WO2019012556A1 - A device for non-invasive treatment of neurodegenerative diseases - Google Patents

Abstract

The present invention provides a device for treating neurodegenerative diseases including but not limiting to Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis using electric field. More specifically, the invention provides a device which generates focused AC/DC electric field specifically targeting dense insoluble deposits of amyloid-beta (A) peptide outside and around neurons in the brain of a patient. This mode of action serves as a non-invasive therapeutic tool for disruption of the inherent -sheet conformation known to be the reason for fibril formation.

Description

"A DEVICE FOR NON-INVASIVE TREATMENT OF NEURODEGENERATIVE

DISEASES"

RELA TED APPLICATION

This application claims the benefit of the Indian Provisional application number 2017310248 filed on July 13, 2017: each of which is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to a device for non-invasive treatment of neurodegenerative diseases using electric field. More specifically, the invention relates to a non-invasive method and a device for treating neurodegenerative diseases such as Alzheimer's disease, and Parkinson's disease, wherein the disease is characterized by the formation of protein or peptide aggregates in suffering patients.

BACKGROUND OF THE INVENTION

Most of the neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis are characterized by selective loss or degeneration of specific regions of the brain, and accumulation of protein or peptide aggregates, also known as amyloids, in neurons or other cells or extracellularly in patients. Among these, Alzheimer's disease, and Parkinson's disease are age-related diseases characterized with progressive loss of neurons with increasing age.

Alzheimer's disease is the most prevalent form of dementia in the elder population and as per WHO estimates, it affects nearly 48 million people worldwide. It is characterized by the presence of dense insoluble deposits of amyloid-beta (Αβ) peptide outside and around neurons. These deposits, often referred to as "plaques", are a result of abnormal aggregation of Αβ peptide into toxic aggregates. As per the amyloid cascade hypothesis, amyloid precursor protein (APP), (a transmembrane protein found in brain cells which helps in synaptic activities and also protects brains cells from injuries) is cleaved by enzyme a-secretase, and the cleaved fragments are exported to extra-cellular space where they re-fold to form soluble oligomers. These soluble oligomers coalesce to form insoluble fibrils which aggregate further to form amyloid plaques.

Approximately hundred potential drugs for treatment of Alzheimer's disease have failed between 1998 and 2011. And the ones that have been approved by Food and Drug Administration have not shown much promise and have several side-effects. There are two types of drugs for treatment of Alzheimer's disease; memantine and four acetylcholinesterase inhibitors (rivastigmine, galantamine, tacrine, and donepezil). At times they are often used in combination. These drugs however have failed to cure the disease or even prevent its progression and can only provide symptomatic treatment.

Several small molecule therapeutic strategies have been suggested for treating Alzheimer's disease such as:

(a) stabilizing the native state: unfolding and thus aggregation is inhibited by using proteins or small molecules that bind to the native folded state of protein and prevent it from unfolding;

(b) sequestering monomeric peptide: antibodies with help of protein-engineering as well as designed peptides have been used to bind to aggregation-prone regions of monomeric peptides to prohibit it from self-assembling;

(c) β -sheet breakers that terminate fibril elongation: β -Sheet formation is fundamental for amyloid aggregation; fibrils grow by adding new strands via hydrogen bonding to the sheet; in this approach the fibrils are capped by adding peptide/pseudo-peptides which prevents fibril growth;

(d) inhibiting gamma (y)-secretase or β-secretase or activation of alpha secretase to prevent abnormal cleavage amyloid precursor protein (APP);

(e) removing existing amyloid plaques using vaccines or antibody based approaches; and

(f) microglial activation using scanning ultrasound: Microglia act like microphage and digest amyloid plaques by phagocytosis.

Though these strategies have shown promise, but they show poor ability to penetrate the blood- brain barrier and are often highly toxic. The electric field studies on biomolecules (like DNA and proteins) have shown permanent or induced dipole formation along the direction of applied field, indicating that electric field interaction could modulate their dipole moments. Against this background, a high level of interest remains in finding new and improved therapeutic strategies for the treatment of Alzheimer's disease. In particular, a need remains in for relatively inexpensive and non-invasive approaches for treating Alzheimer's, that also avoid the limitations of drug therapy.

The present invention provides an inexpensive and non-invasive technique for treating

Alzheimer's disease using electric field. The present invention discloses a device that generates focused AC/DC electric field specifically targeting dense insoluble deposits of amyloid-beta (Αβ) peptide outside and around neurons in the brain of an Alzheimer's patient.

OBJECT OF THE INVENTION

Accordingly, the main object of the invention is to provide a device for non-invasive treatment of neurodegenerative diseases which are characterized by formation and deposit of amyloids, using electric field.

Another object of the invention is to provide a device for treating neurodegenerative diseases characterized by formation and deposit of amyloids, wherein the device provides a non-invasive method by providing electric impulse to specific regions of the brain of a patient.

