Patent Application
20090105781
The present invention is directed generally to methods of regulating gene expression in tissue (e.g., injured or diseased tissue) by applying to such tissue electric and/or electromagnetic fields generated by specific and selective signals, for treating such tissue, as well as to devices for generating such fields. The present invention is directed particularly to methods of down-regulating matrix metalloproteinase (MMP) gene expression in tumor cells by applying to such tumor cells electric and/or electromagnetic fields generated by specific and selective signals, for preventing the growth and/or spread of tumors, as well as to devices for generating such fields.
The bioelectrical interactions and activity believed to be present in a variety of biological tissues are one of the least understood of the physiological processes. However, there has recently been much research into these interactions and activity regarding the growth and repair of certain tissues. In particular, there has been much research into stimulation by electric and/or electromagnetic fields and its effect on the growth and repair of bone and cartilage, and on the regulation of the expression of various growth factors. Researchers believe that such research may be useful in the development of new treatments for a variety of medical problems.
Osteoarthritis, also known as degenerative joint disease, is characterized by degeneration of articular cartilage as well as proliferation and remodeling of subchondral bone. The usual symptoms are stiffness, limitation of motion, and pain. Osteoarthritis is the most common form of arthritis, and prevalence rates increase markedly with age. It has been shown that elderly patients with self-reported osteoarthritis visit doctors twice as frequently as their unaffected peers. Such patients also experience more days of restricted activity and bed confinement compared to others in their age group. In one study, the majority of symptomatic patients became significantly disabled during an 8-year follow-up period. Massardo et al., Ann Rheum Dis 48: 893-7 (1989).
Nonsteroidal anti-inflammatory drugs (NSAIDs) remain the primary treatment modality for osteoarthritis. It is unknown whether the efficacy of NSAIDs is dependent upon their analgesic or anti-inflammatory properties or the slowing of degenerative processes in the cartilage. There is also a concern that NSAIDs may be deleterious to patients. For example, NSAIDs have well known toxic effects in the stomach, gastrointestinal tract, liver, and kidney. However, aspirin inhibits proteoglycan synthesis and normal cartilaginous repair processes in animals. One study in humans suggested that indomethacin might accelerate breakdown of hip cartilage. All adverse effects appear more commonly in the elderly—the very population most susceptible to osteoarthritis.
In the disease commonly known as osteoporosis, bone demineralizes and becomes abnormally rarefied. Bone comprises an organic component of cells and matrix as well as an inorganic or mineral component. The cells and matrix comprise a framework of collagenous fibers that is impregnated with the mineral component of calcium phosphate (85%) and calcium carbonate (10%) that imparts rigidity to the bone. While osteoporosis is generally thought to afflict the elderly, certain types of osteoporosis may affect persons of all ages whose bones are not subject to functional stress. In such cases, patients may experience a significant loss of cortical and cancellous bone during prolonged periods of immobilization. Elderly patients are known to experience bone loss due to disuse when immobilized after fracture of a bone, which may ultimately lead to a secondary fracture in an already osteoporotic skeleton. Diminished bone density may lead to vertebrae collapse, fractures of hips, lower arms, wrists, and ankles, as well as to incapacitating pains. Alternative nonsurgical therapies for such diseases are needed.
Matrix metalloproteinases (MMPs) are proteolytic enzymes that have been known for a long time to be associated with cancer-cell invasion and metastasis. High levels of MMP-1, MMP-3, and MMP-13 are associated with shorter disease-free survival in patients dying from metastatic disease (Nikkola, J., et al., Int. J. Cancer: 97, 432-438, 2002). The MMPs are a family of 16 or more zinc-dependent endoproteinases whose enzymatic action removes the physical barrier to invasion by degradation of the extracellular matrix (ECM) components such as collagens, lamimins, and proteoglycans. Consequently, high levels of MMPs are found at the invasive front of tumors (Kleiner, D. E. and Stetler-Stevenson, W. G., Cancer Chemother. Pharmacol. Suppl. 43, S42-S51, 1999). The MMPs also regulate various cell functions in cancer such as cancer-cell growth, differentiation, apoptosis, migration and invasion, and regulate tumor angiogenesis (Egeblad, M. and Werb, Z., Nature Reviews/Cancer, 2: 161-174, 2002). Not only do tumor cells produce MMPs but they also induce MMP production by host tissue stromal cells (Stamenkovic, I., Seminars in Cancer Biology, 10: 415-433, 2000). Synthetic inhibitors of MMPs have been developed and are in early clinical trial. However, the interaction of such inhibitors with chemo-radiotherapy is unknown (Giavazzi, R. and Taraboletti, G., Critical Reviews in Oncology/Hematology, 37: 53-60, 2001).
Pulsed electromagnetic fields (PEMF) and capacitive coupling (CC) have been used widely to treat nonhealing fractures (nonunion) and related problems in bone healing since approval by the Food and Drug Administration in 1979. The original basis for the trial of this form of therapy was the observation that physical stress on bone causes the appearance of tiny electric currents that, along with mechanical strain, were thought to be the mechanisms underlying transduction of the physical stresses into a signal that promotes bone formation. Along with direct electric field stimulation that was successful in the treatment of nonunion, noninvasive technologies using PEMF and CC (where the electrodes are placed on the skin in the treatment zone) were also found to be effective. PEMFs generate small, induced currents (Faraday currents) in the highly-conductive extracellular fluid, while CC directly causes currents in the tissues; both PEMFs and CC thereby mimic endogenous electrical currents.
