WO2011049680A1 - Cryosensitizing agents for enhancement of cryoablation - Google Patents

Cryosensitizing agents for enhancement of cryoablation

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WO2011049680A1
WO2011049680A1 PCT/US2010/048079 US2010048079W WO2011049680A1 WO 2011049680 A1 WO2011049680 A1 WO 2011049680A1 US 2010048079 W US2010048079 W US 2010048079W WO 2011049680 A1 WO2011049680 A1 WO 2011049680A1
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cell
vitamin
freeze
freezing
cells
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PCT/US2010/048079
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French (fr)
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John M. Baust
John G. Baust
Anthony T. Robilotto
Kristi K. Snyder
Daniel Klossner
Buskirk Rob G. Van
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Baust John M
Baust John G
Robilotto Anthony T
Snyder Kristi K
Daniel Klossner
Van Buskirk Rob G
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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61BDIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B18/00Surgical instruments, devices or methods for transferring non-mechanical forms of energy to or from the body
    • A61B18/02Surgical instruments, devices or methods for transferring non-mechanical forms of energy to or from the body by cooling, e.g. cryogenic techniques
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/28Compounds containing heavy metals
    • A61K31/282Platinum compounds
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/33Heterocyclic compounds
    • A61K31/335Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/337Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/33Heterocyclic compounds
    • A61K31/395Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine, rifamycins
    • A61K31/495Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine, rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/513Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim having oxo groups directly attached to the heterocyclic ring, e.g. cytosine
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/59Compounds containing 9, 10- seco- cyclopenta[a]hydrophenanthrene ring systems
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/70Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7048Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin digitoxin or digoxin
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K45/00Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K9/00Medicinal preparations characterised by special physical form
    • A61K9/0012Galenical forms characterised by the site of application
    • A61K9/0019Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A61K9/0024Solid, semi-solid or solidifying implants, which are implanted or injected in body tissue
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K9/00Medicinal preparations characterised by special physical form
    • A61K9/14Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/141Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers
    • A61K9/145Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers with organic compounds
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61MDEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M37/00Other apparatus for introducing media into the body; Percutany, i.e. introducing medicines into the body by diffusion through the skin
    • A61M37/0069Devices for implanting pellets, e.g. markers or solid medicaments
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61BDIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B18/00Surgical instruments, devices or methods for transferring non-mechanical forms of energy to or from the body
    • A61B18/02Surgical instruments, devices or methods for transferring non-mechanical forms of energy to or from the body by cooling, e.g. cryogenic techniques
    • A61B2018/0212Surgical instruments, devices or methods for transferring non-mechanical forms of energy to or from the body by cooling, e.g. cryogenic techniques using an instrument inserted into a body lumen, e.g. catheter
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61MDEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M2205/00General characteristics of the apparatus
    • A61M2205/36General characteristics of the apparatus related to heating or cooling
    • A61M2205/3606General characteristics of the apparatus related to heating or cooling cooled
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61MDEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M5/00Devices for bringing media into the body in a subcutaneous, intra-vascular or intramuscular way; Accessories therefor, e.g. filling or cleaning devices, arm-rests
    • A61M5/44Devices for bringing media into the body in a subcutaneous, intra-vascular or intramuscular way; Accessories therefor, e.g. filling or cleaning devices, arm-rests having means for cooling or heating the devices or media

Abstract

This invention identifies a novel class of compounds to act as tissue sensitizing agents. The cryosensitizing agents may include vitamins such as Vitamin D3 or cell stress response modulators such as TRAIL, alone or in combination, including each of their natural or synthetic derivatives and/or analogs. While the cryosensitizing agents act as adjuvants to effect precision in cryotherapeutic procedures, the agents are minimally toxic to non-targeted tissues as well as the patient as a whole. This allows for complete target tissue ablation, including cancerous and benign tumors, within the iceball formation. The molecular agents can be an injectable, implantable, liquid, semisolid, or solid that is non-toxic to cells, tissues, and organs at normal body temperature and become toxic when used simultaneously, pre-treatment and/or post-treatment, at subfreezing temperatures. The present invention will increase the ablative capacity of cryosurgery especially at elevated subfreezing temperatures and improve damage associated with post-thaw apoptosis and necrosis.

Description

CRYOSENSITIZING AGENTS FOR ENHANCEMENT OF CRYOABLATION

RELATED APPLICATIONS

[0001] The present application claims priority to U.S. Provisional Patent Application Serial No. 61/240,863 filed on September 9, 2009 and titled Cryosensitizing Agents for the Enhancement of Cryoablation, which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

[0002] The present invention relates generally to the technology field of medical treatments and ablation procedures and, in particular, to an agent for use in cryo-therapeutic procedures.

BACKGROUND OF THE INVENTION

[0003] Since the mid 1960s, techniques for treating tumors by localized freezing have steadily improved as new instrumentation has been developed and imaging techniques have evolved to control the procedure. As a result, the complications of cryoablation of tumors have been reduced and the efficacy of the technique has increased. Even with optimal application of these techniques, however, some cells survive in the periphery of the frozen volume of tissue, the region in which lethal temperatures are not produced and lead to an undesired persistence of disease.

[0004] At present, cryoablation of tissue is achieved through the application of devices which freeze the target tissue. One of the issues with cryoablation is that for eradication of tissue, temperatures well below freezing must be attained to achieve complete cell destructions. Given the thermal gradient within the iceball during a cryoablation procedure, it is difficult to achieve the necessary temperatures throughout the entire targeted tissue without freezing beyond the target edge which affects non-targeted tissues. This complete freeze is necessary to attain complete ablation of the targeted tissue.

[0005] Specifically, the precision of thermal therapies is compromised by the physics of heat transfer which results in a temperature gradient between the energy producing (heat) and absorbing (cold) sites. This gradient yields a non-uniform zone of lethality within the targeted tissue. Due to the temperature gradient across the treatment zone (e.g. a tumor), cryosurgery does not provide the physician with a highly predictable cancer-kill within the ½ cm to 1 cm margin of the tumor. To overcome this limitation, freezing is often extended ½ cm to 1 cm beyond the margin of the targeted tumor. Such an extensive "positive freeze margin" is problematic for such reasons as restricting the ability to ablate benign tissue (i.e. the prostate, liver tissue adjacent to the bile duct and gall bladder, kidney tissue adjacent to the collecting ducts, breast tissue adjacent to the intercostals of the chest, etc.).

[0006] Sublethal freezing at the tumor margin results in an imprecise "zone of lethality." While we often discuss the "ice ball" in terms of length and width, the freeze zone is three dimensional. Assuming for simplicity sake that the freeze zone is a sphere, its volume is a cubic function (V = 4/3πΓ3). If a 3 cm tumor (volume =14.1 cc) is targeted and a ½ cm positive freeze margin is utilized (volume total = 22.4 cc), 37% of the frozen tissue mass will be benign (non-targeted) tissue. If a 1 cm positive freeze margin (volume = 33.5 cc) is utilized, 58% of the frozen tissue mass will be benign (non-targeted) tissue.

[0007] Particularly, in prostate cancer cryoablation, freezing beyond the prostate has been utilized to assure complete cancer eradication results in the freezing, therefore damaging the adjacent neurovascular bundles and rectal wall. In the treatment of cardiac arrhythmias, freezing can extend beyond the cardiac tissue causing damage to the vagus and phernic nerves which may result in major complications. Management of this collateral damage involves monitoring the freezing process and terminating the process prior to reaching non- targeted structures. This results in the potential for incomplete ablation. The other approach is to disregard the collateral damage and aggressively freeze to ablate the targeted tissue. Post-treatment, the damage is monitored in another manner some time following the ablation procedure.

[0008] Previously, in the treatment of patients with prostate cancer, attention has been focused on the periphery of the frozen zone and the use of agents that will increase the sensitivity of cells to freezing. For this purpose, cryoadjunctive agents, such as cancer chemotherapeutic drugs and other chemical agents or irradiation have been utilized. Reports have indicated synergistic effects of administering low-dose 5-fluorouracil (5-FU) chemotherapy prior to cryotherapy, resulting in improved cell ablation for the treatment of advanced stage prostate cancer in vitro and in animal models. Further studies have investigated the use of Taxotere® and cis-platinum as cryosensitizers. Therefore, by pre- treating cell populations prior to freezing, apoptotic pathways are induced to yield superior cryoablative efficacy.

[0009] In the case of prostate cancer, the reported lethality range is currently on the order of -40°C for cryoablation alone. The -40°C isotherm is located well within the iceball rim, millimeters in width depending on the device utilized. Several studies have been reported using chemotherapeutic agents, including 5-fluorouracil (5-FU), Taxotere®, cis-platimun, and other chemo-agents as adjunctive agents in combination with cryotherapy. Other studies have reported using cell death activation agents including Tumor Necrosis Factor (TNF) alpha and TNF-related apoptosis inducing ligand (TRAIL) in a similar manner. In both in vitro and in vivo settings, these agents have sensitized cells to freezing in the temperature range of -10°C and below. One of the issues with this approach is that the use of chemotherapeutic agents as sensitizers involves the commonly known complications of patient toxicity associated with chemotherapy (e.g. illness, loss of hair, overall cytotoxicty to the patient, target tissue refractory nature). As such, in considering agents to make cryoablation more effective, a new set of morbidity factors associated with the use of chemotherapeutic agents have been taken into account. In the case of TNF alpha, clinical trials were halted by the FDA due to patient toxicity and a series of deaths related to standalone TNF therapy; thus, its use as a cryosensitizer is not possible.

[0010] Cells treated with chemotherapy-based sensitizers, also demonstrate problematic drug resistance. As such, the chemo-agents may yield reduced efficacy and insufficient cancer treatment, especially for hormone -refractory tumors. Further, the timing in use of these agents in relation to the application of freezing remains unclear and clinical benefits have been limited. For instance, since the majority of malignant prostate tumors are androgen-dependent, androgen ablation therapy is primarily used to reduce gland volume to a convenient size for cryosurgical ablation. Although androgen ablation is quite successful within the first two years, the disease eventually progresses to a hormone refractory state due to selection of tumor cells that have adapted to survive without androgen signaling. Currently, there are limited effective therapeutic options for this aggressive, metastatic, advanced stage disease.

[0011] Strategies to improve cryosurgical options are necessary in order to treat new prostate cancer patients and prevent disease recurrence. Improvements will avoid injury to important adjacent structures surrounding the prostate and limit the aggressiveness of freezing. Improved measures will increase the volume of tissue destroyed by bringing the cell-lethal temperature closer to the 0°C isotherm.

[0012] Given the issues associated with cryoablation and other combination therapy approaches, there exists a need for improvements in cryo-therapeutic and cryosurgical procedures. The discovery of cryo-agents and their implementation with developing cryo- technology will eliminate adverse side-effects of the procedure, and will more definitively sensitize targeted tissue. The agents of the present invention will beneficially serve as sensitizing agents to targeted tissue while alleviating toxic effects upon normal cell tissue, and being non-toxic to non-targeted tissues within the immediate area of the targeted tissue as well as to the patient as a whole. Further, the agents of the present invention will allow for complete target tissue ablation within the iceball, also known as the treatment zone, allowing for reduced damage to surrounding healthy tissue (e.g. colorectal damage to sounding tissue structures including neurovascular bundles, rectal wall, vagus nerve, etc.).

[0013] Thus, the successful expansion of cryosurgery across diverse medical specialties will depend in part on improved freeze lethality at the margin of the freeze zone. Technology that provides improved and predictable targeted lethality will be advantageous within the cryosurgical markets while simultaneously improving the use of heat-based therapies as well. Therefore, the discovery and use of sensitizer agents will provide for the priming of target cells and tissues to an enhanced sensitivity to further the damaging effects of freezing. The goal would aim to make the ice lethal throughout the iceball, and lethal to targeted cells in the 0 to -5°C range and colder.

