WO 2002098501 A2
METHOD AND APPARATUS FOR TREATING TUMORS USING LOW
STRENGTH ELECTRIC FIELDS
FIELD AND BACKGROUND OF THE INVENTION The present invention relates to methods of using low-strength electric fields to treat tumors. More particularly, the present invention relates to methods and an apparatus which utilize low strength pulsed electric fields on the order of 20-70 V/cm, with or without adjunct chemotherapy to treat or cure various tumor and cancerous tissue. Cancer is second only to heart disease as a cause of death, accounting for
22 % of all deaths (Fraumeni JF, Devesa SS, Hoover RN, Kinlien LJ. Epidemiology of cancer. In: Cancer - principles and practice of oncology, DeVita VT, Hellman S, Rozenberg SA, (eds.) pp. 150, Lippincott J.R. Co., Philadelphia, 1993.). Colon cancer, melanoma and breast cancer are three particularly problematic types of cancer.
Melanomas are aggressive, frequently metastatic tumors derived from either melanocytes or melanocyte related nevus cells ("Cellular and Molecular Immunology" (1991) (eds) Abbas A. K., Lechtman. A. H., Pober, J. S.; W.B. Saunders Company, Philadelphia: pages 340-341) which make up approximately 3 % of all skin cancers. Of particular concern is the current worldwide increase in melanoma which is unsurpassed by any other neoplasm with the exception of lung cancer in women ("Cellular and Molecular Immunology" (1991) (eds) Abbas, A. K., Lechtiman, A. H., Pober, J. S.; W.B. Saunders Company Philadelphia pages: 340-342; Kirkwood and Agarwala (1993) Principles and Practice of Oncology 7: 1-16). The aggressiveness of melanoma is such that even when melanoma is apparently localized to the skin, up to 30 % of the patients will develop systemic metastasis and the majority will die.
Breast cancer is a significant health problem for women in the United States and throughout the world. Although advances have been made in detection and treatment of the disease, breast cancer remains the second leading cause of cancer-related deaths in women, affecting more than 180,000 women in the United States each year. For women in North America, the life-time odds of getting breast cancer are now one in eight.
Colon cancer is the second most frequently diagnosed malignancy in the United States as well as the second most common cause of cancer death. About 100,000 new cases of colon cancer are diagnosed yearly, with about 50,000 deaths. The five-year survival rate for patients with colorectal cancer detected in an early localized stage is 92%; unfortunately, only 37 % of colorectal cancer is diagnosed at this stage. The survival rate drops to 64 % if the cancer is allowed to spread to adjacent organs or lymph nodes, and to 7 % in patients with distant metastases. Recurrence following surgery (the most common form of therapy) is a major problem and is often the ultimate cause of death. In spite of considerable research into therapies for the disease, colon cancer remains difficult to diagnose and treat.
The leading cause of cancer death in general is due to growth of metastases since, in the majority of cases, by the time a malignancy has been diagnosed, metastases have already spread to other sites (for review see Fidler and Balch, 1987. Curr. Prob. Surg. 24: 137). Whereas metastatic primary tumors can in many cases be surgically removed, greatly contributing to satisfactory therapeutic outcomes, metastases, such as disseminated micrometastases, can be difficult or impossible to locate and/or reach and thus surgical removal such metastases is usually not an option. Thus, metastases pose the most serious challenge to cancer therapy and are the main cause of failure of treatment of this disease. Therefore, prevention of metastasis is necessary to improve the prognosis of cancer patients. In order to treat cancer effectively efficient removal of the primary tumor mass and prevention of secondary tumor growth, and eradication of metastatic cells must be achieved. Furthermore, significant prevention of recurrence of cancer growth can be achieved by generation of anti-cancer immune responses.
Surgical excision of tumors is the most widely employed therapeutic modality for the treatment of cancer, in which the primary goal is the complete eradication of local and regional tumor. This involves removal of adequate margins of normal tissue surrounding the tumor, and radical wide excision in order to prevent local recurrence. However, despite major advances in the surgical pre- and postoperative care of patients, surgical treatment of malignant neoplasms remains highly limited (Eilber FR. Principles of cancer surgery. In: Cancer Treatment, Haskell CM, (ed.) 5th ed., pp. 47, W.B. Saunders Co. Philadelphia, 2001). Surgical techniques are effective only in the area of the primary tumor or regional lymphatics and do not affect neoplasms located outside the operative field. Furthermore, due to anatomic location, many tumors cannot be treated by surgical resection because removal of an adequate margin of normal tissue cannot be achieved. Also, surgical treatment is often not an option for tumors intimately involving major blood vessels or essential organs. As well, many patient present problematic medical histories, such as cerebrovascular or cardiovascular accidents, or uncontrolled diabetes, rendering them poor surgical candidates because of their high postoperative mortality rate.