The endogenous electrical currents, originally thought to be due to phenomena occurring at the surface of crystals in the bone, have been shown to be due primarily to movement of fluid containing electrolytes in channels of the bone containing organic constituents with fixed negative charges, generating what are called “streaming potentials.” Studies of electrical phenomena in bone have demonstrated a mechanical-electrical transduction mechanism that appears when bone is mechanically compressed, causing movement of fluid and electrolytes over the surface of fixed negative charges in the proteoglycans and collagen in the bone matrix. These streaming potentials serve a purpose in bone, and, along with mechanical strain, lead to signal transduction that is capable of stimulating bone cell synthesis of a calcifiable matrix, and, hence, the formation of bone. Studies of electrical phenomena in cartilage have demonstrated a mechanical-electrical transduction mechanism that resembles those described in bone, appearing when cartilage is mechanically compressed, causing movement of fluid and electrolytes over the surface of fixed negative charges in the proteoglycans and collagen in the cartilage matrix. These streaming potentials serve a purpose in cartilage similar to that in bone, and, along with mechanical strain, lead to signal transduction that is capable of stimulating chondrocyte synthesis of matrix components.
The main application of direct current, CC, and PEMFs has been in orthopedics in healing of nonunion bone fractures (Brighton et al., J. Bone Joint Surg. 63: 2-13, 1981; Brighton and Pollack, J. Bone Joint Surg. 67: 577-585, 1985; Bassett et al., Crit. Rev. Biomed. Eng. 17: 451-529, 1989; Bassett et al., JAMA 247: 623-628, 1982). Clinical responses have been reported in avascular necrosis of hips in adults and Legg-Perthes's disease in children (Bassett et al., Clin. Orthop. 246: 172-176, 1989; Aaron et al., Clin. Orthop. 249: 209-218, 1989; Harrison et al., J. Pediatr. Orthop. 4: 579-584, 1984). It has also been shown that PEMFs (Mooney, Spine 15: 708-712, 1990) and CC (Goodwin, Brighton et al., Spine 24: 1349-1356, 1999) can significantly increase the success rate of lumbar fusions. There are also reports of augmentation of peripheral nerve regeneration and function and promotion of angiogenesis (Bassett, Bioessays 6: 36-42, 1987). Patients with persistent rotator cuff tendonitis refractory to steroid injection and other conventional measures, showed significant benefit compared with placebo-treated patients (Binder et al., Lancet 695-698, 1984). Finally, Brighton et al. have shown in rats the ability of an appropriate CC electric field generated by electric signals to both prevent and reverse vertebral osteoporosis in the lumbar spine (Brighton et al., J. Orthop. Res. 6: 676-684, 1988; Brighton et al., J. Bone Joint Surg. 71: 228-236, 1989).
More recently, research in this area has focused on the effects stimulation has on tissues. For example, it has been conjectured that direct currents do not penetrate cellular membranes, and that control is achieved via extracellular matrix differentiation (Grodzinsky, Crit. Rev. Biomed. Eng. 9:133-199, 1983). In contrast to direct currents, it has been reported that PEMFs can penetrate cell membranes and either stimulate them or directly affect intracellular organelles. An examination of the effect of PEMFs on extracellular matrices and in vivo endochondral ossification found increased synthesis of cartilage molecules and maturation of bone trabeculae (Aaron et al., J. Bone Miner. Res. 4: 227-233, 1989). More recently, Lorich et al. (Clin. Orthop. Related Res. 350: 246-256, 1998) and Brighton et al. (J. Bone Joint Surg. 83-A, 1514-1523, 2001) reported that signal transduction of a capacitively coupled electric signal is via voltage gated calcium channels, whereas signal transduction of PEMFs or combined electromagnetic fields is via the release of calcium from intracellular stores. In all three types of electrical stimulation there is an increase in cytosolic calcium with a subsequent increase in activated (cytoskeletal) calmodulin.
Much research has been directed at studying tissue culture in order to understand the mechanisms of response. In one study, it was found that electric fields increased [3H]-thymidine incorporation into the DNA of chondrocytes, supporting the notion that Na and Ca2+ fluxes generated by electrical stimulation trigger DNA synthesis. Rodan et al., Science 199: 690-692 (1978). Studies have found changes in the second messenger, cAMP, and cytoskeletal rearrangements due to electrical perturbations. Ryaby et al., Trans. BRAGS 6: (1986); Jones et al., Trans. BRAGS 6: 51 (1986); Brighton and Townsend, J. Orthop. Res. 6: 552-558, 1988. Other studies have found effects on glycosaminoglycan, sulphation, hyaluronic acid, lysozyme activity and polypeptide sequences. Norton et al., J. Orthop. Res. 6: 685-689 (1988); Goodman et al., Proc. Natl. Acad. Sci. USA 85: 3928-3932 (1988).
It was reported in 1996 by the present inventors that a cyclic biaxial 0.17% mechanical strain produces a significant increase in TGF-β1 mRNA in cultured MC3T3-E1 bone cells in a cooper dish (Brighton et al., Biochem. Biophys. Res. Commun. 229: 449-453, 1996). Several significant studies followed in 1997. In one study it was reported that the same cyclic biaxial 0.17% mechanical strain produced a significant increase in PDGF-A mRNA in similar bone cells (Brighton et al., Biochem. Biophys. Res. Commun. 43: 339-346, 1997). It was also reported that a 60 kHz capacitively coupled electric field of 20 mV/cm produced a significant increase in TGF-β1 in similar bone cells in a cooper dish (Brighton et al., Biochem. Biophys. Res. Commun. 237: 225-229, 1997). Recently it was reported that an appropriate capacitively coupled electric signal up-regulated aggrecan mRNA and collagen type II mRNA in bovine articular cartilage chondrocytes grown in cell culture (Wang, W., et al, Clinical Orthop. and Related Research, 427S: S163-S173, 2004). Most recently it was reported that a different appropriate capacitively coupled electric signal up-regulated bone morphogenetic proteins (BMPs) mRNAs in cultured bone cells (Wang, Z., et al, J. Bone and Joint Surgery Am., 88: 1053-1065, 2006), and yet another report on up-regulation of bovine articular cartilage, this time in full-thickness explants, by a capacitively coupled electric field (Brighton, C. T., et al, Biochem. And Biophysical Res. Communications, 342:556-561, 2006).
