This present disclosure provides devices, systems, and methods relating to modulating the temperature of a target tissue in a subject for various therapeutic purposes. In particular, the present disclosure provides devices, systems, and methods for the focal delivery of cystostatic hypothermia to a target tissue to treat a disease or condition in a subject (e.g., cancerous tumors).
Patients with malignant brain tumors such as glioblastoma multiforme (GBM) have a median survival of 15-18 months and only 7% survive 5 years after diagnosis and treatment. This is in part because of the limitations of standard-of-care treatment (surgery, chemotherapy, and radiotherapy). For example, surgical resection can only remove visible and bulk mass and cannot reach any remaining cells that may have infiltrated deeper. These remaining cells are the source of GBM recurrence which happens in nearly all patients with GBM. Chemotherapies are hindered by the blood-brain-barrier, neurotoxicity, the evolution of resistance. Radiotherapy lacks tumor specificity and leads to indiscriminate damage tumor and healthy brain alike. To circumvent these limitations, we provide here a device and method using focal, cytostatic hypothermia to stunt the growth of tumors and its use as an adjuvant to facilitate tumor killing.
Embodiments of the present disclosure include a thermoelectric cooling device for reducing temperature of a target tissue in a subject. In accordance with these embodiments, the device includes a thermoelectric modulator, a heat exchange system functionally coupled to the thermoelectric modulator, and at least one thermally conductive probe functionally coupled to the thermoelectric modulator. In some embodiments, activation of the thermoelectric modulator transfers heat from a target tissue to the heat exchange system, thereby reducing the temperature of the target tissue.
In some embodiments, the thermoelectric modulator comprises a Peltier module. In some embodiments, the Peltier module comprises a Peltier cell coupled to the at least one thermally conductive probe. In some embodiments, the thermoelectric modulator comprises a plurality of Peltier modules, wherein each Peltier module comprises a Peltier cell coupled to the at least one thermally conductive probe. In some embodiments, the Peltier module comprises a thermally conductive base plate positioned between the Peltier cell and the at least one thermally conductive probe.
In some embodiments, the thermoelectric modulator comprises applying electrical power to the thermoelectric modulator to cause heat to be transferred from the target tissue to the heat exchange system.
In some embodiments, the heat exchange system comprises a fan to facilitate the transfer of heat from the target tissue. In some embodiments, the heat exchange system comprises a fluid block to facilitate the transfer of heat from the target tissue. In some embodiments, the fluid block comprises a piping system and a fluid. In some embodiments, the piping system is coupled to the thermoelectric modulator and the fluid flows through the piping system, thereby facilitating the transfer of heat from the target tissue.
In some embodiments, the fluid comprises a biocompatible coolant.
In some embodiments, the thermoelectric modulator comprises a power source.
In some embodiments, the heat exchange system comprises a magnetic motor. In some embodiments, the heat exchange system comprises a non-magnetic motor.
In some embodiments, the at least one thermally conductive probe is in direct contact with the target tissue. In some embodiments, the at least one thermally conductive probe is comprised of a biocompatible material.
In some embodiments, the device further comprises a target tissue temperature monitoring device configured to be in direct contact with the target tissue.
In some embodiments, the device further comprises a control system. In some embodiments, the control system is configured to modulate the temperature of the target tissue by adjusting one or more device parameters to increase or decrease heat transfer from the target tissue.
In some embodiments, the temperature of the target tissue is reduced to about 20° C. to about 35° C.
In some embodiments, the target tissue comprises a tumor. In some embodiments, the target tissue comprises a glioblastoma.
Embodiments of the present disclosure also include a method of treating a target tissue in a subject. In accordance with these embodiments, the method includes activating any of the devices described above. In some embodiments, activation of the device transfers heat from the target tissue to the heat exchange system, thereby reducing the temperature of the target tissue.
Embodiments of the present disclosure also include a method of treating a tumor in a subject. In accordance with these embodiments, the method includes implanting a thermoelectric cooling device in a subject, the device comprising a thermoelectric modulator, a heat exchange system functionally coupled to the thermoelectric modulator, and at least one thermally conductive probe functionally coupled to the thermoelectric modulator and in direct contact with the tumor. In some embodiments, the method includes activating the thermoelectric modulator such that it facilitates the transfer of heat from the tumor to the heat exchange system, thereby inducing cytostatic hypothermia in the tumor.
In some embodiments, the method includes inducing cytostatic hypothermia in the tumor by reducing the temperature of the tumor to about 20° C. to about 35° C.
In some embodiments, treating the tumor includes cycling between activating the thermoelectric modulator for a period of time and deactivating the thermoelectric modulator for a period of time.
In some embodiments, treating the tumor includes adjusting one or more device parameters to modulate heat transfer from the target tissue, thereby increasing or decreasing the temperature of the target tissue. In some embodiments, modulating heat transfer decreases the temperature of the tumor as compared to a base temperature. In some embodiments, modulating heat transfer increases the temperature of the tumor as compared to a base temperature.
In some embodiments, the method further includes subjecting the subject to a magnetic resonance imaging (MRI) procedure.