Yet another object of the present invention is to provide a device for treating neurodegenerative diseases characterized by formation and deposit of amyloids which is inexpensive, non-invasive, highly effective, and eliminates the requirement of drugs which have severe side effects and are ineffective.

SUMMARY OF THE INVENTION

The present invention provides a device for treating neurodegenerative diseases including but not limiting to Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis using electric field. More specifically, the invention provides a device which generates focused AC/DC electric field specifically targeting dense insoluble deposits of amyloid-beta (Αβ) peptide outside and around neurons in the brain of a patient. This mode of action serves as a noninvasive therapeutic tool for disruption of the inherent β-sheet conformation known to be the reason for fibril formation.

Accordingly, in the main embodiment of the invention, a device is provided for treating neurodegenerative diseases using electric field comprising of: a) at least one electrode assembly comprising at least two electrodes placed parallel to each other separated by a distance of at least 17 cm;

b) at least one power module comprising of a battery and a charging circuit unit, wherein the charging circuit module comprises of at least two capacitors, at least one voltage rectifier, and at least one Zener diode; the charging circuit module has an electricity input unit connected to the mains electricity power, and an electricity output unit connected the battery; and the battery is connected to the electrode assembly for supplying power;

c) at least one electronic oscillator to produce a periodic, oscillating electronic signal comprising of at least one transistor, and at least one transformer, preferably a flyback transformer to generate high voltage electric signals; and

d) at least one voltage booster module comprising of plurality of diodes and plurality of capacitors.

The device typically comprises of an electrode assembly adapted to be placed on the head of a patient secured with an insulating material; power is supplied to the electrode assembly through a battery; the battery in turn is connected to a charging circuit unit which sources low power electricity sufficient enough to charge the battery; at least one oscillator and at least one voltage booster are connected in line with the battery for amplifying the voltage received, and this amplified voltage is provided to the electrodes in a continuous manner. An electric field is generated by the electrodes in receiving voltage.

BRIEF DESCRIPTION OF THE DRAWINGS

A complete understanding of the system and method of the present invention may be obtained by reference to the following figures: Figure 1 depicts a schematic illustration of a device for non-invasive treatment of neurodegenerative diseases using electric field.

Figure 2 depicts a schematic illustration of the power module comprising of at least one battery and a charging circuit unit.

Figure 3 depicts a schematic representation of a circuit diagram of electronic oscillator and voltage booster modules of the present invention.

Figure 4 depicts a graphical representation of aggregation kinetics of Αβι₆-42 determined using ThT fluorescence assay as a function of time at 500 μΜ concentration.

Figure 5 depicts a graphical representation of aggregation kinetics of full length peptide Αβι-42 determined using ThT fluorescence assay as a function of time at 100 μΜ concentration.

Figure 6 depicts a schematic illustration of electric field circuit diagram of DC electric field setup employed for testing effect of electric field on aggregation of Αβι-42 and Αβι₆-22 peptides.

Figure 7 depicts a qualitative analysis of Αβι₆-22 aggregation using 90° light scattering assay after incubation inside DC electric field.

Figure 8 depicts a qualitative analysis of Αβι₆-22 aggregation using 90° light scattering assay after incubation inside AC electric field.

Figure 9 depicts a qualitative analysis of full length peptide Αβΐ-42 aggregation using 90° light scattering assay after incubation inside DC electric field.

Figure 10 depicts a qualitative analysis of full length peptide Αβΐ-42 aggregation using 90° light scattering assay after incubation inside AC electric field.

Figure 11 depicts a qualitative analysis of Αβι₆-22 aggregation using ThT fluorescence assay after incubation inside DC electric field.

Figure 12 depicts a qualitative analysis of Αβι₆-22 aggregation using ThT fluorescence assay after incubation inside AC electric field. Figure 13 depicts a qualitative analysis of full length peptide Αβΐ-42 aggregation using ThT fluorescence assay after incubation inside DC electric field.

Figure 14 depicts a qualitative analysis of full length peptide Αβΐ-42 aggregation using ThT fluorescence assay after incubation inside AC electric field.

Figure 15 depicts a circular dichroism spectra of full length Αβΐ-42 peptide (10 μΜ) incubated for 24 hours in the absence (0 Vcm^"1), or in the presence of electric field (150 Vcm^"1, and 300 Vcm^"x). The spectra provide data related to extent of β-sheet formation by the peptide in the absence or in the presence of electric field. The spectra are representative of one set of experiments and are obtained after subtraction of the circular dichroism contributed by the PBS buffer. The peptide exposed to 0 Vcm^"1 (negative control) have expected predominant β-structures. There is a difference in the intensity of the negative peak s in 215-220 nm region of the control and the DC field exposed samples which signifies that electric field is indeed retarding the β-structures formation of the peptide.

