Melanoma Research 1996, 6, pp. 427~433

Enhanced effects of multiple treatment electrochemotherapy

M. J. Jaroszeski*, R. Gilbert, R Perrott and R. Heller

Department of Chemical Engineering, University of South Florida, Tampa, FL 33612, USA. (R. Gilbert, R. Heller).

Department of Surgery, College of Medicine, University of South Florida, 12901 Bruce B. Downs Boulevard, Tampa, FL 33612-4799, USA. Tel: ( + 1) 813 974 4662; Fax: ( + 1) 813 974 2669 (M. J. Jaroszeski, R. Perrot, R.Heller).

Electrochemotherapy has been demonstrated to be an effective treatment for cutaneous cancers. The treatment includes administering a chemotherapeutic agent followed by electric pulses which are applied directly to the tumour. The pulses facilitate delivery of drug through the plasma membrane. Enhanced delivery Is restricted to the area that has been electrically treated. Currently, electrochemotherapy is administered as a single treatment. Complete response rates are high; however, partial responses are obtained In a fraction of the treated tumours. An issue associated with this is whether or not multiple treatments would result In an improved therapy for these partially responding tumours. A multiple treatment electrochemotherapy study was Implemented In order to address this issue. The study utilized subcutaneously Induced murine B16 melanoma tumours in C57B1/6 mice. Results showed large tumour volume reductions in multiple treatment groups. In addition, a twofold increase In tumour doubling time and greater percentages of complete responses were found as a result of multiple treatment. These results will be utilized to augment existing clinical trials with respect to retreating tumours that have partially responded to a single electrochemotherapy treatment.

Key words: bleomycin, electrochemotherapy, electropermeablilization, electroporation, melanoma, multiple treatment.

Introduction

The delivery of chemotherapeutic agents to tumour cells can be augmented by exposing the tumour to electric pulses following the administration of drug. This combined therapy is called electrochemotherapy (ECT). The treatment has been used to successfully treat cutaneous malignancies.^1-6 It is performed by first administering a drug intratumorally or intravenously and then delivering pulses directly to the tumour. The pulses used for ECT are noncytotoxic; however, they do affect the tumour cells at the membrane level. They induce tumour cell membranes to become permeabilized by a process called electroporation.^7-9 Therefore, drug that is in the extracellular space is free to diffuse into permeabilized tumour cells. The permeabilized state is short-lived. Normal membrane fluidity allows electrically treated cells to return to their normal state within a period of a few minutes to 1hr after pulsation has ceased ⁽¹⁰⁾

Electrochemotherapy has been used in animal models for the treatment of melanoma,^(11-14)  lung carcinoma, ⁽¹⁵⁾ mammary tumours, ^(l6) fibrosarcoma, ^(l4) sarcoma,⁽¹³⁾  Ehrlich-Lettre ascites carcinoma, ^{(l4, l7, l8)} hepatocellular carcinoma ^(l9-22) and glioma .⁽²³⁾ Almost all of these studies have used the chemotherapeutic agent bleomycin. This drug has been shown to have the greatest increase in cytotoxicity when used in ECT protocols. Response rates in these animal studies ranged from 70 to 85%.

Clinical trials have been implemented based on data from the animal model work. Trials for the treatment of melanoma, ^{(l2 )}basal cell carcinomal^(1-3) and head and neck squamous cell carcinoma^(4-6) have been conducted. All of these ECT trials have used bleomycin as the chemotherapeutic agent. Objective response rates range from 70 to 80% and complete response rates vary from 50 to 60%. Thus far, ECT has been administered as a single treatment in the clinic. The high complete response rates are encouraging. However, the issue of obtaining complete responses from tumours that partially respond to ECT has not yet been addressed.

In order to resolve this issue, a study was designed to investigate whether or not there is a benefit to performing multiple ECT treatments to recurrent sites or umours that partially respond. A murine C57B1/6 model with subcutaneously induced B16 murine melanoma tumours was used for the investigation. This aggressive tumour model was selected for use because it has shown a high tumour recurrence rate in ECT studies ^(ll-l3) The study also investigated the use of reduced drug doses with multiple treatments.
 

Materials and methods

Cell line and culture methods

Murine B16 melanoma cells (CRL 6322; American Type Culture Collection, Rockville, MD, USA) were used to induce tumours. The cell line was grown in McCoy's SA medium (Mediatech, Washington, DC, USA) supplemented with 10% (v/v) fetal bovine serum (Hyclone, Logan, UT, USA) and 90,ug/ml gentamycin sulphate (Gibco, Grand Island, NY, USA). Cells were grown in a humidified atmosphere that contained 5% CO2. Nearly confluent cultures were prepared for use by detaching with cell dissociation solution (Sigma, St Louis, MO, USA), a nonenzymatic reagent. Viability was determined for harvested cell batches using the trypan blue exclusion dye method. All cells used for this study were > 90% viable.