In some embodiments, the subject is receiving chemotherapy. In some embodiments, the chemotherapy includes administration of temozolomide.
In some embodiments, the subject is receiving cancer immunotherapy. In some embodiments, the cancer immunotherapy comprises administration of CAR T cells, checkpoint inhibitors, or antibody-based treatment.
Section headings as used in this section and the entire disclosure herein are merely for organizational purposes and are not intended to be limiting.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present disclosure. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.
The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms “a,” “and” and “the” include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments “comprising,” “consisting of” and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.
For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise-Indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if a concentration range is stated as 1% to 50%, it is intended that values such as 2% to 40%, 10% to 30%, or 1% to 3%, etc., are expressly enumerated in this specification. These are only examples of what is specifically intended, and all possible combinations of numerical values between and including the lowest value and the highest value enumerated are to be considered to be expressly stated in this disclosure.
“Subject” and “patient” as used herein interchangeably refers to any vertebrate, including, but not limited to, a mammal (e.g., cow, pig, camel, llama, horse, goat, rabbit, sheep, hamsters, guinea pig, cat, dog, rat, and mouse, a non-human primate (e.g., a monkey, such as a cynomolgus or rhesus monkey, chimpanzee, etc.) and a human). In some embodiments, the subject may be a human or a non-human. In one embodiment, the subject is a human. The subject or patient may be undergoing various forms of treatment.
“Treat,” “treating” or “treatment” are each used interchangeably herein to describe reversing, alleviating, or inhibiting the progress of a disease and/or injury, or one or more symptoms of such disease, to which such term applies. Depending on the condition of the subject, the term also refers to preventing a disease, and includes preventing the onset of a disease, or preventing the symptoms associated with a disease. A treatment may be either performed in an acute or chronic way. The term also refers to reducing the severity of a disease or symptoms associated with such disease prior to affliction with the disease. Such prevention or reduction of the severity of a disease prior to affliction refers to administration of a treatment to a subject that is not at the time of administration afflicted with the disease. “Preventing” also refers to preventing the recurrence of a disease or of one or more symptoms associated with such disease.
“Therapy” and/or “therapy regimen” generally refer to the clinical intervention made in response to a disease, disorder or physiological condition manifested by a patient or to which a patient may be susceptible. The aim of treatment includes the alleviation or prevention of symptoms, slowing or stopping the progression or worsening of a disease, disorder, or condition and/or the remission of the disease, disorder or condition. In some embodiments, the treatment comprises the treatment, alleviation, and/or lessening of pain.
Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. For example, any nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, neurobiology, microbiology, genetics, electrical stimulation, neural stimulation, neural modulation, and neural prosthesis described herein are those that are well known and commonly used in the art. The meaning and scope of the terms should be clear; in the event, however of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.
Even after standard-of-care treatment (e.g., surgery, chemotherapy, and radiation), patients with glioblastoma (GBM) have a median survival of 15-18 months. Furthermore, only 7% of patients survive 5 years after their diagnosis. This is likely due to the ineffectiveness of current therapies in eradicating GBM. This results in GBM recurrence, of which >80% of recurrent GBMs are local (3-5) and only 20-30% can be resected again before recurring once more. Unfortunately, patients have few alternative therapeutic options. Chemotherapies are hindered by the blood-brain-barrier and the evolution of resistance. Similarly, monoclonal antibodies that reduce angiogenesis may mildly improve symptoms but have little effect on survival.
This dismal performance has necessitated exploring alternate strategies outside the realm of pharmaceuticals and into that of physical phenomena. The first, albeit mild, success was through enabling sustained release of a chemotherapeutic (via an intratumorally implanted Gliadel wafer). The second used electroceutical “tumor-treating field therapies” (TTFTs) which may have had a mild effect on overall survival but is controversial. Recently, a novel device composed of aligned nanofibers to promote directional GBM migration received FDA breakthrough status, underscoring the need for new approached to this disease.
There are also alternative strategies, one of which involves adaptive therapy. With this strategy, the goal of therapy becomes to maintain a tumor burden by adjusting therapeutic dose. This is because when chemotherapy is administered, only chemosensitive cells perish. This provides an empty and nutrient rich field for more aggressive chemoresistant cells to proliferate. By controlling the dose of chemotherapy, the load of chemosensitive can be ‘maintained’ so that they keep a check on chemoresistant cells. This strategy has prolonged survival in animal models. Merging the idea of maintaining a tumor burden with the use of a physical force, a focal hypothermia approach was developed.
Hypothermia has neuroprotective properties after brain injuries and can reduce tumor division. It reduces metabolism, oxygen and glucose consumption, and reduces edema, excitotoxicity, and free-radical formation. However, whole-body hypothermia can weaken the body's immune system and enables tumor proliferation. Instead, there is significant promise in focal hypothermia. Cortical cooling devices successfully halt seizures in primates and intraoperatively in patients. For tumor therapies, focal hypothermia has been used to ablate tumor mass via subzero temperatures and cryosurgery. In the 1950s, cryoprobes tethered to large refrigeration machines were transiently implanted to ablate intracranial tumors. While these were safe, they were ultimately unsuccessful.