Figure 16 depicts an FTIR spectra of full length Αβΐ-42 peptide (10 μΜ) incubated for 24 hours in the absence (0 Vcm^"1) which is the negative control, or in the presence of electric field (150 Vcm^"1, and 300 Vcm^"1). In FTIR study the control peptide sample with a peak position at 1639 cm^"1 suggests the transformation from alpha helix/random coil to beta sheet structures. Peak position of the peptide samples exposed to electric field is dominant at around 1650 cm^"1 which is a region for alpha helix. This signifies that both intensities of electric field 150 Vcm^"1 and 300 Vcm^"1 are retarding the beta sheet formation during an incubation period of 24 hours.

Figure 17 depicts changes in the hydrodynamic radius (RH) of Αβΐ-42 aggregates monitored by DLS as a function of time. Samples containing ΙΟΟμΜ peptide was exposed to different external electric field strengths and the change in the size distribution of the aggregates formed was monitored over time at 0^th (initial) and 24^th hour.

Figure 18 depicts extent of fibrilization using ThT fluorescence assay upon exposure to external DC electric field 150Vcm^_1 on pre- formed 100 μΜ Αβΐ-42 peptide aggregates. The data clearly indicates that in presence of electric field the extent of fibrilization is significantly reduced. Figure 19 depicts effect of electric field on Αβΐ-42 aggregation in human cerebrospinal fluid. Higher ThT fluorescence is observed at Oh of incubation in CSF. After 12h of incubation in the field, peptide exposed to EF shows a gradual decline in the fluorescence intensity, and after 24h, ThT fluorescence has reduced drastically compared to the control. Field-induced structural effects were predominant at lower field strengths after 12 h, while effects of higher fields were more pronounced after 24h.

Figure 20 depicts TEM of Αβΐ-42 after 24h of incubation in ambient conditions and in presence of external electric field. The control sample has fibrils as predominant form, whereas, samples exposed to 150 Vcm^"1 has homogenous spherical oligomeric clusters, and 300 Vcm^"1 poly- disperse. Scale bars correspond to 200 nm.

Figure 21 depicts SDS-PAGE of BSA fragments after treatment with trypsin. LI and L2 show SDS-PAGE patterns of digestion of BSA by trypsin not exposed to the field (control). L3 and L4 show digestion by trypsin exposed to 150 Vcm^"1 (DC) for 24h and L5 and L6 show band position of BSA fragments after treatment with trypsin exposed to 300 Vcm^"1 (DC) for 24h.

Figure 22 depicts representative graph of an MTT assay. Viability (mean ± S.D. from n = 7) is depicted as percentage of cells without Αβ. The graph represents the mean ± S.D. from two independent experiments.

Figure 23 depicts estimation of monomer percentage at 280 nm (tyrosine estimation) by measuring the intensity of 100 μΜ Αβΐ-42 peptide in solution. Absorbance at 280 nm is measured with strength of external electric field as the only variable at 0th and 24th h of incubation. The higher intensity of the 0th h sample (black) signifies large population of monomers.

Figure 24 depicts stability of the field exposed Αβΐ-42 samples. Different color codes are used for different external field strengths at the 0th, 12th and 24th hour after removing from the field.

DETAILED DESCRIPTION OF THE INVENTION

The present invention now will be described hereinafter with reference to the accompanying drawings, in which some, but not all embodiments of the invention are shown. Indeed, the invention may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements. Like numbers refer to like elements throughout.

The present invention provides a device for treating neurodegenerative diseases including but not limiting to Alzheimer's disease, Parkinson's disease, Huntington's disease, dementia and amyotrophic lateral sclerosis which are characterized by loss neurons, and formation and deposits of abnormal protein or peptide aggregates known as amyloids resulting in fibril formation, insoluble amyloid-beta (Αβ) formation. More specifically, the present invention provides a noninvasive and inexpensive technique for treating these neurological disorders using electric field for disrupting the Αβ fibrils by disrupting the inherent β-sheet conformation.

In the main embodiment of the present invention, the invention provides a device 100 for treating neurodegenerative diseases comprising of: at least one electrode assembly 101 comprising of at least two electrodes placed parallel to each other separated by a distance of at least 17 cm; one power module 102 comprising of a battery 201 and a charging circuit unit, wherein the charging circuit module comprises of at least two capacitors, at least one voltage rectifier, preferably a full-wave bridge rectifier, and at least one Zener diode; the charging circuit module has an electricity input unit connected to the mains electricity power, and an electricity output unit connected the battery; and the battery is connected to the electrode assembly for supplying power; at least one electronic oscillator 103 to produce a periodic, oscillating electronic signal comprising of at least one transistor 301, and at least one transformer, preferably a flyback transformer, comprising of a primary 302, secondary 303, and feedback 304 coils to generate high voltage electric signals; and at least one voltage booster 104 module comprising of plurality of diodes 305 and plurality of capacitors 306; wherein: the electrode assembly 101 is adapted to be placed on the head of a patient secured with an insulating material; the power is supplied to the electrode assembly through the power module via the electronic oscillator and the voltage booster.