[0014] To overcome these past limitations, a new cryoadjuvant will desirably render ice within the entire freeze zone lethal. Freezing within the distal edge of the hyperachoic rim of the freeze zone will desirably be totally ablative. The cryo-adjuvants will desirably be small molecule agents, or analogs thereof, minimally toxic at physiological doses, commonly consumed orally or injected systemically. The combination of freezing at any subzero temperature with exposure to these agents will therefore create a totally lethal combination following just a single freeze. In effect, the surgical precision of cryosurgery may be dramatically increased giving rise to the concept of a precise "cryo-scalpel." [0015] Furthermore, the invention will facilitate the precise eradication of tissue, decrease hospitalization time, limit postoperative morbidities, shorten return to daily functions and work, and further reduce the overall treatment cost. Desirably, these improvements to the cryo-therapeutic procedure, including application of cryosensitzing agents, will advantageously provide better health treatment options and eliminate unnecessary health effects and time delays that negatively impact healthcare overall.

SUMMARY OF THE INVENTION

[0016] In one embodiment of the invention, a cryosensitizing therapy has an increased cryoablation efficacy, the cryosensitizing therapy increasing sensitivity of cells to freezing injury and comprising one or more adjuvants having a composition including a vitamin, an apoptotic initiator, a free-radical scavenger, a cell-survival pathway inhibitor, a cell death pathway initiator, an ion channel blocker, unfolded protein response (UPR) initiator, an antiinflammatory agent, an apoptotic modulator, a nucleotide modulator, an antioxidant, or a natural dietary supplement(s). In one embodiment, the adjuvants take the form of Vitamin D3, Vitamin D3 analogs, resveratrol or resveratrol analogs. In another embodiment, the adjuvant is a TRAIL polypeptide, ligand, antibody, or protein utilized in combination with a second adjuvant. In one aspect, the adjuvant may be natural or synthetic, utilized alone or in combination with another adjuvant.

[0017] One embodiment of a method of using the invention includes a method of enhancing cryoablation techniques, such techniques comprising the steps of: providing a cryoadjuvant; providing a cryoablation device in the application of freezing target tissue; dosing said target tissue with said cryoadjuvant; contacting said target tissue with said cryoablation device; and freezing said target tissue with said cryoablation device. The cryoablation technique may utilize one or more cryogenic instruments including cryoprobes, cryoneedles, cryocatheters, cryoballoons, or cryoclamps, alone or in combination. The use of such instrumentation may be dependent on the target treatment tissue, the location and peripheries of the target site, as well as the type of thermal therapy or other alternative therapy and its defined protocols. In one aspect, the steps of dosing and freezing are applied in synergistic combination to effectively ablate the target tissue as determined by a treatment protocol. In another aspect, the step of dosing may occur prior to, in conjunction with, or subsequent the step of freezing.

[0018] Another embodiment of the invention utilizes a method of treating one or more cells with a substrate to result in cell ablation, the substrate having minimal toxicity to the cells when utilized alone and minimal toxicity to a patient. Specifically, the method comprises the steps of: providing a substrate having minimal toxicity to said cells; sensitizing a target site with the substrate; and administering a therapy to eradicate one or more of the target sites; wherein the target site includes one or more cells, tissues, or organs. In one embodiment, the therapy is a cryo-procedure such as cryoablation. In another embodiment, the therapy is a hyperthermal ablation procedure such as radiofrequency ablation. Another embodiment utilizes the adjuvant prior, during, or subsequent a therapy.

[0019] In a specified cryo-application, the substrate is a cryosensitizer to selectively target one or more desired cell populations while being non-lethal to surrounding non- targeted cells and tissues. The cryosensitizer comprises a composition that initiates or inhibits apoptosis, a cell survival response, or an unfolded protein response. The method may also utilize a substrate at a sufficient concentration for delivery and sensitization of the target site(s). Further, the method may include a step of packaging the substrate in a form which allows direct or systemic injection, oral administration, spray, topical administration, or through a means which integrates said substrate with a cryoinstrument. In addition, the method may integrate a substrate with a cryo-instrument, such as in coating the cryoinstrument with the substrate.

[0020] One embodiment of the invention utilizes a substrate having a composition that inhibits apoptosis and interacts with a polypeptide, DNA sequence, RNA sequence, protein, or derivative thereof in an apoptotic pathway. In another embodiment, the substrate may be an antioxidant. Another embodiment of the substrate inhibits, maintains, or potentiates activity of a polypeptide, DNA sequence, RNA sequence, protein, or derivative thereof.

[0021] While one embodiment encompasses the substrate as an antioxidant, the substrate may also be selected from the group comprising a vitamin, an apoptotic initiator, a free-radical scavenger, a cell-survival pathway inhibitor, a cell death pathway initiator, an ion channel blocker, an unfolded protein response (UPR) initiator, an anti-inflammatory agent, an apoptotic modulator, a nucleotide modulator, a natural dietary supplements, or a cytotoxic chemical, any of which acts to increase sensitivity of said target site to freezing injury. The method can also have a substrate that includes one or more of: Vitamin D3 and analogs therefrom including cholecalciferol, (24R)-24,25-dihydroxyvitamin D3, la, 25- dihydroxy vitamin D3, loc-hydroxy vitamin D3 and 25-hydroxycholecalciferol, calcidol and calcitriol; resvertrol and analogs therefrom including 3,5-dihydroxystilbene, 3,3',4,5'- tetrahydroxystilbene, 3,4,4', 5-tetrahydroxystilbene, 3,3',5,5'-tetrahydroxystilbene, 3,3',4,5,5'- pentahydroxystilbene, 3,5-dimethoxystilbene, 3,4',5-trimethoxystilbene, 3,3',4,5'- tetramethoxystilbene, 3,4,4', 5-tetramethoxystilbene, 3,3',5'5'-tetramethoxystilbene, and 3,3',4,5,5'-pentamethoxystilbene 3,3',4'-5-trans-tetrahydroxystilbene (piceatannol), 3, 3', 5,5'- trans-tetrahydroxystilbene and 3,3',4',5,5'-trans-pentahydroxystilbene; apoptotic modulators including a TRAIL polypeptide, Bcl2 inhibitor, SIRT-1, PARP, TRADD, AKT, Bid, caspase 8,9,and 3, P53, integrin, Bcl2, pTEN, calpain inhibitors and initiators; UPR modulators including tunicamycin, salubranol, GRP78, CHOP ATF-6, XBP-1, eIF2-p or PERK; ion channel blockers including dronedarone, celivarone, and azimide; nucelotide modulators including DNA, RNA, siRNA, micorRNA, cDNA, RNAi sequences targeting the activation of inhibition of cell death or survival response pathways; and natural dietary supplements including Vitamin E, Vitamin C, Vitamin D, Beta Carotene (Vitamin A), super oxidedismutase, selenium, melatonin, zinc chelators, and/or calcium chelators.

[0022] Another method of the invention includes a step of administering a therapy which comprises a step of targeting at least one of a prostate, liver, kidney, pancreas, lung, bone, skin tissue, brain, breast, digestive system; one or more cardiovascular tissues and structures, heart, blood vessels, circulatory systems; lymphatic systems, lymphatic tissues; or reproductive systems including testis, uterus, endometrial lining, uterine fibroids or ovaries. Where the therapy is cryoablation at a temperature of less than about 1°C within the target site, the cryo-ablation procedure avoids damage or causes minimal damage to non-targeted surrounding cells and tissues. In one aspect, an imaging apparatus visualizes a demarcation at an edge of a target site during the therapeutic procedure. Such a demarcation is a hyperachoic ridge when visualized with an ultrasound imaging apparatus. The visualization of this demarcation allows an operator of the system to control administration of the therapy at the target site and minimize damage to non-treatment areas. Thus, CAT scan, MRI, fluoroscopy, and Xray instrumentation may be utilized in designating time periods or treatment depending on the observed and visualized status of a target site during the treatment procedure. In another embodiment, the procedure includes a step of reducing post-thaw damage to non-targeted surrounding tissues.

[0023] Various embodiments of the present invention take the form of a substrate utilized as an adjuvant to enhance ablation of a targeted cell, tissue, or organ structures, the substrate comprising a composition including one or more of a vitamin, an apoptotic initiator, a free- radical scavenger, a cell-survival pathway inhibitor, a cell death pathway initiator, an ion channel blockers, an unfolded protein response initiator, an anti-inflammatory agent, an apoptotic modulator, a nucleotide modulator, an antioxidant, or a natural dietary supplement, any of which acts to increase sensitivity of cells to freezing injury. In one aspect, the substrate is a comprised of a composition including small molecules which are minimally toxic to cells, tissues, and organs under normothermic conditions (body temperature), but become toxic to target tissue when utilized in conjunction with thermal therapies including, but not limited to cryotherapy and hyperthermia. In another aspect, the small molecules are derived from natural or synthetic chemistry derivatives within a class of agents, the natural or synthetic chemistry derivatives utilized alone or in combination.

[0024] Embodiments of the invention use the substrate in the treatment of at least one carcinoma, but may include treatment of any number and various types of carcinomas or other types of undesired tissue. In one aspect, the target cell (cell, tissue, or organ structure) includes the carcinoma and both may be treated individually or simultaneously, before, during, or after an adjuvant therapy. The substrate may target a cell, tissue, or organ structure that includes at least one of a prostate, liver, kidney, pancreas, lung, bone or skin tissue, brain, breast, or digestive system; or may target a cell, tissue, or organ structure including one or more cardiovascular tissues and structures, blood vessels, or heart; lymphatic systems or lymphatic tissues; or reproductive systems including testis, uterus, endometrial lining, uterine fibroids or ovaries.

[0025] In one embodiment, the substrate takes the form of an injectable formulation that enhances freeze damage from physical effects of ice growth in cells of a prostate capsule or other target tissue, the injectable formulation comprising at least one of the following: a steroid-based vitamin, an apoptotic initiator, a free-radical scavenger, a cell-survival pathway inhibitor, a cell death pathway initiator, an ion channel blockers, an unfolded protein response (UPR) initiator, an anti-inflammatory agent, an apoptotic modulator, a nucleotide modulator, an antioxidant, or a natural dietary supplement. In another embodiment, the injectable formulation provides for post-thaw activation of cellular necrosis and apoptosis in said cells of the prostatic capsule and other target tissue(s). In one aspect, the substrate is an injectable formulation that prevents post-surgical impotence which could potentially result due to freeze damage of one or more nervous tissues of the prostatic capsule and the other targeted tissues. Further, another aspect allows the injectable formulation to protect non-targeted cells from post-thaw damage.

[0026] Advantageously, this medical substrate represents an important step in targeted adjuvant therapies. Though the medical substrate has been developed to enable and improve some of the approaches used to target or ablate tissue, other therapeutic measures may integrally make use of the substrate pre-treatment, simultaneously, and/or post-treatment. Thus, the invention facilitates other improvements in ablation techniques, including in fields of cryotherapy or thermal ablation, and as utilized in conjunction with such medical devices or components associated with ablative treatments.

BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The invention is best understood from the following detailed description when read with the accompanying drawing figures. It is emphasized that the various features are not necessarily drawn to scale. In fact, the dimensions may be arbitrarily increased or decreased for clarity of discussion. FIGS. 1 - 12 depict characterizations of the herein disclosed agent.

[0028] FIG. 1 (PRIOR ART): The use of Taxotere® as a neo-adjunctive treatment.

[0029] FIG. 2: The use of Vitamin D3 as a neo-adjunctive therapeutic agent was evaluated to identify whether treatment efficacy differed for androgen-dependent and - independent prostate cancer cell lines.