Instead of ablation, the present disclosure explored the use focal hypothermia to identify a window of “cytostatic hypothermia” wherein tumor division is halted but healthy brain is left unharmed. As described further herein, the depth and duration of hypothermia on multiple human GBM lines in vitro was tested, which was followed by an examination of the effects of hypothermia on chemotherapy and CAR T immunotherapy in vitro, ultimately leading to the development of devices, systems, and methods to deliver hypothermia in vivo.
Embodiments of the present disclosure provides devices, systems, and methods relating to modulating the temperature of a target tissue in a subject for various therapeutic purposes. In particular, the present disclosure provides devices, systems, and methods for the focal delivery of cystostatic hypothermia to a target tissue to treat a disease or condition in a subject (e.g., cancerous tumors). As shown in
In some embodiments, the implantable portion (110) is connected in a thermally conductive manner to the heat exchange system (120), optionally using a thermal connector element (116). In some embodiments, the thermal transfer element is a wire formed of a heavy-gauge solid copper wire. In other embodiments, wire can be a rigid rod-like element formed of a material with low thermal resistance, or the wire can be multiple types of thermally conductive material coupled together. Wire (116) can also include a connector for detaching the heat exchange system (120) from implantable portion (110). In other embodiments, the thermal connector element (116) is not limited to a wire, but can use other types of conductive, convective, or radiative connection. In some embodiments, the probe can also have insulating sleeves/sections so that cooling can be limited in normal brain tissue or parts of the tumor that have smaller diameter.
In some embodiments, the heat exchange system (120) includes at least one thermoelectric modulator (122). In some embodiments, the heat exchange system (120) is configured to be separate from the thermoelectric modulator (122). In some embodiments, the heat exchange system comprises a magnetic motor. In other embodiments, the heat exchange system comprises a non-magnetic motor (e.g., for MRI compatibility). In some embodiments, the thermoelectric modulator (122) is MRI-compatible in that it lacks magnetic and/or metallic components that would be considered by one of ordinary skill in the art to be problematic for use in an MRI machine. The thermoelectric modulator (122) is generally configured to transfer heat from a target tissue to the heat exchange system (120), thereby reducing the temperature of the target tissue. The thermoelectric modulator (122) can be any suitable device for modulating temperature (increasing and/or decreasing), such as a cold plate, heat exchanger, heat pump, thermoelectric cooler (TEC), etc., or a combination thereof. The thermoelectric modulator (122) can also employ any suitable mechanism for transferring heat, such as liquid cooling, convection cooling, and the like (see, e.g.,
In some embodiments, to increase the range of the heat differential, a heat sink (124) can be attached to the hot side of the thermoelectric modulator (122), which maintains the temperature of the hot side at a level within an operating range of the TEC. Heat sink (124) can also be convection-cooled with an integrated fan (126), and/or as shown in
The heat sink (124) can be designed for cooling by any means, including but not limited to natural convection, liquid cooling, a remotely located fan, or any other type of cooling. The heat exchange system (120) can also include a plate (128) on the cold side of the thermoelectric modulator (122), including, for example, positioned between one or more Peltier modules and at least one thermally conductive probe. Base plate (128) can be, for example, a copper plate, which can optionally be integrated into a non-conductive assembly structure (see, e.g.,
In some embodiments, the thermoelectric cooling device (100) includes at least one Peltier module (see, e.g.,
In some embodiments, the Peltier module comprises a thermally conductive base plate (128) positioned between the Peltier cell and the at least one thermally conductive probe (122). In some embodiments, the heat exchange system (120) is provided in a portable form that is fully implantable within a subject (see, e.g.,
In some embodiments, a desired target tissue temperature can be achieved either by providing one or more target tissue temperature monitoring devices and a controller. In the example embodiment of
In some embodiments, the thermoelectric cooling devices (100) are integrated into a system for delivering focal hypothermia to a desired target tissue (e.g., brain tumor). As shown in
In some embodiments, the target temperature of a tissue is in the range of approximately 20° C.-35° C. In some embodiments, the target tissue is reduced to about 20° C. to about 30° C. In some embodiments, the target tissue is reduced to about 20° C. to about 25° C. In some embodiments, the target tissue is reduced to about 25° C. to about 35° C. In some embodiments, the target tissue is reduced to about 30° C. to about 35° C. In some embodiments, the target tissue is reduced to about 20° C., about 21° C., about 22° C., about 23° C., about 24° C., about 25° C., about 26° C., about 27° C., about 28° C., about 29° C., about 30° C., about 31° C., about 32° C., about 33° C., about 34° C., or about 35° C.
In particular, it has been demonstrated that the thermoelectric devices and systems disclosed herein can halt growth of multiple human tumor cell lines using temperatures of approximately 25° C.-35° C., and significantly slow down even the most resilient rat tumor cell line, at a target temperature of approximately 20° C.-30° C. This temperature was also shown not to indiscriminately kill cells in the region of the cancerous cells, and therefore has a reduced risk of damage to healthy cells. Further, as most brain cells do not divide, the disclosed range can be a therapeutic window for hypothermia in which GBM growth is halted and yet cells are not killed. In other words, the probe does not act as an ablation mechanism.