U.S. Provisional Patent Application Ser. No. 60/826,926, filed Sep. 26, 2006; U.S. patent application Ser. No. 10/257,126, filed Oct. 8, 2002; PCT/US01/05991, filed Feb. 23, 2001; and U.S. Provisional Patent Application Ser. No. 60/184,491, filed Feb. 23, 2000 are each incorporated by reference herein.
There is a great need for methods and devices for treating tissue (e.g., diseased or injured tissue), as well as for treating diseases (e.g., osteoarthritis, osteoporosis, cancer, and other diseases). In particular, there is a need for methods and devices that treat tissue and/or diseases by selectively up-regulating or down-regulating the expression of certain genes. More particularly, there is a need for methods and devices that apply treatments (e.g., for peripheral vascular disease, cardiovascular disease, macular degeneration, wound healing, tendon and ligament healing, rheumatoid arthritis, bone healing (e.g., fresh fractures, fractures at risk, delayed healing and nonunion, bone defects, spine fusion, and as an adjunct in any of the above), and/or osteonecrosis), and/or that prevent tumor growth and spread, by selectively down-regulating expression of specific targeted genes, namely, matrix matelloproteinases (MMPs). The present invention is directed to down regulation matrix matelloproteinases (MMPs) in tumor.
The present invention relates to regulating the expression of genes in tissue by applying to such tissue electric and/or electromagnetic fields generated by specific and selective signals. In particular, the present invention relates to methods of regulating the expression of genes in tissue by applying such fields to such tissue, and to devices employing such methods.
In an embodiment of the invention, a method is provided for treating tissue (e.g., injured or diseased tissue), and/or for treating diseases or other conditions (e.g., peripheral vascular disease, osteoarthritis, osteoporosis, cancer, and/or other diseases or conditions), such method preferably includes the steps of (1) providing electric and/or electromagnetic fields that regulate gene expression in targeted tissue, which fields are generated by specific and selective signals, and (2) exposing such targeted tissue to such fields so as to regulate gene expression therein. As contemplated by the invention, a “specific and selective” signal is preferably a signal that (a) has predetermined characteristics (such as, for example but not limited to, amplitude, duration, duty-cycle, frequency, and/or waveform) such that the signal preferably creates an electric and/or electromagnetic field that will up-regulate or down-regulate expression(s) of a targeted gene or targeted functionally complementary genes (e.g., the specificity of the signal is established by which and how many characteristics the signal has and by the predetermined settings of those characteristics), and (b) can preferably be chosen to create such an electric and/or electromagnetic field that will up-regulate or down-regulate expression(s) of a targeted gene or targeted functionally complementary genes in order to achieve a desired response (e.g., a biological and/or therapeutic response) (e.g., the selectivity of the signal is established by the fact that it can be chosen to achieve the desired response). In a related embodiment of the invention, a device is provided for employing the methods of the invention described herein to apply electric and/or electromagnetic fields, generated by specific and selective signals, to up-regulate and/or down-regulate expression(s) of targeted gene(s).
In another embodiment of the invention, a method is provided for determining the specific and selective signal that regulates expression of a particular gene, such method preferably including (1) methodically varying (e.g., preferably by sequential dose-response curves) a first characteristic (e.g., duration, amplitude, duty cycle, frequency, waveform, and/or other characteristic) of a starting signal known to change in a desired manner (e.g., increase or, e.g., decrease) or suspected to change in a desired manner cellular production of a given protein until a desired (e.g., preferably optimal) setting of the first characteristic is determined (based on measured amount of gene expression); (2) methodically varying a second characteristic of the signal (preferably in the same dose-response manner as with the first characteristic) while maintaining the first characteristic at the determined desired setting of the first characteristic, until a desired (e.g., preferably optimal) setting of the second characteristic is determined (based on measured amount of gene expression); (3) optionally methodically varying additional characteristics of the signal (preferably in the same dose-response manner as with the other characteristics), one at a time, while maintaining at their determined desired settings the characteristics for which desired settings have been determined, until desired (e.g., preferably optimal) settings of each of the additional characteristics are determined (based on measured amount of gene expression). It should be understood that each of the determined settings can be reviewed and/or adjusted at the end of the process, or during the process, to ensure their optimum (or at least desired) nature is maintained or established. It should be further understood that the characteristics need not be addressed in any particular order to achieve the present invention, but rather the total number of characteristics addressed can be adjusted, the type of characteristics can be different than those described, and the order in which the chosen characteristics are addressed can be changed, without departing from the scope of the invention. It should be further understood that one or more characteristics can be methodically varied simultaneously, rather than only one characteristic being methodically varied at a time.
In a preferred exemplary embodiment of this aspect of the invention, a method for determining a specific and selective signal that regulates expression of a particular gene preferably includes (1) methodically varying (e.g., preferably by performing sequential dose-response curves) an amplitude of a starting signal known to change in a desired manner (e.g., increase or, e.g., decrease) or suspected to change in a desired manner cellular production of a given protein until an optimal amplitude is determined (i.e., the amplitude corresponding to the maximum (or, e.g., minimum) amount of gene expression observed), (2) methodically varying (preferably in the same dose-response manner as with the amplitude) the duration of the signal for at the determined amplitude until an optimal duration is determined (i.e., the duration of time, applied at the determined amplitude, corresponding to the maximum (or, e.g., minimum) amount of gene expression observed), (3) methodically varying (preferably in the same dose-response manner as with duration and with amplitude) the frequency of the signal for the determined duration of time at the determined amplitude until an optimal frequency is determined (i.e., the frequency, applied at the determined amplitude and for the determined duration of time, corresponding to the maximum (or, e.g., minimum) amount of gene expression observed); (4) methodically varying (preferably in the same dose-response manner as with duration and with amplitude and with frequency) the duty cycle of the signal for the determined duration of time at the determined amplitude and for the determined frequency until an optimal duty cycle is determined (i.e., the duty cycle, applied at the amplitude and for the determined duration of time and at the determined frequency, corresponding to the maximum (or, e.g., minimum) amount of gene expression observed); (5) methodically varying (preferably in the same dose-response manner as with duration and with amplitude and with duty cycle and with frequency) the waveform of the signal for the determined duration of time at the determined amplitude and for the determined duty cycle and at the determined frequency until an optimal waveform is determined (i.e., the waveform, applied at the amplitude and for the determined duration of time and at the determined frequency and for the determined duty cycle, corresponding to the maximum (or, e.g., minimum) amount of gene expression observed).