Also, in many cases, tumor excision can not performed without causing unacceptable levels of impairment of physiologic functions or cosmetic damage.
Chemotherapy alone or in combination with surgery is commonly the most efficient anti-cancer remedy (Haskell CM. Principles of cancer chemotherapy. In: Cancer Treatment, Haskell CM, (ed.) 5th ed. pp. 62-86, W.B. Saunders Co. Philadelphia, 2001). However, chemotherapeutic agents often cause severe and unacceptable side-effects, such as bone marrow and lymphoid organ damage resulting in immunosuppression, thereby rendering subjects highly vulnerable to lethal opportunistic infections, as well as various other types of organ toxicities. Thus, the use of cytotoxic drugs is limited only to tolerated doses. One way to reduce minimal therapeutic doses of chemotherapeutic agents would be to enhance the efficiency of uptake of chemotherapeutic drugs into cancer cells.
During the last two decades, various techniques based on biological, chemical and physical processes have been developed for facilitating incorporation of macromolecules into cells. Methods for intracellular delivery of exogenous substances based on biological phenomena have employed molecules controlling the activity of specific membrane channels in various cell types (Heppel and Weisman, 1985. J Membr Biol. 86:189), pore-forming toxins (Ahnert-Higler et al., 1989. Methods Cell Biology 31 :63) and liposome- endocytosis mediated delivery of compounds (Friend et al., 1996. Biophys Acta 1278:41). Some permeabilization methods are based on chemical modification of cell membranes by various substances, most commonly via the use of detergents as permeabilizing agents. Other methods based on chemically induced permeabilization include protease digestion or stimulation of DNA binding to the cell surface by formation of neutral complexes of DNA with various molecules. Physical methods of introducing molecules into cells include application of hypotonic stress (Poulin et al., 1993. J Biol Chem. 268:4690), cell bombardment by coated molecules (Salford et al., 1993a), microinjection (Soreg and Seidman, 1992. Methods Enzymol. 207:225), electroporation (Potter, 1993. Methods Enzymol. 217:461), and exposure to pulsed low electric fields (LEFs) (Rosenberg and Korenstein, 1997. Bioelectrochemistry and Bioenergetics 42:275).
Electroporation involves formation of a reversible, high permeability plasma membrane state in cells or bacteria exposed to 50-200 μs pulses of high- strength electric fields in the range of 500-5000 V/cm. At a threshold value of about 1 V across the cell membrane, a sudden increase in membrane permeability is observed which is thought to be mediated by stabilization of transient membrane defects and to their expansion to large metastable hydrophilic pores. Both transient and stable pores can be the sites of extrinsic material entry into the cell (Hapala, 1997. Crit Rev Biotechnol. 17: 105; Rols and Teissie, 1990. Biophys J. 58: 1089; Rols and Teissie, 1998. Biophys J. 75: 1415). Electroporation also appears to involve stimulation of biological endocytosis in areas of destabilized membrane structure (Rols et al., 1995. Biochem Biophys Acta 1 1 1 1 :45). While electroporation, also termed electropermeabilization or electroinjection, has been generally used as a method of transfecting cells with nucleic acids, this method has also been used to load cells with a variety of other molecules, including proteins (Lambert et al., 1990. Biochem Cell Biol. 68:729), such as phalloidin (Hashimoto et al., 1989. J Biochem Biophys Methods 19: 143) or antibodies (Chakrabarti et al., 1989. J Biol Chem. 264: 15494).