The power module comprises of a battery 201 and charging circuit unit 202, which is connected to the main electricity power supply. The charging circuit unit module comprises of at least two capacitors, at least one voltage rectifier, and at least one Zener diode; wherein the charging circuit unit is connected to the mains electricity power through an electricity input unit and it is connected to the oscillator through an electricity output unit; the first capacitor 203 helps in limiting the current from the mains and the battery, and is provided to the voltage rectifier 204 for providing DC voltage, the Zener Diode 205 and the second capacitor 206 filter the noise on the DC voltage; and the resultant rectified current exits the charging circuit unit through the electricity output unit to reach the oscillator. This would charge the battery inside when plugged into mains. This may be optionally accompanied with a switch to enable charging. This setup of charging circuit unit is functional for a DC voltage power supply.

Figure 1 depicts a schematic illustration of a device for non-invasive treatment of neurodegenerative diseases using electric field, wherein the device comprises of an electrode assembly 101 safely secured and adapted to be worn on head of a patient, a power module 102, at least one electronic oscillator 103, and at least one voltage booster 104 module.

Figure 2 depicts a schematic illustration of the power module 102 comprising of at least one battery 201 and a charging circuit unit 202, wherein the charging circuit unit comprises of at least one initial capacitor 203 which limits the current from the mains and the battery, at least one voltage rectifier 204 for providing DC voltage, the Zener Diode 205 and the second capacitor 206 filter the noise on the DC voltage.

In an alternate embodiment, in the present invention the charging circuit unit comprises of at least two capacitors, and at least one Zener diode, wherein a voltage rectifier is not used in case of an AC voltage setup. A voltage rectifier is required only in DC voltage setup.

The DC current reaching the oscillator is enabled through a transistor 301, generally an npn transistor, which allows the current to flow through transformer primary coil 302, inducing voltage in a secondary coil 303 and the secondary coil in return induces voltage in feedback coil 304. This counter voltage in the feedback coil causes the transistor to stop conducting and a magnetic field in the ferrite core to collapse via electrical energy from secondary coil. This process helps the transistor 301 to conduct again, repeating the process and creating pulsed DC. The changing magnetic field induces high voltage in the secondary coil.

This voltage is now further boosted using a voltage booster 104 module comprising of plurality of diodes 305 and plurality of capacitors 306. Preferably, a voltage triple booster is used which triples the available voltage from secondary winding of transformer. This high voltage is passed onto the wires connected to the electrode assembly 101 comprising typically of two parallel electrodes, which are separated, by a distance -17 cm.

Figure 3 depicts a schematic representation of a circuit diagram of electronic oscillator 103 and voltage booster 104 modules of the present invention.

These parallel electrodes are insulated by thick plastic lamination and are adapted to be worn on the head of a patient, typically secured to a head-gear or a helmet.

The efficiency of the method has been tested in vitro using electric field on the shortest amyloid forming peptides, Αβι₆-42, and on full length amyloid peptide Αβΐ-42. The following examples illustrate the efficiency of the device on the peptides describes above.

EXAMPLES

The present disclosure includes the following non-limiting Examples. EXAMPLE 1

Preparation, purification and aggregation of amyloid-beta (Αβ) peptides

Αβΐ-42 peptide was synthesized on an automated peptide synthesizer (Protein technologies, Tucson, AZ) using Fmoc chemistry, and KLVFFAE (Αβ_16-42) peptide was synthesized manually using Fmoc chemistry. All other reagents used were of the highest purity (> 99%).

Peptide Synthesis: Fmoc chemistry was employed for manual peptide synthesis. The weighed resin (dry) was swollen overnight by soaking in N, N-dimethylformamide (DMF). The Fmoc group was removed by treatment with piperidine, followed by repeated washing with DMF. It was followed by attachment of the required N-terminus protected amino acid. The amino acid was activated prior to the coupling cycles by Diethyl ether, 0-Benzotriazole-N,N,N,N-tetramethyl-uronium-hexafluoro- phosphate (HBTU), Hydroxybenztriazole (HOBT) and Ν,Ν-diisopropylethylamine (DIPEA) dissolved in DMF. After the attachment of the first amino acid, the entire procedure discussed above was repeated for the following amino acids.

Purification of peptide:

Shimadzu LC-20AD prominence Liquid Chromatography was used to purify synthesized peptides. The gradient run in HPLC was done by running a gradient of acetonitrile and water with a flow rate of 0.5 mL/min. The mass of the synthesized peptides was characterized by ESI-MS (Waters, Q-Tof Premier). Based on the mass/charge ratio of the corresponding synthesized peptides the presence of the desired molecule was concluded.