In another exemplary embodiment of this aspect of the invention, a method for determining a specific and selective signal that down-regulates MMP expression preferably includes (1) methodically varying (e.g., preferably by performing sequential dose-response curves) an amplitude of a starting signal known to decrease or suspected to decrease cellular production of MMP until an optimal amplitude is determined (i.e., the amplitude corresponding to the minimum amount of MMP expression observed), (2) methodically varying (preferably in the same dose-response manner as with amplitude) the duration of the signal at the determined amplitude until an optimal duration is determined (i.e., the duration of time, applied at the determined amplitude, corresponding to the minimum amount of MMP expression observed), (3) methodically varying (preferably in the same dose-response manner as with duration and with amplitude) the frequency of the signal for the determined duration of time at the determined amplitude until an optimal frequency is determined (i.e., the frequency, applied at the amplitude and for the determined duration of time, corresponding to the minimum amount of MMP expression observed); (4) methodically varying (preferably in the same dose-response manner as with duration and with amplitude and with frequency) the duty cycle of the signal for the determined duration of time at the determined amplitude and at the determined frequency until an optimal duty cycle is determined (i.e., the duty cycle, applied at the amplitude and for the determined duration of time and at the determined frequency, corresponding to the minimum amount of MMP expression observed); (5) methodically varying (preferably in the same dose-response manner as with duration and with amplitude and with duty cycle and with frequency) the waveform of the signal for the determined duration of time at the determined amplitude and for the determined duty cycle and at the determined frequency until an optimal waveform is determined (i.e., the waveform, applied at the amplitude and for the determined duration of time and for the determined duty cycle and at the determined frequency, corresponding to the minimum amount of MMP expression observed). It should be understood that each of the determined settings can be reviewed and/or adjusted at the end of the process, or during the process, to ensure their optimum nature is maintained or established. It should be further understood that the characteristics need not be addressed in any particular order to achieve the present invention, but rather the total number of characteristics addressed can be adjusted, the type of characteristics can be different than those described, and the order in which the chosen characteristics are addressed can be changed, without departing from the scope of the invention. It should be further understood that one or more characteristics can be methodically varied simultaneously, rather than only one characteristic being methodically varied at a time.
In an embodiment of the invention, a method is provided for treating tissue and/or a disease and/or condition (e.g., for treating peripheral vascular disease, cardiovascular disease, macular degeneration, wound healing, tendon and ligament healing, rheumatoid arthritis, bone healing (e.g., fresh fractures, fractures at risk, delayed healing and nonunion, bone defects, spine fusion, and as an adjunct in any of the above), and/or osteonecrosis, and/or for preventing tumor growth and spread), such method including (1) providing electric and/or electromagnetic fields that regulate VEGF expression in targeted tissue, which fields are generated by specific and selective signals suitable for generating such fields, and (2) exposing such targeted tissue to such fields so as to down-regulate MMP expression therein. MMP is a target gene of choice because, among other reasons, it is an important growth factor, and possibly the most important growth factor, in preventing tumor growth and spread.
In an exemplary embodiment of this aspect of the present invention, a method is provided for down-regulating MMP expression in tumor cells, such method preferably including (1) providing electric and/or electromagnetic fields that down-regulate MMP expression in tumor cells, which fields are generated by specific and selective signals suitable for generating such fields in tumor cells, and (2) exposing tumor cells to such fields (preferably via electrodes) so as to down-regulate MMP expression in the tumor cells. A desired (e.g., preferably effective for, and more preferably optimal for, generating an electric and/or electromagnetic field that down-regulates MMP expression in tumor cells) specific and selective signal is determinable by applying a method of the invention described above to perform sequential dose-response curves on chosen characteristics of a signal (e.g., duration, amplitude, frequency, and duty cycle), by which curves the effects of the resultant electric and/or electromagnetic field are measured. The signal presently determined by the inventor to be most effective at generating a field that most effectively down-regulates MMP expression in tumor cells generates a capacitively coupled electric field with an amplitude of between 1 and 80 mV/cm inclusively, a duration of between 30 minutes and 24 hours inclusively, a frequency of between 30 and 120 kHz inclusively, and a duty cycle of between 5 and 100% inclusively, with a sine wave waveform. In particular, the present invention relates to down-regulating MMP gene expression in tumor cells via the application of fields generated by such signals. This method is useful for, among treating other diseases or conditions, preventing tumor growth and/or spread.
These and other aspects of the present invention will be elucidated in the accompanying drawings and following detailed description of the invention.
The invention will be described in detail below. Those skilled in the art will appreciate that the description given herein is for exemplary purposes only and is not intended in any way to limit the scope of the invention.
The present invention is based on the determination that the expression of certain genes can be regulated by applying electric and/or electromagnetic fields generated by specific and selective signals. In other words, it has been determined by the present inventors that there is a specific signal that generates an electric and/or electromagnetic field for regulating each gene in tissue (e.g., capillaries and blood vessels, the retina, healing wounds, tendons, ligaments, bone, cartilage, tumor cells, and other tissue cells), and that these specific signals are capable of regulating particular genes in such tissue. In particular, gene expression governing the growth, maintenance, repair, and degeneration or deterioration of tissue can be regulated in accordance with the invention by applying electric and/or electromagnetic fields generated by specific and selective signals so as to produce a salutory clinical effect. Such determinations are useful in the development of treatments for certain medical diseases and/or conditions (including but not limited to peripheral vascular disease, cardiovascular disease, macular degeneration, wound healing, tendon and ligament healing, rheumatoid arthritis, bone healing (e.g., fractures, fresh fractures, fractures at risk, delayed union, nonunion, bone defects, spine fusion), osteonecrosis, osteoarthritis, osteoporosis, and/or cancer, and for preventing tumor growth and spread, and as an adjunct in the treatment of any one or more of the above), as well as in the development of devices employing such methods.
