Therapeutic applications of calcium electroporation to effectively induce tumor necrosis

Abstract

The present inventors have shown that electroporation with calcium ions are efficient on cutaneous and subcutaneous nodules. In particular the present inventors here disclose that a solution comprising calcium ions (Ca.sup.2+) with a concentration of at least 0.1 M is extremely useful in a method of treating a neoplasm, such as cancer with means for causing transient permeabilization of the cell membranes of at least part of the neoplasm before, during and/or after administration of said solution, wherein said solution is administered with a ratio of 0.2 to 0.8 of the volume of said part of the neoplasm.

Claims

1. A method of treating a neoplasm in a subject comprising: a) administering to a subject having a neoplasm a solution comprising calcium ions (Ca.sup.++) with a concentration of at least 0.1 M, wherein said solution is contacted with at least a part of said neoplasm, and wherein the volume of the solution has a ratio of 0.2 to 0.8 of the volume of said part of the neoplasm; and b) inducing transient permeabilisation of the cell membranes in said part of said neoplasm, wherein said transient permeabilisation commences before, during, or after said administration of said solution.

2. The method according to claim 1, wherein the calcium ions induce necrosis in at least 30% of the part of the neoplasm exposed to said transient permeabilisation of the cell membranes.

3. The method according to claim 1, wherein the transient permeabilisation of the cell membranes is induced by electroporation, sonoporation, a hydrodynamics-based procedure or a magnetic field.

4. The method according to claim 1, wherein the transient permeabilisation of the cell membranes is induced by an electroporation of 200-2000 V/cm; pulse length 0.1-10.0 ms; pulse number 2-20; and pulse frequency 1 Hz-5 kHz.

5. The method according to claim 1, wherein the transient permeabilisation is induced by sonoporation.

6. The method according to claim 1, wherein the subject is a mammal.

7. The method according to claim 1, wherein the neoplasm is a solid tumor.

8. The method according to claim 1, wherein the neoplasm is a solid tumor having a minimum diameter of 0.5 cm.

9. The method according to claim 1, wherein the solution is administrated by intratumoral injection or infusion.

10. The method according to claim 1, wherein the solution further comprises a chemotherapeutic agent or a cytotoxic agent.

11. The method according to claim 1, wherein the solution is free from a chemotherapeutic agent or a cytotoxic agent.

12. The method according to claim 1, wherein step a), alone step b) alone, or both of steps a) and b) are repeated one or more times.

Description

FIGURE LEGENDS

(1) FIGS. 1-2

(2) Calcium Overloading Induces Cell Death In Vitro

(3) Cell viability in three cell lines, DC-3F, a transformed Chinese hamster lung fibroblast cell line (.square-solid.), K-562, a human leukemia cell line (.box-tangle-solidup.), and Lewis Lung Carcinoma, a murine lung carcinoma cell line (.circle-solid.) after treatment with increasing calcium concentrations either electroporated (black) or not electroporated (white). MTT viability assay was performed respectively 1 (FIG. 1) and 2 days (FIG. 2) after treatment. Results are depicted as percentages of controls (electroporated or non-electroporated cells in 0 mM calcium) (meanss.d., n6). EP, electroporation.

(4) FIGS. 3-4

(5) Calcium Overloading Induces Cell Death In Vivo

(6) H69 (Enhanced Green Fluorescent Protein (EGFP) transfected human small cell lung cancer cell line) tumors induced on nude mice were treated with isotonic calcium-chloride (168 mM) and electroporation (D), calcium-free physiological saline and electroporation (C), isotonic calcium-chloride alone (B), or calcium-free physiological saline alone (A). Tumor size (FIG. 3) and fluorescence intensity in bioimager (FIG. 4) were measured before treatment and 3 times a week after treatment, (means+s.d., n=3-9). EP, electroporation; NC, normalised counts.

(7) FIG. 5

(8) Fluorescence Intensity Images

(9) Representative images of fluorescence intensity in the tumors treated with isotonic calcium-chloride (168 mM) and electroporation, calcium-free physiological saline and electroporation, isotonic calcium-chloride alone, or calcium-free physiological saline alone. Placement of the mouse in the scanner and location of the tumor is shown in the top right corner, intensity bar is shown as a logarithmic scale. EP, electroporation; NC, normalised counts.

(10) FIG. 6

(11) Calcium Overloading Induces Tumor Necrosis

(12) The fraction of necrosis in tumors before treatment, 2 hours, 1, 2, and 6 days after treatment with calcium electroporation determined by stereological point counting, (median, individual data points (.box-tangle-solidup.), n=4 for treated tumors and n=2 for untreated tumors).

(13) FIG. 7

(14) Light Microscope Images

(15) Representative HE-sections of tumors 2 hours, respectively 6 days after calcium electroporation. EP, electroporation.

(16) FIG. 8

(17) Calcium Overloading Induces ATP Depletion

(18) ATP level in DC-3F cells 1 hour (dark grey/left bars), 4 hours (grey/middle bars) and 8 hours (light grey/right bars) after treatment with calcium electroporation, calcium alone or electroporation alone, control (untreated cells), negative control (dead cells). ATP level is shown in percent of control (means+s.d., n=6). EP, electroporation.

(19) FIG. 9

(20) Schematic View of the Effect of Calcium Overloading

(21) Electroporation (EP) generates reversible pores in the cell membrane (1) allowing influx of calcium and sodium, and efflux of potassium and possible also of ATP. Changes in the intracellular ion concentrations lead to high ATP consumption by Ca.sup.2+-ATPases (in the plasma and endoplasmic reticulum membranes) and Na.sup.+/K.sup.+-ATPases (2). Calcium overload may induce permeability transition pore (PTP) opening in the mitochondrial membrane resulting in loss of ATP production due to loss of the electrochemical gradient (3), and activation of lipases and proteases, and generation of reactive oxygen species (ROS) (4). This results in severe ATP depletion and necrosis of the cell.

(22) FIGS. 10-11

(23) Calcium Overloading Induces Necrosis in Various Tumor Types

(24) Calcium overloading induces tumor necrosis in LPB (murine sarcoma) tumors (10A), B16 (murine melanoma) tumors (10B), MDA-MB231 (human breast cancer) tumors (11A), HT29 (human colon cancer) tumors (11B), and in SW780 (human bladder cancer) tumors (11C).

(25) The fraction of necrosis in tumors before treatment, 2 hours, 1, 2, and 6 days after treatment with calcium electroporation was determined by stereological point counting (median, individual data points (.box-tangle-solidup.), n=3-4 for treated tumors and n=2 for untreated tumors).

(26) FIG. 12

(27) Calcium in Combination with Sonoporation Induces Cell Death

(28) CT26 (murine colon carcinoma cell line) tumors induced on Balb/c mice were treated with isotonic calcium-chloride (168 mM) and sonoporation (CaCl.sup.2+SP), sonoporation alone (SP), isotonic calcium-chloride alone (CaCl.sub.2), or untreated. Tumor size were measured before treatment and every second day after treatment, (means+s.d., n=S). SP, sonoporation.

(29) FIG. 13

(30) Calcium Electroporation Using Different Calcium Sources

(31) Cell viability of DC-3F cells (a transformed Chinese hamster lung fibroblast cell line) after treatment with 1 mM calcium chloride (prepared by SAD, Denmark) or 1 mM calcium glubionate (Sandoz, Holzkirchen, Germany) and electroporation (8 pulses of 991-1s, 1 Hz and increasing field strength). MTT viability assay was performed 1 day after treatment. Results are depicted as percentages of untreated controls (meanss.d., n=6).

(32) FIG. 14

(33) Effect of Electroporation with Calcium and Bleomycin

(34) Cell viability of DC-3F cells (a transformed Chinese hamster lung fibroblast cell line), K-S62 cells (a human leukemia cell line) and Lewis Lung Carcinoma (LLC; a murine lung carcinoma cell line) after treatment with 0.25 mM calcium and/or 0.01 M bleomycin and electroporation with 8 pulses of 99 s at 1.2 kV/cm (DC-3F and K-562) or 1.4 kV/cm (LLC) and 1 Hz. MTT viability assay was performed 2 days after treatment. Results are depicted as percentages of controls electroporated without calcium and bleomycin (means+s.d., n=6).