Preparation of peptide monomers:

Lyophilized peptide was desiccated until reconstitution using 10% ammonium hydroxide at 0.5 mg/ml followed by lyophilization to remove any trace of salts. All peptides were then dissolved in hexafluoroisopropanol for 2 hours in order to have monomeric samples. Then the solvent was removed completely by evaporation inside a fume hood overnight leaving a thin film of peptide which was then sealed and kept at -20 °C. The peptide was dissolved using PBS (pH 7.4) buffer when needed.

Pre-aggregation test:

The absence of any pre-aggregates in peptide stock solution of all the samples were ensured by 90o static light scattering (excitation A = 450 nm, slit width = 2.5 nm; emission A = 450 nm, slit width = 2.5 nm) using a spectrofluorometer (Jasco FP 8500).

Kinetic aggregation experiment: Monomer solution of both the peptides, Αβι₆-42 and Αβΐ-42, and the phosphate buffer used for preparing the experimental samples contained 10 μΜ final concentration of Thioflavin T (ThT). All the samples were kept at -20 °C before starting the experiments. The experiment was initiated by placing a Corning3881, 96-well plate, each well containing a total volume of 200 μΐ_^, in room temperature without shaking. Fluorescence measurements were recorded every 2 hours for 24 hours through the bottom of the plate by placing the plate in a Tecan infinite200 plate reader (Tecan, Crailsheim, Germany). ThT was excited at 440 nm and the emission was recorded at 485 nm. The half time (ti/2) was estimated by fitting the following sigmoidal function to each curve: (t) = .F0 †g - k t - £1/2»

Where F0 is the baseline before aggregation, A is the amplitude, and k is the apparent elongation rate constant.

[The sigmoidal aggregation curves were normalized in originPro (OriginLab corporation, Northampton, MA) and fitted to the following formula: y = (Al - A2)/{1 e((t - tl/2)/ds) } 1 A2

Where, Al and A2 are the minimum and maximum fluorescence, ti/2 is the halftime of the reaction, and dx is the inverse apparent elongation rate.]

Figure 4 depicts a graphical representation of aggregation kinetics of Αβ₁₆-42 determined using ThT fluorescence assay as a function of time at 500 μΜ concentration.

Figure S depicts a graphical representation of aggregation kinetics of full length peptide Αβ_1-42 determined using ThT fluorescence assay as a function of time at 100 μΜ concentration.

These aggregation kinetic experiments of Αβ₁₆-42 and Αβΐ-42 peptides were used to determine the half time of peptide aggregation. All samples contain 10 μΜ ThT, 50 mM phosphate buffer, pH 7.4. Data in the figures represents the average value of four independent experiments each performed in ambient condition.

EXAMPLE 2 The effect of application of electric field on the aggregation properties of amyloid-beta (Αβ) peptides

Electric Field setup:

For the DC field setup, two parallel aluminum electrodes separated by a distance of 1 cm were anchored to a plate; which were then connected to a full-wave bridge rectifier. The voltage was regulated using a single phase variable auto transformer. The AC field setup is similar to DC field setup but without a full-wave bridge rectifier.

Figure 6 depicts a schematic illustration of electric field circuit diagram of DC electric field setup employed for testing effect of electric field on aggregation of Αβι.42 and Αβ₁₆-₂₂ peptides.

Electric Field Exposure:

For Αβΐ6-42, experiments were performed with peptide concentration of 500 μΜ at ambient temperature for a duration of 16 hours, in two different field setups: 150 Vcm^"1, 300 Vcm^"1 DC and 150 Vcm^Hz^"1, 300 Vcm^Hz^"1- AC, while for full length peptide Αβΐ-₄₂,100 μΜ peptide concentration was used for all the experiments and were incubated of 24 hours. In case of the control experiment no external field was applied.

90° Light Scattering Assay:

The extent of peptide self-assembly in the field exposed peptide solution was estimated by measuring the hydrodynamic radius (RH) at room temperature. Samples (600 μΐ) were placed directly in a fluorescence cuvette (Helma, Sigma Aldrich) with 1 cm path length and 90° scatter intensity in kilocounts/s was collected in 1000 sees acquisition time at regular interval of time (excitation A = 450 nm, slit width = 2.5 nm; emission A = 450 nm, slit width = 2.5 nm) using a Jasco FP 8500 spectrofluorometer.

Figure 7 depicts a qualitative analysis of Αβι₆-22 aggregation using 90° light scattering assay after incubation inside DC electric field. Figure 8 depicts a qualitative analysis of Αβι₆-22 aggregation using 90° light scattering assay after incubation inside AC electric field.

Figure 9 depicts a qualitative analysis of full length peptide Αβΐ-42 aggregation using 90° light scattering assay after incubation inside DC electric field.

Figure 10 depicts a qualitative analysis of full length peptide Αβΐ-42 aggregation using 90° light scattering assay after incubation inside AC electric field.