In some embodiments, therapy is applied to a target tissue in a consistent and constant manner, with minimal deactivation (e.g., 20-24 hours/day). In other embodiments, cooling is applied to the target tissue in a cyclical manner (e.g., on for a duration, off for a duration). In further embodiments, the cooling element can also include specific temperature cycling, where the cooling source can be actively set to varying temperatures, thus possibly “heating” the tumor. The higher temperature setting can be in the above-mentioned ranges and even include temperatures higher than the body resting temperature, without departing from the definition of the cooling source or departing from the scope of the invention. In other words, the cooling source can also deliver heating to the tumor.
It is to be noted that, although the devices and methods disclosed herein are described with reference to brain tumors, it will be understood by a person of skill in the art that these devices and methods can also be applicable to tumors occurring in other parts of the body.
Embodiments of the present disclosure include methods of treatment using the thermoelectric cooling devices and systems described herein. Although exemplary methods have been described with respect to the treatment of a brain tumor (e.g., glioblastoma), the devices and systems provided herein can be used to modulate (e.g., increase and/or decrease) the temperature of any target tissue. Additionally, as described further herein, treating a target tissue with the devices and systems of the present disclosure can include administering treatment according to a treatment regimen or protocol established based on various factors, including but not limited to, the type of tissue being treated, specific patient characteristics, the severity of the disease/condition, and the like. In some embodiments, methods of treatment using the thermoelectric cooling devices and systems described herein include treating a solid tumor (e.g., cancers of the lung, breast, prostate, colon, pancrease, rectum, bladder, and the like), and/or treating a liquid tumor (cancers of the blood and bone marrow, such as lymphomas and leukemias).
Accordingly, a particular treatment regimen or protocol can be established for any patient in need thereof, and can include, for example, cycling between decreases and increases in target tissue temperature for a particular period of time, as well as cycling between periods of treatment and non-treatment (on/off cycles any given period of time). Further, as described further herein, treatment can include combination therapies with other treatment modalities. For example, in the context of tumor treatment, a particular treatment regimen or protocol can include combination treatment with an anti-cancer agent (e.g., chemotherapy and/or cancer immunotherapy).
In accordance with the above embodiments, the present disclosure includes methods for treating a tumor in a subject. The method can include implanting a thermoelectric cooling device in a subject. The device can be comprised of a thermoelectric modulator, a heat exchange system functionally coupled to the thermoelectric modulator, and at least one thermally conductive probe functionally coupled to the thermoelectric modulator and in direct contact with the tumor. In some embodiments, the method includes activating the thermoelectric modulator such that it facilitates the transfer of heat from the tumor to the heat exchange system, thereby inducing cytostatic hypothermia in the tumor.
In some embodiments, the method includes inducing cytostatic hypothermia in the tumor by reducing the temperature of the target tissue to about 20° C. to about 35° C. In some embodiments, the target tissue is reduced to about 20° C. to about 30° C. In some embodiments, the target tissue is reduced to about 20° C. to about 25° C. In some embodiments, the target tissue is reduced to about 25° C. to about 35° C. In some embodiments, the target tissue is reduced to about 21° C. to about 30° C. In some embodiments, the target tissue is reduced to about 22° C. to about 30° C. In some embodiments, the target tissue is reduced to about 23° C. to about 30° C. In some embodiments, the target tissue is reduced to about 24° C. to about 30° C. In some embodiments, the target tissue is reduced to about 25° C. to about 30° C. In some embodiments, the target tissue is reduced to about 30° C. to about 35° C. In some embodiments, the target tissue is reduced to about 20° C., about 21° C., about 22° C., about 23° C., about 24° C., about 25° C., about 26° C., about 27° C., about 28° C., about 29° C., about 30° C., about 31° C., about 32° C., about 33° C., about 34° C., or about 35° C.
In some embodiments, treating the tumor includes cycling between activating the thermoelectric modulator for a period of time and deactivating the thermoelectric modulator for a period of time. In some embodiments, the period of time of activation is minutes, hours, days, or weeks. In some embodiments, the period of time of deactivation is minutes, hours, days, or weeks.
In some embodiments, treating the tumor or other target tissue includes adjusting one or more device parameters to modulate heat transfer from the tumor or other target tissue, thereby increasing or decreasing the temperature of the tumor. In some embodiments, modulating heat transfer decreases the temperature of the tumor or target tissue as compared to a base temperature. In some embodiments, modulating heat transfer increases the temperature of the tumor or target tissue as compared to a base temperature. The device parameters that can be adjusted to modulate the temperature of a target tissue include, but are not limited to, power input to the device (e.g., degree of activation of one or more Peltier modules), the degree of activation of the heat exchange system (e.g., more or less heat transfer), conductance of the probes, the number of probes activated in a multi-probe configuration, and the like.
