WO2016130385A2 - Tissue ablation by rapid and sustained alteration in membrane potential - Google Patents

Abstract

A method for the ablation of undesirable tissue, such as cells of a cancerous or non-cancerous tumor, infected tissue, adipose tissue, wound, etc., comprising the injection of at least one ion channel or ion pump blocker into said undesirable tissue then applying a low voltage electrical current into said undesirable tissue to trigger a rapid and sustained change in membrane potential (Vm) of the targeted cells, causing said undesirable tissue to die by a process of programmed cell death (apoptosis) followed by immune system action on any surviving cells in the undesirable tissue. Alternatively, depending on the tissue type, a mechanical vibration or an osmotic force may be used instead of a low voltage electrical current.

Description

TISSUE ABLATION BY RAPID AND SUSTAINED ALTERATION IN MEMBRANE POTENTIAL

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to tissue ablation devices and methods, particularly the ablation of undesirable tissue, such as a cancerous or non-cancerous tumor, infected tissue, adipose tissue, wound, etc.

2. Brief Discussion of the Related Art

Each cell in the human body has a potential difference with its extracellular milieu owing to a difference in the concentrations of the main physiological ions (Na⁺, K⁺, Ca²⁺, and CI^") between the cell and the extracellular milieu. The potential difference can be measured across the plasma membrane and represents the membrane potential (V_m) of the cell. V_m changes in response to a change in conductance (or permeability) of one or more of the major ion types, and also depends on the concentrations of the major ions inside the cell and in the extracellular milieu, as shown in the Goldman-Hodgkin-Katz equation below (Goldman, 1 43; Hodgkin and Katz, 1949):

V_m = RT/F In ((RNa+ [Na⁺]₀ + P_K+ [K⁺]₀ + P_a- [Cf]₀) / ( _Na+ [Na⁺]_; + P_K+ [K⁺]_; + P_a- [CI^"],)) where R is the ideal gas constant, Tis the temperature, and F is the Faraday constant. The role of V_m is different in excitable and non-excitable cells.

Excitable cells, such as neurons and muscle fibers, function primarily by rapid changes in V_m that are followed by an immediate return to the resting V_m. The role of V_m in non-excitable cells is not as clear. There is, however, an increasing body of knowledge that suggests that V_m may have important regulatory roles in non-excitable cells.

It is known that different tissues have different resting V_m values, and abnormal tissues, such as cancer and wounds, have V_m values that are vastly different from those of the corresponding normal tissues. The V_m of adipose tissue is also vastly different from those of normal tissues. Some examples of the differences in V_m between normal and abnormal tissues include the following:

(1) The V_m of cells in proliferating breast cancer is more depolarized than cells in normal breast tissue (Marino et al., 1994);

(2) The V_m of proliferating hepatocellular carcinoma cells is more depolarized than normal hepatocytes (Binggeli and Cameron, 1980; Stevenson et al., 1989);

(3) The V_m of cells in proliferating, neoplastic adrenocortical tissue is more depolarized than cells in normal adrenocortical tissue (Lymangrover et al., 1975); (4) The V_m of proliferating fibrosarcoma cells is more depolarized than normal fibroblasts (Binggeli and Weinstein, 1985);

(5) The V_m of cells in proliferating skin cancer is more depolarized than benign skin cells (Melczer and Kiss, 1957; Woodrough et al., 1975);

(6) The V_m of cells in proliferating ovarian cancer cells is more depolarized than normal ovarian cells (Redmann et al., 1972); and

(7) The V_m of adipocytes, at 34 mV, is significantly more depolarized than other cells in

normal human tissue (Al-Hilli and Willander, 2009).

Clarence D. Cone Jr. was a pioneer in elucidating the role of V_m in cancer pathophysiology. His finding that sarcoma cells attained hyperpolarized V_m prior to entering the M phase of mitosis was published in 1969 (Cone, 1969). Shortly after, he showed that mitosis was inhibited by hyperpolarized R values (Cone, 1970). He later theorized that proliferation of cancer cells is dependent on V_m (Cone, 1971), a theory that was also supported by earlier findings correlating V_m and malignant transformation of cells (Tokuoka and Mori oka, 1957; Johnstone, 1959). Such early findings suggested important regulatory roles of V_m in non-excitable cells.

Unlike the rapid V_m changes that are characteristic of excitable cells, V_m changes in non-excitable cells are slow (Lobikin et al., 2012), but appear to be vital in regulating important cell functions, such as differentiation, proliferation, and metastasis (Binggeli and Weinstein, 1986; Schwab et al., 2007; Blackiston et al., 2009; Sundelacruz et al., 2009). Indeed, we have determined that a rapid and sustained change in V_m results in the death of non-excitable cells by apoptosis.

There is abundant evidence that V_m is involved in the etiology and pathophysiology of cancer (Kunzelmann, 2005; Fiske et al., 2006; Stuhmer et al., 2006; Prevarskaya et al., 2010; Becchetti, 2011; Brackenbury, 2012), and it is, therefore, not surprising that a lot of effort is directed to ion channels, pumps, and transporters for more effective cancer treatments. It has been shown that blockage of voltage-gated potassium (K_v) channels reduces proliferation of cancer cells (e.g., Fraser et al., 2000; Ouadid-Ahidouch et al., 2000; Abdul and Hoosein, 2002; Chang et al., 2003; Menendez et al., 2010). Human Ether a go-go (hERG) K⁺ channels are expressed in many cancers (Ouadid-Ahidouch et al., 2001; Farias et al., 2004; Pardo et al., 2005; Hemmerlein et al., 2006; Ousingsawat et al., 2007; Ortiz et al., 2011; Rodriguez-Rasgado et al., 2012). Inhibition of hERG reduces proliferation of the cancer cell lines in which it is expressed, whereas