[0030] FIG. 3: Tissue engineered matrices containing untreated or 2-day vitamin D3 treated LNCaP LP and LNCaP HP were frozen using a single freezing cycle with a single, centrally placed 17-gauge cryoprobe. [0031] FIG. 4: Tissue engineered matrices containing untreated or 2-day Vitamin D3 treated LNCaP LP and LNCaP HP were frozen using a double freezing cycle (B) with a single, centrally placed 17-gauge cryoprobe.

[0032] FIG. 5: Levels of pro-caspase-3, pro-caspase-8, and pro-caspase-9 levels were determined by western blot for LNCaP LP and LNCaP HP cell lines that were frozen and cell ly sates collected at regular intervals up to 24-hours post- freeze (5A); or first treated for 2 days with 50nM Vitamin D3 and then frozen at -15°C (5B).

[0033] FIG. 6: Caspase-3 (6A), caspase-8 (6B), and caspase-9 (6C) activity was determined for LNCaP LP and LNCaP HPcell lines treated with Vitamin D3 for 2-days prior to freezing.

[0034] FIG. 7: Bcl-2 involvement (7A) in freeze response was evaluated for control (untreated) cells and cells treated with 50nM Vitamin D3 for 2 days prior to freezing. Bcl-2 involvement in post-freeze viability was assessed using the Bcl-2 inhibitor HA 14-1 (25 μΜ) during freezing (7B).

[0035] FIG. 8: Impact of apoptotic mechanism for Vitamin D3 (VD3) as analyzed using caspase inhibitors (20μΜ) in combination with Vitamin D3 treatment (50nM) two days prior to freezing for LNCaP LP.

[0036] FIG. 9: Impact of apoptotic mechanism for Vitamin D3 (VD3) as analyzed using caspase inhibitors (20μΜ) in combination with Vitamin D3 treatment (50nM) two days prior to freezing for LNCaP HP.

[0037] FIG. 10: The use of TRAIL as a neo-adjunctive therapeutic agent was evaluated to identify whether treatment efficacy differed for PC-3 cell lines.

[0038] FIG. 11: Tissue engineered matrices containing untreated or 1-day TRAIL treated PC-3 cells were frozen using a single freezing cycle with a single, centrally placed 17- gauge cryoprobe.

[0039] FIG. 12: The use of Vitamin D3 in combination with TRAIL as a pre-treatment to the cryo-procedure.

DETAILED DESCRIPTION

[0040] In the following detailed description, for purposes of explanation and not limitation, exemplary embodiments disclosing specific details are set forth in order to provide a thorough understanding of the present invention. However, it will be apparent to one having ordinary skill in the art that the present invention may be practiced in other embodiments that depart from the specific details disclosed herein. In other instances, detailed descriptions of well-known devices and methods may be omitted so as not to obscure the description of the present invention.

[0041] Because prostate adenocarcinoma is the second most common cause of cancer-related death in men, improved disease eradication procedures are required to increase a patient's quality of life. Prostate cryosurgical ablation therapy has become a common choice procedure. Furthermore, the age-related transformation from an androgen-dependent (AD) to an androgen- independent (AI) phenotype results in a major prostate cancer treatment challenge. Research indicates that Vitamin D3 demonstrates increased cryosensitizing efficacy against AI cells through its antineoplastic and antimetastatic abilities to increase apoptosis, inhibit cell proliferation, reduce cell migration, and induce differentiation, as associated with androgen signaling pathways. The use of Vitamin D3 as a neo-adjunctive agent prior to cryosurgery has also suggested an increased treatment efficacy for androgen-independent prostate cancer. A series of studies using LNCaP LP (AD) and LNCaP HP (AI) cell lines identify the cellular responses to these treatments; Vitamin D3 surprisingly is a highly effective cryosensitizer for AI cells due to increased mitochondrial-mediated apoptotic activity as a result of Vitamin D3-mediated reductions in Bcl-2 expression. Further investigation indicates that Vitamin D3 neo-adjunctive prostate cryosurgical treatment can reduce disease recurrence for prostate cancer patients.

[0042] In an embodiment of the invention, classes of compounds have been identified that act as sensitizing agents at a target tissue site, but remain non-toxic to minimally toxic in non-targeted tissues within the immediate area of the targeted tissue as well as to the patient as a whole. The class of agents comprises vitamins and various natural and synthetic derivatives and analogs that are highly effective cryosensitizers yet have minimal effect on tissue not targeted before, during, and after cryoablation. Studies show that the use of Vitamin D3, or its analogs, results in complete lethality for prostate cancer cells in the range of about 0°C to -15°C, and even more precisely in the range of about -2 to -5°C. This allows for complete target tissue ablation within the iceball, and thus reduces damage to colorectal tissue and other surrounding tissue structures, including neurovascular bundles, rectal wall, and vagus nerve.

[0043] Specifically, the agents currently utilized which exhibit an increase in cell sensitivity to freezing include cholecalciferol (Vitamin D3), its analogs and derivatives, alone or in combination with apoptotic inducing or inhibiting agents such as TRAIL. While Vitamin D3 is thought to have a beneficial effect on the prevention of diverse types of cancer, including the breast and the colon, the range and characteristics of cellular effects of Vitamin D3 allow it to be utilized as an adjunctive agent in the present invention. Its use in cryosurgery or as an adjunctive treatment to other forms of therapy, including chemo- treatments or thermal therapy, increases the efficacy of the specific therapeutic treatment chosen and reduces the incidence of persistent disease.

[0044] In difficult clinical circumstances, such as androgen-independent prostate cancer, Vitamin D3 is useful as an adjunctive agent to cryosurgical treatment. Research into chemotherapeutic agents identifies the stable metabolite of Vitamin D3, 1,25- dihydroxyvitamin D3 (calcitriol), as demonstrating in vitro and clinical efficacy against androgen-independent prostate cancer with use as an adjuvant to cryotherapy. Vitamin D3 use demonstrates an increase in apoptosis, inhibition of cell proliferation, and reduction in cell migration. However, these discoveries eclipse other findings that mutations in the Vitamin D3 gene lead to increased risk for the development and progression of prostate cancer, which can be prevented or reversed by high dose applications of Vitamin D3. Furthermore, Vitamin D3 treatment induces differentiation in androgen-independent prostate cancer cells, leading to abrogation of aggressive characteristics toward those of early stage disease. Thus, the efficacy of Vitamin D3 treatment upon androgen-independent prostate cancer and its ability to affect proliferation, apoptosis, and differentiation implies that a correlation exists between Vitamin D3 and androgen signaling pathways, especially since they are both members of the steroid nuclear hormone receptor superfamily.

[0045] Research investigating this possibility demonstrates that Vitamin D3 utilizes the androgen receptor non-genotropic signaling pathway to achieve its antineoplastic and antimetastatic properties. For androgen-independent cells that lose androgen receptor expression and receive unregulated growth signaling from growth factor pathways (such as the PI3K/Akt pathway), Vitamin D3 acts as a molecular brake that induces differentiation, reduces metastatic characteristics, and limits growth factor signaling pathways.

[0046] The ability of Vitamin D3 to inhibit growth factor signaling pathways demonstrates the most exciting possibilities for its use as a neo-adjunctive agent. In one aspect, Vitamin D3 is able to inhibit the mitochondrial protein Bcl-2 from suppressing cytochrome c release (which then activates apoptotic caspase cascades). The transition to androgen-independence (and thus loss of androgen receptor) is accompanied by an increase in Akt signaling that leads to an increase in Bcl-2 expression and treatment resistance due to strong antiapoptotic signals. Agents targeting and reducing Bcl-2 levels (thus making cancer cells more susceptible to apoptosis -inducing agents) demonstrates an increase in treatment efficacy in vitro and in vivo. Thus, compared to traditional agents, Vitamin D3 exhibits an increased neo-adjunctive efficacy for cryosurgery due to its ability to reduce Bcl-2 expression.

[0047] The novelty of the present invention presents the use of Vitamin D3 as a neo-adjunctive agent in combination with cryosurgery, or another therapy, that will increase treatment efficacy for androgen-independent prostate cancer and various other disease states. Specifically, the use of Vitamin D3 as a cryosensitizer increases cryoablation efficacy due to increased mitochondrial-mediated apoptotic activity as a result of Vitamin D3-mediated reductions in Bcl-2 expression following combination treatment.

Materials and Methods

Cell Culture

[0048] The human prostate cancer cell line, LNCaP, was obtained from the American Type Culture Collection (ATCC). The LNCaP HP (high passage) cell line was obtained by repeated culture (over 60 passages) of the LNCaP cell line (hereafter called LNCaP LP [low passage]) in low-hormone medium (RPMI-1640 supplemented with 10% charcoal stripped serum [Biomeda] and 1% penicillin/streptomycin [Life Technologies]). Cultures were maintained at 37°C, 5% C02/95% air in RPMI-1640 growth medium (Caisson Labs) supplemented with 10% fetal calf serum (Atlanta Biologies, Inc.) and 1% penicillin/streptomycin (Life Technologies). Cultures were grown in Falcon 75 cm2 T-flasks with medium exchange every 3 days. Subcultures were prepared in Costar® 96-well, removable strip plates at 18,000 cells/well, and experiments were performed 2 days following subculture.

[0049] For tissue engineered prostate studies, rat tail type I collagen solution (BD Bioscience, Bedford, MA) was used to form gel matrices. Cells, 2.5 x 106 cells/mL, were directly suspended in the collagen solution prior to gel solidification in 35 mm Petri dishes. The matrices were cultured 24 hr prior to freezing and media was replenished each day.

Freezing Protocol

[0050] A circulating, temperature-controlled bath provided subzero temperatures, and Costar® strip wells (ΙΟΟμΙ medium/well) were placed into an aluminum block that was partially submerged in the bath. The actual temperature reached in each strip well was measured with thermocouple readings taken at regular intervals. To prevent super-cooling of cell cultures, ice nucleation was initiated by contact with a wire cooled in liquid nitrogen following a fixed cooling period. Following ice nucleation after 3 minutes, cell cultures were allowed to freeze for 12 minutes (15 minutes total). Cultures were allowed to thaw at room temperature before being returned to 37°C. Where indicated, cells were treated with: (1) 0^g/ml or 1/0 μg/ml Taxotere® (Aventis Pharmaceuticals) for two days prior to freezing. (2) 50nM Vitamin D3 (1,25-dihydroxycholecalciferol) (Calbiochem) for two days prior to and during freezing. (3) 25 μΜ Bcl-2 inhibitor HA 14-1 (Sigma) immediately before freezing.

[0051] For the engineered matrices, an argon-based cryosurgical system with 17-gauge argon/helium needle cryoprobes was used for the freezing process. Another cryosystem may include the use of supercritical nitrogen, or variation thereof. Briefly, a single cryoprobe was placed into the center of the engineered prostate model and a single or double freeze cycle was initiated consisting of a 10 min freeze followed by 20 min of thawing at 37°C. The temperature profile of the freeze zone was recorded with an array of copper-constantan (type T) thermocouples placed equidistant and extending radially from the probe tip using an Omega TempScan 1100 (Omega, Stamford, CT). Once thawed, samples were returned to culture for further assessment. Cell Viability

[0052] Cell viability was assessed using a 1:20 dilution of alamarBlue® (Trek Diagnostics) in HBSS every other day following the freezing insult. Diluted alamarBlue® remained on cell cultures for 1 hour at 37°C followed by evaluation of fluorescence using a Tecan SPECTRAFluorPlus plate reader (TECAN Austria GmbH) with an excitation of 530nm and emission of 590nm. Subsequently, cell cultures were aspirated and returned to normal culture.

Data Analysis

[0053] Fluorescence units were converted to percent survival based on an experimental control (37°C) before freezing. Calculations of standard error were performed and statistical significance was determined by single-factor Analysis of Variance (ANOVA).

