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Patent application title: METHOD AND DEVICE FOR TREATING MICROSCOPIC TUMORS REMAINING IN TISSUES FOLLOWING SURGICAL RESECTION
Paul Goldfarb (San Diego, CA, US)
Dietmar Rabussay (Solana Beach, CA, US)
IPC8 Class: AA61M3700FI
USPC Class:
604 21
Class name: Means for introducing or removing material from body for therapeutic purposes (e.g., medicating, irrigating, aspirating, etc.) infrared, visible light, ultraviolet, x-ray or electrical energy applied to body (e.g., iontophoresis, etc.) with tubular injection means inserted into body
Publication date: 2012-06-07
Patent application number: 20120143120
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This invention concerns treating apparently normal tissue surrounding
sites of cancerous tumors so as to reduce both the probability of a
recurrence of cancer at and near the site of a cancerous tissue, and to
reduce the amount of apparently healthy tissue that is usually excised
along with the tumor, thereby providing a substantial benefit to the
cancer patient by eliminating or delaying tumor recurrence and sparing
normal tissue for its functionality and for avoiding unnecessary
1-22. (canceled)
23. A method of treating microscopic residual tumors remaining in a tissue following surgical resection of a tumor mass, the method comprising: (a) excising tumor mass from tissue containing the tumor; (b) providing a source for applying an electroporative electric pulse to said tissue previously adjacent to and about said tumor mass; (c) providing an agent capable of reducing tumor growth; (d) administering said agent to said tissue of step (b); and (e) applying the electroporative electric pulse by said source to said tissue, thereby delivering said agent into said cells, where at least one electroporative electric pulse provides treatment of said tissue of step (b) to kill residual tumors.
24. The method of claim 23 wherein said agent is selected from the group consisting of Bleomycin, cisplatin, a polypeptide, an antibody, RNAi, an antisense nucleic acid, an expressible gene encoding a therapeutically active polypeptide, a chemokine, and a cytokine.
25. The method of claim 24 wherein said agent is delivered into said mammalian tissue either intravenously or injected directly into said tissue previously adjacent to and about said tumor mass.
26. The method of claim 24 wherein said RNAi, anti sense nucleic acid, and expressible gene are formulated in poly-L-glutamate solution.
27. The method of claim 23 wherein said tumor is selected from the group consisting of a cancer cell in cutaneous tissue, a cancer cell located on the head or neck of a mammal, and a melanoma cell.
28. The method of claim 23 wherein said electroporative electric pulse comprises a pulse having a nominal field strength selected from the group consisting of 1500 V/cm, 1200 V/cm, between 800 and 1500 V/cm, between 200 and 800 V/cm.
[0001] This application claims the benefit, under 35 U.S.C. §119(e), of: U.S. Provisional Patent Application No. 60/778,740, which was filed on Mar. 3, 2006, was of same title and named Paul Goldfarb and Dietmar Rabussay, as inventors. The entirety of this application and document is incorporated by reference.
[0002] This invention relates to electroporation systems, devices and methods of using such devices for treating tissues surrounding sites of tumors. More specifically, this invention relates to the debulking of tumor masses, sparing of tissue surrounding tumors and reducing tumor mass size and recurrence rates by treating apparently non-cancerous tissues with electroporative pulses and anticancer agents.
[0003] The following description includes information that may be useful in understanding the present invention. It is not an admission that any such information is prior art, or relevant, to the presently claimed inventions, or that any publication specifically or implicitly referenced is prior art.
[0004] Except for vascular disease, cancer is the most frequent cause of death in industrialized countries. The traditionally accepted paradigm for treating cancers and tumors that comprise a single relatively well defined tissue mass has included the use of radiation and chemotherapy typically in association with surgical removal. In cases where surgical resection is an option, surgery is usually the most effective form of treatment. However, the effectiveness of surgical treatment depends on the complete removal of malignant tissue, encompassing the main tumor mass as well as branches and micrometastases which are frequently present in the vicinity of the main tumor.
[0005] When removing the main tumor, the surgeon also attempts to eliminate such local or regional micrometastases by resecting apparently normal tissue surroundings the tumor. That tissue is referred to as "margin tissue" or simply as "margin." To what extent margin tissue is removed is subject to the surgeon's judgment. Typically, a margin of 0.5-2 cm around the entire tumor is acceptable. However, more extensive resections are not uncommon for invasive tumors. Even after careful resection of the tumor and margin, most types of tumors recur with a frequency of 10-40%. The recurrence rate depends on multiple factors including tumor size, type and location of the tumor, condition of the patient, etc. In order to reduce the rate of recurrence, surgery is usually followed by radiation and/or chemotherapy. Despite such secondary treatments recurrence rates are still uncomfortably high.
[0006] In addition to surgery and radiation, other methods of local tumor control are in use. These include Radiofrequency (RF) Ablation, Photodynamic Therapy (PDT), Cryotherapy (CRYO), Chemo-Radiation (CR), Brachytherapy (BT), Galvanotherapy (GT), and others. Surgery, RF, PDT, CRYO and CR rely on the complete removal or destruction of the tumor and margin tissues, whereas radiation, BT, and GT leave treated normal tissue more or less intact, although radiation and BT may cause severe scarring, fibrosis and vascular and neural damage. In any event, removal, scarring, and physical damage of healthy tissue can result in substantial disfigurement and substantial loss of physical use of body parts and/or functionality thereof. For example, most of the above listed current adjuvants to surgery for destroying or removing tumor masses cause nonspecific damage to normal tissues surrounding the tumor. Excision of a tumor mass can be completely debilitating to function where tumors must be removed from organs including the tongue, vocal chords, rectum, labia, penis, or to fine muscle and visual structures of the face tissue.
[0007] To avoid such disfigurement and preservation of function and to ensure that tissues surrounding the tumor are cleared of such cancerous cells, the present invention is provided as an adjuvant or neo-adjuvant to surgery as there is still a need in the oncology arts for a device and method of sparing apparently healthy tissues that lie adjacent to tumors and to reduce tumor mass, growth of the tumor, and recurrence rates.
[0008] In a first embodiment, the invention comprises a method of reducing the probability of recurrence of cancer cell growth in a tissue. In a preferred embodiment, the method includes providing to the cells of said tissue both an electroporating pulse of electric energy and a medicament. In a related embodiment the medicament is preferably provided to the tissue immediately prior to or simultaneously with the electroporating pulse.
[0009] In a second embodiment, the invention comprises a method of treating residual cancerous cells remaining in tissues following surgical resection. Preferably, the invention provides for controlling further spreading of cancer by subjecting microscopic nodules or other forms of cancerous tissue to the medicament in an electroporating electric field. In a related embodiment, such treatment can be an adjuvant to surgery in that it can be applied either prior to or after tumor removal. In some circumstances, especially where the cancerous cells have not yet formed into a fibrous mass, no surgical procedure may be employed. In such case, the EP treatment provides a method to reduce tumor mass and terminate or delay further growth of cancerous cells in the tissue. In still a further related embodiment, the invention methods provide for debulking of larger tumor masses by causing through the effect of an anticancer agent, such as Bleomycin, a `softening` of the tumor tissue such that the tumor mass will be easier to remove from surrounding healthy or normal tissue.
[0010] In a third embodiment, the invention method provides for decreasing the amount of normal tissue surrounding a tumor site, i.e. the "margin" tissue, that must be removed at the time of tumor excision and thereby spare normal tissue and consequently provide for greater retention of tissue function, and appearance.
[0011] In still other embodiments, the invention provides an instrument that is capable of providing said electroporating energy pulses to the tissue surrounding an excised tumor on demand and in an easily operable manner. In related embodiments, the invention device provides for both administration of an anti-cancer agent and administration of electric fields sufficient to cause electroporation of the tissues in the margin region. Further embodiments of the invention device are provided below.
[0012] Other features and advantages of the invention will be apparent from the following drawings, detailed description, and appended claims.
[0013] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the U.S. Patent and Trademark Office upon request and payment of the necessary fee.
[0014] FIG. 1 is a graph showing wound strength as measured by the breaking point of healing skin samples treated in vivo with saline alone, saline plus electroporation, Bleomycin alone, and Bleomycin plus electroporation. Underlying fat and muscle tissue were also treated in these experiments.
[0015] FIGS. 2A-F are micrographs of trichrome stained tissues showing comparisons between (A) untreated tissue at day 2 post incision, and (B) tissue treated with saline plus electroporation at day 2 post incision, (C) saline plus electroporation at week 2, (D) Bleomycin plus electroporation at 2 weeks, (E) saline plus electroporation at 3 weeks, and (F) Bleomycin plus electroporation at 3 weeks.
[0016] FIGS. 3A-D are stained (Trichrome) micrographs of collagen deposition in skin incisions after three weeks. FIGS. 3A and C are bright field images for Bleomycin and Saline treated skin tissues, respectively, while FIGS. 3B and C are polarized light images showing Bleomycin and Saline treated skin tissues, respectively.