implantation of CHO cells over-expressing ERG (hERG analog) in mice induces tumor formation (Pardo et al., 1999). In synchronized SH-SY5Y cells, hERG expression is reduced to less than 5% in the Gi phase, compared to unsynchronized controls, suggesting that hERG expression and function is cell cycle-dependent (Meyer and Heinemann, 1998). Inhibiting hERG expression in MCF-7 cells, with astemizole, increases the proportion of cells in Gi phase and reduces the proportion in S phase (Borowiec et al., 2007), whereas activating hERG results in hyperpolarization late in the Gi phase (Ouadid-Ahidouch et al., 2001). The hyperpolarization, in turn, activates Ca²⁺-activated K⁺ (K_Ca) channels (Ouadid-Ahidouch et al., 2001), due to elevated intracellular Ca²⁺ (Nilius and Wohlrab, 1992; Ouadid-Ahidouch and Ahidouch, 2008). There are many other ion channels that are expressed in cancer cells. Several ion channel blockers have, therefore, been advanced as potential cancer treatments. Using ion channel blockers to treat cancer is, however, complicated by various factors, which is why there are no channel-blocking cancer cures six decades after knowing that ion channels regulate cancer cells. The confounding factors include the following:

(1) No ion channel blocker has resulted in the complete inhibition of cancer proliferation in vitro.

(2) Even more significantly, no ion channel blocker has led to complete cancer cell death in vitro.

(3) Ion channel blockers do not synchronize the V_m values of the cells in a tumor. A

cancerous tumor consists of stem cells, proliferating cells, and malignant (or potentially malignant) cells. Proliferating cells typically have depolarized V_m values and are the target of ion channel modulators. The modulators do not target the cancer stem cells and the malignant (or potentially malignant) cells because these cells have relatively hyperpolarized V_m values. Additionally, the non-proliferating cancer cells may not express the targeted ion channel at their particular stage in the cell cycle.

(4) Ion channel blockers cannot affect cells in the V_m range of normal tissue without causing massive adverse effects.

(5) The action of ion channel blockers is not specific to the undesirable tissue and would, therefore, cause significant undesirable effects. Ions channels and pumps are highly conserved, and most tissues in the human body share the same core ion channels and pumps. Where different ion channels are expressed, they are hardly ever unique to a particular tissue. For example, the relatively rare ion channel, hERG, expressed by some cancers, is also expressed in the heart, and blocking it would result in ventricular arrhythmia.

The same is true for ion pump blockers.

Ion channel blockers have been known to inhibit proliferation of cancer cells for at least 45 years (Cone, 1970), but due to the limitations outline above, and others, they have not successfully been used as treatments for cancer. The current invention allows for the use of ion channel modulators to rapidly kill cancer cells and cells in other undesirable tissues, such as adipose tissue and wound. Unlike electrical ablation methods in the prior art that use high-voltage electricity to ablate undesirable tissue, the current invention accomplishes ablation of undesirable tissue by rapidly altering the V_m values of the targeted tissue by means of a low-voltage electrical current (alternatively, a mechinal vibration or osmotic force) and at least one ion channel blocker, or pump blocker, to sustain the change in V_m.

The use of electricity for tissue ablation has been reported in the prior art, but the current invention is significantly different from the prior art and constitutes a marked improvement of the tissue ablation achievable by the prior art. Here are some of the pertinent prior art that use electricity for tissue ablation and how the current invention is different and improves on them.

(1) In US 8048067 B2, Davalos and Rubinsky disclose a new method for the ablation of undesirable tissue, such as cells of a cancerous or non-cancerous tumor, that involves the placement of electrodes into or near the vicinity of the undesirable tissue and the application of high -voltage electrical pulses, causing irreversible electroporation of the cells throughout the entire area of the undesirable tissue. The electric pulses irreversibly permeate the cell membranes, thereby invoking cell death. The method disclosed by Davalos and Rubinsky is similar to other methods that use electrical pulses, with a few differences in the amplitude, duration, shape, and number of repeats of the electrical pulses. The method is similar with the others in that they all use high voltages. The voltages range from a minimum of about 189 V/cm for reversible electroporation and a minimum of about 680 V/cm for irreversible electroporation. The inventors disclose other differences as outlined below. Electroporation pulses are defined as those electrical pulses that through a specific combination of amplitude, shape, time, length, and number of repeats produce no other substantial effect on biological cells than the permeabilization of the cell membrane. The range of electrical parameters that produce electroporation is bounded by: a) parameters that have no substantial effect on the cell and the cell membrane, b) parameters that cause substantial thermal effects (Joule heating) and c) parameters that affect the interior of the cell, e.g. the nucleus, without affecting the cell membrane. Joule heating, the thermal effect that electrical currents produce when applied to biological materials has been known for centuries. It was noted in the previous paragraph that electrical thermal effects which elevate temperatures to values that damage cells are commonly used to ablate undesirable tissues. The pulse parameters that produce thermal effects are longer and/or have higher amplitudes than the electroporation pulses whose only substantial effect is to permeabilize the cell membrane. There are a variety of methods to electrically produce thermal effects that ablate tissue. These include radiofrequency (RF), electrode heating, and induction heating. Electrical pulses that produce thermal effects are distinctly different from the pulses which produce