(35) FIG. 15

(36) Calcium Electroporation of Canine Tumor

(37) Pictures showing a canine tumor on the heel joint of a 7 year old Danish Pointer Dog before treatment and 1 day and 11 days after treatment with 168 mM calcium chloride in a total volume equivalent to 50% of the tumor volume and electroporation.

(38) FIG. 16

(39) Calcium Electroporation of Brain Tumor

(40) N32 (a rat brain glioma derived tumor cell line) tumors induced in rat brains are treated with calcium alone (14 l, 168 mM) or calcium electroporation (14 l 168 mM calcium chloride and 32 pulses of 100V for 100 s and 1 Hz). MRI is performed before treatment and at day 1, 6, and S after the treatment. Light microscope images of H&E stained sections of the tumors after termination of the experiment is also shown.

(41) FIG. 17

(42) Normal Tissue ResponseSkin

(43) MDA-MB231 (a human breast cancer cell line) tumors induced on nude mice were treated with calcium chloride (100-500 mM calcium and injection volume equivalent to 20%-80% of the tumor volume) and electroporated or not, treated with physiological saline and electroporated or not, or untreated but with needle inserted in the tumor without injection and with electrodes put on the tumor without pulses applied. Seven days after the treatment corebiopsies of the skin above the tumor were formalin fixed, paraffin-embedded and the presence of inflammation was estimated in H&E stained sections of the skin. The presence of inflammation in the skin dermis and subcutis was scored from 1-3 (1=minimal, 2=moderate and 3=severe). The results are presented as mean+s.d., n=5-6.

(44) FIG. 18

(45) Normal Tissue ResponseMuscle

(46) MDA-MB231 (a human breast cancer cell line) tumors induced on nude mice were treated with calcium chloride (100-500 mM calcium and injection volume equivalent to 20%-80% of the tumor volume) and electroporated or not, treated with physiological saline and electroporated or not, or untreated but with needle inserted in the tumor without injection and with electrodes put on the tumor without pulses applied. Seven days after the treatment the muscle located below the tumor was formalin fixed, paraffin-embedded and the fraction of necrosis was estimated in H&E stained sections of the muscle. The results were grouped into 3 groups (0=no necrosis, 1=scattered, solitary necrotic myocytes, 2=focal areas of coagulation necrosis). The results are presented as mean+s.d., n=s6.

EXAMPLES

Example 1

(47) In Vitro Electroporation

(48) Materials and Methods

(49) Three cell lines are used for in vitro experiments, DC-3F, a transformed Chinese hamster lung fibroblast cell line; K-562, a human leukemia cell line; and Lewis Lung Carcinoma, a murine lung carcinoma cell line. DC-3F cells were tested for mycoplasma in January 2011, K-562 cells were tested for mycoplasma in 2008 prior to freezing and thawing just before experiments were performed, and Lewis Lung cells were tested by rapid MAP27 panel (Taconic, Hudson N.Y.) in July 2011 without signs of infection. Cells are maintained in RPMI 1640 culture medium (GIBCO, Life Technologies, Carlsbad, Calif.) with 10% fetal calf serum (GIBCO, Life Technologies, Carlsbad, Calif.), penicillin and streptomycin at 37 C. and 5% C02. After harvesting, cells are washed and diluted in HEPES buffer containing 10 mM HEPES (Lonza, Basel, Switzerland), 250 mM sucrose and 1 mM MgCl.sub.2 in sterile water. 270 l cell suspension (6.110.sup.6 cells/ml) and 30 l CaCl.sub.2, or in the case of controls, HEPES buffer, are electroporated in 4 mm wide cuvettes with aluminium electrodes (Molecular BioProducts, Inc., San Diego, Calif.). Cooled cells (8 C.) are exposed to 8 pulses of 1.2 kV/cm with pulse duration of 99 s (DC-3F and K562 cells) or 8 pulses of 1.4 kV/cm with pulse duration of 99 s (Lewis Lung Carcinoma) using a BTX T820 square wave electroporator (BTX, Harvard Apparatus, Holliston, Mass.). The electroporation parameters are chosen after optimization to obtain high permabilisation and cell survival. After 20 min at 37 C. and 5% CO.sub.2 cells are diluted in RPMI 1640 culture medium with 10% fetal calf serum and penicillin-streptomycin and seeded in 96-well plates at a concentration of 3.110.sup.4 cells/100 l. After respectively 1 and 2 days incubation MTT assay is performed using a MULTISKAN ASCENT ELISA reader (Thermo Labsystems, Philadelphia, Pa.).

(50) Difference between electroporated and non-electroporated cells in identical buffer is assessed using two-way analysis of variance (ANOVA) with post least-squares-means test with Bonferroni correction for multiple comparisons.

(51) Results

(52) To test the effect of calcium overloading in vitro three cell lines from different species and of different tissue origin (DC-3F, a transformed Chinese hamster lung fibroblast cell line; K-S62, a human leukemia cell line; Lewis Lung Carcinoma, a murine lung carcinoma cell line) were electroporated in buffers containing increasing calcium concentrations (0 to SmM). Cell viability was determined respectively 1 (FIG. 1) and 2 (FIG. 2) days after treatment. This shows that calcium electroporation, induces dose dependent cell death in all three cell lines. In contrast, incubation at high calcium concentrations has no effect on cell viability in non-electroporated cells. There is a dramatic decrease in viability for all electroporated cell lines with EC.sub.50 being 0.57 mM Ca.sup.2+ (range 0.35-0.79 mM) whereas EC.sub.50 is not reached without electroporation. Viability decreases significantly (p<0.01) starting from 0.5 mM for all cell lines treated. As expected, due to differences in e.g. cell size and homogeneity there is a differential effect of the electroporation procedure alone on the different cell lines, as electroporation in buffer without calcium reduce viability by respectively 0% (DC-3F), 6% (Lewis Lung Carcinoma) and 23% (K-562). Consequently, values are listed as a percentage of electroporated respectively non-electroporated controls. The decrease in viability after calcium electroporation is similar to the effect induced by electroporation with the chemotherapeutic agent bleomycin in concentrations from 0.1 M (data not shown).

Example 2

(53) ATP Assay

(54) Materials and Methods

(55) DC-3F cells are electroporated as described in Example 1 with 1 mM calcium. Cells electroporated with HEPES buffer, non-electroporated cells with 1 mM calcium, and untreated cells are used as controls. Cell death induced by irreversible electroporation (8 pulses of 6.6 kV/cm with pulse duration of 99 s) is used as negative control. Cells are seeded in 96-well plates at a concentration of 3.110.sup.4 cells/100 l. Cells are lysed using Cell-Based Assay Lysis Buffer (Cayman Chemical, Ann Arbor, Mich.) and ATP content is determined after 1, 4 and 8 hours incubation by adding 100 l rL/L Reagent (ENLITEN ATP assay, Promega, Madison, Wis.) and measuring light emission using a luminometer (LUMIstar, BMG biotechnology, Ortenberg, Germany).

(56) Difference in ATP level after different treatments is assessed using two-way ANOVA with post least-squares-means test with Bonferroni correction for multiple comparisons.

(57) Results

(58) Since Calcium electroporation (Ca.sup.2+-EP) treatment leads to highly efficient cell death independently in different cell lines and also leads to uniform necrosis across tumors within 6 days (see Example 4), a condition fundamental for cell survival must be involved. Previous work from this group showed that ATP decreased significantly in tissue exposed to high voltage pulses. Determination of ATP levels in tumor cells after treatment show that Ca.sup.2+-EP treatment results in an immediate and severe drop in ATP level, which stays low, at 10.3% (p<0.0001) of control levels up to 8 hours after treatment (FIG. 8). Cells treated with electroporation alone exhibit a similar drop in ATP level but with a marked recovery 4 hours after treatment to levels significantly higher than Ca.sup.2+-EP treated cells (p<0.0001). Calcium without electroporation does not affect ATP levels. Cells electroporated with calcium-free physiological saline show a similar drop and recovery in ATP level as cells treated with electroporation alone (data not shown).