[0017] FIGS. 4A-D are micrographs showing samples of muscle tissue at 3 weeks post incision; (A) saline alone, (B) saline plus electroporation, (C) Bleomycin alone, and (D) Bleomycin plus electroporation.
[0018] FIG. 5 is a cross sectional figure of one embodiment of the invention device showing an embodiment comprising a handle comprising a thumb wheel for raising and lowering an array of plungers which actuate loading or dispensing of a substance through the electrode needles.
[0019] FIG. 6 is a drawing depicting an exploded view of one embodiment of the invention.
[0020] FIG. 7 is a cross sectional drawing of one embodiment of the invention device wherein the plungers are actuated by a screw driven by clockwise (raising plungers) and counter clockwise (lowering plungers) rotation of the housing 50.
[0021] FIGS. 8A and B are perspective drawings of one embodiment of the invention wherein rotation of the housing 50 will drive the array of plungers back and forth. Figure A shows the embodiment with the tray 20 while Figure B shows the main body of the embodiment without the tray 20.
[0022] FIG. 9 is a perspective drawing showing one embodiment of the invention wherein the array of plungers is actuated via a wing nut.
[0023] FIG. 10 shows an example of the array of the plurality of electrode needles from the underside of the substrate 22.
[0024] FIGS. 11 A, B, C, and Dare photographs of Group 2 cohort animals showing the test animal with tumor (A), the tumor surgically exposed prior to complete tumor removal (B), the open wound bed after tumor removal but prior to sham EP (C), and the surgical/treatment site after 3 weeks post treatment (D). With this Group 2, no electroporation was performed and the tumor recurred even though the tumor had been completely removed.
[0025] FIGS. 12 A, B, C, and D are photographs of Group 1 cohort animals showing the test animal with tumor (A), the tumor surgically exposed prior to complete tumor removal (B), the open wound bed after tumor removal but prior to treatment with EP (C), and the surgical/treatment site after 3 weeks post treatment (D). As observed with Bleomycin-EP treatment no tumor recurred at the site of treatment.
[0026] FIGS. 13 A, B, and C are photographs of Group 7 cohort animals showing the test animal with tumor (A), after partial tumor removal (B), and the surgical/treatment site after 3 weeks post treatment (C). The mouse at 3 weeks showed no tumor recurrence even though the tumor was only partially removed.
[0027] FIGS. 14 A, B, and C are photographs of Group 4 cohort animals showing the test animal with tumor (A), after partial tumor removal (B), and the surgical/treatment site after 3 weeks post treatment (C). Without EPT the tumor continued to grow.
[0028] FIG. 15 shows a bar graph of the data presented in Table V. PTE refers to partial tumor excision, CTE refers to complete tumor excision, Bi.v. means Bleomycin administered to test animals intraveneously, EP means electroporation, i.t.B refers to Bleomycin administered intratumorally, and PEP means partial electroporation.
[0029] As those in the art will appreciate, the following description describes certain preferred embodiments of the invention in detail, and is thus only representative and does not depict the actual scope of the invention. Before describing the present invention in detail, it is understood that the invention is not limited to the particular device arrangements, systems, and methodologies described, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the invention defined by the appended claims.
[0030] Electroporation Therapy (EPT), also known as Electrochemotherapy (ECT), is a method to treat localized cancerous lesions and tumor masses. The method comprises administering certain chemotherapeutic drugs, most commonly Bleomycin, either intratumorally or intravenously. In most cases electroporation (EP) is performed by inserting arrays of needle electrodes and delivering pulsed electrical fields emanating from these electrodes directly to the cancerous cell mass. Pulse parameters are generally within the following ranges: field strength 200-2000 V/cm; pulse length 0.1-10.0 ms; pulse number 2-20; and pulse frequency 1-5 Hz. Applying such electric fields results in permeabilization of tumor cell membranes which allows anticancer drugs to enter cells and to cause up to 5000-fold greater cytotoxicity due to the drug uptake than observed in the absence of electroporation. EPT has been shown to be effective against many types of solid tumors in animals and several types of tumors in humans. In fact, several clinical studies are presently ongoing, including a Phase III study evaluating the safety and efficacy of EPT for the treatment of squamous cell carcinomas of the head and neck. However, such treatment studies are not designed or intended to treat or test the affect of EPT on noncancerous margin tissue coincident to cancerous cells.
[0031] EPT is somewhat unique among the many methods employed for local and regional tumor control in that it is far less damaging to normal tissue than to malignant tissue. Results from in vitro as well as in vivo animal and human studies indicate that EPT using a drug, such as Bleomycin, effectively destroys most tumors via apoptotic and necrotic mechanisms while causing only a minor effect (inflammation, minor necrosis) on normal tissues surrounding the tumor, particularly when Bleomycin concentration, doses, and pulse parameters are within appropriate ranges. The margin tissue surrounding tumors that is typically amenable to treatment include, without limitation, those of organs including breast, prostate, tongue, penis, labia, rectum, vocal chords, liver, squamous cell carcinoma of the head and neck (SCCHN), cutaneous and other tumors. EPT can be performed on such tissues whether the tumor mass has been removed or not. In such cancers the patient would be helped substantially by treating the tissues in a manner wherein healing can proceed largely unimpaired and there is a possibility of eliminating microscopic metastases and tumor branches trending into the tissues that give rise to local recurrences. As is also understandable with respect to the invention methods, electroporation treatments with agents such as Bleomycin provide for a measurable degree of selectivity in targeting cancerous cells. This methodology is also pertinent to treatments with other agents that have specific activities against particular disease states, such as highly localized infectious diseases and other disease states where there are foci of infected or diseased tissues wherein a cell, or group of cells that are infected or diseased, can be targeted by the agent to destroy the affected cells. In such case electroporation of such cells and surrounding tissue will allow the agent to reach its intended target in the infected or diseased cells, yet not have a detrimental effect on uninfected or normal cells as empirically observed. The molecular and cellular mechanisms underlying the different response of abnormal and normal cells, respectively, is presently unknown. Additionally, use of the invention methods and device is applicable to disease states where sites of cancerous cells are distributed in areas not easily amenable to surgery. For example, skin tissues of the face containing microscopic or focal disease sites can be treated with the invention device in the same manner as used for treating a margin tissue bed resulting in substantially less scarring than would be caused by surgery. Thus, the invention methods can be used as an adjuvant to surgery or even a neo-adjuvant. Specifically, cells of tumor mass can be electroporated along with surrounding normal tissue and thereby provide for a method of debulking the tumor mass in providing for a mechanism to hinder, terminate or otherwise reduce the growth and/or recurrence rates of the diseased cells. In such case, tumor tissue can be removed either before or after EPT, or can even be allowed to remain unexcised following EPT, in which cases the effect of an agent such as Bleomycin will provide for cell death by apoptotic and/or necrotic mechanisms and softening of the tumor cell mass followed by further necrosis. Normal tissue in the vicinity will remain largely unaffected.
[0032] Typically, treatment of a tumor with EPT includes a Bleomycin dose of 1 Unit/ccm tissue to be treated, injected intratumorally at a concentration of 4 Units/ml. The pulsed fields are generated such that the field is usually applied in six separate pulses at 4 Hz, one each in 6 different field orientations, each of 100 usec duration, at a nominal field strength of anywhere between 200 and 2000V/cm but, generally between 600 and 1500 V/cm, more usually between 600 and 1400 V/cm and even more usually between 1200-1300 V/cm across the tumor mass. Increasing Bleomycin dose or concentration and increasing the intensity of the electroporating pulse beyond the standard parameters can result in more severe effects on normal tissue. At very high field strengths and/or pulse lengths EP by itself (i.e., for example, without Bleomycin) can induce irreversible cell membrane deterioration, leading to the destruction of tumor as well as normal cellular tissue falling within the effective electric field. In still further related embodiments, a device for operating the methods of the invention can itself be operated using pulsed field strengths of between 1-600 V/cm when injecting DNA as a therapeutic agent as set forth in U.S. Pat. No. 6,528,315 which reference is herein incorporated in its entirety by reference. In such embodiment, the DNA ideally codes for a polypeptide that provides for a therapeutic anticancer effect on the tumor-containing tissue being treated.
[0033] With regard to other agents, including genes, proteins, cytokines, chemokines, steroids, antibodies, RNAi, and antisense nucleic acids, the same high pulsing parameters can be used (i.e., for example, 800-1500 V/cm or, alternatively, 1-600 V/cm, more preferably 200 to 600 V/cm, and still more preferred 400-600 V/cm. It is further contemplated that these and any other agents can be used where such agents promote wound healing without promoting growth of malignant cells. With regard to using such agents in EPT, lower-end field strengths as low as 50V/cm can be used to electroporate cells within tissue containing microscopic lesions and diseased cells where the tissues have not formed an abnormal tissue mass.
