electropermeabilized cells in vitro with possible applications in electrochemotherapy.

Electrochemotherapy is a technique where electric pulses in combination with chemotherapeutic agents are applied to tumor cells. In general, patients find electrochemotherapy tolerable, in spite of unpleasant sensations associated with contraction of muscles located beneath or in the vicinity of the electrodes. These contractions are due to the intensity of the electric pulses required for effective electropermeabilization of tumor cell membranes. Since a train of eight electric pulses with repetition frequency of 1 Hz is usually applied to the tumors, each pulse in the train excites underlying nerves and provokes muscle contractions. Therefore, for patients involved in electrochemotherapy, the use of pulses with repetition frequency higher than the frequency of tetanic contraction would represent reduced number of muscle contractions and associated unpleasant sensations. Our results of the uptake of Lucifer Yellow into electropermeabilized cells in vitro show that with increased repetition frequency the uptake stays at similar levels even at frequencies up to 8.3 kHz. On the basis of these results the possibilities for the clinical use of pulses with high repetition frequency in electrochemotherapy are considered.

Electrochemotherapy on liver tumours in rabbits.

Electrochemotherapy (ECT) is a new therapeutic approach combining the effects of a low-permeant cytotoxic drug, bleomycin (BLM), administered i.v. and cell-permeabilizing electric pulses (EPs) locally delivered to tumours. The transient permeabilization of the cell membrane by the EPs allows free access of BLM to its intracellular targets, largely enhancing BLM's cytotoxic effects. ECT efficacy has been proved so far on transplanted subcutaneous murine tumours and on subcutaneous metastases in humans. Here, we present the first study of the effects of ECT on tumours transplanted to livers in rabbits. We used a recently developed EP applicator consisting of an array of parallel and equidistant needles to be inserted in tissues. Effects of EPs alone or of ECT were assessed by histological analysis, tumour growth rates and survival of the treated animals. A transient blood hypoperfusion was seen in the electropulsed areas, with or without BLM, related to EP-dependent vasoconstriction but this had no major effects on cell survival. Long-term effects depended on the presence of BLM at the time of EP delivery. Almost complete tumour necrosis was observed after ECT, resulting from both BLM direct cytotoxic effects on electropermeabilized tumour cells and indirect effects on the tumour vessels. A large reduction in tumour growth rate and significantly longer survival times were scored in comparison with control rabbits. Moreover, ECT of liver tumours was well tolerated and devoid of systemic side-effects. When ECT was associated with a local interleukin 2-based immunotherapy, increased local anti-tumour effectiveness as well as a large decrease in the number of metastases were observed. Thus, ECT could become a novel treatment modality for liver tumours and other solid internal malignancies.

Effective treatment of cutaneous and subcutaneous malignant tumours by electrochemotherapy.

Electrochemotherapy (ECT) enhances the effectiveness of chemotherapeutic agents by administering the drug in combination with short intense electric pulses. ECT is effective because electric pulses permeabilize tumour cell membranes and allow non-permeant drugs, such as bleomycin, to enter the cells. The aim of this study was to demonstrate the anti-tumour effectiveness of ECT with bleomycin on cutaneous and subcutaneous tumours. This article summarizes results obtained in independent clinical trials performed by five cancer centres. A total of 291 cutaneous or subcutaneous tumours of basal cell carcinoma (32), malignant melanoma (142), adenocarcinoma (30) and head and neck squamous cell carcinoma (87) were treated in 50 patients. Short and intense electric pulses were applied to tumours percutaneously after intravenous or intratumour administration of bleomycin. The tumours were measured and the response to the treatment evaluated 30 days after the treatment. Objective responses were obtained in 233 (85.3%) of the 273 evaluable tumours that were treated with ECT. Clinical complete responses were achieved in 154 (56.4%) tumours, and partial responses were observed in 79 (28.9%) tumours. The application of electric pulses to the patients was safe and well tolerated. An instantaneous contraction of the underlying muscles was noticed. Minimal adverse side-effects were observed. ECT was shown to be an effective local treatment. ECT was effective regardless of the histological type of the tumour. Therefore, ECT offers an approach to the treatment of cutaneous and subcutaneous tumours in patients with minimal adverse side-effects and with a high response rate.

Antimetastatic effects of electrochemotherapy and of histoincompatible interleukin-2-secreting cells in the murine Lewis lung tumor.

Murine Lewis lung (3LL) tumors are characterized by the appearance of lung metastases after a regular period following their s.c. transplantation. We tested the respective efficiencies of various antitumor treatments: (i) electrochemotherapy (ECT), i.e. the systemic injection of bleomycin associated with electric pulses, locally delivered, that permeabilizes the tumor cells; (ii) intratumoral injection of histoincompatible cells that have been engineered in vitro to secrete high amounts of interleukin-2; and (iii) the combination of these two treatments. The growth of the s.c. transplanted tumors was followed up and the number of lung metastases was counted 14 days after the treatment. ECT alone resulted in the reduction of both the size of the tumor and the number of lung metastases. This latter effect can be partially explained by the effects of ECT on the s.c. tumor mass from which 3LL cells escape to colonize the lungs. The injection of IL-2-secreting cells alone had no effect on the s.c. mass and only a limited effect on the number of lung metastases. However, the combined treatment ECT plus IL-2-secreting cells resulted in antimetastatic effects potentiation that could result from stimulation of a non-specific immune response through an increase of NK activity.