Animals and tumour induction

Female C57B1/6 mice were used for this study. All mice were 7-8 weeks old at the time of tumour induction. Tumours were induced by injecting 106 B16 murine melanoma cells subcutaneously into the shaved left flank. The cells were in a volume of 0.05 ml. Tumours were allowed to grow for 7-10 days which resulted in nodules that had dimensions ranging from 0.5 to 0.75 cm. Tumours of this size were elevated above the skin and easily measured.

Tumour treatment

Animals received bleomycin (Blenoxane, a generous gift from Bristol-Meyers Squibb, Princeton, NJ, USA) by intratumoral injection. This injection route was selected based on previous studies which showed that it was as effective as systemic.administration.12 Injections were carried out using a 30 gauge needle. Bleomycin doses of 0.25, 0.08, 0.025 and 0.0125 units were used in this study. These doses were administered in a volume of 0.05 ml sterile injectable saline. Electrical treatment was administered using a BTX T820 generator (Genetronics Inc., San Diego, CA, USA) 10 min after bleomycin injection. Electrical energy was transmitted directly to tumours with the aid of custom-built electrodes. The electrodes were constructed of two stainless steel plates that were mounted on a plastic Vernier caliper.2 3 The plates were arranged parallel to each other so that they could be placed on opposite sides of protruding tumours. In addition, their positions on the caliper allowed the spacing between the plates to be precisely adjusted and measured. The face of each plate measured 1 cm by 1 cm. A thin layer of electrocardiography paste (Parker Laboratories Inc., Orange, NJ, USA) was placed on the electrode faces to assure uniform electrical contact with the tumours. Each electrical treatment consisted of eight rectangular direct current pulses that were 100 ps in duration. The electric field strength and duty cycle were 1500 V/cm and 1 s, respectively. All animals that received electric pulses were anaesthetized with a 45 mg/kg dose of sodium pentobarbital prior to treatment.

Electric field monitoring

Each electric pulse was monitored using a digital storage oscilloscope (Philips Model PM3375; Philips, Eindhoven, The Netherlands). Voltages were monitored at the generator output, and the current resulting from each pulse was measured using a standard current monitor (Pearson Electronics, Inc., Pal Alto, CA, USA). The oscilloscope was used to store voltage and current traces during experimentation. A personal computer and IEEE-488 interface were used to transfer, store and analyse digitized trace data.

Tumour measurements

Treatment results were assessed based on tumour volume. These volume determinations were made at periodic intervals post-treatment as well as immediately prior to treatment. Volumes were determined by measuring the longest tumour dimension (a) and the next longest dimension (b) that was perpendicular to a. Tumour volume (V) was computed using the formula: V= (ab^2 )(22/7)/6. Measurements were made using a digital Vernier caliper.

The mean (SD) tumour volume was calculated for each treatment group at periodic intervals during the followup period. The time required for the volume of each tumour to double was also determined. This information was compiled into a mean tumour doubling time for each treatment group. Objective responses were also scored after 21 days as follows: (i) progressive disease (PD), tumour volume increased; (ii) stable disease (SD), less than 50% decrease in tumour volume; (iii) partial response (PR), greater than 50% decrease in tumour volume; and (iY) complete response (CR), no palpable tumour.
 

(please see journal paper for results and discussion  "Melanoma Research 1996, 6, pp. 427-433" )
 

Acknowledgements

This work was supported by grants from Genetronics, Inc., San Diego, CA, USA. Additional support was pro vided by the Departments of Chemical Engineering and Surgery of the University of South Florida and by a Small Business Innovative Research Grant (#1 R43 CA62905 01A2) from the National Cancer Institute awarded to Genetronics, Inc. (Dr Ken Dev, principal investigator) in collaboration with the University of South Florida. The authors would like to acknowledge Nancy Van Voorhis for her assistance with cell culture.
 

References
1 Heller R. Jaroszeski MJ, Glass LF, Messina JL, Rapaport DP, DeConti RC, et al. Phase I/II trial for the treatment of 19.
cutaneous and subcutaneous tumours using electrochemotherapy. Cancer 1996; 77: 964-971.

2. Heller R. Treatment of cutaneous nodules using Electrochemotherapy J Florida Med Assoc 1995; 82: 147-150.

3. Glass LF, Fenske NA, Jaroszeski MJ, Perrott R. Harvey DT, Reintgen DS, et al. Bleomycin-mediated electrochemother apy of basal cell carcinoma. J Am Acad Dermatol 1996; 34: 82-86.
432 Melanoma Research _ Sol 6 _ 1996

4. Mir LM, Belehradek M, Domenge C, Orlowski S. Poddevin J Jr, Schwab G. et al. Electrochemotherapy, a novel antitu mour treatment: first clinical trial. CR Acad Sci Paris 1991; 313: 613-618.