[0034] As mentioned earlier, the present rationale for cancer therapy involves in one embodiment surgical removal of the tumor mass (`debulking`) to reduce biological stress on the patient, surgical elimination of margin tissue to eradicate micrometastases and invading tumor tissue, and radiation and/or chemotherapy to control local, regional and systemic spread of the tumor. While surgical removal of the tumor itself generally provides relief for the patient, without severe sequelae, the subsequent steps of margin resection, radiation and chemotherapy can be very detrimental to the patient. Physiological and mechanical function, look, quality of life and eventual outcome can be severely affected by these treatments. Therefore, it would be ideal to combine surgical removal of the tumor with a treatment method that (a) prevents tumor recurrence due to micrometastases and invasive tumor tissue more effectively than present methods, and (b) minimizes the loss of surrounding margin tissue. In the present invention, a novel application of EPT as a surgical adjuvant or neo-adjuvant is provided to achieve either reduction of tumor mass, prevention of tumor recurrence and minimizing the need to excise margin tissue or both.
[0035] Turning now to the invention method, the current invention is intended to provide a novel method for lowering the probability of recurrence of tumor growth in otherwise normal tissues surrounding a site of excised cancerous cells. In other words, the invention method comprises treating "margins" of tissue surrounding a site of cancer cells, such cancerous cells typically formed at a distinct tissue site. In a preferred embodiment, the invention method provides for reducing the amount of tissue that must be excised along with the tumor and its cancerous cells and making radiation or chemotherapy superfluous. The method comprises applying an electroporative pulse of electric energy to the tissues surrounding the tumor site. In addition to the application of an electroporative pulse, the method further comprises providing, preferentially either prior to or simultaneously with the electric pulse, a formulation comprising an anticancer medicament. In a preferred embodiment, such medicament comprises a biologically active molecule. For example, the medicament can comprise a nucleic acid encoding an expressible polypeptide, such as, for example, a nucleic acid in an expression vector and comprising a gene sequence for a cytokine or chemokine, antibody, or enzyme. Another example comprises antisense DNA or RNA, or interfering RNA (RNAi). Alternatively, the medicament can comprise a polypeptide or an organic molecule such as, for example, a cytokine, chemokine, antibody, Cisplatin or Bleomycin, or any other molecule having an anticancer activity. Further, any of such compounds disclosed above can be administered in a formulation that can comprise any combination of pharmaceutically acceptable salts, buffers and other excipients as are well known in the art. For example, formulations for nucleic acids can comprise, for example, said nucleic acid and poly-glutamic acid (poly-L-glutamate) as described in U.S. Pat. No. 7,173,116 herein incorporated in its entirety by reference.
[0036] In a further embodiment, the invention method comprises applying the electroporative pulse using a device capable of providing such electroporative pulses to localized areas at the tumor site. In a preferred embodiment such a device can include an array of elongate needle-like electrodes. Alternate embodiments can use short non-penetrating electrodes or semi-penetrating microneedle electrodes depending upon the strength of the electric pulse required and upon the delivery mode of anticancer agent. In such embodiment, the device would be designed without a need for a plunger and delivery needles. Instead, the treatment methods can be applied using an electroporation device comprising an electrode array only, such array comprising the non-penetrating or semi-penetrating electrodes. In such a case, drug would be delivered by separate means such as a syringe. In a further preferred embodiment with respect to any of the physical arrangements of the invention device, during performance of the treatment procedure, the electrodes are positionable with respect to the tissues to be treated to create an electric field having a field strength and energy sufficient to electroporate cells in said tissue within a specified area and depth. Typically, the device will be positioned to impart an electroporative pulse to all tissues within a preselected margin of tissue around (in three dimensions) the tumor excision site. Depending upon the size of the excised tumor and the size of the electrode array, the surrounding margin tissue bed can be electroporated either entirely in one electroporative pulse application or may require a multiplicity of electroporative pulse applications by the positioning and repositioning of the electrode array so as to completely encompass the tumor site margin tissue in electroporating energy fields. In some embodiments, the electrodes can serve as delivery needles for the agent intended to be electroporated into the cells, while in other embodiments, the agent can be administered to the tissue independently from the administration/positioning of the electrodes in the tissue.
[0037] Additionally, in order to be sure that the invention method is capable of exposing healthy non-cancerous tissues to electroporative pulses without substantially damaging said healthy tissues, a study was conducted on the recovery characteristics of healthy tissue surrounding an open wound after exposure to electroporative pulses. As disclosed below, treatment of healthy tissues with electroporative pulses, or electroporative pulses and an anticancer agent, imparted no measurable significant effect to the cells of the treated area and the open wounds healed similar to untreated and/or normal tissues. Since healthy tissues can be exposed to an electroporative pulse without substantial detriment, use of electroporation in the treatment of tissues surrounding a tumor for the purpose of allowing surgeons to excise a narrower margin of healthy tissue is now possible.
General Treatment Procedure
[0038] One embodiment of the invention methods is to treat surgical wound margins or, more generally, tumor margins and in the process (a) achieve a superior recurrence rate (i.e., less frequent recurrence) compared with conventional surgical tumor and margin resection with or without secondary treatment; (b) reduce or eliminate the need for surgical resection of margin tissue while maintaining equal or better recurrence rates as with traditional surgical margin removal, and thus preserve functional tissue; and (c) reduce or eliminate the need for radiation or chemotherapy subsequent to surgical tumor resection (with or without margin resection) while maintaining equal or superior recurrence rates as with traditional surgical margin resection and secondary treatments such as, e.g., radiation, and chemotherapy.
[0039] The following examples are intended to illustrate but not limit the invention. While they are typical of those that might be used, other procedures known to those skilled in the art may alternatively be used.
Example I
Treatment of Apparently Healthy Tissue Surrounding an Open Wound with Electroporation Pulses and/or Anticancer Agent
[0040] In order to successfully use electroporation in areas of open wounds comprising for the most part normal tissue, we conducted a series of experiments to show that electroporation of normal tissue, with or without anti-cancer agent, can be performed without significantly affecting the healing processes in cutaneous, subcutaneous and muscle tissues.
[0041] In this experiment eighteen transcutaneous incisions were made into the dorsal muscles in each of eight pig test animals grouped 1-6. In study groups 1-5, longitudinal incisions were made while in group 6 the incisions were transverse to the longitudinal orientation of the muscle fiber. At each incision site, either nothing, normal saline, or Bleomycin in solution was injected such that injections were placed to a 1 cm depth and spaced at 1 cm distances from one another with three injections, one on each side of each incision within 3 cm of each other and one in the center of the incision. Bleomycin, an anti-cancer agent, was used wherein each injection consisted of 0.125 ml (4 units/ml saline). The incisions were then treated with bipolar pulses of 530 V, 100 μsec each, from a pulse generator using a 6-needle electrode array of 0.5 cm diameter (nominal field strength for each pulse was 1,233 V/cm).
[0042] Specifically, 8 male Yorkshire/Hampshire cross swine (40-50 kg) were acclimated 5-7 days prior to surgery. Incision locations were marked on the dorsum using sterile surgical markers. Each incision was 5 cm long and made to a depth of 5 mm into the muscle. All animals in groups 1-5 received 18 longitudinal incisions each, while animals in group 6 received transverse incisions, in accordance with Table I. Each study group comprised 24 incisions.
TABLE-US-00001 TABLE I Group Incision Injection Electroporation Group 1 (NL) Longitudinal None Group 2 (SL) Longitudinal Saline Group 3 (SEL) Longitudinal Saline X Group 4 (BL) Longitudinal Bleomycin Group 5 (BEL) Longitudinal Bleomycin X Group 6 (BET) Transverse Bleomycin X
[0043] Injections of a total volume of 1.5 ml of saline or Bleomycin (6 U) were administered at 12 sites per incision, 6 in the muscle and 6 in the skin. Incisions treated with electroporation were electroporated using a 0.5 cm diameter, 1.5 cm long needle array. On each side of the muscle incision, electroporation was given 3 times in the central 3 cm of the incision, each electroporation site being 1 cm apart from the other. Skin was not electroporated. The epimysium was closed with 4-0 Vicryl absorbable interrupted sutures. The skin was closed with 4-0 nylon interrupted sutures. Swine were sacrificed at 2 d, 1 wk, 2 wks, or 3 wks following surgery. Histology specimens containing skin and at least 5 mm depth of underlying muscle were taken from the center of the incision and immediately placed in 10% neutral buffered formalin. An additional control specimen was taken from non-incised skin on the dorsum. After staining and sectioning, skin, subcutaneous tissue, and muscle were graded histologically on a 0-3 scale using the following parameters: Healing, Granulations/Fibrosis, PMN's, Lymphocytes, Histiocytes, Necrosis, Hemorrhage and Atypical Cells.
