ENHANCING EPITHELIAL INTEGRITY BY A SEQUENCE OF MAGNETIC PULSES

Abstract

The invention provides a method of noninvasively treating a condition associated with reduced integrity or increased permeability of an epithelial layer by a series of magnetic pulses having a magnitude of up to 3 T, wherein the magnetic pulses are designed to induce an electric field having a magnitude of up to 250 Volt per meter adjacent to a target treatment area, the condition particularly being ocular condition associated with reduced barrier function of the cornea. The invention provides a device for effecting the tissue treatment, creating series of pulses exhibiting a rate of change of more than 200 T/s in the absolute value.

Claims

1. A magnetic device for treating an epithelial layer, comprising a magnetic pulses generator, the pulses having a magnitude of up to 3 T at the distance of up to 10 cm from the generator, and a duration of from 50 s to 2000 s, wherein said magnitude increases during said pulse at a rate of change of at least 200 T/s in the absolute value.

2. The device of claim 1, wherein the magnitude is from 0.05 T to 2 T.

3. The device of claim 1, wherein the rate of change is from 2,000 to 20,000 T/s in the absolute value.

4. The device of claim 1, producing the pulses at a rate of up to 100 pulse/s.

5. The device of claim 1, wherein the magnetic pulses generator is designed to induce a suitable electric field around a treatment area.

6. The device of claim 6, wherein the electric field is adapted to affect nerve fibers in the treatment area.

7. The device of claim 7, wherein the electric field is adapted to affect nerve fibers in the medium to long term, by adjusting one or more electric field parameters selected from the group consisting of intensity, direction, pulse rate and pulse duration.

8. The device of claim 7, wherein the electric field is optimized to deliver a magnitude of 20-250 Volt/meter (V/m) in a target nerve fiber.

9. The device of claim 6, wherein the electric field has magnitude of 70-150 V/m.

10. The device of claim 1, wherein said epithelial layer is associated with the cornea of a human eye.

11. The device of claim 11, wherein the electric field is adapted to form a substantially circular pattern around the eye.

12. A method of noninvasively treating an epithelial layer, comprising creating near the epithelial layer magnetic pulses having a magnitude of up to 3 T and a duration of from 50 ms and 2000 ms, wherein the magnitude increases during the pulse at a rate of change of at least 200 T/s in the absolute value.

13. The method of claim 12, wherein the rate of change is from 2,000 to 20,000 T/s in the absolute value.

14. The method of claim 12, wherein the pulses are produced at a rate of up to 100 pulse/s.

15. The method of claim 12, wherein the pulses are created in a series lasting from 0.5 to 20 s.

16. The method of claim 1124, wherein the magnetic field pulses induce an electric field in tissue, the electric field of up to 250 Volt/meter in the absolute value.

17. The method of claim 14, wherein the pulses are created in a series followed by a break without a magnetic signal.

18. The method of claim 16, wherein the magnetic field pulses induce in tissue an electric field of up to 250 Volt/meter in the absolute value in neurons pathways at the vicinity of the eye.

19. The method of claim 14, comprising treating corneal cells selected from the group consisting of epithelial cells, endothelial cells, nerve cells, and stromal cells.

20. The method of claim 16, wherein the effect of one treatment lasts for at least 1 week.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The principles and operation of the system, apparatus, and method according to the present invention may be better understood with reference to the drawings, and the following description, it being understood that these drawings are given for illustrative purposes only and are not meant to be limiting, wherein:

[0019] FIG. 1 is a text book drawing that shows an imposed depiction of a perpendicular magnetic field and a circular electric field around the eye, as shown in research conducted using elements of the present invention;

[0020] FIG. 2 is a prior art (P. J. Maccabee Et. Al., page 44, FIG. 6) diagram depicting a magnetic coil 1 with an arrow showing direction of electric current, and a coronal cross section of the brain showing the circular direction of an induced electric field;

[0021] FIG. 3 is a text book drawing showing tissues, and in particular tissues around the eye, with a depiction of neurons that may be effected by a treatment as described herein, as described in research conducted using elements of the present invention; and

[0022] FIG. 4 is a diagram describing test results showing a lasting effect following a (single) treatment, for example, within up to 3 months, even though multiple treatments may be performed, as described research conducted using elements of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

[0023] It has now been found that certain types of magnetic field are particularly efficient in reducing the permeability of the cornea and enhancing the corneal integrity. In some embodiments, a magnetic signal may be provided in pulses comprising the field strength of between 0.2 T to 2 T, and with an amplitude change of between 7,000 to 15,000 T/s, to substantially reduce the permeability of a compromised cornea, for example when repetitively applied for between 200-500 micro seconds pulses at a rate of between 15 to 50 pulse per second. In one example, as applied to a rabbit cornea, a magnetic signal provided in pulses comprising the field strength of about 0.2 T and the amplitude change of about 13,000 T/s substantially reduced the permeability of a compromised rabbit cornea toward fluorescein, for example when repetitively applied for around 100 ms at a rate of 20 pulse/s.