(59) Here we show that calcium electroporation leads to acute ATP depletion and cell death (in vitro) as well as massive tumor necrosis in vivo (Example 4). As illustrated in FIG. 9, ATP depletion in relation to raised intracellular levels of free calcium may be caused by greatly increased activity of the Ca.sup.2+-ATPase leading to high consumption of ATP. Furthermore, a high intracellular calcium level may induce opening of permeability transition pores (PTP) in the mitochondrial membrane, resulting in loss of the electrochemical gradient, the driving force for ATP production, thereby uncoupling mitochondrial formation of new ATP. Other cellular effects associated to calcium overload include activation of lipases and proteases, and generation of reactive oxygen species (ROS), which may also contribute to cell death. Finally, the electroporation procedure itself may lead to increased ATP consumption as the influx of sodium (either directly or due to sodium calcium exchange) may increase the activity of the Na.sup.+/K.sup.+-ATPase. Furthermore, a direct loss of ATP through the permeabilisation structure is also a possible contributor. Altogether, this may result in cell damage and cell death (FIG. 4). Severe calcium overload causes cell death. Depending on the cellular ATP level, cells undergo either apoptosis or necrosis. If the majority of the mitochondria remain capable of ATP synthesis, the ATP loss may have a transient nature favouring the apoptotic pathway. On the other hand, if the ATP depletion is too severe for apoptosis to occur, the cell will undergo necrosis.

Example 3

(60) Tumor Volume and Tumor Intensity

(61) Materials and Methods

(62) In vivo experiments are performed in accordance with European Convention for the Protection of Vertebrate Animals used for Experimentation and with approval from the Danish Animal Experiments Inspectorate.

(63) H69, a human small cell lung cancer cell line stably transfected with EGFP regulated by the cytomegalo-virus (CMV) promoter, is used for the in vivo experiments. The cells were tested by rapid MAP27 panel (Taconic, Hudson N.Y.) before use without signs of infection. Cells are maintained in vitro as described in Example 1. 1.510.sup.6 cells/100 l PBS are injected subcutaneously in both flanks of NMRI-Foxn1.sup.nu mice (Harlan, Indianapolis, Ind.) that are 9-11 weeks old. Tumor pieces are transplanted from donor mice to the right flank of nude mice. HYPNORM-DORMICUM (fentanyl/fluanisone/midazolam; VetaPharma, Leeds, U K and Roche, Basel, Switzerland) is used for anesthesia complemented with RIMADYL (Carprofen) (Pfizer ApS, Ballerup, Denmark) as well as lidocaine (Region Hovedstadens Apotek, Herlev, Denmark) in the incision. Mice are randomised at an average tumor size of 6.2 mm (range 5.5-6.9 mm) in largest diameter and tumors are treated with 1) injection of isotonic calcium-chloride solution (168 mM CaCl.sub.2) and electroporation (8 pulses of 1.0 kv/cm for 100 s and a frequency of 1 Hz) using a 6 mm plate electrode and a square wave electroporator (CLINIPORATOR, IGEA, Modena, Italy), 2) calcium-free physiological saline injection and electroporation (same parameters as above), 3) injection of isotonic calcium-chloride, or 4) calcium-free physiological saline injection. It is confirmed by atomic absorption spectrophotometry (SOLAAR AAS spectrophotometer, Thermo Fisher, Tewksbury, Mass.) that no calcium is present in the physiological saline used (data not shown). Tumor volume is calculated as ab.sup.2n/6, where a is the largest diameter and b is the largest diameter perpendicular to a. Initially, tumors are injected with a volume equivalent to the tumor volume but as the Ca.sup.2+-EP group shows skin necrosis, the injected volume is changed to half of the tumor volume for all the groups. The solutions are injected through the side of the firm tumor and the needle is moved around inside the tumor to secure injection all over the tumor. Tumor size measurements using a Vernier caliper and bioimaging scanning using the OPTIXOPTIX MX-2 optical molecular image system (ART, Saint-Laurent, Quebec, Saint-Laurent, Quebec) with a scan resolution of 1.5 mm are performed before treatment and three times a week after treatment. Background fluorescence is measured on the opposite flank and then the background fluorescence level is subtracted from fluorescence intensity of tumors. Finally, all fluorescence intensity below 100 normalised counts (NC) is filtered away using ART, Saint-Laurent, Quebec OPTIX Optiview version 2.02 software (ART, Saint-Laurent, Quebec).

(64) The differences in tumor volume and fluorescence intensity between tumors in the 4 treatment groups are evaluated as repeated measurements, validated and analysed with an exponential decrease model with Bonferroni correction for multiple comparisons using SAS software version 9.1 (SAS, Cary, N.C.). Group, days and mouse are considered as factors and baseline levels of tumor volume or fluorescence intensity are used as covariant. The fluorescence intensity values are log transformed before the analysis.

(65) Results

(66) After showing a robust anti-cancer effect in vitro, the effect of calcium electroporation in vivo is tested. Fluorescent H69 tumors, a human small cell lung cancer cell line are treated with an isotonic calcium-chloride injection and electroporated (Ca.sup.2+-EP) or in the case of controls, injected with calcium-free physiological saline and electroporated (NaCl-EP), or injected with isotonic calcium-chloride (Ca.sup.2+) or calcium-free physiological saline (NaCl) without electroporation (FIG. 3). Strikingly, Ca.sup.2+-EP treatment eliminates 89% (8/9) of the treated tumors. Ulceration occurs in all Ca.sup.2+-EP treated tumors, with healing at an average of 18 days (range 9-24 days). Tumor volume is measured including the ulceration, giving the impression that tumor volume is increasing just after treatment, however, fluorescence intensity shows acute decrease in activity after treatment. Volume of tumors treated with Ca.sup.2+-EP is significantly different from control tumors treated without electroporation (p<0.0001) as well as from tumors treated with NaCl-EP (p<0.01). In all non-electroporated tumors volume continues to increase with a doubling time of respectively 3.9 days (NaCl) and 6.3 days (Ca.sup.2+). Tumors treated with NaCl-EP decrease in size in the first days after treatment but started to increase in size again around day 7, except two tumors that were eliminated. This indicates that electroporation alone can modulate tumor growth.

(67) Optical bioimaging is used in vivo to consecutively track the amount of tumor tissue as the H69 tumor cell line is stably transfected with Enhanced Green Fluorescent Protein (EGFP). Fluorescence intensity of Ca.sup.2+-EP treated tumors decrease drastically after treatment and stay at background levels for the remainder of the experiment, being significantly different from tumors treated with NaCl-EP (p<0.01) and from control groups treated without electroporation (p<0.0001). As expected, fluorescence intensity of the non-electroporated tumors rises over time. The fluorescence intensity of tumors treated with NaCl-EP decreases 2-3 days after treatment and is significantly different from tumors treated with NaCl (p<0.05), thereafter the fluorescence intensity increase and is not significantly different from non-electroporated tumors (FIG. 4-5).

Example 4

(68) Histology

(69) Materials and Methods

(70) At an average tumor size of 6.1 mm (range 5.8-6.6 mm) in largest diameter, the tumors are treated with 168 mM CaCl.sub.2 injection and electroporation as described in Example 3. Tumors are removed at skin level before treatment, 2 hours, 1, 2 and 6 days after treatment, fixated in formalin (10% neutrally buffered) and paraffin embedded. Subsequently, tissue sections with a thickness of 3 m are HE-stained according to the routine procedure of the department. The fraction of necrosis within the tumor is estimated from HE-sections by stereological point counting using a light microscope, evaluated by a pathologist, blinded with respect to treatment status.

(71) The difference in fraction of necrosis is assessed using one-way ANOVA with post least-squares-means test with Bonferroni correction for multiple comparisons.

(72) Results

(73) Histological analysis is performed on tissue sections of formalin-fixed, paraffin embedded tumors treated with Ca.sup.2+-EP. Sections are stained with hematoxylin/eosin (HE) and the fraction of necrosis is estimated. The analysis of Ca.sup.2+-EP treated tumors shows progressive necrosis, which is highly significant 2 days after treatment (p<0.0001) and complete 6 days post treatment (FIG. 6-7).

Example 5

(74) Case StudyApplied on Human Patient

(75) In a patient with multiple basocellular carcinomas, where all known treatment options had been tried and the patient was still in need of treatment, calcium electroporation was tried.

(76) In this patient, lesions that were verified by biopsy to contain basocellular carcinoma were treated with different doses of calcium chloride or calcium glubionate with electroporation. Three lesions were treated, measuring 0.8 to 2 cm, by 0.1-0.2 mm deep.

(77) After local anaesthesia (Mir et al), calcium was injected intratumorally using a thin needle and 1 ml syringe. Volumes of resp. 0.1, 0.7 and 3.7 times the calculated tumor volume (in this case calculated as diameter times perpendicular diameter times depth due to the shape of the tumors) was injected.