[0044] Mechanical testing was performed on strips of skin removed from the center of the incision at three different time points, namely, Days 7, 14, and 21. Skin strips were wrapped in gauze soaked in saline and tested the same day for incision breaking strength using an Instron 900 (Instron Corp. Norwood, Mass.) with a 100 pound load cell run at 0.5 in/min. Breaking strength was determined by plotting the stretch versus the strain for each incision up to the breaking point. Breaking strength of skin incisions increased over time in Group 1 (no saline or Bleomycin) from ˜1800 kPa at 1 week to ˜2500 kPa at 2 weeks and ˜5000 kPa at 3 weeks post-incision. Injecting saline (Group 2) yielded similar results. Group 3 (saline injection with electroporation) was not significantly different from Group 2 at any time indicating that electroporation did not cause adverse effects in breaking strength. At 1 week, all three Bleomycin treated groups had significantly (i.e., 90%) reduced breaking strengths compared to all three non-Bleomycin groups, indicating that Bleomycin had delayed dermal wound healing. Electroporation had no adverse effects on breaking strength as Groups 4 and 5 were not significantly different from each other. By week 2, there were no significant differences between Groups 1-5. Only the transverse incisions with Bleomycin and electroporation exhibited lower breaking strengths. This effect carried through to week 3 where Group 6 had significantly lower breaking strength than Groups 1-5.
[0045] As shown in FIG. 1, there was no substantial difference between saline alone, saline plus electroporation, Bleomycin alone, or Bleomycin plus electroporation after three weeks post incision. Even the strength of healed transverse incisions is sufficient not to be of clinical concern in terms of risk to the patient.
Histological Evaluation
[0046] Histology specimens at four different time points, namely, Days 2, 7, 14, and 21 containing skin and at least 5 mm of muscle were taken from the center of the incisions and immediately placed in 10% neutral buffered formalin. Histological studies showed statistically significant changes (Bleomycin groups only) present at day 7. All non-treated tissue and saline injected tissue remained normal in appearance. By day 21, all Bleomycin injected samples were comparable with saline-injected controls. For example, when comparing saline-injected v.s. saline-injected plus electroporated tissue samples, the only effects of electroporation were slight increases in the number of skin histiocytes at 1 week and a reduction in skin lymphocytes at 2 weeks. There were no effects of electroporation on any of the other parameters such as healing of skin, subcutaneous tissue and muscle. Further, when comparing Bleomycin v.s. Bleomycin plus EP treated tissue samples there were no significant differences at 2 days or 1 week. At 2 weeks, electroporation caused a significant increase in muscle necrosis but that difference disappeared by 3 weeks. At 3 weeks electroporation caused a reduction in skin histiocytes but showed increased granulation tissue/fibrosis in the muscle, evidence of healing. There were no sustained significant differences in healing of skin, subcutaneous tissue or muscle caused by electroporation even in the presence of Bleomycin.
[0047] As shown in FIGS. 2 A-F, wound healing as compared between untreated tissue, saline plus electroporation treated tissue, and Bleomycin plus electroporation treated tissue, becomes histologically equivalent by week three. Specifically, by week three saline plus electroporation is equivalent to Bleomycin plus electroporation (compare E and F). By week 2 saline plus electroporation shows more advanced healing than Bleomycin and electroporation (compare C and A). By week 3 saline plus electroporation appears equivalent to Bleomycin plus electroporation at week 2 (compare E and D).
[0048] With regard to other histological features as shown in FIGS. 3 A-D, collagen is deposited in the healing wound but maybe at a slower rate for Bleomycin treated samples than samples treated with saline. Specifically, with no electroporation, incisions in saline treated animals (C and D) show deep blue collagen fibers in continuity with the collagen fibers of the dermis while incisions of Bleomycin treated animals (A and B) show pale blue collagen fibers with fewer zones of continuity with the dermal collagen. With respect to effect, if any, on the muscle tissue, as shown in FIGS. 4A-D, at 3 weeks the micrographs indicate that electroporation does not cause significant differences in the healing of muscle tissue compared to saline or Bleomycin alone.
[0049] Considering the effect, if any, of longitudinal vs transverse incisions, in the presence of saline vs Bleomycin, there were no significant differences at 2 days or 1 week. At 2 and 3 weeks the only significant histological differences were in the amount of granulation tissue/fibrosis at all three tissue layers studied. In skin, subcutaneous tissue and muscle, longitudinal incisions had more granulation tissue/fibrosis than transverse incisions in every case where there were significant differences. This is expected as longitudinal incisions generally heal better than transverse incisions. The scores for healing of skin, subcutaneous tissue and muscle were not significantly different for longitudinal and transverse incisions.
[0050] Electroporation of the muscle caused no significant alteration in the breaking strength of porcine dermis in the presence or absence of Bleomycin at weeks 2 and 3 and arguably even within week 1. However, the presence of Bleomycin regardless of whether EP was used, was associated with a reduction in the breaking strength of porcine dermis up to 1-2 weeks post treatment when compared to incision only, or to incision injected with saline, with or without EP. With respect to histology, electroporation of the tissues resulted in no significant differences in healing of the skin, subcutaneous tissue or muscle. Thus, electroporation therapy does not significantly adversely affect healing of muscle or skin. This appears to apply also where EPT is used for treatment of tissue surrounding tumors, and thus the methods of the invention provide an alternative to surgical resection or other therapeutic interventions in the treatment of wound margins aimed at reducing tumor recurrence.
Example II
Hypothetical Treatment Regimen
[0051] It is contemplated that surgery using the invention method and device will generally follow a protocol likely to employ the following steps:
[0052] 1. The main tumor mass will be surgically resected as usually practiced by a surgeon skilled in the art. The surgeon will either resect the primary tumor mass only or will also resect a surgical margin. Alternatively, the main tumor may be ablated by one of many ablative therapies, such as RF (radio frequency) ablation, PDT (photodynamic therapy), cryotherapy, chemo-radiation, brachytherapy, or even galvanotherapy, with or without ablation of margin tissue.
[0053] 2. After surgical resection (or ablation) of the tumor mass, with or without resection (or ablation) of margin tissue, the entire tissue surrounding the resection (or ablation) site will be treated by EPT employing Bleomycin or other chemotherapeutic or biological drugs. Drug-EPT treatment will be performed to a depth as determined by the surgeon according to theoretical considerations, scientific studies, or practical experience.
Variation A. Protocol for Resecting a Tumor with Standard Margins
[0054] 1. In this protocol variation, after the tumor has been removed along with standard margins, the entire wound surface (tumor margin bed) is treated to an appropriate depth, typically 1 cm with a standard EPT regimen typically comprising local injection of an anticancer agent followed by electroporation using at least a multi electrode array equipped device (for example, a six needle array or other electrode arrangement of an invention device such as disclosed in FIGS. 5-10) and, for example, pulsing in at least three field orientations with an appropriate voltage. By field orientations is meant the spatial and directional orientation of the electric field generated by the electric pulse between any two oppositely charged electrodes. In this embodiment, the protocol Variation A can be performed without the use of (or alternatively a reduced use of) traditional post-surgical secondary treatment. In such instance, the invention method is intended to achieve equal or better recurrence rates compared to complete surgical margin resection and secondary treatment. [0055] 2. Following EPT of the tumor margin bed, if desired, the patient can be further treated with conventional secondary treatment, such as radiation or chemotherapy, and thereby provide additional potential protection against tumor recurrence compared with no EPT treatment of the margin tissues.
[0056] The outcome of performing steps 1 and 2 above is intended to comprise improved cure rate or time to recurrence with some degree of preservation of tissue over conventional therapies but margin tissue would have been removed nonetheless.
Variation B. Protocol for Resecting a Tumor with No Margin Resection
[0057] 1. In this protocol Variation method, following tumor removal and reduced or no margin removal, the entire wound surface is treated to an appropriate depth, typically 1 cm with a standard EPT regimen typically comprising local injection of an anticancer agent followed by electroporation using a multi electrode array (for example, a six needle array or an array as disclosed in the attached figures) and, for example, pulsing in at least three field orientations with an appropriate voltage. [0058] 2. The intended outcome following this protocol and additional conventional secondary treatment is complete or partial preservation of margin tissue and potentially improved cure rate or time to recurrence.
[0059] In alternate embodiments of the above protocols, no traditional secondary treatment is provided, yet the intended outcome is complete or partial preservation of margin tissue and improved cure rate or time to recurrence.
Example III
[0060] Turning now to a disclosure of an electroporation device suitable to treat margin beds of resected tumors, in a first embodiment the device is intended to provide the capability to deliver to the margin tissue an anticancer agent, e.g., for example, Bleomycin, evenly distributed throughout the margin bed. In a second embodiment, the device provides for the capability of delivering to the margin tissue a plurality of pulses of electric energy sufficient to cause electroporation of cells throughout the margin bed to a depth of between 1 and 1.5 cm.