In some embodiments, the method further includes subjecting the subject to a magnetic resonance imaging (MRI) procedure. As would be recognized by one of ordinary skill in the art based on the present disclosure, one advantage of the systems and devices of the present disclosure is that they can be configured to be MRI-compatible (e.g., reduced metal and/or magnetic components). That is, subjects who may receive therapy with the thermoelectric devices provided herein include subjects with brain tumors; since these subject often require monitoring with an MRI procedure, it is particular advantageous to have a treatment device that is MRI-compatible so that once implanted, it does not have to be removed for imaging. As described further herein, one or more components of the thermoelectric devices of the present disclosure can be configured to be MRI-compatible compatible (e.g., reduced metal and/or magnetic components).
In some embodiments, the subject is receiving or is scheduled to receive another form of treatment for a particular disease or condition. In some embodiments, the subject is receiving or is scheduled to receive chemotherapy. In some embodiments, the chemotherapy includes administration of temozolomide. In some embodiments, the subject is receiving or is scheduled to receive cancer immunotherapy. In some embodiments, the cancer immunotherapy comprises administration of CAR T cells, checkpoint inhibitors, or anti-body-based treatment. Chemotherapy and/or cancer immunotherapy can be administered before, during, or after treatment with the thermoelectric devices provided herein. In some embodiments, the subject is receiving or is scheduled to receive radiotherapy. In some embodiments, focal hypothermia can sensitize tumor cells to radiotherapy. As described further herein, treatment with the thermoelectric devices provided herein have been surprisingly been shown to be compatible with these common cancer treatment modalities, and as such, treatment with the thermoelectric devices of the present disclosure can be used in combination with other treatments in order to provide enhanced therapy.
As shown in
Hypothermia as a therapy is an age-old concept but as a therapeutic for cancer it has only been used at subzero temperatures to obliterate cells. Data provided herein demonstrated cytostatic temperatures across multiple GBM lines ranging from 20-25° C. (
Additionally, experiments described herein investigated the effect of intermittent or cycled hypothermia to begin to determine the daily dose of hypothermia required to maintain cell arrest. Results demonstrated that cell arrest for LN-229 may be achieved with 16 h/day, but other cell lines will require 20-22 h/day (
Experiments were also conducted to investigate mechanistic changes and effects of hypothermia. First, these data were consistent with other findings that hypothermia halts cells in the G2-phase of the cell cycle. Ultimately, as the cells are unable to complete division, they undergo apoptosis. In some experiments (e.g.,
An acute increase followed by decrease of cytokine production was also observed. The early increased production is likely a stress response coupled with the presence of metabolic resources enabling cytokine production. The subsequent decrease in cytokine production could be due to reduced efficiency of synthetic pathways under hypothermia, or due to reduced metabolic resource usage to power those pathways. The reduction in IL-6 and IL-8 may also be beneficial in cancer as these cytokines are typically associated with aggressiveness and infiltration. However, the absence of anti-inflammatory cytokine secretion under cytostatic hypothermia is promising in certain therapeutic contexts.
Next, the effect of hypothermia as an adjuvant to a standard-of-care GBM therapy was assessed (i.e. TMZ chemotherapy). TMZ undergoes pH-dependent hydrolysis to form its active ion which methylates guanine residues in the DNA. Attempted correction of this results in double-stranded breaks which halt cell division and ultimately result in apoptosis. Some cell lines, such as T98G, are known to be resistant to TMZ due to the synthesis of an enzyme, MGMT. In the present disclosure, it was observed that, depending on the tumor line, hypothermia might either have no adverse effect or a synergistic effect with TMZ (
As the future of brain tumor therapy seems to lie in modulating the immune response, experiments were conducted to investigate whether immunotherapy could still be used under cytostatic hypothermia. To address this, CAR T cells were used as a model for immunotherapy as they have a direct killing mechanism. It was hypothesized that immune killing efficacy would reduce or completely subside under hypothermic conditions. It was observed that the CAR T cells retained their cytotoxic ability especially at earlier time points. With enough of these effector cells, the tumor was mostly obliterated over 4 days under cytostatic hypothermia (versus 2 days under normothermia). Additionally, by providing only 4 hours of normothermia, the efficacy was improved. These results are interesting as they can be used in at least two approaches. First, an ideal hypothermia duration could be determined to keep tumor growth at check, while providing short windows of normothermia to enable CAR T mediated killing. Second, since CAR T cells seem to function well in the first 24 hours even under hypothermia, multiple doses of CAR T cells could be given while keeping the tumor under constant cytostatic hypothermia. Additionally, data described herein demonstrates that human GBM halt around 25° C. (although rat F98 cells require between 20-25° C.). It should be noted that, until now, all hypothermia data has typically been obtained in the context of intermittent hypothermia or continuous hypothermia for a very short period. However, embodiments of the present disclosure can include cycling hypothermia with normothermia or using adjuvant therapies to reduce the total length of therapeutic hypothermia.