As used herein, the term “signal” refers to any signal, including but not limited to mechanical signals, ultrasound signals, electromagnetic signals and electric signals.
As used herein, the term “field” refers to an electric and/or electromagnetic field within targeted tissue, regardless of type or method of generation. Examples include but are not limited to combined field, pulsed electromagnetic field, or generated by direct current, capacitive coupling or inductive coupling.
As used herein, the term “remote” is used to mean acting, acted on or controlled from a distance. As used herein, the phrase “remote regulation” refers to controlling from a distance (e.g., to “remotely regulate gene expression” refers to regulating the expression of a gene from a distance). As used herein, the phrase “to provide remotely” refers to providing from a distance. For example, providing a specific and selective signal from a remote source can refer to providing the signal from a source at a distance from a tissue or a cell, or from a source outside of or external to the body.
As used herein, the term “regulate” means to control gene expression, and is understood to include both up-regulate and down-regulate. As used herein, the term “up-regulate” means to increase expression of a gene, and the term “down-regulate” means to inhibit or prevent expression of a gene.
As used herein, the phrase “functionally complementary” refers to two or more genes whose expressions are complementary or synergistic in a given cell or tissue.
As used herein, the term “tissue” refers to an aggregate of cells together with their extracellular substances that form one of the structural or other materials of a patient. As used herein, the term “tissue” is intended to include any tissue of the body including but not limited to capillaries, blood vessels, muscle and organ tissue, wound tissue, tumor tissue, bone tissue, or cartilage tissue. Also, the term “tissue” as used herein may also refer to an individual cell.
As used herein, the term “patient” refers to an animal, preferably a mammal, more preferably a human.
The present invention provides treatment methods and devices that target certain tissues and/or diseases and/or conditions, such as, for example, that target tumor tissues, cells, and tumorous diseases. In particular, gene expression associated with the repair process in tissue, or, for example, associated with tumor growth and spread, can be regulated by the application of signals, and electric and/or electromagnetic fields generated by such signals that are effective for regulating gene expression in the target tissue. Gene expression can be up-regulated or down-regulated by the application of electric and/or electromagnetic fields generated by signals that are specific and selective for regulating expression of each gene or each set of functionally complementary genes so as to produce a desired (e.g., preferably beneficial) clinical effect. For example, an electric and/or electromagnetic field generated by a particular specific and selective signal may up-regulate a certain desirable gene expression, while the same or another field generated by a particular specific and selective signal may down-regulate a certain undesirable gene expression. A certain gene expression may be up-regulated by an electric and/or electromagnetic field generated by one particular specific and selective signal and down-regulated by an electric and/or electromagnetic field generated by another specific and selective signal. Those skilled in the art will understand that certain tissues and/or diseases and/or conditions can be treated by regulating expression of those genes governing the growth, maintenance, repair, and degeneration or deterioration of the implicated tissues. Those skilled in the art will further understand that certain tumor tissues can be targeted for treatment by down-regulating those genes governing the growth and spread of tumor tissues.
The methods and devices of the present invention are based on identifying those signals that are specific and selective for generating electric and/or electromagnetic fields that regulate the gene expression associated with treating certain tissue, diseases, and/or conditions. For example, electricity in its various forms (e.g., capacitive coupling, inductive coupling, combined fields) can specifically and selectively regulate gene expression in tissue by varying one or more characteristics (e.g., frequency, amplitude, waveform, or duty cycle) of the signal generating the applied electric and/or electromagnetic field for the gene of interest. Other characteristics (e.g., the duration of time applied) of the signal can also influence the capability of the field to regulate gene expression in targeted tissue. Specific and selective signals may generate fields for application to each gene systematically or otherwise until the proper combination of characteristics (e.g., frequency, amplitude, waveform, duty cycle, and duration) is found that provides the desired effect on gene expression.
It is to be understood that a variety of diseased or injured tissues, conditions, or disease states (including, e.g., a variety of tumors or tumorous tissues or disease states) can be targeted for treatment because the specificity and selectivity of a signal that generates an electric and/or electromagnetic field to regulate expression of a certain gene can be influenced by several factors. In particular, for example, a signal of appropriate frequency, amplitude, waveform, and/or duty cycle can be specific and selective for generating an electric and/or electromagnetic field that regulates the expression of certain genes and thus provide for targeted treatments. Temporal factors (e.g., duration of time exposed to the field) can also influence the specificity and selectivity of a signal that generates an electric and/or electromagnetic electric field for regulating expression of a particular gene. That is, the regulation of gene expression may be more effective (or made possible) by applying an electric and/or electromagnetic field for a particular duration of time. Therefore, those skilled in the art will understand that the present invention provides for varying the frequency, amplitude, waveform, duty cycle, and/or duration of application of a signal that generates an electric and/or electromagnetic field until the field is found to regulate certain gene expressions more effectively in order to provide for treatments targeting a variety of diseased or injured tissue or diseases (including, e.g., a variety of tumors or tumorous tissues or disease states).