electroporation. The distinction can be recognized through their effect on cells and their utility. The effect of the thermal electrical pulses is primarily on the temperature of the biological material, and their utility is in raising the temperature to induce tissue ablation through thermal effects. The effect of the electroporation parameters is primarily on the cell membrane, and their utility is in permeabilizing the cell membrane for various applications. Electrical parameters that only affect the interior of the cell, without affecting the cell membrane, were also identified recently. They are normally referred to as "nanosecond pulses". It has been shown that high amplitude, and short (substantially shorter than electroporation pulses - nanoseconds versus milliseconds) length pulses can affect the interior of the cell and in particular the nucleus without affecting the membrane. Studies on nanosecond pulses show that they are "distinctly different than electroporation pulses" (Beebe, Fox, Rec, Somers, Stark, and Schoenbach, 2001). Several applications have been identified for nanosecond pulses. One of them is for tissue ablation through an effect on the nucleus (Schoenbach, Beebe, and Buescher, 2002). Another is to regulate genes in the cell interior (Gunderson et al., 2003). Electrical pulses that produce intracellular effects are distinctly different from the pulses which produce electroporation. The distinction can be recognized through their effect on cells and their utility. The effect of the intracellular electrical pulses is primarily on the intracellular contents of the cell, and their utility is in manipulating the intracellular contents for various uses - including ablation. There are several significant differences between irreversible electroporation and the current invention, including (i) Irreversible electroporation uses very high voltage pulses, in the order of 680 V/cm or higher, whereas the current invention uses low voltage pulses, in the order of 0.1 to 10 V in the immediate vicinity of the undesirable tissue; (ii) Irreversible electroporation uses electric pulses to create holes in the plasma membrane for random substances to flow into cells and kill them, whereas the current invention uses ion channel blockers or pump inhibitors to prevent ions from flowing into and/or out of cells in response to the applied low- voltage electrical pulse; (iii) Irreversible el ectrop oration relies on leakiness to kill cells of an undesirable tissue, whereas the current invention relies on a rapid and sustained change in the V_m of the cell, e.g. from 20 mV to -140 mV, to kill the cells of any undesirable tissue; (iv) Irreversible electroporation can be used to treat mainly cancerous tumors and very limited amounts of other undesirable tissues, whereas the current invention can be used to ablate any type of undesirable tissue, e.g., cells of a cancerous or non-cancerous tumor, infected tissue, adipose tissue, wound, etc.; (v) Cell death from irreversible electroporation is by leakiness, whereas cell death by the current invention is by apoptosis; (vi) Because cell death, with the current invention, is by apoptosis, it allows the immune system to recognize and destroy cells of the undesirable tissue, and this is not true for irreversible electroporation; (vii) For irreversible electroporation to completely ablate a tumor, all the cells of the tumor must be targeted and successfully pulsed with high voltage, whereas with the current invention an undesirable tissue can be completely ablated by targeting only a portion of the tissue, with a spread in apoptotic signals and the immune system completing the job; (viii) Unlike irreversible electroporation, there is no scarring with the current invention; (ix) Irreversible electroporation cannot be used in patients with pacemakers, whereas the current invention can; (x) Electrochemotherapy cannot be used to treat metastasized cancer, whereas, by activating the immune system, the current invention can be used to treat metastasized cancer by targeting at least one of the tumors and allowing apoptotic spread and the immune system kill the rest of the cancerous cells - even in distant tumors; (xi) Irreversible electroporation cannot be used to treat highly undifferentiated cancers or cancers that have infiltrated normal tissue to an inoperable extent, whereas the current invention can; (xii) Irreversible electroporation cannot be used to ablate adipose tissue, as in liposuction, whereas the current invention can; and (xiii) Irreversible electroporation cannot be used in the central nervous system (CNS), to treat CNS lesions, whereas the current invention can. These differences also apply to other forms of high-voltage ablation, such as the 'nano-knife' and others. The current invention is, therefore, safer, more efficient, and has broader clinical applications that electroporation.

(2) The second category of electrical tissue ablation, electrochemotherapy, has been used for the ablation of cancerous tumors in combination with a chemotherapeutic agent. It is essentially a reversible electroporation that uses high voltage to create holes in the plasma membrane, so that chemotherapeutic agents can be delivered into the cell cytoplasm (Heller, Gilbert, and Jaroszeski, 1999). See also US5468223 A to Mir. It is a drug delivery method, rather than a primary electrical ablation. Tissue electroporation is now becoming an increasingly popular minimally invasive surgical technique for introducing small drugs and macromolecules into cells in specific areas of the body. This technique is accomplished by injecting drugs or macromolecules into the affected area and placing electrodes into or around the targeted tissue to generate reversible permeabilizing electric field in the tissue, thereby introducing the drugs or macromolecules into the cells of the affected area (Mir, 2001). The use of electroporation to ablate undesirable tissue was notably used by Okino and Mohri in 1987 and Mir et al. in 1991, but delivery of substances into cells by electroporation has been known for more than a century. They recognized that some drugs for treatment of cancer, such as bleomycin and cisplatin, are effective in killing cancer cells but have difficulties penetrating the cell membrane. The technique is most effective when used in combination with drugs that are cell cycle- dependent because proliferating cells are killed while non-proliferating cells are spared. Okino and Mori and Mir et al. separately determined that electroporation increases the efficacy of cancer drugs that do not readily cross the plasma membrane (Okino and Mohri, 1987); Mir et al., 1991). Mir et al. soon followed with promising clinical trials (Mir et al., 1991). Currently, the primary therapeutic applications of electroporation are electrochemotherapy (ECT) of cutaneous and subcutaneous tumors, which uses electric pulses to transfer a cytotoxic nonpermeant drug into the cytoplasm of cancer cells;

electrogenetherapy (EGT), which uses electric pulses to transfer a therapeutic piece of genetic material into cells; and transdermal drug delivery (Mir, 2001). ECT and EGT studies have been summarized in several publications (Jaroszeski et al., 1999; Heller, Gilbert, and Jaroszeski, 1999; Mir, L. M., 2001; Davalos, R. V., 2002). There are several significant differences between electrochemotherapy and the current invention, including the following: (i) Electrochemotherapy uses very high voltage pulses, in the order of 189 V/cm or higher, whereas the current invention uses low voltage pulses, in the order of 0.1 to 10 V in the immediate vicinity of the undesirable tissue; (ii) Electrochemotherapy uses electric pulses to deliver cytotoxic drugs into a cell, whereas the current invention uses ion channel blockers and/or ion pump inhibitors to prevent ions from moving across the cell membrane in response to the applied low-voltage electrical pulse; (iii)