(78) Immediately after injection (within 45 seconds), electroporation was performed using a square wave pulse generator (CLINIPORATOR, IGEA, Modena, Italy) and needle electrodes with parallel arrays 0.4 cm apart. Eight pulses of a duration of 0.1 ms and 1 kV/cm (voltage to electrode distance ratio) were administered immediately after calcium injection.

(79) Results showed that efficient tumor cell kill was obtained in the case where the ratio of injected volume was 0.7 with respect to tumor volume (asserted by biopsy), that injecting 0.1 fraction of the tumor volume did not lead to changes (biopsy showed basocellular carcinoma as before treatment), and also that the highest volume of calcium led to necrosis of normal skin (clinical observation), indicating that injected volume of calcium is an important parameter for successful calcium electroporation. Healing of the necrotic area ensued, and patient has been seen subsequently with more than one year of follow-up.

Example 6

(80) Hypothetical Example on Volume Test of Injected Solution

(81) Materials and Methods

(82) In vivo experiments are performed in accordance with European Convention for the Protection of Vertebrate Animals used for Experimentation and with approval from the Danish Animal Experiments Inspectorate.

(83) H69, a human small cell lung cancer cell line stably transfected with EGFP regulated by the cytomegalo-virus (CMV) promoter, is used for the in vivo experiments. The cells are tested by rapid MAP27 panel (Taconic, Hudson N.Y.) before use without signs of infection. Cells are maintained in vitro as described in Example 1. 1.510.sup.6 cells/100 l PBS are injected subcutaneously in both flanks of NMRI-Foxn1.sup.nu mice (Harlan, Indianapolis, Ind.) that are 9-11 weeks old. Tumor pieces are transplanted from donor mice to the right flank of nude mice. HYPNORM-DORMICUM (fentanyl/fluanisone/midazolam; VetaPharma, Leeds, U K and Roche, Basel, Switzerland) is used for anesthesia complemented with RIMADYL (Carprofen) (Pfizer ApS, Ballerup, Denmark) RIMADYL (Carprofen) (Pfizer ApS, Ballerup, Denmark) as well as lidocaine (Region Hovedstadens Apotek, Herlev, Denmark) in the incision. Mice are randomised at an average tumor size of 6 mm in largest diameter and tumors are treated with 1) injection of isotonic calcium-chloride solution (16 SmM CaCl2) and electroporation (8 pulses of 1.0 kV/cm for 100 s and a frequency of 1 Hz) using a 6 mm plate electrode and a square wave electroporator (CLINIPORATOR, IGEA, Modena, Italy), 2) calcium-free physiological saline injection and electroporation (same parameters as above), 3) injection of isotonic calcium-chloride, or 4) calcium-free physiological saline injection. Tumor volume is calculated as ab.sup.2n/6, where a is the largest diameter and b is the largest diameter perpendicular to a. Tumors are injected with a volume ratio of 0.2, 0.4, 0.6 or 0.8 compared to the tumor volume. The solutions are injected through the side of the firm tumor and the needle is moved around inside the tumor to secure injection all over the tumor. Tumor size measurements using a Vernier caliper and bioimaging scanning using the OPTIX MX-2 optical molecular image system (ART, Saint-Laurent, Quebec) with a scan resolution of 1.5 mm are performed before treatment and three times a week after treatment. Background fluorescence is measured on the opposite flank and then the background fluorescence level is subtracted from fluorescence intensity of tumors. Finally, all fluorescence intensity below 100 normalised counts (NC) is filtered away using ART, Saint-Laurent, Quebec OPTIX Optiview version 2.02 software (ART, Saint-Laurent, Quebec).

(84) The differences in tumor volume and fluorescence intensity between tumors injected with the different solution volumes in the 4 treatment groups are evaluated as repeated measurements, validated and analysed with an exponential decrease model with Bonferroni correction for multiple comparisons using SAS software version 9.1 (SAS, Cary, N.C.). Group, days and mouse are considered as factors and baseline levels of tumor volume or fluorescence intensity are used as covariant.

(85) Results

(86) Calcium electroporation (Ca.sup.2+-EP) treatment eliminates most of the tumors treated with a volume ratio of 0.2, 004, 0.6 or 0.8 compared to the tumor volume. Ulceration occurs in most of the Ca.sup.2+-EP treated tumors, with faster healing of tumor treated with volume ratios of 0.2 of the tumor volume compared to tumors treated with volume ratios of 0.8 of the tumor volume. Tumor volume is measured including the ulceration, giving the impression that tumor volume is increasing just after treatment, however, fluorescence intensity shows acute decrease in activity after treatment. Volume of tumors treated with Ca.sup.2+-EP is significantly different from control tumors treated without electroporation as well as from tumors treated with NaCl-EP for all 4 injection volumes. Tumor volume of all non-electroporated tumors continues to increase for all injection volumes. Tumors treated with NaCl-EP decrease in size in the first days after treatment but tumors start increasing in size for all 4 injection volumes. This indicates that electroporation alone can modulate tumor growth.

(87) Optical bioimaging is used in vivo to consecutively track the amount of tumor tissue as the H69 tumor cell line is stably transfected with Enhanced Green Fluorescent Protein (EGFP). Fluorescence intensity of Ca.sup.2+-EP treated tumors decrease drastically after treatment and stay at background levels for the remainder of the experiment, being significantly different from tumors treated with NaCl-EP and from control groups treated without electroporation for all 4 injection volumes. Fluorescence intensity of the non-electroporated tumors for all 4 injection volumes rises over time. The fluorescence intensity of tumors treated with NaCl-EP decreases after treatment and thereafter increases for all 4 injection volumes.

Example 7

(88) Hypothetical Example on Concentration Test of Calcium Solution

(89) Materials and Methods

(90) In vivo experiments are performed in accordance with European Convention for the Protection of Vertebrate Animals used for Experimentation and with approval from the Danish Animal Experiments Inspectorate.

(91) H69, a human small cell lung cancer cell line stably transfected with EGFP regulated by the cytomegalo-virus (CMV) promoter, is used for the in vivo experiments. The cells are tested by rapid MAP27 panel (Taconic, Hudson N.Y.) before use without signs of infection. Cells are maintained in vitro as described in Example 1. 1.510.sup.6 cells/100 l PBS are injected subcutaneously in both flanks of NMRI-Foxn1.sup.nu mice (Harlan, Indianapolis, Ind.) that are 9-11 weeks old. Tumor pieces are transplanted from donor mice to the right flank of nude mice. HYPNORM-DORMICUM (fentanyl/fluanisone/midazolam; VetaPharma, Leeds, U K and Roche, Basel, Switzerland) is used for anesthesia complemented with RIMADYL (Carprofen) (Pfizer ApS, Ballerup, Denmark) as well as lidocaine (Region Hovedstadens Apotek, Herlev, Denmark) in the incision. Mice are randomised at an average tumor size of 6 mm in largest diameter and tumors are treated with 1) injection of calcium-chloride solution and electroporation (8 pulses of 1.0 kV/cm for 100 s and a frequency of 1 Hz) using a 6 mm plate electrode and a square wave electroporator (CLINIPORATOR, IGEA, Modena, Italy), 2) calcium-free physiological saline injection and electroporation (same parameters as above), 3) injection of calcium-chloride, or 4) calcium-free physiological saline injection. Four different concentrations of the calcium-chloride solution are used: 100 mM, 220 mM and 500 mM. Tumor volume is calculated as ab.sup.2n/6, where a is the largest diameter and b is the largest diameter perpendicular to a. Tumors are injected with a volume of half the tumor volume. The solutions are injected through the side of the firm tumor and the needle is moved around inside the tumor to secure injection all over the tumor. Tumor size measurements using a Vernier caliper and bioimaging scanning using the OPTIX MX-2 optical molecular image system (ART, Saint-Laurent, Quebec) with a scan resolution of 1.5 mm are performed before treatment and three times a week after treatment. Background fluorescence is measured on the opposite flank and then the background fluorescence level is subtracted from fluorescence intensity of tumors. Finally, all fluorescence intensity below 100 normalised counts (NC) is filtered away using ART, Saint-Laurent, Quebec OPTIX Optiview version 2.02 software (ART, Saint-Laurent, Quebec).