It will be readily apparent to those skilled in the art that other suitable modifications and adaptations of the methods of the present disclosure described herein are readily applicable and appreciable, and may be made using suitable equivalents without departing from the scope of the present disclosure or the aspects and embodiments disclosed herein. Having now described the present disclosure in detail, the same will be more clearly understood by reference to the following examples, which are merely intended only to illustrate some aspects and embodiments of the disclosure, and should not be viewed as limiting to the scope of the disclosure. The disclosures of all journal references, U.S. patents, and publications referred to herein are hereby incorporated by reference in their entireties.
The present disclosure has multiple aspects, illustrated by the following non-limiting examples.
Fully implantable device to deliver focal hypothermia. In exemplary embodiments, the thermoelectric devices and systems of the present disclosure can include three main components: an implantable water-block with internal circulation to transfer heat from a Peltier plate; a multi-probe platform to homogenously apply focal hypothermia in a region of interest; and a multi-Peltier system attached to the platform to enable individual probe control for therapy and research. Combining all the described subcomponents results in a fully implantable thermoelectric device, as shown in
Biomedical applications of focal hypothermia typically include an element/probe that is cooled either via circulating liquid or through the thermoelectric effect. In the former, cold liquid is run through a pipe that directly or indirectly contacts the region of interest and heat is transferred from the tissue to the liquid. In the latter, electricity is applied to a Peltier plate to pump heat from the cold side, in contact with tissue, to the hot side. In both these situations, the larger problem involves how to remove the heat from the warmed liquid or the hot side of the Peltier. Current solutions consist of using external ice-baths or secondary external Peltier systems. However, these can have numerous limitations, such as cooling pipes/heat transfer pipes that pass through the skin cause infections; any liquid/ice-bath requires frequent replacement; and power draws from additional Peltier systems would necessitate large batteries.
Embodiments of the present disclosure, however, address these limitations, by providing a fully implantable system to transfer the heat away from the hot side of a Peltier module. In some embodiments, the thermoelectric cooling devices of the present disclosure include a sheet(s) or block of material (e.g., copper, gold-plated copper, graphite, or titanium) with a pipe(s) running through it as a passage for liquid at body temperature (e.g., 37° C.). The metal, in contact with a Peltier module, takes heat and passes it to the liquid. This liquid is circulated through a flexible piping system that is implanted under the skin to distribute the heat throughout other regions of the body. This could either be through contact of the piping system with the skin, or through one or more remote heat sinks/water blocks implanted in the body. The circulation is enabled through an implanted pump that consists either of a traditional motor or, if MRI-compatibility is desired, an induction or electromagnetic motor or a non-magnetic motor such as a piezoelectric motor. This pump is connected to an internal battery which can be charged through wireless electromagnetic induction (or, in simpler form, through a power cable passing through the skin). Its speed can be controlled to increase/decrease the rate of fluid passing through the heat sink, depending on how much heat needs to be removed.
When electricity is applied to a Peltier module, its efficacy is, in part, determined by how well the heat is removed from the hot side. If there is no heat sink, the temperature can reach >70-80° C. before it stops functioning. By reducing and maintaining the hot-side temperature to near body temperature through this system, the Peltier module can become effective.
As provided in
Additionally, Lab testing of a prototype water-block (
Removing heat from a region of tissue (e.g., brain), through a probe or surface, can be limited by the amount of surface area in contact with the tissue. With reduced surface area, the temperature within the probe or surface must be significantly colder (and more energy required to maintain it) so that the tissue reaches the desired temperature. However, this forms sharp thermal gradients, wherein the tissue adjacent to the probe is much colder so that tissue further away can also reach a desired temperature.
To address this problem of thermal gradients and excessively cold temperatures around probes, a multi-probe heat distribution platform was developed (see, e.g.,
As provided in
In some cases, different regions of tissue may need to be cooled at different intensities. This could be due to contact with tissue (or nearby tissue) that is critical or the need to be more efficient with power usage.
Therefore, instead of having one large cooling Peltier plate in contact with the multi-probe platform, a multi-Peltier module device was developed wherein each probe is controlled by an individual Peltier (
This application is not limited to therapy in patients. It can also be used as a neuroscientific tool to study the effect of cooling and heating on brain function in animals; analogous to a multi-electrode array to read/control individual neurons.