Electrochemotherapy relies on cytotoxic drugs to kill cancer cells, whereas the current invention relies on a rapid and sustained change in the V_m of the cell, e.g. from 20 mV to -180 mV to kill the cells of any undesirable tissue; (iv) Electrochemotherapy is used to treat only cancerous tumors, cutaneous and subcutaneous tumors in particular, whereas the current invention can be used to ablate any type of undesirable tissue, e.g., cells of a cancerous or non-cancerous tumor, infected tissue, adipose tissue, wound, etc.; (v) Cell death from electrochemotherapy is by cytotoxicity, whereas cell death by the current invention is by apoptosis; (vi) Because cell death, with the current invention, is by apoptosis, it allows the immune system to recognize and destroy cells of the undesirable tissue, and this is not true for electrochemotherapy; (vii) For electrochemotherapy to completely ablate a tumor, all the cells of the tumor must be targeted and successfully pulsed with cytotoxic drugs, whereas in the current invention an undesirable tissue can be completely ablated by targeting only a portion of the tissue with a spread in apoptotic signals and the immune system completing the job; (viii) Electrochemotherapy cannot be used to treat metastasized cancer, whereas, by activating the immune system, the current invention can be used to treat metastasized cancer by targeting only one of the tumors and allowing apoptotic spread and the immune system kill the rest of the cancerous cells - even in distant locations from the targeted tumor; (ix) Electrochemotherapy cannot be used to treat highly undifferentiated cancers or cancers that have infiltrated normal tissue to an inoperable extent, whereas the current invention can; (x) Electrochemotherapy cannot be used to ablate adipose tissue, as in liposuction, whereas the current invention can; (xi) Electrochemotherapy is cell cycle-dependent, whereas the current invention is not; and (xii) Electrochemotherapy cannot be used in the CNS, whereas the current invention can. These differences also apply to other forms of high- voltage ablation, such as 'nano-knife' and others. The current invention is, therefore, safer, more efficient, and has broader clinical applications that electrochemotherapy.

In US 7962223 B2, Young et al. disclosed a tissue ablation probe comprising an elongated probe shaft, at least one electrode carried by the distal end of the probe shaft, and a pharmaceutical agent carried by the probe shaft. The device disclosed by Young et al. is vastly different from the current invention in several important ways, including the following:

(i) The pharmaceutical agent in Young et al. is a chemotherapeutic drug; hence, the device is essentially an electrochemotherapeutic device. As such, all of the differences outlined above between the current invention and electrochemotherapy apply.

(ii) Additionally, the current invention discloses a low-voltage ablation means that is not disclosed by Young et al.

(iii) Also, the current invention can be used to ablate any type of undesirable tissue,

whereas the device in Young et al. is primarily a cancer ablation device.

In US 8834461 B2, Werneth et al. disclosed a low-power tissue ablation system. The low-power tissue ablation system disclosed by Werneth et al. is drastically different from the current invention. The low-power ablation system is a thermal ablation system, whereas the current invention uses a low-voltage electrical field and ion channel blocker(s) to ablate undesirable tissue. The current invention does not use heat to ablate tissue and does not generate heat in the ablation process.

SUMMARY OF THE INVENTION

It is an objective of the current invention to present a method to ablate undesirable tissue, such as cells of a cancerous or non-cancerous tumor, infected tissue, adipose tissue, wound, etc. The current invention ablates undesirable tissue regardless of histological type. For example, it is unnecessary to determine the histological type of a cancerous tumor in order to ablate said cancerous tumor.

It is a specific objective of this invention to present a method to completely ablate the cells of a cancerous tumor regardless of the histological type and anatomical location of said cancerous tumor, and whether said tumor is operable or not operable. Only a portion of said cancerous tumor needs to be targeted for the entire tumor to be ablated. In attaining this objective, the cancer cells in the targeted area of said cancerous tumor release an apoptotic signal and die. The apoptotic signal is propagated among like tissue, causing cancer cells beyond the targeted area to die. Additionally, the dead cancer cells are acted upon by the immune system, which now has an easier time to recognize said cancer cells as foreign. The immune system, therefore, would attack and kill said cancer cells that may be present in the blood stream, lymphatic system, or in a metastasized site that may be close or far from the original cancerous tumor.

It is yet a specific objective of this invention to present a method to completely ablate the cells of a non-cancerous tumor regardless of the histological type and anatomical location of said noncancerous tumor, and whether said tumor is operable or not operable. Only a portion of said cancerous tumor needs to be targeted for the entire tumor to be ablated. In attaining this objective, the cells in the targeted area of said non-cancerous tumor release an apoptotic signal and die. The apoptotic signal is propagated among like tissue, causing like cells beyond the targeted area to die also. Additionally, the dead cells are acted upon by the immune system, which removes them.

It is yet a specific objective of this invention to present a method to completely ablate the cells of an infected tissue. In attaining this objective, the cells of the infected tissue release an apoptotic signal and die. The apoptotic signal is propagated among like tissue, causing the infected cells beyond the targeted area to die. For example, we have used the current invention to treat an advanced stage of genital warts due to human papilloma virus (HPV). The infection included several dozens of warts in the pubis, perineum, and peri-anal regions. Only a couple of warts in the pubis were targeted for treatment, but it was sufficient to completely eliminate all of the warts in the pubis, perineum, and peri-anal regions.

It is also a specific objective of this invention to present a method to completely ablate the fat cells (adipocytes) of adipose tissue regardless of the histological type and anatomical location of said adipose tissue, and whether said adipose tissue is operable or not operable by means, such as liposuction. Unlike means, such as liposuction, only a portion of said adipose tissue needs to be targeted for the entire adipose tissue to be ablated. In attaining this objective, the adipocytes in the targeted area of said adipose tissue release an apoptotic signal and die. The apoptotic signal is propagated among like tissue, causing adipocytes beyond the targeted area to die. Additionally, the dead adipocytes are acted upon by the immune system, which removes them.