In contrast to electroporation, incorporation of macromolecules into cells via exposure to LEFs, a methodology developed by the present inventors, utilizes low voltage electric fields. Exposure of cells and vesicles to LEFs leads to efficient intracellular incorporation of various molecules, including carbohydrates, such as polysaccharides, and proteins, such as β-galactosidase (Rosenberg and Korenstein, 1997. Bioelectrochemistry and Bioenergetics 42:275) via an underlying mechanism involving endocytosis-like processes. Exposure of membrane vesicles and cells to such LEFs leads to electrophoretic lateral mobility of charged proteins and lipids in the plane of the cell membrane (Poo, 1981. Bioeng. 10:245; Brumfield et al., 1989 Biophys J. 56:607), and generation of transmembrane potential differences (Farkas et al., 1984. Biophys J. 45:363). It has been shown by the present inventors that exposure of cells in suspension or monolayer to trains of pulsed unipolar electric fields in the range of about 1-100 V/cm (Rosenberg and Korenstein, 1997. Bioelectrochemistry and Bioenergetics 42:275), or to AC fields with peak-to-peak amplitudes of about 1- 60 V/cm leads to efficient uptake of macromolecules having molecular weights ranging from about 1 to 2000 kDa, an exceptionally broad range. Unlike following electroporation, cells exposed to LEFs in vitro maintain high viability due to the magnitudes of the applied electric fields being too low to induce changes in membrane permeability via physical disruption of its integrity (Rosenberg and Korenstein, 1990. Biophys J. 58:823). LEFs have been shown to induce endocytosis, a process which includes a complex sequence of membrane-linked processes resulting in uptake of extrinsic substances involving binding of such substances to the cell surface, formation of endocytotic vesicles and maturation of endocytotic vesicles to lysosomes (Mellman, 1996. Ann Rev Cell Dev Biol. 12:575).
Several prior art approaches employing electric fields have been employed to treat tumors. One approach has employed electroporation in conjunction with netropsin, bleomycin, or melphalan to attempt to increase of the cytotoxicity of these drugs against cultured DC-3F cells (Orlowski S. et al., 1988. Biochem Pharmacol. 37:4727).
Yet an additional approach has utilized electroporation in conjunction with daunorubicin, doxorubicin, etoposide, paclitaxel, carboplatin or cisplatin in order to attempt to potentiate their cytotoxic effect against cultured cells (Gehl J. et al., 1998. Anticancer Drugs 9:319).
Still another approach has employed very high strength electric field electroporation (1 ,300 V/cm) in conjunction with administration of cis- diamminedichloroplatinum(II) in order to attempt to treat SA-1, EAT, or B16 melanoma tumors in mice (Sersa G. et al., 1995. Cancer Res. 55:3450)
An additional approach has used electroporation in conjunction with administration of bleomycin in order to attempt to treat tumors of the female genital squamous cell carcinoma cell line CaSki in nude mice (Yabushita H. et al., 1997. Gynecol Oncol. 65:297).
Another approach has used very high strength electric field electroporation (1,300 V/cm) in conjunction with administration of bleomycin to attempt to treat head and neck squamous cell carcinoma (Belehradek JJ. et al., 1993. Cancer 72:3694). Yet another approach has utilized very high strength electric field electroporation (1,000-1,300 V/cm) in conjunction with administration of bleomycin to attempt to treat head and neck squamous cell carcinoma, and salivary and breast adenocarcinomas (Domenge C. et al., 1996. Cancer 77:956) However, all of the aforementioned prior art approaches suffer from significant disadvantages. For example, all of these prior art approaches have employed electroporation, and hence high-strength electrical fields which, as described hereinabove, are significantly cytotoxic and which, by their extreme nature, are inherently hazardous. Electroporation suffers from the drawbacks of being inefficient in its potentiation of drug uptake, and in being restricted with respect to the range of molecular weights of the molecules whose uptake it is capable of potentiating. Prior art approaches have demonstrated potentiation of the in vivo anti-tumor effect of a restricted number of chemotherapeutic drugs (bleomycin, cisplatin, adriamycin and 5-fluorouracil). Moreover, none of these prior art methods has been shown to be effective in curing cancer at a metastatic stage. Critically, none of these prior art approaches has been shown to be effective against tumor cells in the absence of chemotherapeutic agents. Also none of these prior art approaches has demonstrated the capacity to upregulate anti- tumor immune responses as demonstrated by resistance to challenge. Thus, all prior art approaches have failed to provide an adequate solution for treating tumors using electrical fields.
There is thus a widely recognized need for, and it would be highly advantageous to have, a method and apparatus devoid of the above limitation.
SUMMARY OF THE INVENTION
According to one aspect of the present invention there is provided a method of treating an individual having a neoplastic tumor, the method comprising applying to cells of the tumor electrical field pulses of a strength, a repetition frequency and a pulse width sufficient to induce endocytosis mediated cell death, thereby treating the individual.
According to another aspect of the present invention there is provided a method of treating an individual having a neoplastic pathology comprising applying to cells of a tumor electrical field pulses having a strength of 2-150 V/cm, a repetition frequency of 1 Hz - 100 kHz and a pulse width of 1 μs-100 ms for a time period of 1 second to 60 minutes thereby treating the individual suffering from a neoplastic disorder.