[0061] In a third embodiment, the invention device can provide for delivery of the electroporation pulses to a substantial portion of the margin bed, if not all of the margin bed (depending upon the relative dimensions of the device and the tumor), by a single placement of the invention device such that in order to provide electroporating pulses to the entire margin bed the device should preferably only need to be placed once.
[0062] In further embodiments, the device includes a plurality of electrodes positioned in a geometric array to provide for delivery of a series of electric pulses between selected electrodes of the array. In a related embodiment the device can comprise a plurality of shapes to accommodate various tumor shapes and sizes and further provide an array of electrodes in a preferred geometry.
[0063] In still other embodiments, the electrodes of the invention device can comprise elongate hollow needles such that the electrodes can act as both electrodes and anticancer agent delivery needles. In a particularly preferred embodiment, the invention device can include a sharps cover to cover the electrodes and to provide a mechanism for delivery of anticancer agent to the hollow needles and a means for keeping the electrodes sterile prior to surgery.
[0064] Turning now to specific details of the invention device, FIG. 5 shows invention device 10 comprising a substantially rigid sharps cover 20 which doubles as a therapeutic agent filling tray. The cover/tray 20 further comprises a fill port 21 which can be designed with a fitting capable of connecting with any desired type fitting for attaching to a source of fluid for filling the tray to a desired level of therapeutic substance. The cover/tray 20 snuggly fits the main body of the invention device which itself comprises a substantially rigid substrate 22 through which an array of a plurality of elongate electrodes 23 pass and which are each individually connected to electric leads 24 which terminate on a lateral portion of the device in a plug or connector 25 for attachment to a source of electrical energy. The substrate 22 and the plug 25 are connected to main body substrate 30 which itself comprises an array of a plurality of wells 31 (see FIG. 6). Each well comprises a hollow electrode/needle through which a substance such as a therapeutic substance can be transmitted.
[0065] Fitting with the substrate 30 is substrate 40 which comprises an array of a plurality of plungers 41. The substrate 40 is connected to a mechanism which can actuate the movement of the substrate 40 towards and away from substrate 30 so as to drive into or extract away from substrate 30 the array of plungers 41 of substrate 40. In one embodiment, the substrate 40 is actuated by a central rod 60 connected to substrate 40 on one end and at the other configured into a ratchet and a thumb activated wheel 61. In a related embodiment, substrate 50 further forms an enclosing support structure 63 to surround and enclose central rod 60. The support structure 63 can further comprise user friendly handgrip 64. In operation, the operator would use port 21 to fill the cover/tray 20 to a desired level with a fluid therapeutic agent, such as for example Bleomycin, then use the thumb wheel 61 to draw into the wells 31 the agent (FIG. 6). The operator would then remove the device from the tray 20 using the handle formed by the support structure 63 and place the device at an appropriate position on the tissue or margin bed. The operator would then insert the electrodes, activate the thumb wheel 61 to administer the agent into the tissue followed by activation of the electrodes, the device having previously been attached to a source of electrical energy via the plug 25.
[0066] The invention device further can comprises other various arrangements of components to provide the same result. For example, in one embodiment the device can comprise attached to the substrate 30, an optionally dynamic housing 50. Housing 50 is considered dynamic on an optional basis since depending upon the style of the invention chosen, the housing can rotate clockwise and counter clockwise to raise and lower the plunger instead of using a thumb wheel. In embodiments such as that depicted in FIG. 5, the housing 50 is not dynamic in that although the housing 50 may be capable of rotation, but for the handle and thumb wheel, such rotation in this embodiment servers no utility. Alternatively, as depicted in FIGS. 7 and 8, the housing is dynamic in that substrate 40 is actuated toward or away from substrate 30 by screw 70 which is attached at one end to substrate 40 and to housing 50 on the other end by engagement of said screw 70. The housing 50 portion which interacts with threads of screw 70 is itself formed into screw threads 71 which upon rotation of housing 50 will draw substrate 40 away from or towards substrate 30. In operation, the operator would fill the cover/tray 20 with agent as previously described, then place the discoid device appropriately on the tissue to be treated. After inserting the electrodes into the tissue, the user would, while pressing down on the device, rotate the housing 50 in the direction that will move the plungers toward substrate 30 and thereby expel the agent into the tissues. The operator would then active the electrodes, assuming the device had been previously connected by plug 25 to a source of electrical energy.
[0067] Still other embodiments of the invention are possible, such as for example use of a wing nut 80 attached to screw 70 for actuating the plungers 41 (see FIG. 9). In such embodiment, the housing 50 would not comprise screw threads 71 but instead only a smooth bore past which the threaded central rod 60 can pass. In such embodiment, the operator would not need to rotate the housing but only the wing nut.
[0068] Still other embodiments of the invention are possible, such as use of inanimate means to actuate the array of plungers. For example, rather than employing animate force to work a wing nut, thumb wheel, or drive screw, the plungers can be actuated by a motor driven plunger. In such embodiment a motor can be placed in arrangement with the housing 50 to, for example, cause bidirectional rotation of the housing with respect to the embodiment disclosed in FIGS. 6-8. Likewise a motor can be affixed to gears as in the thumb wheel embodiment to actuate raising and lowering the plungers. Further, as one of ordinary skill in the art would understand, the device can include indicators to provide the operator notice when the plungers have reached their respective maximum of travel into or away from the wells 31.
[0069] Still further embodiments include use of any variety of energizing parameters including use of monopolar and/or bipolar pulses in any form, including without limitation, trains of pulses, exponential decaying pluses, square pulses, etc. Further still, the invention can use pulses having a field strength of between 10 and 1500 V/cm. Moreover, the invention contemplates the capacity to energize each electrode of the array individually or together with any combination of the remaining electrodes. For example, in one embodiment, any 4 adjacent electrodes can be pulsed such that two are positive and an opposing pair of electrodes are both negatively charged. Such pulsing provides "opposed pair" pulsing as disclosed in U.S. Pat. Nos. 5,993,434 and 5,702,359. In yet other pulsing formats, single pairs of electrodes can be energized to impart an electroporating pulse of energy to the tissue until all adjacent pairs of electrodes are energized at least once. Although not shown in the figures, it is to be understood that the invention device as disclosed in its various embodiments includes individually addressed electrode needles. This is accomplished by incorporating within substrate 22 such as by sandwich technique (i.e., substrate 22 can comprise a two layer construction with electric leads placed on a grid in between the two layers) electric lead connections running from the electrodes to the side of the device (as depicted as leads 24) terminating in plug 25 having multiple pin connector capability.
Example IV
[0070] In this example, a series of experiments were performed in mice wherein tumor recurrence rates were determined after surgery alone or after surgery and EPT. Specifically, this experiment comprises an in vivo study to establish the efficacy of EPT on surgical margin treatment in nude mice after subcutaneous introduction of HT-29 human colon carcinoma cells and resection of the tumors that arise from such introduction. The experiment was conducted under humane conditions with respect to treatment of the test animals.
[0071] The experimental design was as follows. Normal Athymic Nude female mice 4-6 weeks of age were placed in 8 groups of six mice each, except for Group 1 which comprised 10 animals and Group 3 comprising 7 animals. The HT-29 cell line is derived from a human colon carcinoma and is an aggressive fast-growing tumor in mice. In each group the animals received HT-29 human tumor cells in the form of two adjacent inocula of 5×106 cells using a 21 gauge needle and syringe. The HT-29 cell line was obtained from ECACC (#HTB-38), grown in McCoy's 5a medium (modified) with 1.5 mM L-glutamine adjusted to contain 2.2 g/L sodium bicarbonate, 90%, and fetal bovine serum, 10%. Cells were divided 1:2 to 1:4 on growth cycles until sufficient cells were available to use in this experiment.
[0072] Following inoculation the tumors became established at the site of the inoculations. The tumor volume on each animal was monitored regularly and calculated using the formula: Tumor volume=(a2×b/2) where `a` is the smallest diameter and `b` is the largest diameter perpendicular to `a`. Tumors were measured three times per week starting from the day the tumors were palpable by hand. The tumors were allowed to progress until the size was between 500 and 1500 cubic millimeters. Surgical tumor removal, partial removal, or no removal was then performed in association with either EPT or no EPT. In these experiments EPT means treatment with Bleomycin and electroporation (EP). The EP, where performed, was conducted with one pulse cycle (i.e., 6 pulses at 4 Hz) per electrode insertion using a standard MedPulser generator (Genetronics, Inc. San Diego, Calif.) and an applicator with a disposable six needle array of 1 cm diameter, a distance between electrodes of 0.86 cm, and 1 cm needle length. Each square pulse had a duration of 100 usec and an applied voltage of 1500 V. For Groups 4-8 and 10 and 11, after intratumoral injection of Bleomycin the needle electrode array was inserted in such a way as to encompass the tumor within the needle array or, in the case of the largest tumors, such that the needles penetrated the edge of the tumor. The needle array was inserted percutaneously and essentially perpendicular to the surface of the flank of the mouse carrying the tumor. Alternatively, in those cases where the tumor was excised first, followed by i.v. injection of Bleomycin and then by EP, the needle electrode array was inserted percutaneously encompassing the surgical wound area (Groups 5-8).