These experiments were carried out as a function of time in presence of field for 100 μΜ Αβι₆-22, and 10 μΜ Αβΐ-42 under quiescent conditions. Bars of different colors in the graphs correspond to exposure of peptides to different external field strengths, 0 Vcm^"1, 150 Vcm^"1, and 300 Vcm^"1 at 0^th hour and after 24 hours of incubation. The negative control is the sample with 0 Vcm^"1 electric field. All samples contain 50 mM phosphate buffer, pH 7.4.

Lower 90° scatter intensity of peptide samples exposed to electric field as compared to the control, in general, shows the ability of electric field to efficiently retard the β-sheet formation. In case of Αβΐ6-22 peptide more retardation in aggregation is seen at 150 Vcm^"1 compared to 300 Vcm^"1.

This clearly indicates that KLVFFAE (Αβ_16-42) peptide, and the full length Αβΐ-42 peptide show decreased aggregation in the presence of electric field.

ThT fluorescence assay:

The Thioflavin (ThT) dye binding assay was performed at room temperature to check the rate of fibril formation in the peptide solution with respect to time. The Αβΐ6-42 sample for ThT measurements was prepared by dissolving 120 μL· of Αβΐ6-42 peptide solution (100 μΜ) with 6 μL· of 1 mM ThT (10 mM) in 480 μL· of phosphate buffer pH 7.4. Similarly, a final concentration of 10 μΜ for full length peptide Αβΐ-42 was used for ThT fluorescence estimation with 10 μΜ ThT and was prepared using 50 mM sodium phosphate buffer. Fluorescence measurements were recorded in a Horiba Florimax-4 spectrofluorometer at 25 °C using quartz cuvette with 1 cm path length. The relative ThT fluorescence of the samples was measured. (Ex=450 nm; slit width=2 nm and Em=460-550 nm; slit=5 nm). Figure 11 depicts a qualitative analysis of Αβι₆-22 aggregation using ThT fluorescence assay after incubation inside DC electric field.

Figure 12 depicts a qualitative analysis of Αβι₆-22 aggregation using ThT fluorescence assay after incubation inside AC electric field.

Figure 13 depicts a qualitative analysis of full length peptide Αβΐ-42 aggregation using ThT fluorescence assay after incubation inside DC electric field.

Figure 14 depicts a qualitative analysis of full length peptide Αβΐ-42 aggregation using ThT fluorescence assay after incubation inside AC electric field.

These experiments were carried out as a function of time in presence of field for 100 μΜ Αβι₆-22, and 10 μΜ Αβΐ-42 under quiescent conditions. Bars of different colors in the graphs correspond to exposure of peptides to different external field strengths, 0 Vcm^"1, 150 Vcm^"1, and 300 Vcm^"1 at 0^th hour and after 24 hours of incubation. The negative control is the sample with 0 Vcm^"1 electric field. All samples contain 50 mM phosphate buffer, pH 7.4.

This data clearly indicates that KLVFFAE (Αβ_16-42) peptide, and the full length Αβΐ-42 peptide show decreased aggregation in the presence of electric field.

Circular Dichroism Analysis:

The spectra were recorded using a Jasco-J 1500 spectropolarimeter with a scanning speed of 2 nm/min. Data points were collected from 260 to 190 nm at room temperature using a 10mm path length quartz cuvette. The final concentration of all peptides used for circular dichroism analysis was 10 μΜ and was prepared using 10 mM phosphate buffer, pH 7.4. Spectra were analyzed after 24 hours of incubation of full length Αβΐ-42 peptide.

Figure 15 depicts a circular dichroism spectra of full length Αβΐ-42 peptide (10 μΜ) incubated for 24 hours in the absence (0 Vcm^"1), or in the presence of electric field (150 Vcm^"1, and 300 Vcm^"x). The spectra provides data related to extent of β-sheet formation by the peptide in the absence or in the presence of electric field. The spectra are representative of one set of experiments and are obtained after subtraction of the circular dichroism contributed by the PBS buffer. The peptide exposed to 0 Vcm (negative control) have expected predominant β-structures. There is a difference in the intensity of the negative peak s in 215-220 nm region of the control and the DC field exposed samples which signifies that electric field is indeed retarding the β-structures formation of the peptide.

Fourier Transform Infrared Spectroscopy (FTIR) analysis:

The samples of full length Αβΐ-42 peptide exposed to electric field were casted on potassium bromide (KBr) pellet immediately after taking out from the field. FTIR spectra was recorded on a Micro FTIR-200 (Jasco Co., Japan) equipped with an MCT detector at 4 cm^"1 resolution. The 16 scan data were collected and the spectra evaluation was carried out by spectral manager for windows software (Jasco Co., Tokyo, Japan). The examined region was 1600^"1 to 1700^"1.