According to yet another aspect of the present invention there is provided a method of treating an individual having a neoplastic and potentially metastatic tumor, the method comprising applying to cells of the tumor electrical field pulses of a strength, a repetition frequency and a pulse width sufficient to induce endocytosis mediated cell death thereby initiating or enhancing immune response to the cells in the individual, thereby treating the individual.
According to further features in preferred embodiments of the invention described below, the electrical field pulses are unipolar and are of a strength of 10-70 V/cm, a repetition frequency of 100 Hz -10 kHz and a pulse width of 1-
200 μs.
According to still further features in the described preferred embodiments the method further comprising exposing the cells of the tumor to a cytotoxic agent concomitant or prior to application of the electrical field pulses. According to still further features in the described preferred embodiments the exposing the cells of the tumor to the cytotoxic agent is effected 0.1-20 minutes prior to the application of the electrical field pulses.
According to still further features in the described preferred embodiments the electrical field pulses are unipolar and are of a strength of 10-40 V/cm a repetition frequency of 300-2000 Hz and a pulse width of 1 -200 μs.
According to still further features in the described preferred embodiments the exposing the cells of the tumor to the cytotoxic agent is effected by administering the cytotoxic agent to the individual.
According to still further features in the described preferred embodiments the administering is effected by directly injecting the cytotoxic agent into or around the tumor.
According to still further features in the described preferred embodiments the cytotoxic agent is selected from the group consisting of bleomycin, 5- fluorouracil, cisplatin, taxol, doxorubicin, cyclophosphamide, methotraxate and carmustine. According to still further features in the described preferred embodiments the cytotoxic agent is provided in a carrier mixture including oleum ricini and ethanol.
According to still further features in the described preferred embodiments the method further comprising determining a volume of the tumor, the volume being for determining the strength, repetition frequency and the pulse of the electrical field pulses applied to the cells of the tumor.
According to still further features in the described preferred embodiments the applying to cells of the tumor the electrical field pulses is effected in the absence of a cytotoxic agent.
According to still further features in the described preferred embodiments the neoplastic pathology is a sarcoma or carcinoma, selected from the group consisting of fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma and neuroblastoma.
According to still another aspect of the present invention there is provided an apparatus for treating an individual suffering from a neoplastic pathology comprising: (a) an electrode system including an anode electrode and at least one cathode electrode configured for placement in contact with a tissue of the individual; (b) a power source being in circuit communication with the electrode system and being for generating an electrical potential across the electrode system; and (c) a controller being for setting the duration, frequency and strength of the electrical potential in a manner enabling the electrode system placed within the tissue to expose cells of the tissue to electrical field pulses having a strength of 2-150 V/cm, a repetition frequency of 1 Hz - 100 kHz and a pulse width of 1 μs-100 ms for a time period of 1 second to 60 minutes.
According to still further features in the described preferred embodiments the controller enables setting of the strength of the electrical potential in increments of 1 mV.
According to still further features in the described preferred embodiments the electrode system includes three cathode electrodes.
According to still further features in the described preferred embodiments the anode electrode and the at least one cathode electrode are needle electrodes. According to still further features in the described preferred embodiments the electrode system includes a support for holding the anode electrode and the cathode electrodes and for simultaneously contacting the anode electrode and the cathode electrodes with the tissue region.
According to still further features in the described preferred embodiments the anode electrode and the cathode electrodes are held by the support in a configuration which enables placement of the anode electrode in a center of the tissue and the cathode electrodes in a periphery of the tissue.
According to still further features in the described preferred embodiments the apparatus further comprising a processing unit being connected to the controller, the processing unit being for determining the duration, the frequency and the strength of the electrical potential according to an input of a volume of the tissue region.
According to still further features in the described preferred embodiments the apparatus further comprising an injector mechanism for injecting a cytotoxic agent into the tissue. According to still further features in the described preferred embodiments the injector mechanism forms a part of the electrode system.
According to still further features in the described preferred embodiments the injector mechanism is at least partially integrated within the anode cathode. According to an additional aspect of the present invention there is provided a method of stimulating an individual's immune response to tumor cell, the method comprising applying to cells of a tumor tissue electrical field pulses of a strength, a repetition frequency and a pulse width sufficient to stimulate an individual's immune response to the cells. According to still further features in the described preferred embodiments the applying to cells of the tumor tissue the electrical field pulses is effected in the absence of a cytotoxic agent.
According to still further features in the described preferred embodiments the applying to cells of the tumor tissue the electrical field pulses is effected in the presence of an administered immunostimulatory agent.