It is yet a specific objective of this invention to present a method to completely ablate the cells of a wound regardless of the histological type and anatomical location of said wound. Only a portion of said wound needs to be targeted for the entire wound to heal. In attaining this objective, the cells in the targeted area of said wound release an apoptotic signal and die. The apoptotic signal is propagated among like tissue, causing cells beyond the targeted area to die.

It is yet a specific objective of this invention to present a method to completely ablate the cells of connective tissue regardless of the histological type and anatomical location of said connective tissue. Only a portion of said connective tissue needs to be targeted for the entire connective tissue to be ablated. In attaining this objective, the cells in the targeted area of said connective tissue release an apoptotic signal and die. The apoptotic signal is propagated among like tissue, causing cells beyond the targeted area to die.

There is no scarring in or around any undesirable tissue ablated by the current invention, as the dead cells are processed and removed by the immune system.

In satisfying these and other objectives, there has been provided, in accordance with one aspect of the present invention, incubating the target area of said undesirable tissue with at least one ion channel blocker and/or ion pump inhibitor and then administering a low-voltage electrical current (alternatively, a mechanical vibration or osmotic force). The voltage administered in the undesirable tissue is lower than that required for electroporation. So, unlike the high-voltage electrical ablation methods that kill cells by electroporation or heat, the current inventions employs a low-voltage current that merely alters the V_m of the targeted cells in the undesirable tissue. The ion channel blocker(s) could be highly nonspecific and, therefore contraindicated as systemic drugs. For example, it has been known for decades that blockers of voltage-gated potassium channels could be effective cancer drugs, by inhibiting the proliferation of cancer cells, but the issue of specificity restricts the use of voltage-gated potassium channel blockers as cancer drugs. Because the pore regions of voltage-gated potassium channels, and other voltage- gated ion channel types, are highly conserved, it is proving to be extremely difficult to design drugs that are specific to a particular type of voltage-gated channel. A non-specific voltage-gated potassium blocker used to treat a cancerous tumor is more likely to stop the heart and adversely affect other tissues and organs than it is to treat the tumor. The current invention overcomes that problem by eliminating the need for specificity of the ion channel blocker. Actually, in the most preferred embodiment, the current invention uses therapeutic doses of highly nonspecific ion channel blocker(s) within the undesirable tissue. The ion channel blocker is directly injected into the undesirable tissue or is placed in a biodegradable capsule, which is then placed in the undesirable tissue.

Where the undesirable tissue is highly vascularized, or is proximal to a highly vascularized tissue, the ion channel blocker is introduced in a gel-like matrix, such as collagen, containing a vasoconstrictor, such as endothelin, or is delivered in a biodegradable capsule. Additionally, the collagen is designed to facilitate the immune response following treatment.

BRIEF DESCRIPTION OF THE DRAWINGS

In order that the invention may be more readily understood, and so that further features thereof may be appreciated, the invention will now be described by way of example with reference to the accompanying drawings, in which Figures 1 and 4 relate to the introduction of ion channel blocker(s) and/or ion pump inhibitor(s) into an undesirable tissue; Figures 2 and 5 relate to the application of low-voltage electrical current to said undesirable tissue, and Figure 3 shows the anatomical relationship of a distant undesirable tissue that may be ablated by treating and ablating a primary undesirable tissue, and in which:

Figure 1 shows the introduction of an ion channel blocker(s) and/or ion pump inhibitor(s) into an exemplary undesirable tissue (cancerous tumor) by means of an injection-type device.

Figure 2 shows the application of a low-voltage current into said exemplary undesirable tissue by means of two exemplary electrodes introduced into said undesirable tissue.

Figure 3 shows the anatomical relationship between two exemplary undesirable tissues

(metastasized cancerous tumors) and the treatment of one, which may lead to the ablation of both.

Figure 4 shows the introduction of an ion channel blocker(s) and/or pump inhibitor(s) into an exemplary undesirable tissue (cancerous tumor) by means of a capsule-type device. Figure 5 shows the application of a low-voltage current into said exemplary undesirable tissue by means of two exemplary electrodes introduced into said undesirable tissue following release of ion channel blocker(s) or pump inhibitor(s) by means of said capsule-type device.

The figures accompanying this document, as well as the detailed description of embodiments, are exemplary only, and are not intended to show the bounds of the current invention. Also, the figures are not to scale.

DETAILED DESCRIPTION OF EXEMPLARY EMBOD EVENTS OF THE INVENTION

In accordance with one aspect of the present invention, is an ion channel blocker(s) and/or pump inhibitor(s) (9). In a preferred embodiment the ion channel blocker is a non-specific blocker of potassium channels, such as tetraethylammonium chloride, or a non-specific blocker of voltage- gated potassium (Kv) channels, such as 4-aminopyridine (4-AP) and kaliotoxin. In one embodiment, the ion channel blockers comprise Kv_xy channel blockers, such as terfenadine, psora 4, margatoxin, linopirdine dihydrochloride, dofetilide, bapta am, astemizole, or in combination with shaker potassium (Ks) channel blockers, such as agitoxin 2, and/or in combination with blockers of KCNQ_X; MinK, HVA(L), LVA(T), CFTR, CICas, SK_X, CLC-x, CLC-K_X, Kir_xy, MaxiK, ROMK, IRK, BIR, RACTK, K(ATP), and AD AC.

In another preferred embodiment the ion channel blocker is a non-specific blocker of sodium channels, such as neosaxitoxin, tetrodotoxin, and saxitoxin. In one embodiment the ion channel blockers comprise epithelial sodium (ENaC) channel blockers, such as amiloride, and/or combination of specific sodium channel blockers, as above with the potassium channel blockers, to ensure complete blockage of sodium into the cells of the undesirable tissue, e.g., a class lx agent or combination of class lx agents.