[0024] When employing a field of at least 0.1 T, changing with a rate of change of at least 200 T/s in the absolute value, various pulse-shaped signals may be employed. It is understood that changing magnetic field comprises an electric component (electromagnetic field), but for technical reasons, the magnetic component was regulated in the generators employed in the development work which resulted in the invention, and it is the magnetic component to which the description of the invention mainly relates. The alternating magnetic field can induce electric fields in tissues [P. J. Maccabee et al.: J. Clin. Neurophysiol. 8(1) (1991) 38-55], as described with reference to FIG. 2, it may affect cells growth, it may modulate vascular tone and permeability by modulating calcium channels in vascular smooth muscle cells [Okano H. et al.: Bioelectromagnetics 20(3) (1999) 161-71]. Determination of potential mechanisms involved in this physiological responses to magnetic field exposure is ongoing, and multiple and variable biological mechanisms have been suggested [see WO 2014/181327 of the same inventors]. The inventors believe that induced electrical fields and electrical currents, affecting various types of tissues, including neurons, as described with reference to FIG. 3, contribute to the overall effects, at least in case of corneal treatment. Both magnetic and electric components may affect the cellular function, for example in epithelium, by altering mechanisms associated with survival, proliferation, orientation, migration, differentiation, cell adhesion, protein phosphorylation, gene expression, metabolic state, RNA & micro RNA expression, and others. For example, in regard to the human cornea, it comprises several layers, such as corneal endothelium, Descemet's membrane, corneal stroma, Bowman's layer, and corneal epithelium, and in addition the human cornea is one of the most richly innervated structures in the body and is densely supplied by sensory and autonomic nerve fibers; the magnetic and electric stimulation of the mentioned tissues may contribute to the whole positive treatment effect.

[0025] The magnetic field employed in a system according to the present invention comprises electromagnetic field of which magnetic component near the treated tissue has a strength of between 0.05 and 3 T, such as at least 0.1 T, or at least 0.2 T, or at least 0.3 T, or at least 0.4 T, or at least 0.5 T. In this context, the strength of the magnetic field means the peak amplitude of the pulse. The magnetic field employed in a system, method, or device according to the present invention includes a strength near the treated tissue of about 0.1 T or 0.2 T or 0.3 T or 0.4 T or 0.5 T or 0.6 T or 0.7 T or 0.8 T or 0.9 T or 1 T or 1.2 T or 1.4 T or 1.6 T, wherein the field amplitude changes in the absolute value at a rate of change of at least 200 T/s. The term about before a value means the value 10%; however, it is understood that all experimental values may vary within certain error, and a range of 10% is usually assumed as such an error. Said strength may be considered in the vicinity of the epithelial tissue to be treated or near the field generator; the distance of the generator may be usually up to 15 cm, such as up to 10 cm, for example in case of treating cornea the distance may be similar to the distance of the eyeglasses from the eye surface, for example, between 0.5 cm to 3 cm. The magnetic field may be an alternating field, and preferably is a pulsed field having a rate between 5 to 500 pulse/s, such as 20 pulse/s. The field is preferably applied repetitively, comprising pulses preferably between 50 and 2000 s long applied as trains in periods of 0.5 to 20 s separated by intervals, possibly of similar duration. The total treatment session time is usually between 1 and 60 minutes. In some embodiments, a treatment session takes up to 30 min, with various arrangements of pulses within the session, comprising a pulse rate of between 0 and 200 pulse/s; a session may include several different sections, for example comprising rates of 20, 0, 50, and 200 pulse/sec.

[0026] The invention provides, in one embodiment, a device for treating eye conditions associated with reduced corneal integrity, which can be worn by a person, in which a magnetic field generator is incorporated. The generator can generate a magnetic field of different parameters. The magnetic field reaches the eye. The decrease of the corneal permeability protects the cornea from physical damages, for example in persons suffering from eye dryness.

[0027] Certain magnetic stimulations decrease the permeability of the cornea, beneficially affecting persons suffering from eye dryness or persons wearing contact-lenses, who are afflicted with decreased lubrication of the eye, known to make the cornea more susceptible to different types of damages. It has been found by the inventors that repetitive magnetic stimulation reduces the corneal permeability as desired; the field preferably comprising changing magnetic field which has a strength of at least 0.1 T, preferably about 1 T or more, and which changes at a rate of change of at least 200 T/s, preferably more than 1000 T/s, preferably provided in pulses at a rate of 5-500 pulse/s, such as 10-100 pulse/s, for example 20 pulse/s; such field enhances the corneal integrity and so protects the eye from damages eventually caused by undesired entry of damaging agents from the environment and/or undesired exit of eye liquids.