According to still further features in the described preferred embodiments the immunostimulatory agent is composed of a mixture of oleum ricini and ethanol.
The present invention successfully addresses the shortcomings of the presently known configurations by providing an apparatus and method highly effective in treating an individual having a neoplastic disorder such as cancer.
BRIEF DESCRIPTION OF THE DRAWINGS
The invention is herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of the preferred embodiments of the present invention only, and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of the invention. In this regard, no attempt is made to show structural details of the invention in more detail than is necessary for a fundamental understanding of the invention, the description taken with the drawings making apparent to those skilled in the art how the several forms of the invention may be embodied in practice. In the drawings:
FIGs. la-b depict the effect of LEF treatment alone on survival of CT-26 tumor bearing mice. Figure l a is a data plot depicting cumulative survival of CT-26 tumor bearing mice. Untreated tumor bearing mice (TB). Figure lb is a table depicting statistical analysis of results obtained. FIG. 2 is a histogram depicting the effect of LEF treatment alone on tumor size in CT-26 tumor bearing mice. Untreated tumor bearing mice (TB). FIG. 3 is a histogram depicting the energy (in Joules) measured between electrodes when applying LEFs.
FIGs. 4a-c depict the effect of various combinations of LEF/bleomycin treatments on CT-26 tumor progression. Figures 4a and 4b are data plots respectively depicting percent survival of tumor bearing mice treated with the indicated combinations of LEF and bleomycin (chemotherapy) treatments. Figure 4c is a table depicting statistical analysis of the results obtained in Figures 4a and 4b. FIGs. 5a-b are a data plot depicting survival and a histogram depicting tumor size, respectively, in CT-26 tumor bearing mice treated with LEF, BCNU or both. TB - untreated tumor bearing mice. BCNU was injected intratumorally at a dose of 35 mg/kg body weight. Cumulative survival data were compiled from two separate experiments and statistical analysis was assessed by the Mantel-Cox test.
FIGs. 6a-b are a data plot depicting survival and a histogram depicting tumor size, respectively, in CT-26 tumor bearing mice treated with LEF, bleomycin or both. TB - untreated tumor bearing mice. Cumulative survival data were compiled from two separate experiments. Number of mice in each group: 9-13. Statistical analysis was assessed by the Mantel-Cox test. FIG. 7 is a data plot depicting survival of CT-26 tumor bearing mice treated with LEF, 5-FU, or both. TB - untreated tumor bearing mice. Mice were treated with 100 mg/kg 5-FU either intravenously (i.v) or intratumorally (i.t). Statistical analysis was assessed by the Mantel-Cox test. Data was obtained from six mice per group.
FIG. 8 is a data plot depicting survival of CT-26 tumor bearing mice treated with surgery or BCNU in conjunction with LEF treatment. TB - untreated tumor bearing mice. Statistical analysis was assessed by the Mantel- Cox test. Data was obtained from 5 mice per group. FIGs. 9a-b are a data plot depicting survival and a histogram depicting tumor size, respectively, in mice cured of CT-26 tumors via low electric field enhanced cancer chemotherapy (LEFCT-EC) with BCNU and subsequently rechallenged with the same initial lethal dose of CT-26 cells. TB mice - mice inoculated for the first time with 105 CT-26 cells. Challenge - mice cured by LEFCT-CT and challenged with 105 CT-26 cells.
FIGs. l Oa-b are a data plot depicting survival and a histogram depicting tumor size, respectively, in mice inoculated with CT-26 cells mixed with splenocytes of mice cured of CT-26 tumors via LEFCT-EC. Mice were inoculated with 105 CT-26 cells only (control; TB mice), or with 105 CT-26 cells mixed with either splenocytes of normal (N; norm) or cured (Im; immune) mice in a ratio of CT-26 cells to splenocytes of 1 : 50 or 1 : 100.