Western Blot

[0054] In order to obtain protein samples for western blot analysis for protein expression, LNCaP LP and LNCaP HP cell cultures were cultured in 100mm Petri dishes and frozen at - 15°C for 15 minutes. Cell lysates (detached and adherent) were collected on ice using cell scraping for control (unfrozen) samples at 1, 3, 6, 12, and 24 hours post-thaw using ice-cold RIPA (Radio-Immunoprecipitation Assay) cell lysis buffer with phosphatase inhibitor (sodium fluoride ImM, sodium orthovanadate ImM, sodium pyrophosphate ImM), leupeptin lug/ml, PMSF (phenylmethylsulphonylfluoride ImM), and lx Halt Protease Cocktail Inhibitor (Pierce). Samples were homogenized by vortex mixing and centrifuged at 16,000 x g for 15 minutes at 2°C. Protein concentrations were quantified with standard bicinchonic acid protein assay (Pierce) and assessed with a Tecan SpectraFluorPlus spectrophotometer. Equal amounts of protein (25μg) were loaded in each lane and separated by 10% SDS-PAGE (Bio-Rad). The proteins were transferred to PVDF membranes (Bio-Rad), blocked with 3% BSA solution containing 0.05% Tween 20, and incubated at 4°C overnight in the presence of ^g/ml of each antibody (mouse monoclonal anti-human β-tubulin [BD Pharmingen], mouse monoclonal anti -human pro-caspase-3 [Cell Signaling], mouse monoclonal anti-human pro- caspase-8 [Cell Signaling], rabbit polyclonal anti-human pro-caspase-9 [Cell Signaling], mouse monoclonal anti-human Bcl-2 [Cell Signaling], or mouse monoclonal anti -human bax [Cell Signaling]). The membrane was washed three times with 0.05% Tween 20 in PBS and exposed with horseradish peroxidase conjugated secondary antibodies. The Fujifilm Las-3000 luminescent image analyzer was used for detection.

Fluorescent Imaging

[0055] To assess freeze-associated cell death pathways, LNCaP LP and LNCaP HP were cultured in Costar® 96- well, removable strip plates and exposed to 15 minute freezing regimens at -15°C. Cultures were monitored by triple labeling using the fluorescent probes (Molecular Probes): Hoechst (blue fluorescence, 0.06μg/μl), propidium iodide (red fluorescence, 0.007μg/μl), and YO-PRO®-l (green fluorescence, 0.8μΜ) to detect living cells, necrotic cells (freeze -ruptured), and apoptotic cells respectively. After a 20 minute incubation period, stained cells were visualized using fluorescence microscopy at control and 24 hours post-thaw time points using a Zeiss Axiovert 200M microscope at 240X magnification.

Caspase Activity Assays

[0056] Protein samples for caspase-3, -8, -9 activity assays were obtained from LNCaP LP and LNCaP HP cultures in 100mm Petri dishes frozen at -15°C for 15 minutes. Cell lysates were collected on ice using a cell scraper for control (unfrozen) samples and at 1, 3, 6, 12, and 24 hours post-thaw using ice-cold RIPA (Radio-Immunoprecipitation Assay) cell lysis buffer without protease or phoshosphatase inhibitors. Protein concentrations were quantified with BCA protein assay (Pierce) and assessed with a Tecan SpectraFluorPlus spectrophotometer. Equal amounts of protein (50μg) were tested in duplicate for caspase activity using the BD ApoAlert™ Caspase Fluorescent Assay Kits for Caspase-3, -8, and -9 which measure the conversion of non-fluorescent substrate to a cleaved, fluorescent form.

Results

Taxotere® Neo-adjunctive Treatment Exhibited Reduced Neo-adjunctive Efficacy with Pmdro gen-Independent Cells LNCaP HP

[0057] The use of neo-adjunctive low-dose chemotherapy (in vitro and in animal studies) prior to cryotherapy exhibits increased efficacy over either treatment alone for the management of prostate cancer. In studying chemocryotherapeutic efficacy between early stage, AD, and late stage, AI, prostate cancers, prostate cancer cell monolayers of AD LNCaP LP and AI LNCaP HP were treated with low-dose Taxotere® (generically known as docetaxel) for two days prior to freezing at -15°C for 15 minutes (FIG. 1). Metabolic indicator alamarBlue® assessed cell viability post-freeze. LNCaP LP and PC-3 AR exhibited susceptibility to low-dose drug exposure (p < 0.05), but biologically insignificant differences occurred between 0.5 μg/ml and 1.0 μg/ml concentrations. Combination drug and freeze treatment achieved total ablation after experiencing apoptotic cell death over the 9-day assessment period. Similarly, LNCaP HP and PC-3 exhibited susceptibility (p < 0.05) to low-dose drug exposure, but biologically insignificant differences occurred between 0.5 μg/ml and 1.0 μg/ml concentrations. However, androgen-independent cell lines exhibited significant (p < 0.05) recovery after 7 days, indicating insufficient ablation. For LNCaP LP, 2-day Taxotere® treatment alone induced a delayed cell death response that reduced cell viability to 40% 7 days after exposure, at which time cell viability began to recover.

[0058] Biologically insignificant differences in cell response occurred for both Taxotere® concentrations, indicating similar efficacy. The combination of chemocryotherapy exhibited superior efficacy over either treatment alone in each cell type. LNCaP LP cells experienced significantly (p < 0.05) different 1-day post-freeze neo-adjunctive viability for the 0.5 μg/ml and 1.0 μg/ml doses, with the greater dose showing increased ablative effects. For combination treatment at the l^g/ml dose 1-day post-freeze, LNCaP LP exhibited 2.5 times greater ablation efficacy than freeze alone. Furthermore, cells treated with combination therapy were unable to recover over the 9-day assessment period, indicating that low-dose Taxotere® in combination with cryotherapy exhibited complete ablation for AD LNCaP LP cells. For LNCaP HP, 2-day Taxotere® treatment alone induced a delayed cell death response that reduced cell viability to 70% for LNCaP HP 7 days after exposure, at which time cell viability began to recover. The chemocryotherapy combination exhibited superior efficacy over either treatment alone. For combination treatment at the 1.0μg/ml dose 1-day post- freeze, LNCaP HP exhibited 1.3 times greater ablation efficacy than freeze alone, indicating that chemocryotherapy was significantly less effective on AI cells compared with AD cell lines. Furthermore, cells treated with combination therapy began to recover after 7- days post-freeze, indicating that low-dose Taxotere® in combination with cryotherapy exhibited incomplete ablation for AI cells.

Vitamin D3 Neo-Adjunctive Therapy Demonstrated Neo-adjunctive Efficacy for

Androgen-Dependent and -Independent Cells

[0059] Since Taxotere® combination freezing treatment (FIG. 1) demonstrated an inability to completely ablate androgen-independent cell lines LNCaP HP and PC-3 as shown by cell repopulation in vitro, the use of Vitamin D3 as a neo-adjunctive agent for cryotherapy was investigated to determine whether improved prostate cancer treatment could be achieved.

[0060] The use of Vitamin D3 as a neo-adjunctive therapeutic agent was evaluated to identify whether treatment efficacy differed for androgen-dependent and androgen-independent cell lines. As shown in FIG. 2, prostate cancer cell monolayers of LNCaP LP and LNCaP HP were treated with 50nM Vitamin D3 (VD3) for two days prior to freezing at -15°C for 15 minutes and a metabolic indicator alamarBlue® was used to assess cell viability post-freeze. LNCaP LP and PC-3 AR (A) treated for 2-days with Vitamin D3 exhibited continuous cell death over the 9-day assessment period, while combination drug and freeze treatments achieved total cell ablation with lack of re-growth. Similarly, Vitamin D3 treated LNCaP HP and PC-3 (B) exhibited continuous cell death over the 9-day assessment period. In contrast to FIG. 1, combination drug and freeze treatments achieved total cell ablation (p < 0.05) with lack of re-growth for androgen-independent cell lines, indicating Vitamin D3 as a promising neo-adjunctive chemotherapeutic agent.

[0061] For LNCaP LP (FIG. 2 Left), 2-day Vitamin D3 treatment alone induced a delayed cell death response that reduced LNCaP LP cell viability to 70% by 9 days after exposure. Cells were unable to recover during the assessment period (when cell recovery was monitored) after exposure to Vitamin D3 alone. A combination of Vitamin D3 and cryotherapy exhibited superior efficacy over either treatment alone. For combination treatment 1-day post-freeze, LNCaP LP exhibited 1.5 times greater ablation efficacy compared to freeze alone. LNCaP LP cell viability dropped to 0% by 3 -days post- freeze and therefore unable to recover over the 9-day assessment period, indicating that Vitamin D3 in combination with cryotherapy exhibited complete ablation for AD LNCaP LP cells. For LNCaP HP (FIG. 2 Right), 2-day Vitamin D3 treatment alone induced a delayed cell death response that reduced cell viability to 83% by 9 days after exposure. The combination of Vitamin D3 and cryotherapy exhibited superior efficacy over either treatment alone. For combination treatment 1-day post-freeze, LNCaP HP exhibited 2.2 times greater ablation than freeze alone, indicating that Vitamin D3 is significantly more effective as compared to Taxotere® (which showed repopulation) use in these treatments. Furthermore, LNCaP HP cell viability declined after combination treatment and cell populations were unable to recover. These data indicate that Vitamin D3 in combination with cryotherapy can complete ablate both AD and AI cells.

Vitamin D3 Neo-adjunctive Therapy Exhibited Efficacy in Tissue-Engineered Matrices

[0062] Because in vitro cell analysis provides optimum conditions for cell repopulation, a tissue engineered prostate model in FIGS. 3 and 4 was utilized to assess post-freeze viability that may more closely simulate clinical cryosurgery. Tissue engineered matrices containing untreated or 2-day Vitamin D3 treated LNCaP LP and LNCaP HP were frozen using a single freezing cycle (FIG. 3) or a double freezing cycle (FIG. 4) with a single, centrally placed 17- gauge cryoprobe. Temperatures were monitored using a thermocouple array. Matrices were frozen for a single or double 10 min freeze followed by return to 37 °C. Twenty-four hours post-thaw, matrices were stained with calcein AM (green, live cells) (Molecular Probes) and propidium iodide (red, dead cells). A 50X panoramic series of fluorescent micrographs taken from the center near the cryoprobe tip (left of images) to the periphery of the ice sphere (right of image) shows that Vitamin D3 treatment prior to freezing demonstrates superior ablation as compared to freeze alone. Note: In the black and white depiction, the dark gray and black regions represent areas of dead cells while the light gray and white areas represent living cells. An increased level of dead cells, dark gray and black cells, appear at warmer temperatures in the Vitamin D3 and freezing treatment compared to freeze alone.

[0063] Overall, data from the engineered model experiments support the viability data in FIG. 2, showing that Vitamin D3 neo-adjunctive treatment is equally effective for AD and AI cells. LNCaP HP displayed increased resistance to freezing alone as shown with cell monolayers. Extensive cell survival was observed to the -30°C isotherm, and sparse cell survival occurred to -40°C. In contrast, AD LNCaP LP displayed cell survival to the -20°C isotherm for freeze alone. [0064] The Vitamin D3 treatment in combination with freezing achieved two effects. First, the treatment reduced the density of surviving cells for each cell line tested. Second, the margin of surviving cells was reduced by half. For example, with freezing alone, LNCaP HP exhibited cell survival at -35°C to -40°C, but Vitamin D3 treatment in combination with freezing limited survival to the -15°C to -20°C range.

[0065] As utilized in a cryosurgical procedure clinically and depicted in FIG. 4, a double freeze cycle demonstrated similar results. The tissue engineered prostate model was used to evaluate cell death resulting from double freeze cycle alone or double freeze cycle for cells treated with Vitamin D3 two days prior to freezing. LNCaP LP and LNCaP HP were frozen as described above but consisting of a second freeze-thaw cycle.