In another preferred embodiment, the ion channel blocker is a non-specific blocker of calcium channels, such as bepridil hydrochloride. In one embodiment the ion channel blocker(s) comprises a combination of L-, T-, N-, P-, Q-, and R-type calcium channel blocker(s), such as co- agatoxin, amlodipine besylate, benidipine hydrochloride, cilnidipine, co-conotoxin, diltiazem hydrochloride, efonidipine hydrochloride monoethanolate, felodipine, flunarizine

dihydrochloride, huwentoxin XVI, israpidine, lercanidipine hydrochloride, loperamide hydrochloride, mibefradil dihydrochloride, nifedipine, protx 1, ruthenium red, and verapamil hydrochloride. Said embodiment comprising a combination of specific calcium channel blockers such as to totally block calcium current in the undesirable tissue.

In yet another preferred embodiment, the ion channel blocker(s) is a non-specific blocker of chloride channels, such as DIDS. In one embodiment the ion channel blocker(s) comprises a combination of selective (specific) chloride channel blockers, such as CaCCinh-Aoi, CFTRin -m, chromanol 293B, DCPIB, GaTx2, glibenclamide, lonidamine, NPPB, and talniflumate. Said embodiment comprising a combination of specific chloride channel blockers such as to totally block chloride current in the undesirable tissue. In yet another preferred embodiment, the ion channel blocker(s) is a non-specific blocker of transient receptor potential (TRP) channels, such as 2-APB. In one embodiment the ion channel blocker(s) comprises a combination of specific blocker(s) of TRPA1, TRPC, TRPM, TRPML, TRPP, and TRPV, such as A967079, AP18, HC030031, GsMTx4, ML204, Pyr3, SKF 96365 hydrochloride, AMTB hydrochloride, ononetin, 9-phenanthrol, TC-1 2000, gadolinium chloride, amiloride hydrochloride, benzamil, EIPA, and phenamil. Said embodiment comprising a combination of specific TRP channel blockers such as to totally block TRP current, in response to alteration of the membrane potential, of cells in the undesirable tissue.

In a most preferred embodiment, the ion channel blocker(s) is a combination of a non-specific blocker of potassium channels with at least one non-specific blocker of calcium, chloride, sodium, or TRP channels in a therapeutic dose.

In another most preferred embodiment, the ion channel blocker(s) is a combination of a nonspecific blocker of calcium channels with at least one non-specific blocker of potassium, chloride, sodium, or TRP channels in a therapeutic dose.

In yet another most preferred embodiment, the ion channel blocker(s) is a combination of a nonspecific blocker of chloride channels with at least one non-specific blocker of potassium, calcium, sodium, or TRP channels in a therapeutic dose.

In another most preferred embodiment, the ion channel blocker(s) is a combination of a nonspecific blocker of sodium channels with at least one non-specific blocker of potassium, chloride, calcium, or TRP channels in a therapeutic dose.

In another most preferred embodiment, the ion channel blocker(s) is a combination of a nonspecific blocker of TRP channels with at least one non-specific blocker of potassium, chloride, sodium, or calcium channels in a therapeutic dose.

In one embodiment, the ion channel blocker(s) is a combination of a specific blocker(s) of calcium channels with at least one specific blocker of potassium, chloride, sodium, or TRP channels subtypes in a therapeutic dose.

In another embodiment, the ion channel blocker(s) is a combination of a specific blocker(s) of potassium channels with at least one specific blocker of calcium, chloride, sodium, or TRP channels subtypes in a therapeutic dose.

In another embodiment, the ion channel blocker(s) is a combination of a specific blocker(s) of chloride channels with at least one specific blocker of calcium, potassium, sodium, or TRP channels subtypes in a therapeutic dose.

In another embodiment, the ion channel blocker(s) is a combination of a specific blocker(s) of sodium channels with at least one specific blocker of calcium, chloride, potassium, or TRP channels subtypes in a therapeutic dose. In yet another embodiment, the ion channel blocker(s) is a combination of a specific blocker(s) of TRP channels with at least one specific blocker of calcium, chloride, sodium, or potassium channels subtypes in a therapeutic dose.

In a preferred embodiment, the therapeutic dosage of ion channel blocker(s) is combined with an adjuvant such as iodine in a therapeutic dose.

In a preferred embodiment, the therapeutic dosage of ion channel blocker(s) is combined with a vasoconstrictor, such as endothelin.

In a preferred embodiment, the ion channel blocker is introduced into the undesirable tissue, prior to application of, or during application of, a low-voltage electrical current, by means of an injection-type device (1). In one embodiment the injection-type device (1) is an

electrotherapeutical apparatus as described by Northcott et al. (1,653,819) and herein

incorporated by reference in its entirety. In yet another embodiment, the injection-type device (1) is an electrochemical apparatus as described by Mir et al. (US 005674267A) and herein incorporated by reference in its entirety. In yet another embodiment, the injection-type device (1) is adapted to deliver blockers using the equipment in minimally invasive and robotic surgery. In yet another embodiment, the channel blocker(s) is introduced into the undesirable tissue by means of a catheter.

Following or during application of the channel blocker(s) and/or pump inhibitor(s), an electrical field is applied to cells of the undesirable tissue. In one embodiment, the electric field is applied by means of needle electrodes. At least two needle electrodes (4 & 5) are introduced into the undesirable tissue, and a low-voltage pulse generator is used to apply an electrical field between them, such that the V_m of the cells in said electrical field is changed from a resting value to a relatively depolarized or hyperpolarized value. The pulse applicator is intended to apply a variable electric field to cells located between a pair of needles 1, 2 . . . n. To achieve this, it comprises a pulse generator, a selector switch and a control unit. The pulse generator comprises a low-voltage power supply, which is connected to the mains supply by a mains cord, and to the selector switch via a switch connected to the generator's positive output, and a capacitor connected in parallel across its positive output and negative output. Because the pulse generator is low-voltage, the power supply could be a battery source, e.g. lithium ion and nickel/cadmium, allowing for a small, compact device.