[0073] Where Bleomycin was used in the experiment, it was injected at 4 U/ml saline (1 U=1 mg) as uniformly as possible into tumor and margin tissues, as indicated, at a dose of 0.25 ml/cm3 volume of tissue to be treated. Alternatively, where indicated, a corresponding amount of Bleomycin was injected into the tail vein.
[0074] The experiment used 8 cohorts of at least 6 mice each. Each cohort was subjected to a treatment regimen of a combination of any of EP, Bleomycin, saline instead of Bleomycin, complete tumor excision, partial tumor excision, and no tumor excision. Tables II and III disclose the treatment regimens.
TABLE-US-00002 TABLE II Excision Remove Partially No Bleomycin Mouse entire remove removal Intraveneous Group # # tumor tumor of tumor delivery EP 1 1-1 X X X 1-2 X X X 1-3 X X X 1-4 X X X 1-5 X X X 1-6 X X X 1-7 X X X 1-8 X X X 1-9 X X X 1-10 X X X 2 2-1 X Saline Sham 2-2 X Saline Sham 2-3 X Saline Sham 2-4 X Saline Sham 2-5 X Saline Sham 2-6 X Saline Sham 3 3-1 X X X 3-2 X X X 3-3 X X X 3-4 X X X 3-5 X X X 3-6 X X X 3-7 X X X 4 4-1 X Saline Sham 4-2 X Saline Sham 4-3 X Saline Sham 4-4 X Saline Sham 4-5 X Saline Sham 4-6 X Saline Sham
[0075] For experiments on animals listed in Table II, the animals were anaesthetized, followed by removal or partial removal of the tumor as indicated, followed in turn by intraveneous (i.v.) injection of Bleomycin or saline. The Bleomycin dose was dependent on tumor size as described above. After 3 to 4 minutes, the animals indicated were treated with EP followed by closure of the wound with clips and surgical adhesive. In cohort 1, the entire tumor was surgically removed (see, for example FIGS. 12B and C) and the animals treated with Bleomycin and EP. In cohort 2, the tumors were removed from the animals but only saline was injected instead of Bleomycin and no EP was performed. Only a "sham" EP procedure was performed in that the electrode array was inserted into the tissue but the electrodes were not pulsed. In cohort 3, the tumors were partially (at least 95%) removed followed by Bleomycin injection and electroporation. In cohort 4, the tumors were again partially removed followed by saline injection and sham EP.
TABLE-US-00003 TABLE III Excision Remove Partially Bleomycin entire remove No removal Intratumoral Group # Mouse # tumor tumor of tumor delivery EP 5 5-1 X X X 5-2 X X X 5-3 X X X 5-4 X X X 5-5 X X X 5-6 X X X 6 6-1 X X X 6-2 X X X 6-3 X X X 6-4 X X X 6-5 X X X 6-6 X X X 7 7-1 X X X 7-2 X X X 7-3 X X X 7-4 X X X 7-5 X X X 7-6 X X X 8 8-1 X X X* 8-2 X X X* 8-3 X X X* 8-4 X X X* 8-5 X X X* 8-6 X X X* In Table III the cohort animals were anaesthetized followed by intratumoral administration of Bleomycin solution at a dose and volume as described above. EP was performed 10 minutes after drug administration followed by tumor removal or partial tumor removal 15 minutes later, followed in turn by wound closure. In cohort 8, EP was performed incompletely (denoted by * in Table III) in that the needle array electrodes were placed such that effective electroporation only occurred in about 75% of the tumor and associated margin tissue. In other words, the EP treatment was offset with respect to the tumor.
[0076] In addition to the above experiments, we also increased the time between completion of EPT and tumor excision to assess both gross consistency of tumor tissue and evaluate it histologically. For this purpose, three additional cohort Groups 9, 10, and 11 were used. The experimental regimen is shown in Table IV.
TABLE-US-00004 TABLE IV Group # Mouse # Excision Bleomycin EP Complete tumor removal 9 9-1 X 9-2 X 9-3 X 10 10-1 X (at 2 hr) X X 10-2 X (at 2 hr) X X 10-3 X (at 2 hr) X X 11 11-1 X (at 24 hr) X X 11-2 X (at 24 hr) X X 11-3 X (at 24 hr) X X
The animals of Group 9 were anaesthetized followed by complete excision of the tumors. The tumors were examined for consistency and preserved in formalin for later histological evaluation, as were the tumor tissues of cohorts 10 and 11. Anaesthetized animals of cohort 10 were subjected to intratumoral administration of Bleomycin solution at a dose and volume as described above. EP was performed 10 minutes after drug administration. Tumors were excised 2 hrs after EP. Animals of cohort 11 were treated as those in cohort 10 except that their tumors were completely removed at 24 hrs after EP. All wounds were closed as described above.
[0077] With respect to the above cohorts 1 to 8 we examined the rate of recurrence of the tumors in the test animals three weeks after treatment. As disclosed in Table V and FIG. 15, the majority of animals receiving Bleomycin and EP were protected from tumor recurrence. (see description of FIG. 15 for definitions of abbreviations therein).
TABLE-US-00005 TABLE V Route of Tumor excision administration of com- Bleomycin % Group # plete partial none EP i.v. intratumoral efficacy* 1 + + + 90 5 + + + 100 7 + + + 100 6 + + + 80 3 + + + 67 8 + +/-** + 67 2 + - 0 4 + - 0 *% efficacy is based on the number of test animals in each Group that did NOT experience tumor growth post treatment **+/- represents a partial EPT procedure wherein the treatment was off-centered from the tumor/tumor bed.
[0078] Specifically, Groups 1 to 8 provide results indicating that treatment of tumor bed tissue provides an unexpected and surprising benefit in protecting the animal from recurrent tumors at the site of the treatment. As shown by the data, excision alone is not effective, whether the tumors were completely or partially removed. This is likely due to the aggressive nature of the tumor type used which left invasive tumor segments and/or micrometastases at or close to the tumor site despite careful surgical removal of the tumor correlating to similar aggressive tumors in man. This mimics situations encountered in human surgical therapy although recurrence rates in humans are generally in the 10 to 40% range and not as high as observed in this mouse experiment. Importantly, as exhibited by the results of the partial tumor removal, use of EPT with Bleomycin is associated with substantial reduction in tumor recurrence even if the surgical procedure failed (in this case on purpose) to excise all of the tumor mass. Further, the route of administration does not appear to play a significant role in efficacy in that administration by i.v. and i.t., respectively, produced similar results. Further still, the efficacy effect was surprisingly provided even after a 15 minute interval between completion of Bleomycin-EP treatment and tumor excision. The data are further supportive of the treatment methods in effectively treating microtumor seeding in the tissue in that the experiment wherein EP was only partially performed without tumor removal, i.e. the effective electrical field was off-centered from the tumor. Here, there was still 67% efficacy indicating there was at least a microregional effect caused by the EPT. In this example study cohorts for testing Bleomycin administration alone without EP or vice versa (i.e. EP without Bleomycin) were not included as Bleomycin administration without EP is known to have no significant antitumoral activity.
[0079] With respect to the tumor cells introduced into the test animals, we allowed the tumor masses to grow to relatively large size. See FIGS. 11A, 12A, 13A and 14A which represent tumors in test animals prior to treatment. FIGS. 11B and 12B represent examples of surgical tumor removal at the stage of having the tumor exposed before the final excision. FIGS. 11C, 12C, 13B, and 14B represent examples of the wound after complete tumor removal prior to Bleomycin-EPT (FIG. 12C) or saline-sham EP treatment (FIG. 11C), or after partial tumor removal prior to saline-sham treatment (FIG. 14B), or 15 minutes after i.t. Bleomycin-EPT (FIG. 13B).
[0080] FIG. 11D shows recurring tumor after complete tumor excision and saline-sham EP treatment and FIG. 14C shows recurring tumor after partial tumor excision and saline-sham EP treatment. FIGS. 12D and 13C show tumor sites free of tumor after tumor excision and treatment with i.v. Bleomycin and EP, or treatment with i.t. Bleomycin and EP with subsequent partial tumor excision, respectively. These procedures have advantages over present standard procedures that treat cancers with anticancer drug i.t. or i.v. Excision of the tumor, either prior to or after drug-EPT treatment prevents formation of a large necrotic mass at the tumor site which the body has to resorb or otherwise eliminate, which empirically takes at least several weeks and enhances the probability of complications. Removal of the tumor prior to or after drug-EPT is likely to expedite wound healing and reduce potential complications. Further with this invention methodology, both intratumoral and intravenous injection of drug can be used.