[0066] Overall, a double freeze cycle (untreated and Vitamin D3 treated) exhibited increased cell death and cryoablative efficacy as compared to a single freeze cycle. Similar to single freeze cycle, Vitamin D3 treatment in combination with double freeze cycle reduced the density of surviving cells and reduced survival margin by half for all cell lines tested. For example, with freezing alone, LNCaP HP exhibited cell survival at -20°C to -25°C, but Vitamin D3 treatment in combination with freezing was able to limit survival to the -10°C to - 15°C range. These tissue-engineered prostate models for single and double freeze cycle corroborated in vitro data suggest use of Vitamin D3 as a neo-adjunctive chemotherapeutic agent in combination with freezing.

Vitamin D3 Treated Androgen-Independent Prostate Cell Lines Exhibited Differentially Increased Freezing-Induced Apoptosis and Necrosis Levels

[0067] The significant efficacy of neo-adjunctive cryotherapy for both AD and AI cell lines prompted the investigation into the differences in cell death mechanisms that might be occurring to achieve ablation. Thus, total levels of apoptotic and necrotic cell death were evaluated in vitamin D3 treated LNCaP LP and LNCaP HP cell lines using qualitative microscopy. Cell cultures were frozen at -15°C and triple-probe fluorescent micrographs were taken using Hoechst fluorescence to identify viable cells, propidium iodide to identify necrotic cells, and YO-PRO®-l to identify apoptotic cells. Total levels of necrotic and apoptotic cell death were evaluated in LNCaP LP and LNCaP HP cell lines treated with 50nM vitamin D3 for 2 days prior to freezing. [0068] For LNCaP LP cells subjected to freeze only, little apoptosis and necrosis in control (unfrozen) samples occurred, but subsequent to freeze at -15°C, apoptosis peaked after 3 hours and remained elevated up to 24 hours post-freeze. Compared with freeze alone, Vitamin D3 treated LNCaP LP cells exhibited increased levels of apoptosis and necrosis for control (unfrozen) samples, while post-freeze time points showed only slightly increased levels. Quantitative analysis of the 3-hour time point indicated more interesting differences between freeze only and freeze with Vitamin D3 treatment. For freeze alone at the 3 hour post-freeze time point, LNCaP LP exhibited 22.1% + 1.2% viability, 58.2% + 0.8% necrosis, and 19.7 + 1.5% apoptosis, while the 3 hour post-freeze sample treated with Vitamin D3 exhibited 14.2% ± 1.1% viability, 60.2% + 0.7% necrosis, and 25.6% + 0.9% apoptosis.

[0069] Compared to AD cells, the AI LNCaP HP cells subjected to freeze alone exhibited less overall apoptosis and necrosis. Little apoptosis and necrosis occurred in control (unfrozen) samples, but subsequent to freeze at -15°C, apoptosis peaked after 6 hours and rapidly declined by 24 hours post-freeze. Compared with freeze alone, Vitamin D3 treated LNCaP HP exhibited significantly (p < 0.05) increased levels of apoptosis and necrosis. Post-freeze time points continued to show dramatically increased levels of apoptosis and necrosis, with peak levels occurring after 3 hours post-freeze, indicating a more rapid apoptotic climax compared with freeze alone. Quantitative analysis of the 3-hour time point revealed the most interesting differences between freeze only and freeze with Vitamin D3 treatment. For freeze alone at the 3 hour post-freeze time point, LNCaP HP exhibited 63.7% + 1.4% viability, 23.4% + 0.6% necrosis, and 12.9% + 1.1% apoptosis, while the 3 hour post- freeze sample treated with Vitamin D3 exhibited 27.3% + 0.6% viability, 50.6% + 1.2% necrosis, and 22.1 + 1.4% apoptosis. Interestingly, the AI LNCaP HP cells exhibited a greater increase in apoptosis than occurred with the AD cells. These data indicate that Vitamin D3 treatment prior to freezing significantly (p < 0.05) increases both necrotic and apoptotic cell cascades.

Vitamin D3 Treatment Increases Mitochondrial-Mediated Apoptosis

[0070] In order to more closely identify a specific caspase cascade, western blot analysis was performed to assess whether changes in pro-caspase-3, pro-caspase-8, and pro-casase-9 expression correlated significantly with prostate cancer cell survival following freezing alone and vitamin D3 neo-adjunctive treatment (FIG. 5). Pro-caspase levels for freeze alone (FIG. 5A) revealed that the AD LNCaP LP showed greater uncleaved control levels of pro-caspase-3, pro-caspase-8, and pro-caspase-9 compared to the AD LNCaP HP cells, which is indicative of a greater overall potential for apoptotic involvement for LNCaP LP. Furthermore, LNCaP LP exhibited decreases in pro-caspase-3, -8, and -9 levels by 3 hours post-freeze indicating possible protein cleavage to an active form. Pro-caspase-9 exhibited greater changes (reductions) in protein levels than pro-caspase-8 post-freeze, which indicates greater caspase-9 based mitochondrial mediated apoptotic involvement. Interestingly, compared to LNCaP LP, LNCaP HP exhibited significantly lower pro-caspase-8 levels, and both pro-capase-8 and pro-caspase-9 showed few significant changes post-freeze.

[0071] Cells treated with Vitamin D3 2-days prior to freezing (FIG. 5B) exhibited significant differences compared to untreated, frozen cells. While all cell lines experienced a decline in pro-caspase-3 levels, few changes post-freeze indicate caspase-3 activation. For LNCaP LP treated with Vitamin D3, reduced pro-caspase-8 expression levels were observed compared with freeze alone. Vitamin D3 treated AD cells, however, exhibited higher pro-caspase-8 expression that decreased up to 6 hours post- freeze followed by an increase by 24 hours, which was the same trend observed for freeze alone. Similarly, LNCaP HP exhibited lower levels of pro-caspase-8, as exhibited by freeze alone. Thus, the data indicate that the caspase- 8 apoptotic pathway may not differ significantly from freeze alone.

[0072] Both Vitamin D3 treated cell lines exhibit similar trends for post-freeze pro-caspase-9 expression that differ significantly from freeze alone. Pro-caspase-9 levels in Vitamin D3 treated control (unfrozen) samples exhibited comparable pro-caspase-9 levels with untreated controls (unfrozen). However, post- freeze Vitamin D3 treated cells exhibited significant declines in pro-caspase-9 expression, indicating that caspase-9 activity may significantly increased post- freeze for AD and AI cells after Vitamin D3 treatment.

Vitamin D3 Treated LNCaP HP and PC-3 Cells Exhibited Greater Post-freeze Caspase Activity Compared with Freeze Alone

[0073] To more accurately portray the actual levels of caspase-cleavage, which would result in apoptotic cascade activation, FIG. 6 (6A, 6B, 6C) illustrates the activity of several specific caspases that were investigated to determine which, if any, caspase-specific pathway may be involved in the different freezing responses of Vitamin D3 treated AD and AI cell lines. Briefly, the levels of caspase-3 (a common effector or "executioner" apoptotic member), caspase-8 (a membrane-mediated apoptotic member), and caspase-9 (a mitochondrial-mediated apoptotic member) activity were collected and evaluated from post- thaw samples at regular intervals over a 24-hour period.

[0074] Caspase-3 activity analysis (FIG. 6A) revealed several differences. For freeze only, LNCaP LP exhibited overall increased levels of caspase-3 activity (compared with LNCaP HP) that peaked at 3 hours post-freeze and maintained at peak activity levels at 6 hours, indicating that freezing rapidly induced apoptotic death cascades. Conversely, LNCaP HP exhibited lower overall levels of caspase-3 activity for freeze alone that peaked after 6-hours post-freeze, which indicated a more delayed activation of cell death cascades. For freeze at - 15°C alone, peak caspase-3 levels for LNCaP LP were 2.8 times greater than peak LNCaP HP levels. However compared to freezing alone, LNCaP HP cells treated with Vitamin D3 prior to freezing exhibited significantly increased (p < 0.05) post-freeze caspase-3 activity. Interestingly for Vitamin D3 treated cells, peak caspase-3 levels for LNCaP LP were 1.7 times greater than peak LNCaP HP levels, indicating that Vitamin D3 treatment induced similar post-freeze caspase-3 activity in AD and AI cell lines. In sharp contrast to freeze alone, Vitamin D3 treated AI cells displayed increased caspase-3 activation that extended to 24-hours post-freeze, indicating that apoptotic cascades were prolonged, while with freezing alone caspase-3 activity declined to near control levels in that same period of time. The data indicated that Vitamin D3 treatment prior to freezing was able to increase peak caspase-3 activity and duration in AI cells more significantly than was observed for AD cells.

[0075] For cells with Vitamin D3 pre-treatment (FIG. 6B), post-freeze caspase-8 activity exhibited trends that were very similar to freeze responses of cells with freezing alone. LNCaP LP cells exhibited greater maximum levels of caspase-8 activity after Vitamin D3 treatment than was observed for LNCaP HP, but the overall caspase-8 activity and changes observed post-freeze were not biologically significant compared with freezing alone. For freeze at -15°C alone, peak caspase-8 levels for LNCaP LP were 7.1 times greater than peak LNCaP HP levels. Similarly, for cells with vitamin D3 pre-treatment, peak caspase-8 levels for LNCaP LP were 7.4 times greater than peak LNCaP HP levels. These data indicate that, although overall caspase-8 activity declines to a lower level for AI cell lines, caspase-8 activity is not different for Vitamin D3 treated cells compared to freeze alone.

[0076] Analysis of caspase-9 activity (FIG. 6C) showed significant differences resulting from Vitamin D3 treatment. LNCaP LP treated with Vitamin D3 exhibited greater maximum levels of caspase activity that peaked 3 hours post-freeze compared to LNCaP HP. Although LNCaP HP overall activity was lower, peak activity results within 3 hours post-freeze, a different result than for freeze alone (6 hours post-freeze). For freeze at -15°C alone, LNCaP LP showed 2.7 times greater caspase-9 activity than LNCaP HP. However for cells with Vitamin D3 treatment prior to freezing, peak caspase-9 levels for LNCaP LP were 1.3 times greater than peak LNCaP HP levels. Furthermore, Vitamin D3 treated LNCaP HP cells showed significant differences in caspase-9 activity post-freeze and significant changes compared to controls over the recovery period following freezing. The elevated caspase-9 activity for AD LNCaP HP indicated that Vitamin D3 treatment prior to freezing was able to increase mitochondrial mediated apoptotic cascades to levels comparable with AD cells.

Vitamin D3 Decreased Post-freeze Expression of the Bcl-2 Anti-apoptotic Protein

[0077] Fluorescent probe analysis demonstrates that Vitamin D3 treatment prior to freezing significantly increases overall apoptotic cell death. Caspase activity assays have discovered significant increases in caspase-9 activity, which may initiate the mitochondrial-mediated apoptotic cell death pathway. These changes induced by Vitamin D3 treatment prompted the investigation into expression levels of the anti-apoptotic mitochondrial protein Bcl-2. Therefore, as shown in FIG. 7, post-freeze Bcl-2 levels were screened for LNCaP LP and LNCaP HP cell lines treated with Vitamin D3 prior to freezing, and those protein levels were compared to time-matched untreated controls exposed to freezing only. For freeze only, LNCaP HP exhibited greatly increased Bcl-2 expression for control (unfrozen) and 1-hour post-freeze sample. By 3 hours post-freeze, Bcl-2 levels in LNCaP HP declined slightly, but Bcl-2 levels were maintained up to 24-hours post-freeze. In contrast, LNCaP LP exposed to freeze only exhibited lower levels of Bcl-2 expression in controls, and Bcl-2 expression increased by 3 hours post-freeze. Subsequently, Bcl-2 expression levels in LNCaP LP declined significantly by 6-hours post-freeze and continued to decline to 24 hours post- freeze. Cells treated with Vitamin D3 prior to freezing treatment exhibited significantly different post-freeze Bcl-2 expression responses. Compared to freeze alone, LNCaP HP exhibited reduced control Bcl-2 levels. Subsequent to freezing, LNCaP HP exhibited a decline in Bcl-2 expression up to 24 hours post-freeze. These data contrast with freeze alone, indicating that AI cell lines demonstrate reduced ability to maintain expression levels of the anti-apoptotic protein Bcl-2 that result in increased post-freeze apoptotic cell death. LNCaP exhibited similar reductions in Bcl-2 expression for both control (Vitamin D3 treated only) and frozen samples which indicates increased post-freeze mitochondrial-mediated apoptotic activity as a result of Vitamin D3 exposure.