Thus, the present invention provides for targeted treatments because it is possible to regulate expression of certain genes associated with a particular diseased or injured tissue, or a particular disease or condition, via the application of electric and/or electromagnetic fields generated by specific and selective signals of appropriate frequency, amplitude, waveform and/or duty cycle for an appropriate duration of time. The specificity and selectivity of a signal generating an electric and/or electromagnetic field may thus be influenced so as to regulate the expression of certain genes in order to target certain diseased or injured tissue, conditions, or disease states for treatment. The present invention thereby provides for a multitude of targeted treatments including the treatment of osteoarthritis, osteoporosis, cancer, peripheral vascular disease, cardiovascular disease, macular degeneration, wound healing, tendon and ligament healing, rheumatoid arthritis, bone healing (e.g., fresh fractures, fractures at risk, delayed healing, nonunion, bone defects, spine fusion, and as an adjunct to any of the above) osteonecrosis, and/or as an adjunct in the treatment of one or any of the above, and in preventing tumor growth and spread.
The present invention further provides devices for the treatment of injured or diseased tissue, conditions, and disease states. In particular, the present invention provides devices that include a source of at least one signal that is specific and selective for generating an electric and/or electromagnetic field that regulates expression of a gene. The devices of the present invention can provide for the production of such signals or fields for application to the targeted tissue by at least one electrode adapted to apply the specific and selective signal.
The devices of the present invention are capable of applying specific and selective signals, and consequently an electric and/or electromagnetic field generated by specific and selective signals, directly to diseased or injured tissue (e.g., tumor or tumorous tissue) and/or indirectly to diseased or injured tissue (e.g., tumor or tumorous tissue) (e.g., the signal can be applied to the skin of a patient near the targeted tissue). The devices of the present invention may also provide for the remote application of specific and selective signals, or an electric and/or electromagnetic field generated by specific and selective signals (e.g., remote application being application of a signal, or an electric and/or electromagnetic field generated by a signal, at a distance from targeted tissue (e.g, diseased or injured tissue) yet which yields the desired effect on the patient's body and/or within the targeted tissue), although it will be appreciated that capacitively coupled devices must touch the patient's skin. The devices of the present invention may include means for attaching electrodes to the body of a patient in the vicinity of injured or diseased tissue, such as in the case of capacitive coupling. For example, self-adherent conductive electrodes may be attached to the skin of the patient on both sides of a fractured bone, or both sides of a knee joint afflicted with osteoporosis as shown in
The devices of the present invention can be employed in a variety of ways. The devices may be portable or may be temporarily or permanently attached to a patient's body. The devices of the present invention are preferably non-invasive. For example, the devices of the present invention may be applied to the skin of a patient by application of electrodes adapted for contact with the skin of a patient for the application of predetermined specific and selective signals, or electric and/or electromagnetic fields generated by predetermined specific and selective signals. Such signals may also be applied via coils in which time varying currents flow, thus producing electric and/or electromagnetic fields that penetrate the tissue and create the electric and/or electromagnetic fields in the targeted tissue. The devices of the present invention may also be capable of implantation in a patient, including implantation under the skin of a patient.
Examples below will illustrate that the methods of the present invention may provide for bone growth and repair via regulation of gene expression in bone cells. The methods of the present invention can stimulate bone growth and repair in the vicinity of fresh fractures and non-union fractures. Bone growth and repair also can be stimulated in the vicinity of osteoarthritis or osteoporosis. A variety of cells can be targeted by the methods of the present invention including capillary and blood vessel cells, muscle cells, organ cells, bone cells, cartilage cells, retinal cells, tendon cells, ligament cells, fibrous tissue cells, stem cells, tumor cells, cancer cells, and other tissue.
Examples below also will illustrate that the methods of the present invention may provide for cartilage growth and repair. Cartilage growth and repair can be stimulated by applying electric and/or electromagnetic fields generated by signals that are specific and selective for the expression of certain genes. For example, the methods of the present invention can stimulate articular cartilage repair in osteoarthritis patients and provide for the regulation of gene expression in cartilage cells. In particular, the methods of the present invention can provide for the up-regulation of genes that repair cartilage (e.g., genes encoding for aggrecan and Type II collagen), down-regulation of genes that destroy cartilage (e.g., genes encoding for metalloproteinase) and the up-regulation of genes that inhibit metalloproteinases that destroy articular cartilage (e.g., genes encoding for tissue inhibitors of metalloproteinase). A variety of cartilage cells can be targeted by the methods of the present invention including articular chondrocytes and including articular cartilage, hyaline cartilage, and growth plate cartilage.
Examples below further illustrate that the methods of the present invention provide for the regulation of gene expression in articular chondrocytes. For example, in the examples below, fetal articular chondrocytes have been exposed to a capacitively coupled 60 kHz electrical field of 20 mV/cm for 0.5, 2.0, 6.0 and 24.0 hours. A statistically significant incorporation of 35SO4/ug DNA (indicating significant proteoglycan synthesis) was found after only 0.5 hours of stimulation. An identical experiment was repeated and the levels of aggrecan mRNA, the messenger for the major cartilage proteoglycan, monitored. After only 0.5 hours of electrical stimulation there was a significant increase (almost 100%) in aggrecan mRNA. Accordingly, temporal factors may influence the specificity and selectivity of a signal that generates an electric and/or electromagnetic field regulating gene expression in articular chondrocytes.
Examples below further illustrate that the methods of the present invention provide for the down-regulation of MMP in tumor cells. By performing sequential dose-response curves on characteristics (e.g., duration, amplitude, frequency, duty cycle, and/or waveform) of a signal, by which curves the effects of the electric and/or electromagnetic field generated by the signal are measurable, a desired (e.g., preferably optimal) signal for down-regulating MMP mRNA in tumor cells is determinable. The signal presently determined by the inventor to be most effective at generating a field that most effectively down-regulates MMP expression in tumor cells generates a capacitively coupled electric field with an amplitude of between 1 and 80 mV/cm inclusively, a duration of between 30 minutes and 24 hours inclusively, a frequency of between 30 and 120 kHz inclusively, and a duty cycle of between 5 and 100% inclusively, with a sine wave waveform. In particular, the present invention relates to down-regulating MMP gene expression in tumor cells via the application of fields generated by such signals. Decreasing MMP gene expression in tumor cells is a viable treatment for preventing tumor growth and/or spread.