5. Belehradek M, Domenge C, Luboinnski B. Orlowski S. Belehradek J. Mir LM. Electrochemotherapy, a new anti tumour treatment: first phase 1-11 trial. Cancer 1993; 72: 3694-3700.

6. Domenge C, Orlowski S. Luboinski B. DeBaere T. Schwaab G. Belehradek J Jr, et al. Antitumour Electrochemotherapy new advances in the clinical protocol. Cancer 1996; 77: 956-963.

7. Zimmermann U. Pilwat G. Riemann F. Dielectric break down of cell membranes. Biophys J 1974; 14: 881 899.

8. TeissieJ, Rols MP. An experimental evaluation of the critical potential difference inducing cell membrane electro permeabilization. Biophys J 1993; 65: 409-413.

9. Weaver JC. Electroporation: a general phenomenon for manipulating cells and tissues. J Cell Biochem 1993; 51: 426-435.

10. Teissie J. Rots MP. Time course of electropermeabilization. In: Allen MJ, Cleary SF, Sowers AK, Shillady DD, eds. Charge and Field Effects in Biosystems: 3. Boston: Birkhau ser, 1992: 285-301.

11. Heller R. Jaroszeski M, Leo-Messina J. Perrott R. Van Voor his N. Reintgen D, et al. Treatment of B16 melanoma with the combination of electroporation and chemotherapy. Bioelectrochem Bioenerget 1995; 36: 83-87.

12. Heller R. Jaroszeski M, Perrott R. Messina J. Gilbert R. Effective treatment of B16 melanoma by direct delivery of bleomycin using Electrochemotherapy Melanoma Res 1996; 7(1), in press.

13. Mir LM, Orlowski S. Belehradek J Jr, Paoletti C. Electro chemotherapy potentiation of antitumour effect of bleo mycin by local electric pulses. EurJ Cancer 1991; 27: 68-72.

14. Sersa G. Cemazar M, Miklavcic D. Antitumour effectiveness of Electrochemotherapy with cis-diamminedichloroplati num (11) in mice. Cancer Res 1995; 55: 3450-3455.

15. Kanesada H. Anticancer effect of high voltage pulses com bined with concentration dependent anticancer drugs on Lewis lung carcinoma. J Jpn Soc Cancer Ther 1990; 25: 2640-2648.

16. Belehradek J Jr, Orlowski S. Poddevin B. Paoletti C, Mir LM. Electrochemotherapy of spontaneous mammary tumours in mice. Eur J Cancer 1991; 27: 73-76.

17. Cemazar M, Miklavcic D, Vodovnik L, Jarm T. Rudolf Z. Stabuc B. et al. Improved therapeutic effect of electroche motherapy with cisplatin by intratumoural drug adminis tration and changing of electrode orientation for electropermeabilization on EAT tumour model in mice. Radiol Oncol 1995; 29: 121-127.

18. Sersa G. Cemazar M, Semrov D, Miklavcic D. Changing electrode orientation improves the efficacy of electroche motherapy of solid turnouts in mice. Biolectrochem Bioe nerg 1996; 39: 61-66.

19. Okino M, Marumoto M, Kanesada H. Kuga K, Mohri H. Electrical impulse chemotherapy for rat solid tumours. Proc Jpn Cancer Congress 1987; 46: 420-420.

20. Okino M, Esato K. The effects of a single high voltage electrical stimulation with anticancer drug on in vitro grow ing malignant tumours. Jpn J Surg 1990; 20: 197-204.

21. Okino M, Tomie H. Kanesada H. Marumoto M, Morita N. Esato K, et al. Induction of tumour specific selective toxicity in electrical impulse chemotherapy_analysis of dose re sponse curve. Oncologia 1991; 24: 71-79.

22. Okino M, Tomie H. Kanesada H. Marumoto M, Esato K, Suzuki H. Optimal electric conditions in electrical impulse chemotherapy. Jpn J Cancer Res 1992; 83: 1095-1101.

23. Salford LG, Persson BRR, Brun A, Ceberg CP, Kongstad

PCH, Mir LM. A new brain tumour therapy combining bleomycin with in vivo electropermeabilization. Biochem Biophys Res Comm 1993; 194: 938-943.

24. Mir LM, Roth C, Orlowski S. Quintin-Colonna F. Fradelizi D,

Belehradek J Jr, et al. Systemic antitumour effects of elec trochemotherapy combined with histoincompatible cells secreting interleukin-2. J Imunother 1995; 17: 30-38.
Multiple treatment electrochemotherapy

25. Mir LM, Orlowski S. Poddevin B. Belehradek J Jr. Electro chemotherapy tumour treatment is imporoved by interleu kin-2 stimulation of the host's defenses. Eur Cytokine Network 1992; 3: 331-334.

26. Mir LM, Roth C, Orlowski S. et al. Potentiation of the antitumoral effect of electrochemotherapy by an immu notherapy with allogeneic cells producing interleukin-2. C R Acad Sci Paris 1992; 314: 499-504.