[0081] With respect to cohorts 9-11, we observed that the consistency of the tumor changed progressively with time after treatment. When the tumor was excised two hours after treatment (cohort 10) its consistency was softer, less well defined and it leaked edemic fluid but relatively little blood. Tumors excised 24 hrs after treatment (cohort 11) had completely softened to a consistency sometimes referred to as liquefactive necrosis. At that time point surgical excision was somewhat difficult because the boundaries of the tumor were ill defined as compared to the solid tumor tissue mass of cohort 9 animals. Interestingly, we also noticed a softening of the tumor even 15 minutes after treatment when we removed tumors from cohorts 5 and 7, which made it slightly more difficult to perform surgical removal with well defined boundaries than in the case of untreated tumors. However, this did not affect the success of the treatment since both cohorts 5 and 7 showed no tumor recurrence. These observations provide proof of the opportunity of using drug, such as Bleomycin or other anticancer agent, in combination with EPT as adjuvant or tumor debulking therapy that can additionally be used alone or in conjunction with other cancer therapies. For example, a tumor could be treated with Bleomycin-EPT, the disintegrated tumor material be removed by a simple minimally invasive procedure, and if desired, followed by conventional treatments such as radiation or chemotherapy, or other tumor therapy modalities. Easy removal of the liquefied tumor material can also allow the wound to heal faster and with fewer complications, especially in the case of large tumors.
[0082] All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods and in the steps or in the sequence of steps of the method described herein without departing from the spirit and scope of the invention. More specifically, the described embodiments are to be considered in all respects only as illustrative and not restrictive. All similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit and scope of the invention as defined by the appended claims.
[0083] All patents, patent applications, and publications mentioned in the specification are indicative of the levels of those of ordinary skill in the art to which the invention pertains. All patents, patent applications, and publications, including those to which priority or another benefit is claimed, are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.
[0084] The invention illustratively described herein suitably may be practiced in the absence of any element(s) not specifically disclosed herein. Thus, for example, in each instance herein any of the terms "comprising", "consisting essentially of", and "consisting of" may be replaced with either of the other two terms. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that use of such terms and expressions imply excluding any equivalents of the features shown and described in whole or in part thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims.
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Angel Power
Location: San Pablo Laguna Philippines
By: Shirley Abuel
One rainy Tuesday afternoon my husband and I went to a nearby town for some particular matter. The place we went to used to be a squatters area so the road was narrow and passable by only one vehicle at a time. The road is somewhat rough so everytime we go there, we make a detour going home because the road a little farther is much worse though it's a short cut going home. Because I was eager to home since myh daugther is alone in the house we went straight ahead. Reaching the middle of the road, we saw that repairs were done and it was better now, only a certain portion before reaching that road was somewhat muddy. We stopped for a while and considered going back rather than get stacked in the mud. But my husband insisted that we go straight ahead despite my vehement refusal. Off we went just to get STACKED in mud. Unknowlingly there was a pit and our front tires was caught. All efforts done to get away from the place was a failure and was already getting d!
ark and I wanted to cry because we know of no one and that place and my daughter is alone. I started to pray to our Lord and the ANGELS. I called them one by one and prayed really hard. I kept on praying for help and for Angels to come. After sone few minutes two men came, offered help but to no avail. Another few came, still failed. I kept on praying. All of a sudden a group of men came more than TEN of them making them almost TWENTY men at that time. I had never felt any fear that time. All they said was "NO PROBLEM. We will lift the front right side of your car to get that tire out of the pit". And THEY JUST DIS THAT. They came just knowing what the problem was. No questions asked. So we were out out of the muddy road. Before we left, I offered them financial consideration but they refused it and told me if I pay them they return the car to the mud. That they came because they wanted to help. I was so thankful considering the house in that place was only few. I knew they were ANGELS heaven sent to me because I called on them. Until up to this time, I ask for their help from the smallest to the biggest favors. Believe in Angels, they are REAL and had helped me a lot of times.
Some may never see with earthly eyes these heavenly beings or know that they exist. Many are unaware of the numerous ways they have protected and guided us. We would like to share with you the stories of people who take pleasure in expressing how real angels are.
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If you like theater, film, or just want to fly a kite, this is your weekend
Our local actors have so much for you to enjoy this weekend. And, here's hoping the weather is good enough for kite-flying.
Hello first weekend of Spring, 2013 - what will you do to celebrate
The weather may not feel like it, but Spring is here and there are lot's of things to do this weekend.
It's a weekend of art, theater, and, another parade
So many things to do this weekend in our area, what will you choose?
Begin your weekend with a parade and end it with a play
Start celebrating St. Paddy's Day, go to a concert, and see a play.
So much music, so many films
As we ease out of February and travel into March, there's so much music to hear and numerous films to see!
A weekend of film, music, and more
See some of the top movies of the year for free; watch classic theatre, hear Handel, or just welcome the Chinese New Year.
It's a weekend with something for everybody
Go " ZooZoo", hear live jazz, celebrate "Black History Month" - all in our area this weekend!
Fun events for a Healthy New Year
This weekend offers fun, healthy events for you and your resolutions!
Happy New Year and mindful resolutions
Begin 2013 in a better state of mind and spirit.
One of the most wonderful holiday movies and a wonderful play are waiting for you
This week offers some wonderful things for you to do!
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SAN FRANCISCO -- A San Francisco man will be in federal court Friday to answer charges he ran a massive online black market for drugs.
This arrest is part of the latest effort by the federal government to crack down on the anonymous online marketplaces that have grown because of virtual currencies and software created to help users browse the web without detection.
In recent months, there have been other arrests; but none as significant as this one.
There's BMR -- Black Market Reloaded. Drugs and even weapons are sold anonymously. If you want pure cocaine, there's Sheep Marketplace. The website Atlantis markets drugs called Sour Diesel or Happy Jack.
But the biggest and most successful of all the online black markets was closed by the feds. And they arrested the young man whom they say ran it.
The 29-year-old entrepreneur looked clean cut and innocent. But the FBI says Ross Ulbricht was secretly living the life of a digital drug lord; operating an online black market called "Silk Road" on the so-called deep web, where fictitious names are used and emails are hard to track.
His pseudonym was Dread Pirate Roberts, like a character in the movie "The Princess Bride."
Court records show Silk Road sold $1.2 billion of illegal drugs in just under three years, generating tens of millions in commissions by being the middleman for those transactions.
Documents say the secret site was used by several thousand drug dealers around the world.
But now Ulbricht is in federal custody on charges related to money laundering, drugs, and computer hacking.
He was arrested Tuesday at the Glen Park branch of the San Francisco Library.
"They heard a crashing sound coming from the science fiction section," library spokesperson Michelle Jeffers said.
According to Jeffers, library staffers went to find out what was happening.
"They walked over to look and they saw a library user being pressed against the window by several other plainclothes, as it turns out, FBI agents," she said.
In recent months, Ulbricht reportedly lived at several addresses.
An apartment on Hickory Street was one of them. Just about 500 feet away is an Internet cafe on Laguna, which he frequented and most likely operated his drug kingdom from his laptop.
Several patrons we spoke to said they recognized his picture but never spoke to him.
All were surprised.
Matt Hayne is a regular here.
"You're sitting here in a cafe and somebody next to you drinking coffee is selling heroin on the other wide of the world?" he said. "It's a little crazy, right?"
Ulbricht has also been charged with trying to hire a hitman to kill a user who threatened to reveal the identities of thousands of other users.
He was arraigned Wednesday, but goes back to federal court on Friday.
drugs, websites, internet, FBI, crime, san francisco news, vic lee
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Was a bit windy so the restaurant dropped the side curtains to make it a bit warmer. Would be the perfect spot to eat al fresco on a warm night. Good service. Tasty, fresh food
Was a bit windy so the restaurant dropped the side curtains to make it a bit warmer. Would be the perfect spot to eat al fresco on a warm night. Good service. Tasty, fresh food
I have dined here on several occasions and have never been disappointed. Service with a smile and nothing is too much trouble. The food is amazing especially the bugs & the pork with prawns, but the coffee is incredible always fast, always good. Keep it up Ellenix.
Attentive staff, amazing food and very timely service. A perfect dining experience for a private function- we will be back for the squid! Delicious!
Amazing staff, so accommodating! Highly recommended for a private function! Oh, and the coffee was amazing! Calamari is off the chain! Not a single fly in sight!