Figure 16 depicts an FTIR spectra of full length Αβΐ-42 peptide (10 μΜ) incubated for 24 hours in the absence (0 Vcm^"1) which is the negative control, or in the presence of electric field (150 Vcm^"1, and 300 Vcm^"1). In FTIR study the control peptide sample with a peak position at 1639 cm^"1 suggests the transformation from alpha helix/random coil to beta sheet structures. Peak position of the peptide samples exposed to electric field is dominant at around 1650 cm^"1 which is a region for alpha helix. This signifies that both intensities of electric field 150 Vcm^"1 and 300 Vcm^"1 are retarding the beta sheet formation during an incubation period of 24 hours.

Table 1. Secondary structure content of full length Αβι-42 peptide samples in presence of different external fields at 10 μΜ concentration

SOOVcm^DC 84 0 16

The spectra were recorded approximately 1 min after dissolution and after 24 hours of incubation of full length Αβΐ-42 peptide at room temperature. The values are as obtained from dichroweb server using the K2D3 algorithm. The output is expressed in percentage of secondary structure present.

The data clearly shows that upon exposure to DC electric field 300Vcm^_1 β-sheets structure is completely eliminated, providing evidence for the efficiency of the treatment method.

Dynamic light scattering (DLS) experiments:

DLS experiments were performed on a Zetasizer Nano ZS (Malvern Instruments Ltd, UK) using a laser source with λ= 633 nm and a detector at a scattering angle of 0=173 degrees. Sample containing 100 μΜ (100 μL) Αβΐ-42 (both EF treated and untreated) after 24 h of incubation were analysed and each measurement was consecutively repeated ten times. Zetasizer program was used for the measurement of average diameter and plotted the information as fractional distribution versus size. DLS experiment was performed to get an insight of the size distribution of structures formed under the influence of field after 24h of incubation

Figure 17 depicts changes in the hydrodynamic radius (RH) of Αβΐ-42 aggregates monitored by DLS as a function of time. Samples containing ΙΟΟμΜ peptide was exposed to different external electric field strengths and the change in the size distribution of the aggregates formed was monitored over time at 0^th (initial) and 24^th hour.

The DLS data clearly shows that both the applied field has a retarding effect on the aggregation of the peptide, with significant shift in the peak position towards left with respect to the control. After 24h incubation in the presence of field, visibly clear relatively smaller structures (most likely small oligomers) are observed, whereas, the structures are of a much larger size, averaging 800 nm in the absence of field (0 Vcm¹). Samples exposed to 150 Vcm¹ have generated more of a mono- disperse population of structures with a major signal around 120nm, while 300 Vcm^_1generated relatively poly-disperse population, with a small contribution even at micro scale (5-6 μπι).

Electric field effect on pre-aggregated peptides:

Experiments were carried out by incubating the peptide samples (100 μΜ Αβΐ-42) for 12 hours in aggregating conditions at pH 7.4 after which external DC electric field 150Vcm^_1 was applied. ThT fluorescence was recorded at regular interval of time in quiescent conditions for a period of 72 hours. The ThT assay measures changes of in fluorescence intensity of ThT upon binding to amyloid fibrils. The enhanced fluorescence can be observed by fluorescence microscopy or by fluorescent spectroscopy. The spectroscopic assay is commonly used to monitor fibrilization over time.

Figure 18 depicts extent of fibrilization using ThT fluorescence assay upon exposure to external DC electric field 150Vcm^_1 on pre- formed 100 μΜ Αβΐ-42 peptide aggregates. The data clearly indicates that in presence of electric field the extent of fibrilization is significantly reduced.

Electric field effect on peptide allowed to aggregate in human cerebrospinal fluid:

In order to mimic and maximally align with clinical conditions, we have extended the study with human cerebrospinal fluid (CSF). CSF is routinely checked for pathological biomarkers for nervous system disorders. Fresh CSF samples were collected from healthy patients, and the Αβι- 42 peptide is added to get a desired 100 μΜ concentration. The samples are then subjected to electric fields for a period of 24h. Effect of electric field on peptide aggregation in CSF is monitored by recording ThT fluorescence ( EX. 440 nm, AE_m: 485 nm) of the samples at regular intervals of time. Samples exposed to the electric field, in general, have reduced ThT fluorescence intensity as compared to the control (0 Vcm^"1). Through this experiment, we wanted to verify whether field based therapy can be successfully employed under clinical conditions.

Figure 19 depicts effect of electric field on Αβΐ-42 aggregation in human cerebrospinal fluid. Higher ThT fluorescence is observed at Oh of incubation in CSF. After 12h of incubation in the field, peptide exposed to EF shows a gradual decline in the fluorescence intensity, and after 24h, ThT fluorescence has reduced drastically compared to the control. Field-induced structural effects were predominant at lower field strengths after 12 h, while effects of higher fields were more pronounced after 24h.

The results of this experiment are in agreement with earlier results, suggesting the effect of electric field on fibril formation and its potential use as a future therapeutic option conclusively

Morphological evolution of Αβι-42 in the presence of external electric field:

We examined the effect of 150 Vcm^"1 DC electric field on aggregation of Αβΐ-42 using negatively stained Transmission Electron Microscope (TEM) in ΙΟΟμΜ solution. Monomers of Αβΐ-42 was dissolved in PBS (pH 7.4) at room temperature 'in-field' to get a 100 μΜ solution which was then incubated for 24 h followed by TEM imaging.