Each electrode 1, 2, . . . n, can be connected either to the positive pole of the low-voltage power supply, or to its negative pole by means of two relays belonging to selector switch. A control unit controls the power supply switch and changeover switch according to the instructions it receives from an operator or via a program. The electric pulse applicator is, thus, able to apply previously determined pulse cycles between needles 1, 2 . . . n in twos and in all possible combinations. These cycles can be determined by any means, particularly experimental, in order to provide the best possible results.

In one embodiment, the relays are formed by a bar relay or REED bulb relay, the excitation for which is produced either by physical displacement of a small magnet whose position is slaved, or by a conventional command using a coil. This displacement is produced by a conventional position slaving system ensured by a coil. Due to this arrangement, the selector switch can be made very compact. So, by closing relay X₁ and relay Y_n, it is possible, when the switch is closed, to send a pulse between electrodes 1 and n, electrode 1 being the positive electrode and electrode n the negative electrode.

In one embodiment, the electric contact is established with the tissue via the electrodes over all their non-insulated length, the produced field thus extending into the depth of the tissue. It is, therefore, possible to subject cells to electric fields, which would not be accessible, at least not easily, from electrodes simply placed on the surface of the tissue.

In one embodiment, the pulses applied to each pair of needles are rectangular pulses having an amplitude of 0.1 to 150 V and a pulse length of 10 to 10⁸ μβ. Where more than a single pulse is applied, these pulses are spaced, for each pair of needles, by an adjustable interval in the range 0.2 to 2 s.

For each pair of needles, it is possible, for example, to apply multiple successive pulses of the same polarity, or several pulses of a first polarity followed later in the cycle by several pulses of the opposite polarity. In the case of electrodes comprising many needles, the pulse sequences between the different pairs of needles can be interleaved. Thus, given the length of each pulse and the interval which must separate two successive pulses applied to a given pair of electrodes, it is possible to excite the different pairs of electrodes one after another while respecting these sequences.

The electric fields thus produced can be approximately uniformly distributed, including depth, since the needles penetrate into the tissue and define a set of volume of tissue, each of these volumes being included between a pair of electrodes. By applying electric fields to these elementary volumes, good uniformity of treatment can be obtained over the targeted volume of undesirable tissue treated.

In one embodiment, each needle 1, 2 . . . n, comprises a base, a head, a connector comprising a flat surface and a base.

The base comprises a stem, which is terminated by a point. One or more parts of the stem preferably comprises an insulating sleeve, made from polytetrafluoroethylene (PTFE), for example, which provides, when inserted into tissue, a means of preventing the application of electric pulses to areas other than the target area in the undesirable tissue.

In an embodiment, the insulating sheath in the base of the needle is removable and can function as a catheter or miniature trocar.

Alternatively, following or during application of the channel blocker(s) and/or pump inhibitor(s), a mechanical stimulus, such as vibration from massage, vortex, sound, etc, is applied to cells of the undesirable tissue. In one embodiment, the mechanical stimulus, such as vibration from massage, vortex, sound, etc, is applied externally, e.g. to stimulate subcutaneous adipose tissue that is being ablated. In yet another embodiment, the mechanical stimulus, such as vibration from massage, vortex, sound, etc, is applied by means of devices used in minimally invasive and robotic surgery.

Also alternatively, following or during application of the channel blocker(s) and/or pump inhibitor(s), an ion flux is applied to cells of the undesirable tissue. In one embodiment, the ion flux is applied by means of an injection-type device. In another embodiment, the ion flux is applied by means of a catheter. In yet another embodiment, the ion flux is applied by means of devices used in minimally invasive and robotic surgery.

'Blocker' is used interchangeably with 'modulator' in this patent application, and the use of one refers to the full scope and definition of both.

Patent citations

US 2007/0043345 Al 12/2007 Davalos and Rubinsky

US 2002/0010491 Al 1/2002 Schoenbach et al.

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US 1,653,819 12/1927 Northcott and James

US 8834461 B2 9/2014 Werneth et al.

US 7962223 B2 6/2011 Young et al.

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Claims

That which is claimed is:

1. A method of ablating undesirable animal tissue by rapid and sustained alteration of the membrane potential of cells in said undesirable tissue, comprising the steps of:

(a) Identifying the tissue to be ablated;

(b) Injecting at least one ion channel blocker and/or ion pump blocker into the tissue to be ablated; and

(c) Applying an electrical pulse, mechanical vibration, or osmotic force in the tissue to be ablated; the amount of the electrical pulse which has been predetermined to be sufficient to induce a rapid change in membrane potential of the cells comprising the tissue to be ablated, but which has been predetermined to be insufficient to cause electroporation and/or thermal damage, such that the cells of the tissue to be ablated are killed by a sustained alteration in membrane potential but not by thermal damage or electroporation.

2. The method of claim 1, wherein the ion channel blocker comprises a non-specific blocker of potassium channels, such as tetraethylammonium chloride; or a non-specific blocker of voltage-gated potassium (Kv) channels, such as 4-aminopyridine (4-AP) and kaliotoxin; or a Kv_xy channel blocker, such as terfenadine, psora 4, margatoxin, linopirdine dihydrochloride, dofetilide, bapta am, astemizole; or a combination with shaker potassium (Ks) channel blockers, such as agitoxin 2; and/or a combination with blockers of KCNQ_x, MinK, HVA(L), LVA(T), CFTR, CICas, SK_X, CLC-x, CLC-K_X, Kir_xy, MaxiK, ROMK, IRK, BIR, RACTK, K(ATP), and AD AC, such as to substantially block the potassium current, in response to the rapid alteration of the membrane potential, in the cells of the tissue to be ablated.