(92) The differences in tumor volume and fluorescence intensity between tumors injected with the different solution volumes in the 4 treatment groups are evaluated as repeated measurements, validated and analysed with an exponential decrease model with Bonferroni correction for multiple comparisons using SAS software version 9.1 (SAS, Cary, N.C.). Group, days and mouse are considered as factors and baseline levels of tumor volume or fluorescence intensity are used as covariant.

(93) Results

(94) Calcium electroporation (Ca.sup.2+-EP) treatment eliminates most of the tumors treated with calcium chloride solutions above 100 mM. Ulceration occurs in tumors treated with the highest calcium chloride concentrations, whereas no ulceration occurs in tumors treated with the low calcium chloride solution. Tumor volume is measured including the ulceration, giving the impression that tumor volume of tumors treated with the highest calcium chloride concentration is increasing just after treatment, however, fluorescence intensity shows acute decrease in activity after treatment. Tumor volume of tumors treated with all the calcium chloride concentrations decrease in size after treatment. Volume of tumors treated with Ca.sup.2+-EP is significantly different from control tumors treated without electroporation as well as from tumors treated with NaCl-EP for all calcium chloride solutions. Tumor volume of all non-electroporated tumors continues to increase for all calcium chloride solutions. Tumors treated with NaCl-EP decrease in size in the first days after treatment and start increasing in. This indicates that electroporation alone can modulate tumor growth.

(95) Optical bioimaging is used in vivo to consecutively track the amount of tumor tissue as the H69 tumor cell line is stably transfected with Enhanced Green Fluorescent Protein (EGFP). Fluorescence intensity of Ca.sup.2+-EP treated tumors decrease drastically after treatment for all calcium chloride solutions and stay at background levels for the remainder of the experiment, being significantly different from tumors treated with NaCl-EP and from control groups treated without electroporation. Fluorescence intensity of the non-electroporated tumors treated with physiological saline or the different calcium chloride solutions rises over time. The fluorescence intensity of tumors treated with NaCl-EP decreases after treatment and thereafter increases.

Example 8

(96) Hypothetical Example on Application of Sonoporation

(97) Materials and Methods

(98) Three cell lines are used for in vitro experiments, DC-3F, a transformed Chinese hamster lung fibroblast cell line; K-562, a human leukemia cell line; and Lewis Lung Carcinoma, a murine lung carcinoma cell line. All cell lines are tested for mycoplasma. Cells are maintained in RPMI 1640 culture medium with 10% fetal calf serum, penicillin and streptomycin at 37 C. and 5% CO2. After harvesting, cells are washed and diluted in HEPES buffer containing 10 mM HEPES, 250 mM sucrose and 1 mM MgCl.sub.2 in sterile. The cell suspension with 0, 0.5, 1 and 3 mM CaCl.sub.2 is added to a sample tube which is placed in the ultrasound exposure chamber and exposed to low-frequency ultrasound at room temperature. A function generator is programmed to provide a wave of selected voltage, duty cycle, burst length, and total exposure time.

(99) After 20 min at 37 C. and 5% CO.sub.2 cells are diluted in RPMI 1640 culture medium with 10% fetal calf serum and penicillin-streptomycin and seeded in 96-well plates at a concentration of 3.110.sup.4 cells/100 l. After respectively 1 and 2 days incubation MTT assay is performed using a MULTISKAN ASCENT ELISA reader (Thermo Labsystems, Philadelphia, Pa.).

(100) Difference between cells treated with and without ultrasound in identical buffer is assessed using two-way analysis of variance CANOVA) with post least-squares-means test with Bonferroni correction for multiple comparisons.

(101) Results

(102) To test the effect of calcium overloading in vitro using ultrasound we use three cell lines from different species and of different tissue origin (DC-3F, a transformed Chinese hamster lung fibroblast cell line; K-562, a human leukemia cell line; Lewis Lung Carcinoma, a murine lung carcinoma cell line) in buffers containing increasing calcium concentrations (0 to 3 mM). Cell viability is determined respectively 1 and 2 days after treatment. This shows that ultrasound of cells in buffer containing calcium, induced dose dependent cell death in all three cell lines. In contrast, incubation at high calcium concentrations has no effect on cell viability in cells treated without ultrasound. There is a dramatic decrease in viability for all cell lines treated with ultrasound. The viability decrease significantly for all cell lines treated with ultrasound. As expected, due to differences in e.g. cell size and homogeneity there is a differential effect of the ultrasound procedure alone on the different cell lines. Consequently, values are listed as a percentage of controls treated with or without ultrasound, respectively. The decrease in viability after treatment with calcium and ultrasound is similar to the effect induced by calcium electroporation.

Example 9

(103) Hypothetical Example on Application of Electromagnetic Field

(104) Materials and Methods

(105) Three cell lines are used for in vitro experiments, DC-3F, a transformed Chinese hamster lung fibroblast cell line; K-562, a human leukemia cell line; and Lewis Lung Carcinoma, a murine lung carcinoma cell line. All cell lines are tested for mycoplasma. Cells are maintained in RPMI 1640 culture medium with 10% fetal calf serum, penicillin and streptomycin at 37 C. and 5% CO2. After harvesting, cells are washed and diluted in HEPES buffer containing 10 mM HEPES, 250 mM sucrose and 1 mM MgCl.sup.2 in sterile water. The cell suspension with 0, 0.5, 1 and 3 mM CaCl.sup.2 is transferred to the electroporation cuvette. A coil system is used to produce sufficiently strong electric fields by electromagnetic induction.

(106) After 20 min at 37 C. and 5% CO.sub.2 cells are diluted in RPMI 1640 culture medium with 10% fetal calf serum and penicillin-streptomycin and seeded in 96-well plates at a concentration of 3.110.sup.4 cells/100 l. After respectively 1 and 2 days incubation MTT assay is performed using a MULTISKAN ASCENT ELISA reader (Thermo Labsystems, Philadelphia, Pa.).

(107) Difference between cells treated with or without electromagnetic field exposure in identical buffer is assessed using two-way analysis of variance (ANOVA) with post least-squares-means test with Bonferroni correction for multiple comparisons.

(108) Results

(109) To test the effect of calcium overloading in vitro using electromagnetic field exposure we use three cell lines from different species and of different tissue origin (DC-3F, a transformed Chinese hamster lung fibroblast cell line; K-562, a human leukemia cell line; Lewis Lung Carcinoma, a murine lung carcinoma cell line) in buffers containing increasing calcium concentrations (0 to 3 mM). Cell viability is determined respectively 1 and 2 days after treatment. This shows that electromagnetic field exposure of cells in buffer containing calcium, induced dose dependent cell death in all three cell lines. In contrast, incubation at high calcium concentrations has no effect on cell viability in cells treated without electromagnetic field exposure. There is a dramatic decrease in viability for all cell lines treated with electromagnetic. The viability decrease significantly for all cell lines treated with electromagnetic field. As expected, due to differences in e.g. cell size and homogeneity there is a differential effect of the electromagnetic field procedure alone on the different cell lines. Consequently, values are listed as a percentage of controls treated with or without electromagnetic field exposure, respectively. The decrease in viability after treatment with calcium and electromagnetic field exposure is similar to the effect induced by calcium electroporation.

Example 10

(110) Hypothetical Example of Calcium Electroporation on Various Tumor Types

(111) Materials and Methods

(112) In vivo experiments are performed in accordance with European Convention for the Protection of Vertebrate Animals used for Experimentation and with approval from the Danish Animal Experiments Inspectorate.

(113) A squamous cell cancer of the head and neck cell line, a breast cancer cell line, a malignant melanoma cell linea colon cancer cell line and/or a sarcoma cell line is used for the in vivo experiments. The cells are tested by rapid MAP27 panel (Taconic, Hudson N.Y.) before use. Cells in 1001-11 PBS are injected subcutaneously in the flank of NMRI-Foxn1.sup.nu mice (Harlan, Indianapolis, Ind.) that are 9-11 weeks old. Mice are randomised at an average tumor size of 6 mm in largest diameter and tumors are treated with 1) injection of isotonic calcium-chloride solution (168 mM CaCl.sup.2) and electroporation (8 pulses of 1.0 kV/cm for 100 s and a frequency of 1 Hz) using a 6 mm plate electrode and a square wave electroporator (CLINIPORATOR, IGEA, Modena, Italy), 2) calcium-free physiological saline injection and electroporation (same parameters as above), 3) injection of isotonic calcium-chloride, or 4) calcium-free physiological saline injection. Tumor volume is calculated as ab.sup.2n/6, where a is the largest diameter and b is the largest diameter perpendicular to a. The injected volume is half of the tumor volume for all the groups. The solutions are injected through the side of the tumor and the needle is moved around inside the tumor to secure injection all over the tumor. Tumor size measurements using a Vernier caliper is performed before treatment and regularly after treatment.