[0028] This invention provides a device, a method, and a system for reducing the corneal permeability and enhancing corneal integrity by applying magnetic pulses comprising a strength of at least 0.1 T and a time change in the absolute value of at least 200 T/s, so assisting clinicians and pharmacologists challenged by eye disorders in which the corneal function is perturbed. The invention enables to treat or to prevent or to mitigate the conditions selected from the group consisting of eye dryness, keratitis sicca, corneal keratitis, corneal epithelial dysfunctions, reduced barrier function of the cornea associated with diabetes, conditions associated with increased corneal permeability due to ageing, minor lesions of the corneal surface, conditions associated with wearing contact lenses, reduced self-healing capabilities of the cornea, penetration of harmful agents to the eye from the contaminated environment, weakened anti-penetration system, and cornea-associated inflammation. The conditions to be handled by the device and method of the invention may be associated with items selected from the group consisting of keratitis caused be contact lenses, epidemic keratoconjunctivitis, aging of the cornea, epithelial corneal dystrophies, atopic keratoconjunctivitis, vernal keratoconjunctivitis, allograft corneal epithelial rejection, limbal chemical burn, epithelial keratitisherpes simplex, epithelial keratitisneurotropic, Sjogren's syndrome, tear production induced by anti-Parkinson agents or anti-spasmotic agents or antiulcer-agents or aqueous tear deficiency medication, staphylococcal belephritis, argon laser burns, Reiter syndrome, rheumatoid peripheral ulcerative keratitis, systemic diseases, and after ophthalmic procedures such as LASIK and others. The device and method of the invention may advantageously handle post-operative conditions which expose the cornea to susceptibility of dry eye such as any refractive type of surgery, other corneal surgeries, cataract surgeries and glaucoma surgeries, systemic diseases and conditions which may cause dry eye including diabetes autoimmune diseases, as well as the conditions associated with treatment including local drops which cause corneal epithelial damage or with systemic treatment which may cause dry eye.

[0029] In exemplifying some embodiments of the invention, ocular penetration of sodium-fluorescein in rabbit eyes following magnetic stimulation at different intensities was used. The model using rabbit eyes and sodium fluorescein was employed by the inventors due to the high ocular safety profile of the compound, its hydrophilic nature, and the fact that corneal staining by this fluorescent dye is the acceptable clinical measure for epithelial damage, as well as the ability to measure its concentration in the anterior chamber with good precision and reproducibility using a fluorometer. The baseline corneal permeability of this hydrophilic substance is very limited, due to the corneal barrier function. It is known that fluorescein does not penetrate or stain live corneal epithelial cells upon topical application, the corneal epithelial defects are readily stained as the dye diffuses between cells into the adjacent intercellular spaces and penetrates into the underlying corneal stroma. Rabbits were either non treated (sham) or treated with magnetic pulses of minimal strength and minimal amplitude time change one hour prior to fluorescein application. Corneal staining by fluorescein as well as fluorescence measurements of anterior chamber fluid were used to quantify the ocular penetration. In order to demonstrate the safety of the procedure, animals were tested for retinal function by electro-retinogram prior to the magnetic treatment, at 1 day, 1 week, 1 month, and 9 weeks following the treatment. Moreover, animals were examined by optical coherence tomography (OCT), histological analysis, and other methods for possible adverse effects on retinal structure.

[0030] The invention enables to enhance the corneal integrity. Ocular surface disease comprises numerous disorders affecting millions around the world, and is a problem encountered routinely in daily practice. A subgroup of these patients suffers from a chronic compromise of the corneal surface, which in turn may result in corneal scarring, infection, thinning and ultimately perforation. In this subgroup, the self-healing capabilities of the cornea are significantly impaired in comparison to normal corneas. A method for enhancing corneal integrity and reducing permeability may serve to help protect these compromised corneas, and may even facilitate accelerated healing. The invention enables to reduce the permeability in cornea subjected to magnetic stimulation, so safeguarding the ocular surface in these delicate situations. It was found that the best results were achieved with magnetic pulses of relatively strong field including relatively quick field amplitude changes, such as at least 0.1 T, for example about 0.2 T or 0.3 T or 0.4 T or 0.5 T or 0.6 T or 0.7 T or 0.8 T or 0.9 T or about 1 T, changing at a rate of change of at least 1000 T/s, such as at least 5,000 T/s or at least 10,000 T/s or at least 15,000 T/s, administered for example at a rate of at least 10 pulse/s, such as about 15 pulse/s or about 20 pulse/s or about 25 pulse/s. This was achieved with pulses of a duration of preferably between 100 and 2000 s, such as between 150 and 1500 s or between 200 and 1000 s.