The methods of the present invention also provide for the treatment of certain diseases. In particular, the methods of the present invention can provide for the treatment of cancer. In a patient with a primary (or even metastatic) cancer, metalloproteinase is at least partly responsible for spread of the cancer. Metalloproteinase enzymatically breaks down fibrous walls or membranes erected by adjacent cells in an attempt to contain the cancer. However, as mentioned above, tissue inhibitors of metalloproteinase may inhibit the production of such metalloproteinases. Accordingly, methods of the present invention can provide for the down-regulation of genes encoding for metalloproteinase and the up-regulation of genes encoding for tissue inhibitors of metalloproteinase (“TIMP”). Those skilled in the art will understand that a variety of other diseases and conditions may be targeted for treatment via the methods of the present invention, such as peripheral vascular disease, cardiovascular disease, macular degeneration, wound healing, tendon and ligament healing, rheumatoid arthritis, bone healing (e.g., fresh fractures, fractures at risk, delayed healing and nonunion, bone defects, spine fusion, and as an adjunct in any of the above), osteonecrosis, tumor growth and spread, and/or other diseases or conditions.
While not limiting the present invention in any way, it is presently believed that those genes that are functionally complementary may respond to identical or substantially similar signals. In other words, a signal may be specific and selective for generating an electric and/or electromagnetic field that regulates expression of functionally complementary genes. With reference to
Those skilled in the art will further understand that the devices of the present invention can be provided in a variety of forms including a capacitively coupled power unit with programmed, multiple, switchable, specific and selective signals for application to one pair or to multiple pairs of electrodes, electromagnetic coils, or a solenoid attached to a power unit with switchable, multiple, specific and selective signals, and an ultrasound stimulator with a power supply for generating specific and selective signals. Generally speaking, device preference is based on patient acceptance and patient compliance. The smallest and most portable unit available in the art at the present time is a capacitive coupling unit; however, patients with extremely sensitive skin may prefer to use inductive coupling units. On the other hand, ultrasound units may be used, and require the most patient cooperation, but may be desirable for use by certain patients.
The invention is further demonstrated in the following examples, which are for purposes of illustration, and are not intended to limit the scope of the present invention.
Chondrocyte cultures were prepared from fetal bovine articular cartilage. Chondrocytes (5×105 cells/cm2) were plated onto specially modified Cooper dishes. The cells were grown to seven days with the medium changed just prior to beginning the experimental condition. The experimental cell cultures throughout these studies were subjected to a capacitively coupled 60 kHz sine wave signal electric field with an output of 44.81 volts peak to peak. This produced a calculated-field strength in the culture medium in the dishes of 20 mV/cm with a current density of 300 μA/cm2. Control cell culture dishes were identical to that of the stimulated dishes except that the electrodes were not connected to a function generator.
Total RNA was isolated using TRIzol, according to the manufacturer's instructions, and reversed transcription using SuperScript II reverse transcriptase was performed. Oligonucleotide primers to be used in the competitive PCR technique were selected from published cDNA sequences. Quantitative analysis of PCR products was performed using ScionImage software.
The optimal signal for generating a field to effect the desired gene regulation was determined as follows. A signal known to increase (or suspected to increase) cellular production of a given protein is taken as the starting signal for determining the specific signal for generating a field to regulate the gene expression (mRNA) of that protein. A dose-response curve is first performed by varying the duration of the signal while holding other chosen signal characteristics constant (e.g., amplitude, duty-cycle, frequency, and waveform). This determines the optimal duration of the starting signal for generating a field to regulate the gene expression of that protein. A second dose-response curve is performed by varying the amplitude for the optimal duration of time. This determines the optimal amplitude for the optimal duration of time as determined by the gene expression of the protein of interest. A third dose-response curve is then performed, this time varying the duty-cycle from 100% (constant) to 1% or less while holding the optimal amplitude and other chosen signal characteristics constant. A dose-response curve is repeated a fourth time (varying frequency) and a fifth time (varying waveform) each time keeping the other chosen signal characteristics constant (preferably at the determined optimal settings). By this method an optimal signal is determined for generating a field that produces the greatest increase in the gene expression of the protein of interest.
Protein expression may be determined by any method known in the art, such as reverse transcriptase PCR, Northern analysis, immunoassays, and the like.
Articular chondrocytes were exposed to a capacitively coupled electric signal of 20 mV/cm at 60 kHz. The results are illustrated in
Articular chondrocytes were exposed to a capacitively coupled electric signal of 20 mV/cm at 60 kHz. The results are illustrated in
With reference to Examples 1 and 2, it is demonstrated that each of those genes encoding aggrecan or Type II collagen can be regulated by an identical 20 mV/cm, 60 kHz capacitively coupled signal. Those skilled in the art will appreciate that each of these gene transcripts regulates cartilage matrix formation and are functionally complementary. Accordingly, the findings of Examples 1 and 2 are believed to support electrical therapy through gene regulation in accordance with the techniques described herein.
Articular chondrocytes were exposed to a capacitively coupled electric signal of 20 mV/cm at 60 kHz. The results are illustrated in
Articular chondrocytes were exposed to a capacitively coupled electric signal of 20 mV/cm at 60 kHz. The results are illustrated in
As noted above, it has been reported that a 60 kHz capacitively coupled electric field of 20 mV/cm produces a significant increase in TGF-β1 in similar bone cells. Brighton et al., Biochem. Biophys. Res. Commun. 237: 225-229 (1997). It was found that there was significant production of TGF-β1, mRNA, but only after 6 hours of stimulation (in contrast to 0.5 hours for aggrecan mRNA and Type II collagen mRNA). The experiment was repeated to determine if the exposure of MC3T3-E1 bone cells to the 20 mV/cm, 60 kHz capacitively coupled electric signal had an effect on the production of PDGF-A mRNA. No effect was found.