(114) The differences in tumor volume between tumors in the 4 treatment groups are evaluated as repeated measurements, validated and analysed with an exponential decrease model with Bonferroni correction for multiple comparisons using SAS software version 9.1 (SAS, Cary, N.C.). Group, days and mouse are considered as factors and baseline levels of tumor volume are used as covariant.

(115) Results

(116) Ca.sup.2+-EP treatment eliminates 89% (8/9) of the treated tumors independently of tumor origin. Ulceration occurs in most of Ca.sup.2+-EP treated tumors, with healing at an average of 18 days (range 9-24 days). Volume of tumors treated with Ca.sup.2+-EP is significantly different from control tumors treated without electroporation (p<0.0001) as well as from tumors treated with NaCl-EP (p<0.01). Tumor volume of all non-electroporated tumors continues to increase with a doubling time of respectively 3.9 days (NaCl) and 6.3 days (Ca.sup.2+). Tumors treated with NaCl-EP decrease in size in the first days after treatment but tumors start increasing in size. This indicates that electroporation alone can modulate tumor growth.

Example 11

(117) Cell Death in Various Tumors

(118) Materials and Methods

(119) In vivo experiments were performed in accordance with European Convention for the Protection of Vertebrate Animals used for Experimentation and with approval from the Danish Animal Experiments Inspectorate.

(120) LPB, a murine sarcoma cell line, B16, a murine malignant melanoma cell line, MDA-MB231, human a breast cancer cell line, HT29, a human colon cancer cell line, and SW780, a human bladder cancer cell line were used for the in vivo experiment. The cells were tested by rapid MAP27 panel (Taconic, Hudson N.Y.) before use without signs of infection. 210.sup.5 cells (B16), 810.sup.5 cells (LPB), 2.510.sup.6 cells (MDA-MB231), and 510.sup.6 cells (HT29 and SW780) in 100 l PBS were injected subcutaneously in the flank of NMRI-Foxn1.sup.nu mice (Harlan, Indianapolis, Ind.) that were 9-11 weeks old. Mice were randomised at a tumor volume above 85 mm.sup.3 and tumors were treated with injection of isotonic calcium-chloride solution (168 mM CaCI2) and electroporation (8 pulses of 1.0 kv/cm for 100 s and a frequency of 1 Hz) using a 6 mm plate electrode and a square wave electroporator (CLINIPORATOR, IGEA, Modena, Italy). Tumor volume was calculated as ab.sup.2n/6, where a is the largest diameter and b is the largest diameter perpendicular to a. The injected volume was half of the tumor volume. The solution was injected through the side of the tumor and the needle was moved around inside the tumor to secure injection all over the tumor. Tumors were removed at skin level before treatment, 2 hours, 1, 2 and 6 days after treatment, fixated in formalin (10% neutrally buffered) and paraffin embedded. Subsequently, tissue sections with a thickness of 3 m were HE-stained according to the routine procedure of the department. The fraction of necrosis within the tumor was estimated from HE-sections by stereological point counting using a light microscope, evaluated by a pathologist, blinded with respect to treatment status.

(121) The difference in fraction of necrosis at different time after treatment was assessed using student's t-test.

(122) Results

(123) Histological analysis was performed on tissue sections of formalin-fixed, paraffin-embedded tumors treated with calcium electroporation. Sections were stained with hematoxylin/eosin (HE) and the fraction of necrosis was estimated. The analysis of calcium electroporation treated tumors shows progressive necrosis in all 5 different tumor types (FIGS. 10-11) the first days after the treatment which is significant in four of the tumor types (p<0.05 for MDA-MB231 at day 2, p<0.05 for HT29 and LPB at day 1, p<0.01 for LPB at day 2, p<0.05 for B16 at day 5-7). In four of the tumor types (LPB, MDA-MB231, HT29, and SW780) re-growth is seen 6 days after the treatment resulting in reduced fraction of necrosis. This could be due to untreated areas of the tumors or too low concentration and/or injection volume of calcium-chloride.

(124) Conclusion

(125) Calcium electroporation induce tumor necrosis in all five treated tumor types which is significant in four of the treated tumor types. Re-growth is seen in four of the tumor types which likely could be avoided by increasing the calcium concentration and/or the injected calcium volume or by re-treating the tumors.

Example 12

(126) Application of Sonoporation

(127) Materials and Methods

(128) All in vivo experiments were approved by the ethics committee of University College Cork and carried out under license (Dr. Patrick Forde B100/4038) issued by the Department of Health, Ireland as directed by the Cruelty to Animals Act Ireland and EU Statutory Instructions.

(129) CT26, a murine colon carcinoma cell line was used for the in vivo experiments. The cells were tested by mycoplasma detection kit (Sigma, St. Louis, Mo.) before use without signs of infection. Cells were maintained in vitro in DMEM culture medium (Sigma, St. Louis, Mo.) with 10% fetal calf serum (Sigma, St. Louis, Mo.), L-glutamine, penicillin and streptomycin at 37 C. and 5% CO.sub.2. All antibiotics were removed from culture media 24 hours before tumour inoculation. 1.010.sup.6 cells/200 l serum free DMEM were injected subcutaneously in the right flank of 6-8 week old female Balb/c mice, in condition weighing 16-22 g. Mice were randomised at an average tumor size of approximately 5.0 mm in largest diameter and tumors were treated with 1) injection of isotonic calcium-chloride solution (168 mM CaCl.sub.2) and sonoporation (3.5 W/cm.sup.2 at 1 MHz and 100% duty cycle for 2 minutes) using a SONITRON 2000 ultrasound apparatus (Rich-Mar, Inola, Okla.), 2) sonoporation alone (same parameters as above), 3) isotonic calcium-chloride solution injection, or 4) no treatment. Tumor volume was calculated as ab.sup.2n/6, where a is the largest diameter and b is the largest diameter perpendicular to a. Tumors were injected with a volume of half the tumor volume and the solutions were injected through the side of the firm tumor and the needle was moved around inside the tumor to secure injection all over the tumor. Tumor size measurements using a Vernier caliper were performed before treatment and every second day after treatment.

(130) The difference in tumor volume between the different treatment groups at different time after treatment was assessed using student's t-test.

(131) Results

(132) After showing a robust anti-cancer effect using electroporation in the presence of calcium, the effect of calcium in combination with sonoporation in vivo was tested. Murine colon carcinoma (CT26) tumors were treated with an isotonic calcium-chloride injection and sonoporated CaCl.sub.2-SP) or in the case of controls, treated with sonoporation alone SP), or isotonic calcium-chloride injection (CaCl.sub.2), or untreated tumors (FIG. 12). Strikingly, CaCl.sub.2-SP treatment eliminates 88% (7/8) of the treated tumors. Volume of tumors treated with CaCl.sub.2-SP is significantly different from all three control groups (p<0.0001 at day 14 after treatment). Tumor volume of all control tumors continues to increase after the treatment.

(133) Conclusion

(134) Sonoporation induced loading of cancer cells with calcium is also a very efficient anti-cancer treatment.

Example 13

(135) Calcium Electroporation with Two Different Calcium Sources

(136) Materials and Methods

(137) DC-3F cells were electroporated as described in example 1 with 1 mM calcium chloride (prepared by SAD, Denmark) or 1 mM calcium glubionate (Sandoz, Holzkirchen, Germany). The calcium concentration was chosen based on the experiment described in example 1 where 1 mM calcium is the lowest concentration inducing high cell death. The cells were electroporated with 8 pulses of 991-1s at 1 Hz and increasing electric field (0-1.6 kV/cm). After 20 min at 37 C. and 5% CO.sub.2 cells were diluted in RPMI 1640 culture medium with 10% fetal calf serum and penicillin-streptomycin and seeded in 96-well plates at a concentration of 3.110.sup.4 cells/100 l. The viability was measured 1 day after incubation by MTT assay using a MULTISKAN ASCENT ELISA reader (Thermo Labsystems, Philadelphia, Pa.).