FIGs 1 la-c depict results from treatment of mice bearing 10 mm diameter CT-26 tumors via BCNU, LEF alone or LEF with BCNU. Figures l la-b are a data plot depicting survival and a histogram depicting tumor size, respectively, and Figure l ie is a table depicting statistical analysis of results obtained in Figure 1 l a. TB mice - untreated tumor-bearing mice, BCNU 35 mg/kg - tumor bearing mice treated with BCNU (35 mg/kg) only, LEF 40 V/cm - tumor bearing mice treated with LEF (40 V/cm, 12 min) only, LEF-chemotherapy - tumor bearing mice treated with LEF (40 V/cm, 12 min) with BCNU (35 mg/kg). FIGs. 12a-c depict results from treatment of mice bearing 15 mm diameter CT-26 tumors via BCNU, LEF alone, LEF with BCNU, or surgery. Figures 12a-b are a data plot depicting survival and a histogram depicting tumor size, respectively, and Figure 12c is a table depicting statistical analysis of results obtained in Figure 12a. TB mice - untreated tumor-bearing mice, BCNU 35 mg/kg - tumor bearing mice treated with BCNU (35 mg/kg) only, LEF 40 V/cm - tumor bearing mice treated with LEF (40 V/cm, 12 min) only, LEF- chemotherapy - tumor bearing mice treated with LEF (40 V/cm, 12 min) with BCNU (35 mg/kg), Surgery - tumor bearing mice treated with surgery only. FIG. 13 is a data plot depicting cumulative survival of C57BL/6 mice bearing melanoma following LEFCT-EC with intratumoral cisplatin administration. Mice bearing 60-70 mm3subcutaneous tumors were subjected to LEF treatment alone or with cisplatin (4 mg/kg) injected intratumorally.
FIG. 14 is a data plot depicting cumulative survival of C57BL/6 .mice bearing melanoma following LEFCT-EC with taxol. Mice bearing melanoma were treated either with taxol (20 mg/kg) alone, taxol in combination with LEF or by intratumoral injection of a mixture of oleum-ricini/ethanol (1 :1 v/v) with LEF.
FIG. 15 is a data plot depicting cumulative survival of C57BL/6 mice bearing melanoma following LEFCT-EC with intratumoral bleomycin administration. Mice bearing melanoma were treated with bleomycin (8 U/kg) intratumorally and/or with LEF.
FIG. 16 is a data plot depicting the effect of LEF with either intratumoral or intraperitoneal administration of bleomycin on cumulative survival of C57BL/6 mice bearing melanoma.
FIG. 17 is a data plot depicting the cumulative survival of mice previously bearing B 16-F10.9 melanoma and cured by LEFCT-EC with cisplatin or taxol following challenge with B 16-F10.9 melanoma cells. Mice cured by LEF-chemotherapy that survived for 120-180 days after initial tumor inoculation, were challenged with 2 x 105 B 16-F10.9 cells subcutaneously. FIGs. 18a-d are photomicrographs depicting infiltration of lymphocytes (Figures 18a-b) and macrophages (Figures 18c-d) into tumor tissue 2 days following LEF-chemotherapy (Figures 18b and 18d).
FIG. 19 is a data plot depicting the cumulative survival of C57BL/6 mice bearing subcutaneous B16-F10.9 melanoma following surgery with taxol or following LEF with taxol. In one treatment group, subcutaneous melanomas were surgically removed from tumor bearing mice, and part of the mice received 20 mg/kg taxol subcutaneously near the site of excision. In another treatment group, tumor bearing mice were treated via LEF with taxol. FIG. 20 is a data plot depicting the cumulative survival of DA3 mammary adenocarcinoma bearing BALB/c mice following treatment via LEF alone or treatment via LEF with taxol.
FIG. 21 is a data plot depicting the cumulative survival of DA3 mammary adenocarcinoma bearing BALB/c mice following treatment via LEF- chemotherapy alone or with bleomycin.
FIG. 22 is a data plot depicting the cumulative survival of BALB/c mice previously cured of DA3 mammary adenocarcinoma via LEFCT-EC treatment with taxol and subsequently challenged with a tumorigenic dose of DA3 cells. The inset table is a statistical analysis of the results shown in the data plot. FIG. 23 is a photomicrograph depicting immune cell infiltration in pulmonary metastases of mice cured of DA3 tumors via LEFCT-EC with taxol.
FIGs. 24a-d are histograms depicting a comparison of the proportions of CD3-, CD4-, CD8-, and CD19-positive splenocytes (Figures 24a-d, respectively) in normal mice, mice treated with LEF alone (ES), mice treated with intratumoral taxol injection (Taxol i.t.), mice treated via LEFCT-EC with taxol (ESC w. taxol), mice treated with intratumoral bleomycin injection (Bleomycin i.t.), and mice treated via LEFCT-EC with bleomycin (ESC w. bleomycin).
FIG. 25 illustrates a device suitable for treating tumor tissue according to the teachings of the present invention. DESCRIPTION OF THE PREFERRED EMBODIMENTS
The present invention is of a method and apparatus which can be used to treat tumors. Specifically, the present invention can be used to treat tumors by exposing tumor cells to a low electric field of a distinct parameter in the presence or absence of cytotoxic drugs.