Thus, a 20 mV/cm, 60 kHz capacitively coupled signal regulates bone cell genes encoding TGF-β1 but fails to regulate genes encoding PDGF-A. It is presently understood that the expression of each of these genes participates in the regulation of different phases and physiologic processes of bone healing and are thus are not functionally complementary.
With reference to
The power unit is preferably small (e.g., 6-8 ounces) and powered by a standard 9-volt battery to emit a 5 volt peak-to-peak, 6-10 mAmp, 20 mV/cm, 60 kHz sine wave signal to the electrodes 12 placed on the skin. As illustrated in the above examples, this signal provided 30 minutes per day with the desired time duration, field amplitude, and duty cycle should significantly up-regulate genes encoding aggrecan and Type II collagen. This treatment should prevent or minimize further articular cartilage deterioration as well as heal articular cartilage that already is damaged or degenerated.
The power unit 14 also may be reconfigured to provide signals specific and selective for other genes. For example, as illustrated in the above examples, the power unit 14 may be reconfigured to provide signals for down-regulating the gene expression of metalloproteinase (MMP) as well as signals for up-regulating genes expressing tissue inhibitors of metalloproteinase (“TIMP”) genes. The power unit 14 may be reconfigured to provide such signals in sequence with the aggrecan/Type II collagen signal. Accordingly, the patient may be treated through the up-regulation of genes that repair cartilage (e.g., aggrecan and Type II collagen genes), down-regulation of genes that destroy cartilage (e.g., metalloproteinase gene) and the up-regulation of genes that inhibit the metalloproteinases that destroy articular cartilage (e.g., tissue inhibitors of metalloproteinase).
With reference to
The power unit 14′ provides a 20 mV/cm, 60 kHz sine wave signal to the electrodes 12 placed on the skin. The signal is provided for 6 hours per day as in example 5. The power unit 14′ is differentiated from power unit 14 in the previous example since the same electrical signal as defined by time duration, field amplitude, and duty cycle is not necessarily applied. This technique should aid in the repair process by up-regulating TGF-β1, a gene important in the cartilage phase of bone repair.
Those skilled in the art will appreciate that the power unit 14′ may be reconfigured to provide other signals specific for certain genes. For example, the power unit 14 may be reconfigured to provide signals for the up-regulation of PDGF-A, basic FGF and BMP-2 genes. The power unit 14 also may be reconfigured to provide in sequence those signals specific and selective for TGF-β1, PDGF-A, basic FGF, and BMP-2 genes. Therefore, the power unit 14 may be reconfigured to provide specific and selective signals that up-regulate genes necessary to heal bone defects.
With reference to
The device 10″ of the invention provides specific capacitively coupled electric fields via electrodes 12 for selectively down-regulating the gene encoding for metalloproteinase as discussed in the above examples and/or selectively up-regulating the gene encoding for tissue inhibitors of metalloproteinase (“TIMP”) The device 10″ can provide the electric field generated by power unit 14″ so as to selectively down-regulate and up-regulate the genes sequentially for specific periods of time per day. The melanoma can be safely excised once the melanoma has been sufficiently encapsulated by the body's own defensive mechanism.
The signal presently determined by the inventor to be preferable for generating a field that effectively down-regulates MMP expression in tumor cells as measured by mRNA production generates a capacitively coupled electric field with an amplitude of between 1 and 80 mV/cm inclusively, a duration of between 30 minutes and 24 hours inclusively, a frequency of between 30 and 120 kHz inclusively, and a duty cycle of between 5 and 100% inclusively, with a sine wave waveform. It is the inventor's current determination that tumor cells exposed to such a field will experience a down-regulation in MMP expression as measured by mRNA production in the cells.
With reference to
The power unit is preferably small (e.g., 6-8 ounces) and powered by a standard 9-volt battery to emit a signal to the electrodes 12 placed on the skin. As discussed in Example 9, it has been determined by the inventor that a signal with characteristics in the preferred ranges will down-regulate MMP expression in tumor cells. This treatment should prevent or minimize tumor growth and spread in the upper arm
Those skilled in the art will also appreciate that numerous other modifications to the invention are possible within the scope of the invention. For example, genes encoding for tissue inhibitors of metalloproteinase (“TIMP”) and other genes may have improved specific dose responses at selective frequencies other than 60 kHz so as to provide specific and selective responses for applied signals at different frequencies with different time durations, field amplitudes, and duty cycles. Also, as noted above, inductively coupled signals, direct coupled signals, and pulsed electromagnetic fields may also be applied in lieu of capacitively coupled signals as described in the examples above. Accordingly, the scope of the invention is not intended to be limited to the preferred embodiments described above.
A signal presently determined by the inventor to be preferable for generating a field that effectively down-regulates MMP expression in tumor cells (such as in the human colon) as measured by mRNA production generates a capacitively coupled electric field with an amplitude of about 20 mV/cm to 80 mV/cm, more preferably 50 mV/cm to 70 mV/cm, and most preferably 60 mV/cm, a duration of about 1-10 hours, more preferably 3-6 hours, and most preferably about 5 hours, a frequency of about 50-70 kHz, more preferably 60 kHz, a duty cycle of 100%, and a sine wave waveform. It is the inventor's current determination that tumor cells exposed to such a field will experience a down-regulation in MMP expression as measured by mRNA production in the cells. This determination is based on experience employing the inventive process herein to down-regulate MMP in bone, cartilage, and endothelial cells versus tumor cells. Indeed, experience has shown that bone cells require a relatively long duration (approaching 24 hours), cartilage cells require a lower duration (about 1-3 hours), and endothelial cells require a slightly higher duration (about 4 hours). Thus, it has been determined that tumor cells would respond to signals having one or more of the parameters described above.
This application is based on and claims the benefit of U.S. Provisional Patent Application No. 60/975,367, filed Sep. 26, 2007, the entire disclosure of which is hereby incorporated by reference.
| Number | Date | Country | |
|---|---|---|---|
| 60975367 | Sep 2007 | US |