(138) The calcium concentration used in vitro is much lower than the calcium concentration used in vivo since the cells treated with calcium electroporation in vitro are in suspension and completely surrounded by the calcium solution whereas cells in a tumor treated with calcium electroporation are close together and the calcium solution is diluted when injected into the tumor.

(139) Results

(140) We have tested if there was any difference in the effect of calcium electroporation using calcium chloride or calcium glubionate. As seen in FIG. 13 there is no difference in the effect of calcium electroporation using the same concentration of calcium chloride or calcium glubionate.

(141) Conclusion

(142) There is no difference in the effect of calcium electroporation using different calcium sources at the same concentration.

Example 14

(143) Effect of Electroporation with Calcium and/or Bleomycin

(144) Materials and Methods

(145) DC-3F, K-562 and Lewis Lung carcinoma cell lines were used for the in vitro experiment. The cell lines were maintained and harvested as described in example 1. Cells were electroporated (8 pulses of 991-1s, 1.2-1.4 kV/cm and 1 Hz) in HEPES buffer (as described in example 1) containing 0.01 m bleomycin and/or 0.25 mM calcium chloride. The low concentration of bleomycin and calcium was chosen to be able to see an additive effect of the drugs. For in in vivo assays the concentration of calcium may be raised according to the present invention. After 2 days incubation MTT assay was performed using a MULTISKAN ASCENT ELISA reader (Thermo Labsystems, Philadelphia, Pa.).

(146) Results

(147) The effect of treatment with bleomycin, calcium, and electroporation was studied (FIG. 14) to test the effect of calcium and bleomycin together. The results indicate that there is an additive effect of calcium and bleomycin in all three tested cell lines.

(148) Conclusions

(149) Calcium and bleomycin in combination could be used clinically and there seems to be an additive effect of the two drugs. Such an additive effect may also be seen with calcium and another drug.

Example 15

(150) Case StudyCalcium Electroporation of Canine Tumor

(151) Materials and Methods

(152) A seven year old Danish Pointer dog with a tumor on the heel joint was treated with calcium electroporation. The tumor was injected with 168 mM calcium chloride solution in a total volume equivalent to 50% of the tumor volume calculated as ab.sup.2n/6, where a is the largest diameter of the tumor and b is the largest diameter perpendicular to a. The calcium chloride solution was injected throughout the tumor to secure an even distribution of calcium in the tumor. After the calcium injection the tumor was electroporated (8 pulses of i oous at 1.0 kV/cm and 1 Hz) using a needle electrode and a square wave electroporator (CLINIPORATOR, IGEA, Modena, Italy). The tumor was photographed before and after the treatment.

(153) Results

(154) The photos of the tumor on the heel joint (FIG. 15) show the tumor before treatment and 1 day and 11 days after the treatment. The photos show that the tumor is reduced in size 11 days after treatment with calcium electroporation and the tumor is seen with clinical signs of necrosis.

(155) Conclusion

(156) Calcium electroporation can induce necrosis in a canine tumor.

Example 16

(157) Calcium Electroporation of Brain Tumor

(158) Materials and Methods

(159) In vivo experiments were performed in accordance with European Convention for the Protection of Vertebrate Animals used for Experimentation and with approval from the Danish Animal Experiments Inspectorate.

(160) N32, a rat brain glioma derived tumor cell line, and 7-11 weeks old Sprague Dawley male rats (Taconic, Hudson N.Y.) were used for the in vivo experiments. The cells were tested by rapid MAP27 panel (Taconic, Hudson N.Y.) before use without signs of infection. Inoculation of 3,000 N32 cells in 5 l at the stereotaxic coordinates; X=2, Y=1, Z=4 was performed and verification of tumor by MRI prior to treatment was performed two weeks after inoculation. HYPNORM-DORMICUM (fentanyl/fluanisone/midazolam; VetaPharma, Leeds, U K and Roche, Basel, Switzerland) was used for anesthesia complemented with RIMADYL (Carprofen) (Pfizer ApS, Ballerup, Denmark) for pain relief after surgery. Tumors were treated two weeks after inoculation with 1) injection of 14 l calcium-chloride solution (168 mM) and electroporation (32 pulses of 100V for 100 s and a frequency of 1 Hz) using an 8-electrode device developed for treatment of brain tumors, a square wave electroporator (CLINIPORATOR, IGEA, Modena, Italy), and a switch box (Sonion), or 2) injection of 14 l calcium-chloride solution (168 mM) and placement of electrodes but no pulses applied. MRI was performed at day 1, 3, 6, and 8 after treatment. After termination of the experiment the rat brains were prepared for immuno-histo-pathological staining by perfusing the rats with isotonic NaCl followed by 4% paraformaldehyde and kept at 5 C. for at least 24 hours. The extend of necrosis, the presence of macrophages and the influence on neurons and glia cells were evaluated by H&E, PAS, NF, and GFAP staining, respectively.

(161) Results

(162) In FIG. 16 is shown MRI images of the tumor treated with calcium electroporation before treatment, 1 day and 8 days after treatment and the control tumor treated with calcium alone before treatment, 1 day and 6 days after treatment. Light microscope images of the H&E stained sections of the brain after termination of the experiment is also shown in the figure. The MRI images of the control tumor treated with calcium alone indicate that calcium alone has no effect on tumor size since the size of the tumor seems to increase 6 days after the treatment. The tumor is also clearly visible on the light microscope image of the H&E stained section of the tumor. The MRI images of the tumor treated with calcium electroporation indicate that the tumor is eliminated 8 days after the treatment which is also seen on the light microscope image of the H&E stained section of the brain where no tumor is seen.

(163) Conclusion

(164) Calcium electroporation is effective in a brain tumor.

Example 17

(165) Normal Tissue Reaction

(166) Materials and Methods

(167) In vivo experiments were performed in accordance with European Convention for the Protection of Vertebrate Animals used for Experimentation and with approval from the Danish Animal Experiments Inspectorate.

(168) MDA-MB231, a human breast cancer cell line was used for the in vivo experiments. The cells were tested by rapid MAP27 panel (Taconic, Hudson N.Y.) before use without signs of infection. Cells were maintained in vitro as described in Example 1. 2.510.sup.6 cells/100 l PBS were injected subcutaneously in the flank of NMRI-Foxn1nu mice (Harlan, Indianapolis, Ind.). Tumor pieces were transplanted twice from donor mice to the flank of nude mice that were 13-19 weeks old. HYPNORM-DORMICUM (fentanyl/fluanisone/midazolam; VetaPharma, Leeds, U K and Roche, Basel, Switzerland) was used for anesthesia complemented with RIMADYL (Carprofen) (Pfizer ApS, Ballerup, Denmark) as well as lidocaine (Region Hovedstadens Apotek, Herlev, Denmark) in the incision. Mice were randomised at a tumor volume above 85 mm.sup.3 and tumors were treated with 1) injection of calcium-chloride solution and electroporation (8 pulses of 1.0 kV/cm for 100 s and a frequency of 1 Hz) using a 6 mm plate electrode and a square wave electroporator (CLINIPORATOR, IGEA, Modena, Italy), 2) calcium-free physiological saline injection and electroporation (same parameters as above), 3) injection of calcium-chloride, 4) calcium-free physiological saline injection, or 5) untreated but with needle inserted in the tumor without injection and with electrodes put on the tumor without pulses applied. Three different concentrations of the calcium[ ]chloride solution were used: 100 mM, 220 mM and 500 mM and four different injection volumes of calcium chloride were used: 20%, 40%, 60% and 80% of the tumor volume. Tumor volume was calculated as ab2n/6, where a is the largest diameter and b is the largest diameter perpendicular to a. The solutions were injected through the side of the firm tumor and the needle was moved around inside the tumor to secure injection all over the tumor. The skin above the tumor and the muscle beneath the tumor were removed 7 days after treatment, fixated in formalin (10% neutrally buffered) and paraffin embedded. Subsequently, tissue sections with a thickness of 31-1m were HE-stained according to the routine procedure of the department. Estimation of normal tissue damages was evaluated semiquantitatively on HE-sections by light[ ]microscopy (LEICA DM 2000 light microscope, Leica Microsystems, Wetzlar, Germany).