The principles and operation of the present invention may be better understood with reference to the drawings and accompanying descriptions.
Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not limited in its application to the details of construction and the arrangement of the components set forth in the following description or illustrated in the drawings. The invention is capable of other embodiments or of being practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting. Numerous cancer treatment modalities are known in the art including various surgical procedures and/or treatments utilizing various cytotoxic drugs.
In attempts to increase the efficacy of cytotoxic drugs against tumor cells researchers have devised methodology for increasing the uptake and thus exposure of these cells to the cytotoxic drugs. Thus, approaches such as electroporation have been used in conjunction with various cytotoxic drugs in order to attempt to potentiate their cytotoxic effect against tumor cells.
Incorporation of molecules into cells via exposure to low electric fields (LEFs), a methodology developed by the present inventors (U.S. Pat. No. 5,964,726) has also been used to introduce cytotoxins into tumor cells.
The use of low strength electric field therapy provides numerous benefits including, but not limited to, very high efficiency of incorporation of molecules, including macromolecules into treated cells, resulting in intracellular concentrations of cytotoxins that are at least several fold higher than their extracellular concentration, potent and specific adjunct anti-tumor activity intrinsic to the LEF itself, and generation of subsequent immunoprotection from re-growth of cured tumors.
In the present study, the present inventors have experimented with various electrical pulse parameters in efforts of improving the therapeutic efficiency of the LEF approach.
As illustrated in the Examples section which follows, the present study uncovered that an electrical pulse of a distinct signature range (as characterized by parameters of strength, duration and frequency) substantially improves the efficacy of cytotoxic agents in treating tumors as compared to prior art electroporation based approaches. Unexpectedly, the present inventors have also uncovered that an electrical pulse of another distinct signature range can be used to treat tumors without having to utilize cytotoxic agents.
Thus, according to one aspect of the present invention there is provided a method of treating an fhdividual suffering from a neoplastic disorder such as for example, cancer.
As used herein, the phrase "neoplastic pathology" refers to any disorder characterized by formation of tissue which includes cells exhibiting abnormal growth (e.g., hyperproliferation) or phenotype; such tissue is also referred to herein as a "tumor". Cancer such as sarcoma or carcinomas or any other hyperproliferative disorders are considered herein as neoplastic pathologies.
As used herein, the term "treating" when used in conjunction with a tumor refers to halting growth of the tumor tissue, reducing its size or eradicating such tumor tissue.
The method according to this aspect of the present invention is effected by applying to cells of the tumor electrical field pulses having a strength of 2- 150 V/cm, a repetition frequency of 1 Hz - 100 kHz and a pulse width of 1 μs- 100 ms for a time period of 1 second to 60 minutes. Preferably, the parameters are selected such that the average power applied to the cells of the tumor over the treatment period in the range of 1.4mW - 2.25 W. Selection of appropriate pulse parameters is preferably effected with respect to the tumor size, type of chemotherapy (when utilized) and type of cells.
Further description relating to selection of pulse parameters is provided in the
Examples section which follows.
Depending on the location and size of the tumor, application of such electrical pulses can be effected using surgically positioned electrodes or preferably by using electrodes which can be directly inserted into the individuals body and positioned in or around the tumor. Further description relating to methodology and electrode configurations suitable for application of electrical field pulses to tumor tissue according to the teachings of the present invention is provided herein below and in the Examples section which follows.
Preferably, the electric pulses applied to the tissue are unipolar, although bipolar pulses can also be used with some embodiments of the present invention.
Preferably, the electrical pulses are of a strength of 2-100 V/cm, a repetition frequency of 100 Hz -1 kHz and a pulse width of 1-200 μs, more preferably, the electrical pulses are of a strength of 5-80 V/cm, a repetition frequency of 200-
800 Hz and a pulse width of 50-200 μs, most preferably, the electrical pulses are of a strength of 10-70 V/cm, a repetition frequency of 300-500 Hz and a pulse width of 100-200 μs.
As shown in the Examples section below, LEFs having a field strength of about 70 V/cm, a repetition frequency of about 500 Hz and a pulse width of 180 μs are effective in treating tumors.
Preferably, the electrical pulses are applied for a duration of 6-24 minutes, more preferably for a duration of 8-16 minutes and most preferably for a period of 10-14 minutes. As demonstrated in the Examples section below, application of LEFs for a period of 12 minutes is particularly suitable for treating various types of tumors.