[0078] In FIG. 7A, Western blot analysis indicated that Vitamin D3 treatment mediated a reduction in Bcl-2 expression levels that might possibly increase susceptibility to freezing. In FIG. 7B, in order to investigate if a reduction in Bcl-2 activity might affect post- freeze survival, LNCaP LP and LNCaP HP cells were exposed to 25 μΜ the Bcl-3 inhibitor HA 14-1 immediately prior to freezing. Overall, addition of the Bcl-2 inhibitor significantly (p < 0.05) reduced post-freeze viability for each cell line, but different responses occurred between them. Compared to freeze alone, LNCaP LP experienced reductions in cell viability of 4.7% + 1.5% in the presence of Bcl-2 inhibitor. Interestingly, LNCaP HP exhibited a much larger reduction in cell viability when frozen with Bcl-2 inhibitor. Compared to freeze alone, LNCaP HP experienced a 20.3% + 2.3% + 1.5% reduction in viability in the presence of Bcl-2 inhibitor. Thus, these data support the possibility that the reduction of Bcl-2 levels by Vitamin D3 treatment differentially affect apoptotic cell death cascades following neo- adjunctive cryotherapeutic treatment.

Caspase Inhibitors Corroborate Mitochondrial-mediated Apoptotic Mechanism for Vitamin D3

[0079] Data demonstrates that apoptosis plays a significant role in the efficacy of the Vitamin D3 and freezing combination (FIG. 6). Further, this increase in cryosurgical efficacy may be through a mitochondrial-mediated apoptotic mechanism (FIG. 7). In order to provide supporting evidence for this mechanism, LNCaP LP and LNCaP HP were treated with Vitamin D3 and caspase inhibitors (caspase-8, caspase-9, or pan-caspase) two days prior to freezing (FIGS. 8 and 9) in order to determine if caspase inhibitor might inhibit Vitamin D3 efficacy. [0080] In FIG. 8, LNCaP LP exhibited increased post-freeze viability for all caspase inhibitors. Compared to freezing alone, post-freeze viability was increased by 14.4% with caspase-8 inhibitor, 24.4% with caspase-9 inhibitor, and 45.1% with pan-caspase inhibitor, indicating that apoptosis plays a large role in post- freeze survival of AD LNCaP LP cells. Additionally, pre-treatment with Vitamin D3 reduced LNCaP LP post-freeze viability by 2.6 times. Addition of caspase-8 inhibitor with Vitamin D3 showed post-freeze viability that was not statistically different from freezing with Vitamin D3 alone. Compared to Vitamin D3 pretreatment alone, however, addition of caspase-9 and pan-caspase inhibitors significantly (p < 0.05) increased post-freeze viability by 1.7 times and 2.3 times, respectively.

[0081] In FIG. 9, as compared to freezing alone for LNCaP HP, post-freeze viability increased by 19.5% for pan-caspase inhibitor, while the caspase-8 and caspase-9 inhibitors showed post-freeze responses showed no statistical difference from freeze alone. Pretreatment with Vitamin D3 reduced LNCaP HP post-freeze viability by 2.2 times, that is comparable to LNCaP LP. Similar to LNCaP LP, the addition of caspase-8 inhibitor with Vitamin D3 showed a post-freeze viability that was not statistically different from freezing with Vitamin D3 alone. Compared to Vitamin D3 pretreatment alone, however, addition of caspase-9 and pan-caspase inhibitors significantly (p < 0.05) increased post-freeze viability by 2.1 times and 2.4 times, respectively. These data collectively indicate that the mitochondrial-mediated apoptotic pathway is an important mechanism for Vitamin D3 efficacy when combined with freezing.

Cryoablation enhances TRAIL-induced cytotoxicity

[0082] In FIG. 10, PC-3 cells were exposed to TRAIL (500ng/ml), freezing (-5, -10, -15, - 20°C), TRAIL prior to freezing, or TRAIL and freezing combined at the same time. Cell viability was then assessed 24-hours post-exposure to evaluate the efficacy of each of the conditions. Again, TRAIL exposure alone resulted in a minimal loss of viability. Freezing alone to temperatures of -5, -10, -15, and -20°C resulted in a 5%, 15%, 35%, and 70% decrease in viability respectively. When TRAIL exposure was followed by freezing, a significant increase in cell death was evident at all temperatures tested. Following this combination, a 25%, 80%, 90%, and 100% decrease in viability was observed when TRAIL was followed by each of the freezing temperatures. In each case, except for those exposed to -20 C, cells began to repopulate over the recovery period. When TRAIL and freezing were applied simultaneously, a greater loss in viability occurred. Applying this combination to prostate cancer (PC-3) cultures led to a 50% initial decrease with the TRAIL and -5°C exposure, while combinations with each of the other temperatures yielded 100% ablation. After 7 days of analysis, the TRAIL/-5°C combination was the only condition that resulted in cell survival. Similar trends were also documented using lower concentrations of TRAIL (Data not shown).

[0083] The relative resistance of prostate cancer cells to the deleterious effects of cryoablation has been demonstrated. By combining chemotherapeutic agents with freezing results in a synergistic enhancement of cell death as compared to the treatments tested with single agents. These studies also revealed that addition of the chemotherapeutic agents prior to freezing resulted in the greatest level of cell death.

Freeze and TRAIL combination induces apoptosis

[0084] The viability data in the previous section revealed that TRAIL cytotoxicity could be significantly enhanced when combined with cryoablation. Additional studies were then designed to examine whether the combination led to an increase in PC-3 cell apoptosis. PC-3 cells were grown in a collagen gel (2mm thick) to form a 3D environment. The cells were then exposed to freezing or the simultaneous combination as described in the materials and methods section. The freezing process, similar to in vivo situations, generates a freeze zone extending from the center of the gel. Furthermore, a temperature gradient is produced with the lowest temperature (-85°C) at the center, and elevated temperatures (0°C) at the periphery.

[0085] While both freezing and the combination reveal similar patterns of cell death directly after exposure, a significant burst of apoptosis was induced at 6-hours post treatment with the combination treatment. The apoptotic event peaked at 12-hours and appeared to lessen by 24-hours, although this may be due to the lack of viable cells present at this time. A similar trend was observed when PC-3 cells were exposed to the TRAIL and freezing to - 15°C, except that the apoptotic induction peaked at 6 hours post-exposure as compared to 12 hours when -10°C was applied. In each study, few viable cells remained at 24-hours-post following exposure to the combination. Effects of freezing and the freezing/TRAIL combination on caspase activation

[0086] Previously, the combination of cisplatin or 5-fluorouracil followed by freezing proved to activate the mitochondrial apoptotic pathway through an increase in the Bax to Bcl- 2 ratio. Studies were performed to determine which apoptotic pathway was activated when TRAIL and freezing were combined. First, PC-3 cells were either exposed to freezing or the combination treatment, and then cell lysates were collected over a 24-hour time course. Western blot analysis revealed that the combination of freezing with TRAIL induced TNF-R associated death domain (TRADD) activation. TRADD (34 kDa) levels decreased by 6- hours after exposure to the combination and continued to decrease over the remainder of the 24-hour time course. Freezing also appears to induce a change in TRADD levels around 6- hours, but this is a minimal decrease as levels return to controls by 24-hours. Protein analysis also shows that procaspase-8 (57 kDa) was cleaved to the intermediate/active (41/43 kDa) by 6 hours following exposure to the combination. As shown in FIG. 11, the cleaved caspase-8 persisted at both the 12 and 24-hour time points. Caspase-8 was also cleaved following freezing alone, but only a small amount of the cleaved form was noted. The results thus far indicated that the combination treatment may induce a caspase-8 mediated death cascade, and also suggested a membrane mediated involvement in freezing-induced cell death. Interestingly, the combined treatment also resulted in an apparent activation of caspase-9. Procaspase-9 (47 kDa) decreased at 6 and 12-hours post-exposure before returning to control levels by 24-hours {See FIG. 11). Freezing alone did not induce a similar change in procaspase-9 levels. Protein analysis also demonstrated downstream apoptotic events including caspase-3 (32 kDa) and Poly-ADP Ribose Polymerase (PARP) (113 kDa) cleavage following exposure to either condition. A greater enhancement of cleavage was promoted by the cytotoxicity induced by the freeze/TRAIL combination. TRAIL alone did not result in caspase activation (data not shown).

Effects of Vitamin D j and TRAIL combined with freezing

[0087] With the observed benefits of Vitamin D3 and TRAIL as cryosensitizers, studies were conducted to evaluate if the combined utilization of these agents in conjunction with freezing could further improve cancer destruction (FIG. 12). Initial studies revealed that the Vitamin D3 and TRAIL (VD3/TRAIL) combination was highly effective at temperatures below -15°C (data not shown). Given this, samples were exposed to the combination and frozen to -5°C. The combination VD3/TRAIL cryosensitization and freezing to -5°C resulted in enhanced cell death compared to any of the treatments individually or in combination.

Discussion

Vitamin D3

[0088] Cryosurgery is an option for treating either early or advanced (localized) prostate cancer and its efficacy has been well documented. For patients with localized (early or advanced stage) prostate cancer, cryosurgery demonstrates superior efficacy and improved long-term disease-free prognosis. Despite successes in disease treatment, cryosurgery still results in low, yet significant, recurrence rates, which are greater for advanced stage carcinomas. Furthermore, the progression of prostate cancer to an AI, treatment resistant form remains a therapeutic challenge.

[0089] The identification of Vitamin D3 as a cryosensitizer with antineoplastic and antimetastatic properties that "cross-talk" with androgen signaling makes it a promising candidate for the treatment of AI prostate cancer. As investigated, Vitamin D3 neo-adjunctive pre-treatment improves cryosurgical success. Vitamin D3 neo-adjunctive treatment prior to freezing yields complete ablation of AD and AI prostate cancer cell lines in vitro, possibly due to reductions in the anti-apoptotic mitochondrial protein Bcl-2 and increased caspase activity.

[0090] In one embodiment, the use of Vitamin D3 as a neo-adjunctive for prostate cryosurgery exhibits efficacy against both AD and AI cell lines. Indeed, Vitamin D3 exhibits efficacy and a favorable toxicity profile that makes it ideal for use as a sensitizing agent. The data from this study indicate that neo-adjunctive use of Vitamin D3 in cryotherapy induces delayed onset cell death yielding complete ablation for AD and AI cell lines. This supports evidence indicating that Vitamin D3 demonstrates efficacy for AI cells. Furthermore, Vitamin D3 treatment prior to freezing demonstrates increased efficacy compared to the conventional chemotherapeutic ageni, Taxotere®. Taxotere® yields complete ablation of AD cells in combination with freezing, while AD cells, however, experienced recovery during the assessment period. [0091] The significant efficacy of Vitamin D3 treatment for AD and AI cell lines prompted the investigation into what changes in cell death pathways might have occurred to differentially reduce AI cell lines post-freeze viability. Vitamin D3 can regulate apoptotic processes, but its role in the freezing response of AD and AI cells is unknown. Fluorescent micrographs provided qualitative assessment showing that levels of apoptosis significantly increase for AI cells treated with Vitamin D3 prior to freezing, while AD cells exhibit biologically insignificant apoptotic increases. Western blot analysis indicates that pro- caspase levels are reduced post- freeze with Vitamin D3 treatment compared to freeze alone.