Host's immune response in electrotherapy of murine tumors by direct current.

Electrotherapy by low level direct current has been demonstrated to have antitumor effects in different murine tumor models and in clinics. Electrotherapy in "field" configuration, where electrodes are placed subcutaneously outside of the tumor in a way that tumor lies in between the electrodes, was performed in immunodeficient nude and immunocompetent mice. Electrotherapy was much more effective in immunocompetent mice based on the observed tumor growth retardation, thus demonstrating that antitumor effectiveness of electrotherapy greatly depends on host's immune response. Further experiments were conducted by combining electrotherapy with concomitant immunotherapy in order to potentiate the antitumor effect of electrotherapy. Immunotherapy consisted of local delivery of genetically engineered cells selected for IL-2 secretion. This combined treatment was much more effective than any of the treatments alone.

First clinical trial of cat soft-tissue sarcomas treatment by electrochemotherapy.

Electrochemotherapy combines bleomycin and local electric pulses that allow cell permeabilization and free access of bleomycin to its intracellular target. We report the first veterinarian clinical trial of electrochemotherapy in 12 cats with spontaneous large soft-tissue sarcomas that suffered relapse after treatment with conventional therapies. Permeabilizing electric pulses were delivered using external surface electrodes, as well as new needle-shaped electrodes that were designed to be inserted in tumours for more effective treatment of several-centimetre-thick tumour nodules. The electric pulses were applied to the tumours several times from 4 to 15-30 min after a bolus intravenous injection of 0.5 mg kg(-1) bleomycin. Tolerance to treatment was excellent without general side-effects. The cats showed local inflammatory reactions for a few days and disease stabilization lasted from 2 weeks to 7 months. One partial regression was observed, and the general absence of nodule volume decrease can be explained by local fibrotic reactions. Histological analysis of biopsies also revealed massive tumour cell death. The cats' lifespan increased (P<<0.001), with a mean survival time of 6.1 months (maximum 18 months) compared with 0.8 months (maximum 1.5 months) for a group of 11 untreated control cats displaying similar carcinological features. Electrochemotherapy is clearly effective as a salvage treatment for large spontaneous solid tumours in adverse clinical situations and this is promising for future applications.

Phase I/II trial for the treatment of cutaneous and subcutaneous tumors using electrochemotherapy.

BACKGROUND: Electroporation is a process that causes a transient increase in the permeability of cell membranes. It can be used to increase the intracellular concentration of chemotherapeutic agents in tumor cells (electrochemotherapy; ECT). A clinical study was initiated to determine if this mode of treatment would be effective against certain primary and metastatic cutaneous malignancies. A group of six patients, three with malignant melanoma, two with basal cell carcinoma, and one with metastatic adenocarcinoma, were enrolled in the study. the treatment was administered in a two-step process. METHODS: Each patient received a 10 unit/m2 dose of bleomycin administered intravenously at 1 to 1.5 units/minute. This was followed by eight 99 microsecond pulses at an amplitude of 1.3 kV/cm administered directly to the tumors 5 to 15 minutes after the bleomycin was completely infused. Pulses were administered after the injection of 1% lidocaine solution around the treatment site. RESULTS: Two of three melanoma patients had objective responses. In these two patients, five of six treated tumors decreased in size, and three completely responded. Untreated tumors displayed continued growth. Objective responses were observed in both basal cell carcinoma (BCC) patients. One patient had partial responses in both treated tumors. The other patient had one of four primary BCCs respond completely, and the remaining three respond partially. Patients with metastatic breast adenocarcinoma showed complete responses in both treated nodules after ECT. All patients tolerated the treatment well with no residual effects from the electric pulses. CONCLUSIONS: ECT was an effective local treatment in the majority of nodules treated. The results thus far are very encouraging and the study is being continued.

Antitumor electrochemotherapy: new advances in the clinical protocol.

BACKGROUND: Electrochemotherapy (ECT) is a new antitumor approach that combines systemic bleomycin (BLM) with electric pulses (EP) delivered locally at the tumor site. These EP permeabilize the cells in the tissue, allow BLM delivery inside the cells, and increase BLM cytotoxicity. As an extension of our initial Phase I trial on patients with head and neck squamous cell carcinoma (HNSCC) permeation nodules, we tested variations of ECT protocol to determine how to improve it. METHODS: Seven patients with multiple and/or large permeation nodules of HNSCC or of salivary or breast adenocarcinoma were treated in 10 sessions. They received BLM followed by runs of four or eight short (100 microseconds) and intense (1000 or 1300 V/cm-1) EP delivered at adjacent positions on the nodules to cover all of the tumor surface. RESULTS: We determined the therapeutic window for EP delivery to be between 8 and 28 minutes after BLM intravenous injection. We showed patient tolerance to a high number of EP, along with ECT feasibility after BLM intraarterial injection or on adenocarcinoma nodules. Clear antitumor effects were obtained, especially in the small nodules. In the largest nodules we observed extended tumor necrosis. CONCLUSIONS: Relatively efficient ECT can be performed for large and think nodules, and ECT remains safe even when a large number of EP are delivered. However, in this study, ECT's effectiveness on large nodules was lower than on the previously treated small nodules, probably due to external electrodes inadequacy. The data reported stimulated us to design a new device for EP delivery.