As described herein, the method of the present invention can be used with or with out administration of a cytotoxic agent. When utilized in conjunction with a cytotoxic agent (low electric field enhanced cancer chemotherapy - LEFCT-EC) which can be administered prior to (preferably 0.1-20 minutes, more preferably 2-4 minutes) or concomitant with application of the electrical pulses, the method of the present invention preferably utilizes electrical field pulses which are unipolar and are of a strength of 10-40 V/cm a repetition frequency of 300-500 Hz and a pulse width of 10-200 μs.
Administration of the cytotoxic agent can be effected systemically (e.g., via intravenous or intramuscular injection) or preferably via direct injection of the cytotoxic agent into or around the tumor or via selective perfusion of the tumor as taught, for example, in Lejeune FJ. et al., 2001. Surg Oncol Clin N Am.
10:821.
Examples of cytotoxic agents which can be used with this embodiment of the present method include, but are not l;imited to, altretamine, amifostine, aminoglutheimide, asparginase, bleomycin, busulfan, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin, dexamethasone, diethylstilbestrol, docetaxel, doxorubicin, epirubicin, estramustine, etoposide, floxuridine, fludarabine, fluorouracil, flutamide, gemcitabine, goserelin, hexamethylmelamine, hydroxyurea, idarubicin, ifosfamide, irinotecan, leucovorin, leuprolide, leucovorim, levamisole, lomustine, mechlorethamine, megestrol, melphalan, mercaptopurine, methotrexate, mitomycin, mitotane, mitoxanthrone, octreotide, paclitaxel, pentostatin, plicamycin, prednisone, procarbazine, streptozocin, tamoxifen, teniposide, thioguanine, thiotepa, topotecan, vinblastine, vincristine, vinorelbine. Such cytotoxins can be used in combination with the LEF methodology of the present invention to treat primary and/or metastatic tumors associated with a neoplastic disorder including, but are not limited to, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma and neuroblastoma
As shown in the Examples section which follows, the present method was highly effective in increasing the efficacy of cytotoxic treatment.
Furthermore, as shown in the Examples section below, administration of a mixture of oleum ricini and ethanol alone in conjunction with LEF treatment according to the teachings of the present invention can be used to treat tumors such as mammary carcinoma.
The Examples section which follows also demonstrates that the method of the present invention can be used as a general method to dramatically potentiate the effect of a range of cytotoxic agents such as bleomycin, BCNU, 5-FU, cisplatin and taxol against various tumor types, including some of the most widespread and lethal cancer types known, such as, for example, colon carcinoma, melanoma and mammary carcinoma.
As mentioned hereinabove and described in the Examples section which follows, the present inventors have also provided conclusive evidence showing that the present methodology can also be used to treat tumor tissue in the absence of cytotoxic drug administration.
This embodiment of the method of the present invention is effected by applying to cells of neoplastic/tumor tissue electrical field pulses of a strength, a repetition frequency and a pulse width sufficient to induce endocytosis mediated death of said cells in the absence of administration of a cytotoxic agent. Thus, contrary to the teachings of the prior art, tumor therapy is achieved by this embodiment of the present approach in the total absence of an administered cytotoxin. Traversing cytotoxin administration is enabled by the electrical pulse parameters employed by this embodiment of the present method, since such parameters induce endocytosis in tumor cells and thus lead to endocytosis mediated transport of extracellular ions and molecules into tumor cells, which transport is not affected by transport equilibriums. The resultant high intracellular concentration of these ions and/or molecules triggers mechanisms which lead to cell death. Treatment of tumor tissue according to this embodiment of the present invention provides numerous benefits. Since such treatment forgoes the use of cytotoxic drugs it avoids the side effects caused by the toxicity of the cytotoxic drug, it enables treatment of individuals sensitive to cytotoxic drug therapy and it substantially reduces the cost associated with tumor therapy. In addition, such treatment also maintains or enhances the immune response directed against the neoplastic tissue and in particular metastases formed from such tissue.
Treatment according to this embodiment of the present invention preferably utilizes electrical field pulses which are unipolar and are of a strength of 10-70 V/cm a repetition frequency of 300-500 FIz and a pulse width of 10-200 μs. Further detail regarding this treatment approach is provided in the Example section which follows.
Thus, the present invention provides methodology which can be used to safely and effectively treat tumors with or without adjunct administration of cytotoxic agents.
As is illustrated in the Examples section which follows, one distinct and important advantage of the present invention lies in its ability to induce or enhance the individual's immune response against tum