[0092] Caspase activity assays then provided qualitative analysis of the observed increases. Caspase-3 activity data indicate that compared to freeze alone at -15°C, Vitamin D3 treated AD cells show smaller relative increases in caspase-3 activity than observed for AI cells. Furthermore, AI cells exhibit increased caspase-3 activation that endurs up to 24 hours post-freeze, which contrasts with freeze alone that shows declining caspase-3 activity by that time. This finding indicates that Vitamin D3 treatment results in greater relative apoptotic activity, and corresponds with viability data showing that AI cells have significantly greater reductions in post-freeze viability compared with AD cells. Importantly, AI cells show caspase-3 activity levels that are comparable with those observed for AD cells, indicating that Vitamin D3 treatment effectively achieves similar total levels of apoptosis in both AD and AI cell lines. Vitamin D3 treatment shows little efficacy in changing caspase-8 activity for AD and AI cell lines, indicating that its mechanism may have little effect on membrane-mediated apoptotic pathways. However, the increases in caspase-9 activity for both AD and AI cell lines indicate that Vitamin D3 may induce mitochondrial-mediated cell death pathways, which is significantly greater for AI cells. Additionally, caspase-9 exhibit peak activity at 3 hours, which indicates a shift to earlier apoptotic activation for AI cell lines (compared with 6 hours for freeze alone).

[0093] Final corroboration of Vitamin D3 mitochondrial-mediated apoptotic mechanism of action is provided by experiments utilizing pre-treatment of cells with caspase inhibitors along with Vitamin D3, since caspase-9 inhibitors, but not caspase-8 inhibitors, are able to reverse the effects of Vitamin D3 pre-treatment. The activation of caspase-9 for Vitamin D3 treated AI prostate cells prompted the investigation into mitochondrial proteins that may mediate these effects. Reports indicate that Vitamin D3 reduces expression of the anti-apoptotic protein Bcl-2, which can lead to cytochrome c release and activation of apoptotic caspase cascades through cleavage of caspase-9. Western blot analysis into expression of Bcl-2 levels for treated and untreated cells indicates that Vitamin D3 significantly reduced Bcl-2 expression for AD and AI cell lines, especially when analyzed post-freeze. These data corroborate findings from other in vitro and in vivo studies that reduction in Bcl-2 levels significantly increase prostate cancer treatment efficacy. Though it may not only be reductions in Bcl-2 levels that correlate with treatment efficacy, the ratio of anti-apoptotic Bcl-2 to pro-apoptotic Bax may be important as well.

[0094] When Bax expression levels for all cell lines for Vitamin D3 treated and untreated cells were evaluated using western blot analysis (data not shown), Bax expression levels remained unchanged after freeze alone, Vitamin D3 treatment alone, or the combination. This indicated that any reduction in Bcl-2 expression reduced the Bcl-2/Bax ratio and resulted in increased apoptotic cell death. Further investigation into the Bcl-2 protein in freezing response using Bcl-2 inhibitors showed that reduction of Bcl-2 function during freezing significantly reduced cell viability for all cell lines, while the greatest effects were observed with AI cells.

[0095] In conclusion, the advantages of using neo-adjunctive agents, particularly Vitamin D3, prior to, during, or subsequent to another therapy, has shown to be effective beyond the level of either treatment alone. The data from this study support the hypothesis that Vitamin D3 exhibits potential for use as an effective neo-adjunctive agent prior to cryosurgery that may increase treatment efficacy for androgen-independent prostate cancer compared to traditional agents. Importantly, Vitamin D3 exhibits superior cryosentization of prostate cancer (androgen-dependent and independent) cells due to increased mitochondrial-mediated apoptotic activity as a result of Vitamin D3-mediated reductions in Bcl-2 expression. Further investigation into Vitamin D3 neo-adjunctive cryotherapy may reveal reduced prostate cancer disease recurrence and an increase in a patient' s quality of life.

TRAIL

[0096] In the current study, we show that applying sub-lethal freezing temperatures with TRAIL significantly reduces PC-3 cell viability when compared to each applied as a single treatment. While TRAIL alone is unable to activate apoptosis, the addition of freezing promotes a significant level of apoptosis. The combination treatment led to caspase-8, -9, and -3 activation indicating involvement of both the membrane and mitochondrial mediated pathways. Subsequent caspase inhibitor analysis revealed that while both pathways were activated, caspase-8 cleavage was necessary for the combined efficacy. Although the freeze/TRAIL combination effectively leads to prostate cancer cell death, the combination is noticeably less effective when applied to normal prostate cells.

[0097] Due to the resistance of prostate cancer cells to most therapies, combined therapies have been utilized to provide an effective ablation procedure. The combination of freezing with chemotherapeutic agents has been more effective than either treatment alone, as with freezing in combination with other cryoadjuvants. Pretreatment of prostate cancer cells with a sub-toxic concentration of these various agents has led to significant increases in cell death when followed by a sub-lethal exposure to freezing. Similarly, in the TRAIL studies indicated in FIG. 10, the combination of freezing and TRAIL resulted in a significant synergistic decrease in cell viability. Interestingly, the simultaneous combination of TRAIL was more effective than the addition of TRAIL prior to freezing.

[0098] It has previously been shown that the application of freezing can induce apoptosis. Prior to these reports, freeze rupture and necrosis were the only forms of cell death thought to be associated with cryoablation. The advent of apoptosis activation has provided a novel opportunity to manipulate freezing-induced cell death in an attempt to improve procedural efficacy. In an earlier study, some cells died through the mitochondrial-mediated apoptotic pathway and a shift in the Bax/Bcl-2 ratio. The cells that survived the freezing episode revealed increased levels of Bcl-2 thereby shifting the ratio towards that of protection. When chemotherapeutic drugs were combined with freezing, Bcl-2 protein levels were prevented from increasing while Bax levels increased significantly. In the current study, the combination of freezing and TRAIL results in a dramatic induction of apoptosis. The molecular studies indicate that the combination results in the activation of membrane- mediated (TRADD, caspase-8) events as well as mitochondrial-mediated (caspase-9) events. Interestingly, whereas caspase-9 activity increased, Bcl-2 levels remained unchanged (Data not shown). Thus, these data support a role for the membrane-mediated pathway in cell death associated with freezing.

[0099] Despite the fact that both pathways were activated with the freeze/TRAIL combination, caspase-8 activation and the membrane-mediated pathway allow for the combination to be successful. Although an increase in caspase-9 activity is observed, the addition of the caspase-9 inhibitor did not provide a significant amount of protection. The cleavage of caspase-8 may have led to downstream cleavage of Bid to tBid stimulating the release of cytochrome c and the resultant activation of caspase-9. Furthermore, neither of the specific inhibitors provided any protection for the cells exposed to freezing alone with only the pan-caspase inhibitor demonstrating protection. The data support the idea that the freeze/TRAIL combination treatment initiates a specific pathway. Since TRAIL alone cannot stimulate the cascade in PC-3 cells, the freezing must be initiating a physical or molecular change in the cells that facilitates the cytotoxic effects of TRAIL. In one aspect, the sublethal application of freezing leads to changes to the cell membrane leading to expression of the death receptors (DR4, DR5).

[00100] Targeted induction of programmed cell death via the extrinsic apoptotic pathway represents an additional unexploited therapeutic strategy that can be combined with cryoablation to destroy cancer cells. The adjuvant strategy is an effective method to reduce the critical freezing temperature thereby reducing the need to extend the freeze zone beyond the target tissue. This can lessen the chance of affecting the surrounding organs. The freeze/TRAIL combination represents a particularly exciting therapeutic model because molecules that directly activate the TRAIL receptors, such as agonistic monoclonal antibodies and recombinant TRAIL are currently being developed. It may be possible to place TRAIL "seeds" in a similar manner to that which is done with brachytherapy, and target them towards the periphery of the diseased tissue along with the cryosurgical application. The adjuvant application of TRAIL may also reduce the harmful cytotoxic side effects typically associated with current chemotherapeutic agents especially since the freeze/TRAIL combo appears to be less toxic to normal prostate cells. Furthermore, knowledge of the molecular mechanisms involved in both the resistance of the cancer along with the efficacy of the therapy will provide significant benefits.

[00101] For instance, small molecules designed to inhibit caspase-8 may be used to protect specific areas to protect them against the destructive measures of the freeze/TRAIL combination. As demonstrated in one aspect, freezing enhances TRAIL-induced cell death by increasing apoptosis through a caspase-8 mediated pathway. Overall, the study described herein provides data illustrating an effective therapeutic model for the cryodestructive treatment of prostate cancer and provides the framework for future studies.

[00102] The invention works to improve destruction of targeted cells and tissues by increasing the sensitivity of the cells to apoptosis induced by TRAIL combined with the destructive stresses and forces of freezing events. The use of both the TRAIL ligand and antibodies to the TRAIL receptor are of particular interest. The tumor cells are exposed to TRAIL or receptor antibodies, either locally or systemically, which results in the sensitization of the cells to undergo cell death when exposed to additional stressor such as freezing (cryotherapy) which also induced apoptosis as well as necrosis and freeze rupture. The killing of the tumor cells by induction of apoptosis through the death domain receptor or ligand TRAIL is greatly enhanced by the synergistic action of both the biochemical and physical stresses imposed on a cell by freezing. When cells are exposed to the combination of TRAIL ligand and freezing, an increase in the extent of cell death occurs. For example prostate cancer cells (PC3) exposed to the TRAIL ligand (lOOng to lOOOng) or freezing to -10°C alone results in minimal death (<20%) where as PC3 cells exposed to the combination of TRAIL and freezing to -10°C results in >90 death.

[00103] As demonstrated in FIG. 12, the use of Vitamin D3 and TRAIL in combination was evaluated to identify whether treatment efficacy differed for prostate cancer cell viability. Prostate cancer cell monolayers were treated with Vitamin D3 and TRAIL prior to freezing at -5°C for 15 minutes. Metabolic indicator alamarBlue® was used to assess cell viability post-freeze. The integration of drug combination and freeze treatments achieved greater levels of cell ablation with slower re-growth as compared to use of each cryosensitizer alone. Various levels of cell death, however, could be achieved based on treatment protocol.

In Conclusion

[00104] Embodiments of the invention identify a novel class of compounds to act as tissue sensitizing agents. The cryosensitizing agents may include vitamins such as Vitamin D3 or cell stress response modulators such as TRAIL, alone or in combination, including each of their natural or synthetic derivatives and/or analogs. While the cryosensitizing agents act as adjuvants to effect precision in croytherapeutic procedures, the agents are minimally toxic to non-targeted tissues, as well as the patient as a whole. The use of the agents and/or their analogs results in complete lethality for prostate cancer in the 0°C to -15°C range. This allows for complete target tissue ablation within the iceball, allowing for reduced damage to sounding tissue structures (e.g. neurovascular bundles, rectal wall, vagus nerve, etc.). Further, these agents are able to be utilized with any number of procedures to effect cell death at various stages and ranges of temperature (i.e. cryoablation, high frequency ablation, radiofrequency ablation, laser ablation, and other various methodologies to ablate tissue).

[00105] The agents comprise natural and synthetic vitamins including steroid-based vitamins, analogs, and derivatives therefrom to sensitize cells to freezing injury for use in conjunction with (before, during, or after) a cryoablation procedure. These agents can also comprise cell stress modulators, apoptotic inducers and inhibitors, antioxidants, free radical scavengers, and others. The cryosensitizer product takes the form of a tablet, liquid, or coating which could be administered orally, by injection, or simultaneously with the application of a cryoablation device. In one embodiment, the product comprises natural or synthetic steroidal vitamin compounds. Current studies have identified Vitamin D3 as a powerful group of cryosensitizers.