This was a terrific find! We were walking down the Main Street and passed this place and decided to give it a try! Really glad we did! The food was outstanding and very well presented! I had the calamari entree which was just perfect! I then went for one of the burgers and was really pleased with the result! A... More
We had breakfast there on a sunday and it was not full yet took nearly an hour for our meals. Two small eggs with a little piece of toast and a little bacon and eggs benedict were small but nice. I got the feeling that there is a clicky set going on with staff and locals verses the tourist. My... More
My favourite place to eat in Lennox! I love coming for my morning coffee...everyone is always so happy to see me and the staff consistently remember my order. I've had many a party and dinner booking at Ellenix and locate all my meetings there. I love the food, and the cocktails are to die for. Andy Wheeler is an innovative... More
We were in Lennox for a family weekend away. Really not sure were to dine, wasn't till our last night we tried Ellenix, it was awesome only wished we discovered it sooner! Yummy Barramundi wings my favourite, prawns also fab!! Mains beautiful seafood with Asian influence lots of fresh ingredients, my husband ordered rib eye and loved it, wot a... More
On a holiday to byron recently we decided to travel south to the beautiful town of lennox head. We stumbled across a this very stylish restaurant. The share plates were delicious including amazing salt and pepper squid and moreton bay bugs which we could not find anywhere in other restaurants in the area. the service was friendly and attentive and... More
I've dined here a number of times and the food has ALWAYS been fabulous. The Panaeng curry on a Tuesday night whether it be lamb shoulder, pork belly or vegetable is by far the most outstanding curry I've ever tasted. Better than anything I've had even in Thailand. A must try.
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Online Dispatching Rules For Vehicle-Based Internal Transport Systems
On-line vehicles dispatching rules are widely used in many facilities such as warehouses to control vehicles' movements. Single-attribute dispatching rules, which dispatch vehicles based on only one parameter, are used commonly. However, multi-attribute dispatching rules prove to be better in general. In this study, we introduce new dispatching rules and evaluate their performance compared to several good dispatching rules in literature, using the experimental design of a real case study. The performance criteria are minimizing the average load waiting time while keeping the maximum load waiting time as small as possible and better utilize vehicles. The experiments show that newly introduced hybrid dispatching rule yields the best performance overall.
Additional Metadata | |
Keywords | centralized control, on-line dispatching, performance, vehicle-based internal transport system |
Persistent URL | hdl.handle.net/1765/1324 |
Citation |
Le-Anh, T., & de Koster, M.B.M.. (2004). Online Dispatching Rules For Vehicle-Based Internal Transport Systems (No. ERS-2004-031-LIS). Retrieved from http://hdl.handle.net/1765/1324


text: If discovering how oil-dependent we have become has caused you alarm or discomfort while learning about Peak Oil you will want to check out this workshop! We will be exploring our personal responses to the issue and how we can empower ourselves and our communities to respond creatively. Get Peak Oil out of the 'too hard' basket! This workshop is on Saturday 25th February from 9:30-5pm at the CERES environment room, Lee St East Brunswick. The cost is $40 full-fare and $25 concession. (Full scholarships available.) Lunch provided. Booking is essential. For bookings and further information contact firstname.lastname@example.com Download the flyer (PDF) About Gaia: GaiaVic Inc. is part of an international network of individuals and Gaia* groups dedicated to living sustainably on the Earth. We are committed to these three objectives: 1. Personal growth: healing and empowering ourselves to fulfill our true personal, community and planetary potential. 2. Community building: creating communities and strengthening those of which we are part. 3. Service to the Earth: enhancing the wellbeing and flourishing of all life. * the living earth. We fulfill these commitments through engaging in Gaia Projects – creating new initiatives with our own members and other organisations.


text:
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No Description
Camera: | Nikon D80 Check out Nikon Nation! |
Lens: | Nikkor 80-200 Check out Nikon Nation! |
Recording media: | RAW (digital) |
Title: | Beauty |
Username: | RAYMO |
Uploaded: | 20 Oct 2008 - 7:15 PM |
Tags: | Close-up / macro, Closeup, Flower, Flowers & plants, Reflector fill, Stamen |
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Votes: | 65 |
Beautiful soft image. Like the presentation.
Lovely light and details Ray.Like the composition also.
Very apt title as it is a beauty, lovely detail & beautifully presented
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- Thursday 12 December 2013
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Patrick Cockburn
Foreign Commentator of the Year (Editorial Intelligence Comment Awards 2013)
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i Newspaper
'War on terror' is a tempting defence, but it isn't that simple
20 January 2013 12:00 AM
World View: We must understand the strange alliances in Mali to unravel its complex, conflicting loyalties
Suicide bomber kills 21 in al-Qa'ida attack on Kirkuk
16 January 2013 07:36 PM
Attack had the hallmarks of al-Qa'ida in Mesopotamia and is aimed at fuelling tensions
As long as the cash rolls in, the West appears untroubled by Gulf monarchies' ideology
15 January 2013 07:09 PM
The West has portrayed Gulf leaders as natural allies in promoting democratic revolutions
Analysis: The murky motives behind Mali's crisis
14 January 2013 10:00 PM
The small Islamist groups would find it near impossible to take the whole of Mali
The war against the Shia catches all in its crossfire
13 January 2013 12:00 AM
World View: Sunni attacks on their Muslim neighbours have left the West with strange bedfellows
At this rate, Syrian president Bashar al-Assad could still hold power in Damascus a year from now
10 January 2013 07:23 PM
In his World View column, our correspondent find links between the conflict in Syria today and the Algerian civil war of the 1990s. And below: the ghosts of Abu Ghraib
This is a war without end unless the government makes concessions
10 January 2013 06:51 PM
The guerrilla war between the Turkish state and Kurdistan Workers Party (PKK) dates back to 1984. The government has often believed itself close to military victory only to see the PKK survive. Its leader, Abdullah Ocalan, has been in a Turkish prison since 1999, but the PKK remains the dominant political force among the Turkish Kurds, who number an estimated 14 million – or 18 per cent of Turkey's population.
Iraqis win $5.8m from US firm in Abu Ghraib torture lawsuit
09 January 2013 07:03 PM
Payout to 71 former prisoners is first of its kind from a defence contractor active in Iraq
Court upholds jail terms for activists in Bahrain
07 January 2013 10:18 PM
The highest court in Bahrain has upheld the jail sentences of 20 opposition leaders accused of conspiracy to overthrow the government during pro-democracy demonstrations which began in February 2011.
Syria is many conflicts rolled into one. It is also at the centre of two regional struggles
30 December 2012 12:00 AM
World View: A military stalemate between the government and rebels will make a negotiated settlement inevitable – but not for a long time
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Five things to do for a fiver in London this December
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After splitting-up with former Indian skipper Mohammed Azharuddin, Sangeeta Bijalni found solace in Salman Khan's family. The 53-year old actress spotted very often with Salman Khan's family, bonding with his sisters Alvira and Arpita and other family members. Whenever Salman Kh
Dil Ko Bewaja Dhadakne Ki Aadat Ho Jati Hai - Tehqiqat - With Eeagle Jhankar
Song: Dil Ko Bewaja Dhadkne Ki Aadat Ho Jati Hai Singer: Abhijeet Singh, Alka Yagnik Cast: Aditya Pancholi, Danny, Farheen, Jeetendra, Mehmood, Ronit Roy, Sangeeta Bijlani Music: Anu Malik Release Date: 1993 Presented by: *****mya*****
sangeeta bijlani
Former Team India skipper and Congress MP Mohammad Azharuddin was spotted with his new girlfriend Shannon Marie Talwar. The couple was spotted holidaying in Paris together. Shannon Marie hails from Los Angeles, USA. She studied at Riverside Community College. She is currently based in Delh
The stars of Bollywood could go more unpredictable with their actions than ever. This is evident from the fact that Sangeeta Bijliani came to attend her ex-beau, Salman Khan's birthday party this Thursday. The party was thrown by Salman on his Galaxy Apartment in Mumbai. It seems that neither San


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The Walking Dead: A TellTale Games Series
(PC, iPad, PS3, Xbox 360, Macintosh, iPhone )
This game is wonderful, a true tribute to the series with few flaws. Character driven, fast combat and believable puzzles all come together to form a unique experience. Check out my full review on my profile!
See all The Walking Dead: A TellTale Games Series Reviews >


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Category Archives for Uncategorized
People knew
Let's look at the list of people who knew about Bora Zivkovic's behaviour. Monica Byrne and Hannah Waters knew, after incidents in 2012 and 2010 respectively. Two others who commented on Monica Byrne's post with their own stories about Bora … Continue reading
The Department of Culture's secretive meetings to define 'small blogs'
Earlier today (Wednesday), a meeting took place between the Department of Culture, Media and Sport, and a number of bloggers. The meeting/consultation was invitation only, nothing about it was advertised on the department's website as far as I could tell, … Continue reading
Why the Daily Mail is evil…
(Talk recorded at The Pod Delusion's 3rd birthday in September.)