Radiation and electro pulse treatment of N32 tumours on flank of rats.

1. Radiation and electro pulse treatment of N32 tumours on flank of rats. Bertil R.R.Persson1), Per E. Engström1), Arne Brun2), and Leif G. Salford3) Departments of Radiation Physics1), Neuropathology2) and Neurosurgery3) Lund University, S‑221 85 LUND, Sweden.

2. Previous studies We have found that a combination of chemo-therapy and intracranial el-pulse treatment in vivo increases the survival of rats with RG2 brain tumours implanted in the brain. Salford et al. Biochem. Biophys. Res. Com. 194 (2) 938-343, 1993, Engström et al Bioelectrochemistry and Bioenergetics47, 163-166, 1998.

3. Experimental set-up of intracranial el-pulse treatment of rat glioma Needle electrodes inserted on each side of the tumour Pulse generator: Delivers 8 pulses of 800 Volt/cm, 1.0 ms duration, 1 pulse per second. 0.3 mg Bleomycin i.v. 4 min prior to electric pulses • Results: • Prolonged survival time (P<0.05) • Mean survival time: • Treated: 12 d • Controls: 7 d • 4 out 40 treated survived >30 d RG2 Tumour (2-3 mm)

4. AIM OF PRESENT STUDY • The purpose of the study was to investigate the effect of • el-pulse treatment (EPT) on rat gliomatumours • In the present study we have applied el-pulses combined with radiotherapy (EPX) for treatment of N32 brain tumours implanted under the skin of the thigh of Fisher-344 rats.

5. Visualisation of drug uptake in electro pulse treated tumour 16 pulses of 1300 V/cm with 1/1000 sec duration applied to the tumour 0.3 mg Bleomycin labelled to 111-indium given i.v. 4 min before EP

6. Tumours on the thigh were produced by the injection of 100 000 N32 glioma cells subcutaneously on Fischer-344 rats. • After 4 weeks, a solid tumour has grown to a size of 1 cm located directly under the skin. • The fur over the tumour was shaven and the tumour was fixed in position between two plate electrodes.

7. Treatment: 16 pulses of 1300 Volt/cm, 1.0 ms duration, 1 pulse per second given immediately followedby or prior to 5 Gy Co-60 gamma radiation Treatment repeated 4 consecutive days El-pulse treatment subcutaneous rat glioma tumours in combination with radiation therapy (EPX) 

8. El-pulse treatment • Electrocardial paste was used to achieve good electric conductivity • between tissue and electrodes. Electric pulses were delivered through • two flat brass electrodes sized 2´2 cm and mounted on a slide calliper • to set the correct distance between the electrodes. • 16 el-pulses with a field strength of 1300 V/cm and a time constant • of 1.0 ms were delivered at approximately one pulse per second. • All animals were treated during four consecutive days. • Animals were sacrificed when tumour volume reached 5 cm3

9. Tumour growth after Radiation therapy prior to El- Pulse Treatment. 2500 3 2000 1500 Treatment Tumor volume / mm four days 1000 Recidive in 2 out of 5 rats 500 Measure of crust Growth before treatment 0 0 20 40 60 80 100 Time after inoculation / Days

10. Tumour growth after El-Pulse Treatment prior to Radiation Therapy

12. Radiation and electro pulse treatment of N32 tumours on flank of rats. Persson, B.R.R.1), Engström, P.E.1), Brun, A2), and Salford, L.G.3) Departments of Radiation Physics1), Neuropathology2) and Neurosurgery3) Lund University, S‑221 85 LUND, Sweden. The therapeutic effect of radiation treatment combined with short electric impulses of high voltage has been studied on subcutaneous implanted N32 rat brain tumours. Sub-optimal, fractionated 60Co-gamma radiation treatment was administered separately or in combination with electric pulses. Electropulsation and/or radiation treatment was repeated on four consecutive days and evaluated by cure rate, tumour growth delay and microscopically examined. All untreated and radiation-treated animals showed progressive disease with neither partial nor complete remission. When radiation and electric pulses were applied concomitantly, a cure rate of 67% was achieved for more than 80 days after treatment. The remaining showed 137% prolonged survival. Animals given only electric pulses resulted in a prolonged survival of 69% and 25% cures (CR>100d). Histopathological and immunohistochemical examinations showed almost instant deteriorating effects on tumour vascularisation and a gradual development of wide-spread tumour cell death over time (up to 72 hrs). We believe the tumouricidal effect arises partly from destruction of the tumour vasculature and partly from cell damage from reactive oxygen species formed by the electric pulses and the radiation treatment.

13. Electro pulsation (EP) is used in a variety of applications to internalise various compounds into the cytoplam of cells to alter the properties of the cell´: • cytotoxic drugs (bleomycin, platinol) • antibodies • nucleic acids (DNA) • restriction nucleases • gene constructs

14. Extracellular (hydophilic) molecules Electrodes _ E _ + + AFTER Electro pulsation: Cell membranes transiently permeabilised BEFORE Electro pulsation Cell membranes intact

15. EP effect on cells EP effect on blood vessels Destruction and long lasting vasoconstriction of fragile tumour vasculature DNA and lipid destruction by Reactive Oxygen Species Release of of tumour specific substances into extracellular space and circulation? Immunologic response? Tumour cellstarvation, toxic effects of retained metabolites

16. Ongoing clinical trials of el-pulse treatment USA , France, Slovenien, and Denmark Cutaneous and subcutaneous malignancies, basal cell carcinoma, malignant or metastatic melanoma, adenocarcinoma, head and neck squamous cell carcinoma Independent clinical trials performed in three countries encompasses 50 patients with 291 cutaneous or subcutaneous tumours (1998). Complete clinical response was achieved in 56% and partial response in 29% of all tumours treated.