[00106] In embodiments of the invention, the cryoadjuvants include a class of small moleculer agents in liquid, semi-solid, or solid form, injectable or implantable. The agents are nontoxic or minimally toxic to cells, tissues, and organs at normal body temperature and become toxic in targeted tissue when used in conjunction with subfreezing temperatures. The invention increases the ablative capacity of cryosurgery, especially at elevated subfreezing temperatures (nominal temperature range of -1 to -40° C). Exposure of targeted cells to the cryoagents enhances cell death mechanisms and improves freeze damage associated with post-thaw apoptosis and necrosis. In addition, a method of using the agents in the treatment of cancer, particularly during transperineal cryoablation of the prostate gland in human males improves curative potential while preventing damage to surrounding tissue.

[00107] In one embodiment, the agent may be administered orally prior to surgery or injected prior to surgery. Thereafter, the cells become hypersensitized. In one aspect, the cells are specifically sensitized to freezing.

[00108] In another embodiment, the invention can be structured by using any apoptotic inducing or inhibiting agents, such as the death domain receptor ligand TRAIL or an antibody to the TRAIL membrane receptor. The application of freezing can be through a closed or open system designed to produce freezing temperatures within a target tissue. One aspect describes an approach and method for synergistic combination of TRAIL and freezing to destroy target tissue, including cancerous and non-cancerous cells and tissues. TRAIL interacts with death domain receptors to destroy its target tissue. The utilization of TRAIL in combination with freezing represents a novel neo- adjunctive approach for the ablation of tissues, including, but not limited to, prostate, kidney, liver, and breast tissues. The utilization of this invention provides for a more efficacious, less toxic and less invasive approach for the eradication of tissue including cancerous and benign tumors.

[00109] The combined application of TRAIL and freezing can be achieved in several temporal configurations including simultaneous application, pre-treatment, or post- treatmentof target tissue with a TRAIL ligand, TRAIL receptor antibody, or freezing. This treatment period can be single or multiple doses administered over a period of minutes to week's prior or subsequent to the application of the other treatment. Additionally the administration of the various TRAIL derivatives can be either targeted for delivery to the desired tissue location directly or systemically.

[00110] One embodiment of the invention integrates a procedure for utilizing the cryosensitizing agents. First, the TRAIL ligand or antibody to the TRAIL receptor is introduced into the target tissue. The application of TRAIL results in the activation of apoptosis within the targeted cells, resulting in sensitization. A heat sink (cryoprobe) is then applied to freeze the targeted tissue. Application of freezing then acts as a second series of stressful events activating both apoptosis and necrosis signaling pathways within the cell. The dosage of both the TRAIL treatment and the freezing can vary substantially while still providing for effective destruction of the target tissue. The simultaneous activation of cell death cascades within the target tissue by the TRAIL and freezing results in amplification of the cell death signaling and the destruction of cells which would otherwise have survived either of the treatments independently.

[00111] In various embodiments, the invention works to improve destruction of targeted cells and tissues by increasing the sensitivity of the cells to apoptosis induced by either Vitamin D3 and TRAIL, the respective analogs or derivatives, combined with the destructive stresses and forces of freezing events. The use of Vitamin D3 and its analogs, as well as the TRAIL ligand and antibodies to the TRAIL receptor are of particular interest. The tumor cells which are exposed to these cryoagents, either locally or systemically, results in the sensitization of the cells to undergo cell death when exposed to additional stressor such as freezing (cryotherapy), thereby inducing apoptosis as well as necrosis and freeze rupture. The killing of the tumor cells by induction of apoptosis through the death domain receptor or ligand TRAIL is greatly enhanced by the synergistic action of both the biochemical and physical stresses imposed on a cell by freezing. When cells are exposed to the combination of cryoadjuvant and freezing an increase in the extent of cell death occurs. For example prostate cancer cells (PC3) exposed to the TRAIL ligand (lOOng to lOOOng) or freezing to - 10°C alone results in minimal death (<20%) where as PC3 cells exposed to the combination of TRAIL and freezing to -10°C results in >90 death.

[00112] In utilizing the medical substrate of the present invention, various methods in the industry may be employed in accordance with accepted cryogenic or thermal applications. As discussed, the embodiments of the invention are for exemplary purposes only and not limitation. Advantageously, this medical substrate represents an important step in targeted adjuvant therapies. Though the medical substrate has been developed to enable and improve some of the approaches used to target or ablate tissue, other therapeutic measures may integrally make use of the substrate pre-treatment, simultaneously, and/or post-treatment.

[00113] Thus, the invention facilitates other improvements in ablation techniques, including in fields of cryotherapy or thermal ablation, such medical devices or components associated with the treatments. The invention facilitates the eradication of tissue and can thereby decrease hospitalization time, limit postoperative morbidities, shorten return to daily functions and work, and further reduce the overall treatment costs.

[00114] The embodiments of the invention may be modified to take the form of any analog or derivative therefrom. Furthermore, the substrate may be applied as a liquid, crystallized component, or as a polymeric or other coating onto any device, container, apparatus, or vessel currently used in industry.

[00115] As presented, multiple embodiments of the invention offer several improvements over standard adjuvant agents currently used in the ablation industry. The previously unforeseen benefits have been realized and conveniently offer advantages for the treatment of multiple disease states. The invention being thus described, it would be obvious that the same may be varied in many ways by one of ordinary skill in the art having had the benefit of the present disclosure. Such variations are not regarded as a departure from the spirit and scope of the invention, and such modifications as would be obvious to one skilled in the art are intended to be included within the scope of the following claims and their legal equivalents.

Claims

In The Claims:
1. A method of enhancing cryoablation techniques, said method comprising the steps of: providing a cryoadjuvant;
providing a cryoablation device in the application of freezing target tissue;
dosing said target tissue with said cryoadjuvant;
contacting said target tissue with said cryoablation device; and
freezing said target tissue with said cryoablation device.
2. The method of Claim 1, wherein said cryoablation technique utilizes one or more cryogenic instruments including cryoprobes, cryoneedles, cryocatheters, cryoballoons, or cryoclamps, alone or in combination.
3. The method of Claim 2, wherein said step of dosing occurs prior to, in conjunction with, or subsequent said step of freezing.
4. A method of treating one or more cells with a substrate to result in cell ablation, said substrate having minimal toxicity to said cells when utilized alone and minimal toxicity to a patient, said method comprising the steps of:
providing a substrate having minimal toxicity to said cells;
sensitizing a target site with said substrate, said target site including one or more cells, tissues, or organs; and
administering a therapy to eradicate one or more of said target sites; wherein said therapy is a cryo-procedure or a hyperthermal ablation procedure.
5. The method of Claim 4, wherein said substrate is a cryosensitizer to selectively target one or more desired cell populations while being non-lethal to surrounding non-targeted cells and tissues.
6. The method of Claim 5, wherein said cryosensitizer comprises a composition that initiates or inhibits apoptosis, a cell survival response, or an unfolded protein response.
7. The method of Claim 4, wherein said step of providing a substrate includes a step of packaging said substrate in a form which allows direct or systemic injection, oral administration, spray, topical administration, or through a means which integrates said substrate with a cryoinstrument.
8. The method of Claim 7, wherein said cryoinstrument is coated with said substrate.
9. The method of Claim 4, wherein said substrate is a composition that inhibits, maintains, potentiates, or interacts with a polypeptide, DNA sequence, RNA sequence, protein, or derivative thereof in a cell survival or death pathway.
10. The method of Claim 4, wherein said step of administering a therapy comprises a step of targeting at least one of a prostate, liver, kidney, pancreas, lung, bone, skin tissue, brain, breast, digestive system; one or more cardiovascular tissues and structures, heart, blood vessels, circulatory systems; lymphatic systems, lymphatic tissues; or reproductive systems including testis, uterus, endometrial lining, uterine fibroids or ovaries.
11. The method of Claim 4, wherein said step of administering said therapy, said therapy is cryoablation at a temperature of less than about 1°C within said target site and said cryoablation avoids damage to non-targeted surrounding cells and tissues.
12. The method of Claim 4, wherein said step of administering a therapy an imaging apparatus visualizes a demarcation at an edge of a target site.
13. The method of Claim 12, wherein said demarcation is a hyperachoic ridge visualized with ultrasound imaging to control administration of said therapy.
14. The method of Claim 4, further comprising a step of reducing post-thaw damage to non- targeted surrounding tissues.
15. A substrate utilized as an adjuvant to enhance ablation of a target site and said target site includes cells, tissues, or organ structures, said substrate comprising a composition including one or more of a vitamin, an apoptotic initiator, a free -radical scavenger, a cell-survival pathway inhibitor, a cell death pathway initiator, an ion channel blockers, an unfolded protein response initiator, an anti-inflammatory agent, an apoptotic modulator, a nucleotide modulator, an antioxidant, or a natural dietary supplement, any of which acts to increase sensitivity of cells to freezing injury.
16. The substrate of Claim 15, wherein said substrate is a comprised of a composition including small molecules which are minimally toxic to said cells, tissues, and organs under normothermic conditions, but become toxic to target tissue when utilized in conjunction with thermal therapies including cryotherapy and hyperthermia.
17. The substrate of Claim 16, wherein said small molecules are derived from natural or synthetic chemistry derivatives.
18. The substrate of Claim 15, wherein said target site is a carcinoma or other undesired tissue.
19. The substrate of Claim 15, wherein said substrate comprises Vitamin D3, Vitamin D3 analogs, resveratrol or resveratrol analogs.
20. The substrate of Claim 15, wherein said substrate includes one or more of:
Vitamin D3 and analogs therefrom including cholecalciferol, (24R)-24,25- dihydroxyvitamin D3, l a, 25-dihydroxyvitamin D3, 1 oc-hydroxy vitamin D3 and 25- hydroxycholecalciferol, calcidol and calcitriol;
resvertrol and analogs therefrom including 3,5-dihydroxystilbene, 3,3',4,5'- tetrahydroxystilbene, 3,4,4', 5-tetrahydroxystilbene, 3,3',5,5'-tetrahydroxystilbene, 3,3',4,5,5'- pentahydroxystilbene, 3,5-dimethoxystilbene, 3,4',5-trimethoxystilbene, 3,3',4,5'- tetramethoxystilbene, 3,4,4', 5-tetramethoxystilbene, 3,3',5'5'-tetramethoxystilbene, and 3,3',4,5,5'-pentamethoxystilbene 3,3', 4' -5-trans-tetrahydroxystilbene (piceatannol), 3, 3', 5,5'- trans-tetrahydroxystilbene and 3,3 ' ,4' ,5 ,5 ' -trans-pentahydroxystilbene;
apoptotic modulators including a TRAIL polypeptide, Bcl2 inhibitor, SIRT-1, PARP, TRADD, AKT, Bid, caspase 8,9, and 3, P53, integrin, Bcl2, pTEN, calpain inhibitors and initiators;
UPR modulators including tunicamycin, salubranol, GRP78, CHOP ATF-6, XBP-1, eIF2-p or PERK;
ion channel blockers including dronedarone, celivarone, and azimide;
nucelotide modulators including DNA, RNA, siRNA, micorRNA, cDNA, RNAi sequences targeting the activation of inhibition of cell death or survival response pathways; or
natural dietary supplements including Vitamin E, Vitamin C, Vitamin D, Vitamin A, super oxidedismutase, selenium, melatonin, zinc chelators, and calcium chelators.
PCT/US2010/048079 2009-09-09 2010-09-08 Cryosensitizing agents for enhancement of cryoablation WO2011049680A1 (en)

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