In vitro tumor growth rate is hampered by varying depths and durations of hypothermia. It was hypothesized that different depths of hypothermia may affect cell growth rates differently. Thus, three human GBM cell lines and one rat GBM line were cultured at 20, 25, and 37° C., while also culturing some at 30° C. (
To assess the effects of hypothermia applied for 24 hours per day, the plates were cycled between 37° C. and 25° C. incubators for varying durations. This demonstrated that even 16 hours of cooling (H16) could significantly reduce growth rate (
Hypothermia halts cell cycle, and affects ATP stock and cytokine production. To begin to understand the mechanism and effects of halted cell division, cell cycle, ATP levels, and cytokine production were assessed under hypothermia. A larger proportion of cells grown for 3 days under 37° C. were found to be in the G1 phase of the cell cycle (
Next, ATP levels were studied to begin exploring metabolic alterations. It was hypothesized that, under cytostatic hypothermia, ATP production and consumption would cease and thus intracellular ATP levels would remain constant. Interestingly, an increase in ATP levels in 3 out 4 cell lines under hypothermia was observed (
Additionally, to assess the effect of hypothermia on cytokine production, IL-6, IL-8, CX3CL1, IL-10, IL-4, and IFNγ were measured. It was hypothesized an ‘acute’ increase in anti-inflammatory cytokines followed by a decrease over time. Interestingly, the latter 3 cytokines were not found in the conditioned media regardless of the temperature (e.g., suggesting anti-inflammatory cytokines were not suddenly increasing). Additionally, inflammatory cytokines IL-6 and IL-8 increased at 3 days of hypothermia but drastically reduced over 7 and 14 days across all 3 cell lines assessed (
Hypothermia treatment with chemotherapy and CAR T immunotherapy treatment. Current standard of care for GBM includes chemotherapy with Temozolomide (TMZ). Additionally, the future of many cancer therapies includes some form of modulating the immune response; an immunotherapy. To assess whether hypothermia would hamper or synergize with Temozolomide, human GBM cells were treated with different TMZ doses either at 37° C. or 25° C. for 3 days followed by washing the cells and growing at 37° C. Interestingly, the responses of the cell lines were different. The addition of TMZ to LN-229 cells under hypothermia reduced cell division more significantly than under 37° C. and compared to cells that did not receive TMZ (
Next, the effect of hypothermia on CAR T immunotherapy against EGFR+CT2A mouse GBM was explored. It was hypothesized that immune cell function would be significantly hampered but may retain some killing activity due to accumulated ATP. It was also hypothesized that killing efficacy would increase if the temperature were cycled between 25 and 37° C. Synthesized CAR T cells were functional and specifically killed EGFR+CT2A cells (and not EGFR−). Absence of the CAR on T cells resulted in no killing of any CT2A cell. Without CAR T cells, CT2A cell growth was inhibited by 25° C. hypothermia both when the hypothermia was applied for 24 h/day and 20 h/day (
Focal hypothermia in vivo prolongs survival of GBM bearing rats. Since hypothermia of 20-25° C. can reduce cell division in vitro, and this temperature range may be safe in vivo, a method was developed to deliver hypothermia in vivo and assess its effect on tumor growth and rat survival. To deliver hypothermia, a Peltier based device was designed and developed that was comprised of an implantable portion (including an intratumoral gold needle, a thermistor 1-mm from probe, and polycarbonate base) and a removable portion (including the Peltier, aluminum heatsink and fan) (
A finite element model was set up to assess the extent of cooling in vivo (
After inoculation with the aggressive F98 tumor cells, tumor take was confirmed in all rats via MRI at week, and the device was implanted on the subsequent day. Once switched on, the device was able to reduce local temperature to at least 25° C., 1 mm away from the intratumoral probe (
In subsequent survival studies, rats receiving hypothermia did not exhibit any obvious signs of distress and ate their normal diet. This translated to longer weight maintenance and gain compared to their control counterparts (
Cell Culture. All cell lines were purchased from either ATCC or the CCF at Duke. To simplify culturing and passaging, all cells were progressively adapted to a unified medium: Dulbecco's MEM (with glucose and Na pyruvate)+NEAA+10% FBS. This might affect growth rates for individual cells and thus future studies can characterize the effects of other media with cytostatic hypothermia. Cells were given at least one passage to recover after thawing and then used within the next 5-10 passages for all experiments. Cells were plated at a density of 5000 cells/well in falcon clear-bottom 96-well plates or 10,000 cells/well in a 24-well plate and left to adhere overnight at 37° C. before any experiment. In certain experiments, cells were left for longer at 37° C. prior to changing incubator temperature. A HeraCell CO2 incubator was used for all experiments and set at either 37, 30, 25, or 20° C.
Imaging and analysis. Imaging was performed with a Leica DMi8 live cell scope with a built-in incubator and CO2 regulator. Leica software was calibrated to the imaging well plate to enable tile scanning. Images were taken at 5× with a 4×4 field in 24-well plates and 2×2 field in 96-well plates. Images were then analyzed through a custom automated imageJ script to quantify the area coverage of a well plate. For immunotherapy experiments, images were taken at 10× with a GFP laser, and the imageJ script was modified to count the number of cells. All analyzed images were then processed through a custom Python script to organize the data for analysis.
Chemo/immunotherapy. Chemotherapy: temozolomide (sigma Aldrich) was dissolved in DMSO and frozen in aliquots and used within 2 months at varying concentrations. In these experiments, tumor was grown in the well plate overnight followed by treatment of 37 deg plate with TMZ for 3 days. The second plate was moved to 25° C. for 5 days. After 5 days, TMZ was added for 3 days. After TMZ treatment, the media was replaced and the plates were moved back to 37° C. with daily imaging.
CAR T cells were made by harvesting splenocytes and isolating T cells. HEK cells were transfected with a plasmid to produce lentivirus with EGFR+. This media was then used to transduce the T cells to express the CAR. CAR was confirmed via flow cytometry and the cells were frozen in liquid nitrogen. For in vitro experiments, all experiments were on days 5 or 6 after transduction.