(169) The presence of inflammation in the skin dermis and subcutis was scored from 1-3 (1=minimal, 2=moderate and 3=severe). Edema in the dermis, extravasations of erythrocytes and vasculitis was noted if present, but not scored.

(170) The presence of necrosis in the muscle samples were scored by counting numbers of necrotic myocytes per 10 HPF (400 magnification). Necrosis was only recorded if the myocytes had lost the nucleus. Increased eosinofilic intensity in the myocyte cytoplasm with a retained nucleus was not recorded as a necrotic fiber. Counting was performed in the area with the most obvious and worst morphological changes.

(171) The results were grouped into 3 groups as follows:

(172) no necrosis=0 necrotic myocytes/10HPF (0)

(173) scattered, solitary necrotic myocytes or 1-4 necrotic myocytes/10HPF (1)

(174) focal areas of coagulation necrosis or 5 necrotic myocytes/10HPF (2)

(175) Interstitial inflammation, internalization of myocyte nuclei, extravasation of erythrocytes and vasculitis was noted if present, but not scored.

(176) The skin biopsies and the muscle samples were not orientated, and hence location in relation to the electrodes not known.

(177) Results

(178) Inflammation in the skin located above the tumor after treatment was estimated (FIG. 17). The inflammation seen in the skin dermis and subcutis is dominated by mast cells and only few mononuclear cells and granulocytes are seen. In general, there is substantial degranulation of the mast cells, but in varying degree across samples. The epidermis is in general reactive with enlargement of the nuclei of the keratinocytes.

(179) In subcutis, fat necrosis and reactive stromal changes could occasionally be observed, often present alongside severe inflammation in the dermis and subcutis and/or necrosis of muscle in the deep part of the biopsies.

(180) As seen in FIG. 17 the presence of inflammation is similar in all the samples not depending on the treatment (untreated, physiological saline injection with or without electroporation, calcium injection with or without electroporation) nor depending on the concentration or injection volume of calcium showing that calcium electroporation is only affecting the surrounding normal skin tissue very little.

(181) In some of the mice treated with the high concentration and volume of calcium ulceration above the tumor was seen but in previously experiments it has been shown that healing of the ulceration occurred in average 18 days (range, 9-24 days) after the treatment. Healing of the ulceration in this experiment was not seen since tumors were removed 7 days after the treatment.

(182) The effect on the muscle tissue below the tumor was also examined. In general, all muscle tissue samples contains an increased number of mast cells lying in between the myocytes, and therefore none of the samples are considered completely normal. Mast cells are present in variable amounts, and with variable degrees of degranulation. In the majority of samples, the myocyte nuclei are enlarged and the chromatin structure reactively changed. A large number of samples show regeneration of myocytes with centrally located nuclei (internalization of nuclei). Focal areas of coagulation necrosis are only present in a minority of samples, but the majority samples show regenerative/reactive changes. In general, cross striation is retained. Extravasation of erythrocytes is only observed in a couple of samples.

(183) FIG. 18 shows the fraction of necrosis in the muscle tissue located below the treated tumor. The fraction of necrosis is grouped into three groups where 0 indicates no necrosis, 1 indicates scattered, solitary necrotic myocytes in the muscle tissue (maximum of 5% necrosis), and 2 indicates minor focal areas of coagulation necrosis (most of them below 10% necrosis and a few with 15-35% necrosis). As seen in the figure there is an increase in the fraction of necrosis at increasing calcium concentrations (FIG. 18) and also a slight increase in the fraction of necrosis at increasing injection volumes (FIG. 18). Even though the fraction of necrosis increases it is still only a minor fraction of the muscle tissue that is affected after calcium electroporation of the tumor above the muscle.

(184) Conclusion

(185) The anti-tumoral effects of calcium electroporation are limited to the relevant tumor cells, while sparing adjacent normal tissue. Skin tissue above the tumor is mildly affected following treatment, where inflammation is observed Inflammation may be attributed to needle insertion, as this was independent of treatment type. Muscle tissue below the treated tumor exhibits a small degree of necrosis, which increases slightly at higher calcium concentrations and injections volumes, yet remains low.

Example 18

(186) Effect of Calcium Electroporation Using Different Calcium Concentrations and Injection Volumes

(187) Materials and Methods

(188) In vivo experiments were performed in accordance with European Convention for the Protection of Vertebrate Animals used for Experimentation and with approval from the Danish Animal Experiments Inspectorate.

(189) MDA-MB231, a human breast cancer cell line was used for the in vivo experiments. The cells were tested by rapid MAP27 panel (Taconic, Hudson N.Y.) before use without signs of infection. Cells were maintained in vitro as described in Example 1. 2.510.sup.6 cells/100 l PBS were injected subcutaneously in the flank of NMRI-Foxn1nu mice (Harlan, Indianapolis, Ind.). Tumor pieces are transplanted twice from donor mice to the flank of nude mice that were 13-19 weeks old. HYPNORM-DORMICUM (fentanyl/fluanisone/midazolam; VetaPharma, Leeds, U K and Roche, Basel, Switzerland) was used for anesthesia complemented with RIMADYL (Carprofen) (Pfizer ApS, Ballerup, Denmark) as well as lidocaine (Region Hovedstadens Apotek, Herlev, Denmark) in the incision. Mice were randomised at a tumor volume above 85 mm3 and tumors were treated with 1) injection of calcium-chloride solution and electroporation (8 pulses of 1.0 kV/cm for 100 s and a frequency of 1 Hz) using a 6 mm plate electrode and a square wave electroporator (CLINIPORATOR, IGEA, Modena, Italy), 2) calcium-free physiological saline injection and electroporation (same parameters as above), 3) injection of calcium-chloride, 4) calcium-free physiological saline injection, or 5) untreated but with needle inserted in the tumor without injection and with electrodes put on the tumor without pulses applied. Three different concentrations of the calcium[ ]chloride solution were used: 100 mM, 220 mM, and 500 mM and four different injection volumes were used: 20%, 40%, 60%, and 80% of the tumor volume. Tumor volume was calculated as ab2n/6, where a is the largest diameter and b is the largest diameter perpendicular to a. The solutions were injected through the side of the firm tumor and the needle was moved around inside the tumor to secure injection all over the tumor. Tumors were removed 7 days after treatment, fixated in formalin (10% neutrally buffered) and paraffin embedded. Subsequently, tissue sections with a thickness of 3 m were HE-stained according to the routine procedure of the department. The fraction of necrosis within the tumor was estimated from HE-sections using a light microscope, evaluated by a pathologist, blinded with respect to treatment status.

(190) Results

(191) The fraction of necrosis in tumors was estimated 7 days after treatment with calcium electroporation using different calcium concentrations (100 mM to 500 mM) and constant injection volume (50% of the tumor volume). This shows that the fraction of necrosis in these tumors is higher than the fraction of necrosis in untreated tumors.

(192) Also in tumors treated with calcium electroporation using different injection volumes of calcium (20% to 80% of the tumor volume) and a constant calcium concentration (168 mM) the fraction of necrosis is higher than the fraction of necrosis in untreated tumors. The fraction of necrosis is highest in tumors treated with a volume of 40% of the tumor volume.

(193) The effect of calcium electroporation using an injection volume of 80% of the tumor volume and a calcium concentration of 500 mM was estimated and in all five treated tumors there is between 80% and 100% necrosis.

(194) Conclusions

(195) The results of this experiment show that calcium electroporation using calcium concentrations between 100 mM and 500 mM and injection volumes between 20% and 80% of the tumor volume induces necrosis in the tumors. It is also seen that both calcium concentration and injection volume is affecting the fraction of necrosis induced by the treatment meaning that the calcium concentration and the injection volume have to be considered when planning the treatment. These results support the previous results showing that injecting volumes equal to the tumor volume leads to necrosis of surrounding healthy, whereas by lowering the volume this is avoided while still maintaining efficient treatment.

REFERENCES

(196) Mashiba et al., [2005] Augmentation of antitumor and antimetastatic effect in combined use of electroporation with calcium chloride. Experimental and Molecular Therapeutics 17: Apoptosis: Therapeutic Applications, Abstract #2251 Mir, L. M. et al. Standard operating procedures of the electrochemotherapy: Instructions for the use of bleomycin or cisplatin administered either systemically or locally and electric pulses delivered by the Cliniporator by means of invasive or non-invasive electrodes. Eur J Cancer Suppl 4, 14-25 (2006).