Systemic antitumor effects of electrochemotherapy combined with histoincompatible cells secreting interleukin-2.

Electrochemotherapy is an antitumor treatment that combines a cytotoxic drug with the local administration of electric pulses delivered at the tumor site. We previously found that in mice the cure rate of subcutaneous transplanted tumors treated by electrochemotherapy is increased by repeated systemic interleukin-2 (IL-2) injections. Moreover, histoincompatible cells engineered to secrete IL-2 allow the rejection of syngeneic tumor cells when both cells are inoculated together. In this study of preestablished tumors in mice we show that after electrochemotherapy, delayed peritumoral injections of histoincompatible IL-2-producing cells result in the cure of almost all the tumors. Moreover, this combined local treatment leads to cures of untreated, contralaterally transplanted tumors. This systemic antitumor immunity also resulted in complete protection of the cured mice against further inocula of the tumor cells. These results, which were obtained using allogeneic as well as xenogeneic IL-2-secreting cells, suggest that electrochemotherapy combined with such cellular immunotherapy might be a useful approach for the treatment of metastasizing cancers.

Electrochemotherapy, a new antitumor treatment. First clinical phase I-II trial.

BACKGROUND: Electrochemotherapy is a new antitumor treatment consisting of electrical pulses administered to the tumor several minutes after intravenous injection of bleomycin. In mice, important antitumor effects were observed on subcutaneously transplanted tumors and on spontaneously occurring mammary carcinomas. Cures were obtained after one single treatment combining bleomycin and electric pulses. In humans, permeation nodules seemed an adequate oncologic situation to assay this new procedure. The authors report the first Phase I-II trial of electrochemotherapy. METHODS: Eight patients with 40 permeation nodules of head and neck squamous cell carcinomas were treated with 10 mg/m2 bleomycin intravenous bolus, followed by four or eight short (100 microseconds) and intense (1300 V/cm) pulses administered through two external electrodes located on each side of the treated nodule. RESULTS: An instantaneous painless contraction of the underlying muscles was regularly observed. Neither local nor general side effects were observed, and electrochemotherapy was well tolerated. In addition, a clear local antitumor efficacy was found: 23 (57%) nodules were in clinical complete response within a few days. CONCLUSION: The absence of toxicity, the good tolerance by the patients, and the net antitumor effects observed are encouraging for additional electrochemotherapy developments in clinical oncology.

Electrochemotherapy tumor treatment is improved by interleukin-2 stimulation of the host's defenses.

We have recently described a new antitumor treatment, electrochemotherapy (ECT), based on the large local potentiation of the effects of the chemotherapeutic agent bleomycin (BLM) by electric pulses (EP) delivered at the tumor site. We demonstrate here that the host's immune response participates in cure achievement. We obtain an increase of the rate of completely cured animals by injecting mice with interleukin-2 (IL-2).

Electrochemotherapy potentiation of antitumour effect of bleomycin by local electric pulses.

In cell culture the cytotoxicity of some anticancer drugs, especially bleomycin, can be greatly enhanced by exposing cells to non-cytotoxic electric pulses. Nude or conventional mice bearing subcutaneous transplanted tumours were treated with intramuscular doses of bleomycin followed by local delivery of electric pulses similar to those used in vitro. Tumors were reduced and even eradicated after this electrochemotherapy. Thus the antitumour effects of bleomycin in mice can be considerably potentiated by local electric pulses.

Electrochemotherapy of spontaneous mammary tumours in mice.

Electrochemotherapy delivers external electric pulses to the tumour site to induce local potentiation of the antitumour activity of intramuscular injections of bleomycin. C3H/Bi mice with spontaneous mammary carcinomas received weekly injections of 50 micrograms bleomycin followed by electric pulses 30 min later. All the 38 tumours treated exhibited at least a partial regression. 23 complete remissions were observed, 3 of which were cures. One difficulty in assessing the cure rate in this model is that frequent parallel or sequential tumours cause early death. Electrochemotherapy appears similarly efficient in spontaneous tumours as in previously studied transplanted tumours.

[Electrochemotherapy, a new antitumor treatment: first clinical trial]. / L'électrochimiothérapie, un nouveau traitement antitumoral: premier essai clinique.

Electrochemotherapy (ECT) is a new antitumor treatment which consists in delivering electric pulses to the tumor some minutes after an intravenous injection of bleomycin. We report here the first clinical trial of ECT, applied to patients with permeation nodules of head and neck squamous carcinomas. ECT was well tolerated by patients, no serious incident occurred and a clear antitumor efficiency was found.