3. The method of claim 1, wherein the ion channel blocker comprises a non-specific blocker of sodium channels, such as neosaxitoxin, tetrodotoxin, and saxitoxin; epithelial sodium (ENaC) channel blockers, such as amiloride; a combination of specific sodium channel blocker(s) with potassium channel blocker(s), to ensure complete blockage of sodium into the cells of the undesirable tissue, for example, a class lx agent or combination of class lx agents, such as to substantially block the sodium current, in response to the rapid alteration of the membrane potential, in the cells of the tissue to be ablated.

4. The method of claim 1, wherein the ion channel blocker comprises a non-specific blocker of calcium channels, such as bepridil hydrochloride; or a combination of L-, T-, N-, P-, Q-, and R-type calcium channel blocker(s), such as co-agatoxin, amlodipine besylate, benidipine hydrochloride, cilnidipine, co-conotoxin, diltiazem hydrochloride, efonidipine hydrochloride monoethanolate, felodipine, flunarizine dihydrochloride, huwentoxin XVI, israpidine, lercanidipine hydrochloride, loperamide hydrochloride, mibefradil

dihydrochloride, nifedipine, protx 1, ruthenium red, and verapamil hydrochloride, such as to substantially block the calcium current, in response to the rapid alteration of the membrane potential, in the cells of the tissue to be ablated.

5. The method of claim 1, wherein the ion channel blocker comprises a non-specific blocker of chloride channels, such as DUDS; or a combination of selective (specific) chloride channel blocker(s), such as CaCQn -_Aoi, CFTRin_h-m, chromanol 293B, DCPIB, GaTx2, glibenclamide, lonidamine, PPB, and talniflumate, such as to substantially block the chloride current, in response to the rapid alteration of the membrane potential, in the cells of the tissue to be ablated.

6. The method of claim 1, wherein the ion channel blocker comprises a non-specific blocker of transient receptor potential (TRP) channels, such as 2-APB; or comprises a

combination of specific blocker(s) of TRPA1, TRPC, TRPM, TRPML, TRPP, and TRPV, such as A967079, API 8, HC030031, GsMTx4, ML204, Pyr3, SKF 96365 hydrochloride, AMTB hydrochloride, ononetin, 9-phenanthrol, TC-1 2000, gadolinium chloride, amiloride hydrochloride, benzamil, EIPA, and phenamil such as to substantially block the TRP current, in response to the rapid alteration of the membrane potential, of cells in the tissue to be ablated.

7. The method of claim 1, wherein there comprises a therapeutic dose of:

(a) a combination of a non-specific blocker of potassium channels with at least one nonspecific blocker of calcium, chloride, sodium, or TRP channels;

(b) a combination of a non-specific blocker of calcium channels with at least one nonspecific blocker of potassium, chloride, sodium, or TRP channels;

(c) a combination of a non-specific blocker of chloride channels with at least one nonspecific blocker of potassium, calcium, sodium, or TRP channels;

(d) a combination of a non-specific blocker of sodium channels with at least one nonspecific blocker of potassium, chloride, calcium, or TRP channels;

(e) a combination of a non-specific blocker of TRP channels with at least one nonspecific blocker of potassium, chloride, sodium, or calcium channels;

(f) a combination of a specific blocker(s) of calcium channels with at least one specific blocker of potassium, chloride, sodium, or TRP channels subtypes;

(g) a combination of a specific blocker(s) of potassium channels with at least one

specific blocker of calcium, chloride, sodium, or TRP channels subtypes;

(h) a combination of a specific blocker(s) of chloride channels with at least one specific blocker of calcium, potassium, sodium, or TRP channels subtypes;

(i) a combination of a specific blocker(s) of sodium channels with at least one specific blocker of calcium, chloride, potassium, or TRP channels subtypes;

(j) a combination of a specific blocker(s) of TRP channels with at least one specific blocker of calcium, chloride, sodium, or potassium channels subtypes.

8. The method of claim 1, wherein the ion channel blocker(s) and/or ion pump blocker(s) is administered with an adjuvant, such as iodine, in a therapeutic dose.

9. The method of claim 1, wherein the ion channel blocker(s) and/or ion pump blocker(s) is administered with a vasoconstrictor, such as endothelin, in a therapeutic dose.

10. The method of claim 1, wherein the ion channel blocker(s) and/or pump blockers are administered in a gel, or gel-like, formulation to slow their diffusion in the tissue to be ablated.

11. The method of claim 1, wherein the ion channel blocker(s) and/or pump blockers are administered in a degradable capsule formulation to delay and slow their diffusion in the tissue to be ablated.

12. The method of claim 1, wherein the osmotic force is a hypertonic solution, such as a concentrated NaCl solution, in a therapeutic dose.

13. The method of claim 1, wherein the osmotic force is a hypotonic solution, such as H₂0.

14. The method of claim 1, wherein the mechanical vibration is applied on the skin, for

example to ablate subcutaneous fat.

15. The method of claim 1, wherein the mechanical vibration is applied to deeper tissues, by invasive or minimally invasive means.

16. The method of claim 1, wherein the mechanical vibration is sonically applied to the tissue to be ablated, for example by ultrasound.

17. The method of claim 1, wherein the mechanical vibration is by non-invasive, penetrating, wave or radiation pulse(s).

18. The method of claim 1, comprising continuously detecting the electrical activity of the tissue to be ablated to ensure that a membrane potential altering pulse(s) has been administered and that the change in membrane potential is sustained.

19. The method of claim 1, wherein the electrical pulse(s) is applied for a duration in a range of from about 5 microseconds to about 600 seconds, and the pulse(s) produces a voltage gradient in a range that is insufficient to cause tissue death by electroporation or by thermal damage.