Device manufacturing. Device design and manufacturing was done in collaboration with the Pratt Machine Shop at Duke University. Custom designs were made and transferred to 3D CAD drawings. Raw polycarbonate, copper, and aluminum material was purchased from McMaster and parts were machined using a CNC machine. Other components include Peltier plates from TeTech, fans from Sunon (purchased through supplier: Digikey), thermistors from Amphenol Advanced Sensors, 24K Gold from Hauser & Miller, thermal paste (Kryonaut) from Thermal Grizzly, screws from McMaster, and connectors from JST Sales America (purchased through supplier: Digikey).
The implant consists of a gold needle, copper base, and thermistor that warps around brass screw posts. This and other components were sterilized through UV for 30 minutes followed by EtOH overnight.
Animals. All animal procedures were approved by the Duke IACUC. Fischer (CDF) rats and RNU rats were purchased from Charles River at 6-9 weeks of age. All procedures began at 10 weeks of age. The animals were induced with 5% isoflurane and maintained at 2%. A central incision was made on the scalp and the skin mildly retracted. A 0.6 mm conical burr was used to drill at −0.5 AP and +3 ML to a depth of 0.8-0.9 mm. A Hamilton syringe loaded with at least 5 uL of F98 or U87MG cells was centered to drill site and then the tip was cleaned of any droplets. The syringe was lowered to a depth of 1.5 mm from the outer table. Infusion was begun with a pump at 0.5 uL/min for 10 min. Upon 1 minute after completion, the syringe was slowly retracted and the rat scalp sutured. The rat was then placed in the custom cage.
One week after, an MRI was taken to confirm tumor-take. The subsequent day, the rats were again induced under anesthesia and the scalp exposed. This time, extra effort was put into retraction, scraping off the peritoneum, slightly separating the termporalis muscles, and hemostasis. Once the cranium was dry and absent of blood, additional burr holes were made using conical drill bits. This included one burr hole with 0.6 mm tip for thermistor, −1.5 mm from tumor inoculation. Following this, 1.0 mm conical burr was used −6 mm from thermistor for titanium screw (TS) 1, −4 mm from TS1 for TS2, −10 mm from thermistor for TS3, and +6 mm from tumor inoculation for TS4. The original tumor inoculation burr hole was expanded to 1.4 mm. Screws (modified to be 1 mm in length) were then twisted into their holes at a depth of 0.6 mm. Next, the sterile implant was gently inserted and held down while dental cement was added to the sides. As quickly as possible to hold the implant, a UV light was shone. Following this, layers of dental cement were added around the screws and implant and skull to secure the implant to the skull. Upon completion, stitches were used to gently approximate the skin (including around the arms of the implant) while keeping the surface of the implant exposed.
The rat was monitored while waking up and for 2 hours after to ensure full recovery. Two days after recovery, the rat was put under anesthesia to screw on the remaining device and connect the cable to the slip-ring on the cage. For studies where MRI was possible, an additional MRI was taken 5 days after implant (with the device screwed off). After this, the device was switched on, and temperature was monitored through an Arduino connected to a computer.
The rat temperatures were intermittently monitored throughout the day through a local network. This enabled rapid responses to any sudden changes in temperature, usually due to some transient failure of the device which was rectified in future iterations. One gradual source of inefficiency was the buildup of fur on the intake of the heat sink and inside the fan. This needed to be intermittently removed with tweezers.
Rat weight was measured every 3-7 days and every day when weight started falling. The procedure involved transiently disconnecting the rat from the tether, moved to an empty cage, and subtracting the weight of the cable/components/and cage. the rat lost 15%
MRI. For MRI, the cooling portion of the device was detached under anesthesia. The rats were imaged in a Bruker 7T MRI machine. T1 and T2 weighted images were taken. Contrast was also added for the T1 images through a tail vein catheter inserted under anesthesia right before the MRI.
Euthanasia/perfusion/histology. Euthanasia criteria included: 15% weight loss from initial weight (post device implant), and signs of porphyrin staining and distress. When a rat reached these criteria, they were put under anesthesia. A thoracotomy was performed and the rats were then transcardially perfused with saline (250 mL) followed by fresh 4% formalin (250 mL). The animals were decapitated, and the skull and implant was carefully removed. The brain was left for 24 hours overnight in formalin. The following day they were transferred to 30% sucrose and left until the brain sunk. For histology, the brain was snap frozen in liquid nitrogen, cryosectioned at 12 um slices, stored on slides and placed in −20° C. The slides were stained with H&E and imaged under the microscope.
Computational modelling. Modelling was performed on COMSOL v5.2.
Statistical analysis. All statistical analysis was performed on Graphpad prism v8.2.
This application claims priority to and the benefit of U.S. Provisional Patent Application No. 62/923,081 filed Oct. 18, 2019, which is incorporated herein by reference in its entirety for all purposes.
Filing Document | Filing Date | Country | Kind |
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PCT/US2020/056078 | 10/16/2020 | WO |
Number | Date | Country | |
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62923081 | Oct 2019 | US |