[0031] The term strength in regard to the magnetic field is used in the same sense as the term intensity, intending the field magnitude measured in the units of tesla (T). The terms amplitude time change, or velocity of the field strength change, or velocity of magnitude increase, or rate of change relate to the time derivative of the magnetic field magnitude, regardless the direction and the sign, and it can be obtained either by differentiating a known time signal shape (time development of the magnetic magnitude/strength), or they can be roughly assessed from the pulse magnitude and duration, as experts will acknowledge. For example, a biphasic pulse having a duration of about 100 s and a maximal intensity of about 0.2 T, will comprise a maximum rate of change of about 12,500 T/s [0.2*2/(100*10.sup.6)]; of course, the rate of change (dB/dt) goes through all the values from 12500 to +12500 during the pulse.

[0032] The system and device of the invention enable a noninvasive treatment by magnetic pulses which enhance the barrier function of a cellular layer such as of an epithelium. In a preferred embodiment, the system and device of the invention assist in protecting the cornea by retaining ocular surface integrity via reducing the corneal permeability. The magnetic treatment according to the invention protects epithelial barrier (function and structure), particularly it decreases corneal permeability, preferably in handling dry eye and conditions manifested by the dry eye syndrome, or in handling other conditions where corneal permeability is increased, including operations, diseases, trauma desiccation, and others. The treatment may reduce eye dehydration, pain, discomfort, and infection, and it may handle the situations when the barrier functions are compromised, such as in cases of reduced blinking.

[0033] The invention will be further described and illustrated in the following examples.

Examples

[0034] Method:

[0035] The application of magnetic pulses was found to protect corneal epithelium in a dry eye model in rabbits. To investigate the effect of the pulses on corneal epithelial permeability in a short-term dry eye model in rabbits, thirteen New Zealand white rabbits were used. One eye of each rabbit was treated with magnetic stimulation (Rapid2 stimulator, Magstim) at 20 pulse/s, the field strength of about 0.8 T, the treatment session of about 10 minutes. The other untreated eye served as a control. One hour later, rabbits were anesthetized and eyes were kept open for two hours to induce acute corneal desiccation. To evaluate corneal barrier, five microliters of sodium fluorescein (10%) were instilled on rabbit corneas. Extent of fluorescein corneal staining was evaluated using IMAGE J. Fluorescein penetration through the corneal barrier was assessed by determining fluorescein concentration in a 100 microliter sample withdrawn from the anterior chamber 1 hour following fluorescein installation. To determine duration of the therapeutic effect, five rabbits underwent acute corneal desiccation once a week for five weeks, and monitored for fluorescence in corneal staining and penetration to the anterior chamber. Histopathology and optical coherence tomography (OCT) were used to evaluate the safety of the treatment.

[0036] Results:

[0037] Compared with untreated control eyes, the magnetic-pulse treated eyes showed a significant decrease in fluorescein concentration in the anterior chamber (147 ng/ml [SE=19] vs 54 ng/ml [SE=9], p=0.00005) and in percentage of corneal surface stained with fluorescein (2% [SE=4.5] versus 17.4%[SE=3.4], p=0.001, wherein SE is standard error). As can be seen with reference to FIG. 4, Therapeutic effect was maintained for 3 months, with significantly lower fluorescein corneal staining in eyes treated with RMS versus control non-treated eyes. OCT and histopathology analysis revealed no gross or microscopic pathological changes in any of the eyes that underwent treatment or the contralateral eyes.

[0038] Conclusions:

[0039] In a preclinical study, the inventors demonstrated that treatment with the magnetic pulses protected the epithelial layer in the dry spots, preventing loss of the epithelial cells and maintaining the function of the corneal barrier under extreme desiccation conditions. One short (12 minutes) magnetic treatment supported corneal barrier integrity under acute dry eye conditions for at least 3 months. The treatment was safe; there was no damage in any ocular tissue as determined by imaging and pathological analyses. These findings demonstrate that the treatment according to the invention may present a novel means not only for protecting the corneal epithelium from desiccation in the patients with dry eye, but also for protecting a cellular barrier such as an epithelial barrier in general.

[0040] While the invention has been described using some specific examples, many modifications and variations are possible. It is therefore understood that the invention is not intended to be limited in any way, other than by the scope of the appended claims.