Figure 20 depicts TEM of Αβΐ-42 after 24h of incubation in ambient conditions and in presence of external electric field. The control sample has fibrils as predominant form, whereas, samples exposed to 150 Vcm^"1 has homogenous spherical oligomeric clusters, and 300 Vcm-1 poly- disperse. Scale bars correspond to 200 nm.

In the case of control at 0 Vcm^"1, thin fibrils with a diameter ranging from 10-15 nm was observed predominantly throughout the grid. Narrow, elongated thread like structures (protofibrils) suggests that the oligomers formed by the peptide monomers are on a productive pathway for fibril formation as explained in the earlier reports.

The sample exposed to 150 Vcm^"1 however, has well defined homogenous spheres as the dominant species. The average diameter of the oligomers is in the range between 20-35 nm, which is comparable to the reported dimensions of oligomers before they form highly toxic protofibrils.

Effect of electric field on functions of normal proteins and cells:

The goal of this study was to experimentally verify the possibility of electric field elicited toxicity by analyzing the effect of the external electric field on other native proteins. The interaction of another β-sheet containing protein; trypsin, with electric fields of varying strengths was investigated after 24hours incubation. Trypsin is a commonly available serine protease in the body and cleaves protein next to lysine and arginine residues. The proximity with His-57 makes the serine residue more reactive, which exhibits enhanced reactivity because of H-bonding to a negative aspartate residue Asp- 102, resulting in a charge relay system. Any change in the secondary structure of trypsin affects the catalytic triad and thus its functionality. Trypsin is incubated in the presence of external electric field (DC) strength of 150 Vcm^_1and 300 Vcm^"1 for 24 h followed by verification of its activity against bovine serum albumin (BSA) protein (50 mg mL¹).

Figure 21 depicts SDS-PAGE of BSA fragments after treatment with trypsin. LI and L2 show SDS-PAGE patterns of digestion of BSA by trypsin not exposed to the field (control). L3 and L4 show digestion by trypsin exposed to 150 Vcm- 1 (DC) for 24h and L5 and L6 show band position of BSA fragments after treatment with trypsin exposed to 300 Vcm-1 (DC) for 24h.

The addition of trypsin (control) resulted in several fragments (LI and L2 of Figure 21). The field- exposed and non-exposed trypsin were compared, and the bands formed by the field-exposed trypsin (L3 and L4 for 150 Vcm^"1 and L5 and L6 for 300 Vcm^"1) show no noticeable difference in the band position of BSA fragments, suggesting negligible effect on overall function of the trypsin molecule upon exposure to the applied field. L7 and L8 show band position of standard trypsin and undigested BSA alone.

Cellular toxicity of Field exposed peptide samples:

HEK293 cells were harvested from culture plate and 10,000 cells were seeded per well in a 96- well plate in 10% Fetal Bovine Serum (FBS) and was incubated at 37°C. The toxicity of the structures formed under EF was observed after 24h by the standard MTT (3-[4,5-dimethyl-thiazol- 2yl]-2,5-diphenyl tetrazolium bromide) assay.

Figure 22 depicts representative graph of an MTT assay. Viability (mean ± S.D. from n = 7) is depicted as percentage of cells without Αβ. The graph represents the mean ± S.D. from two independent experiments.

After 24h of incubation both 150 Vcm^"1 and 300 Vcm^"1 DC treated Αβΐ-₄₂ (10 μΜ) show <25% cell death due to oligomer illicit toxicity. However, for the control sample, ~60% cells were killed. This may be attributed to the formation of highly toxic large oligomers and protofibrils. This observation holds significance, largely because exposure to EF is not producing any new toxic species of Αβ that might cause abnormal cell death. Secondly, EF is successfully retarding the aggregation of Αβ peptide thus reducing the number of toxic oligomers and protofibrils in the solution. The fresh sample act as a positive control for the field treated samples, where it shows that the monomers and small oligomers formed in the initial phase of aggregation is relatively nontoxic to the studied cells. However, this experiment is of suggestive nature, and more elaborate and extensive testing is necessary before confirming the therapeutic potential of field based treatments.

Monomer Estimation Assay:

Generation of Αβΐ-42 monomers is a physiologically relevant event, whereas their aggregation into oligomers and fibrils is pathogenic. Ultracentnfugation can be used to separate amyloid fibrils and their oligomeric intermediates from their respective monomers. Adopting this method, Ultracentrifugation of the peptide solution was performed at 12000 g for 20 min, after 24 hours of incubation. This results in pelleting of the large aggregates, thus, leaving only the soluble Αβ monomers suspended in the supernatant. The supernatant of external EF treated samples, however, has a greater absorbance at 280nm suggesting a higher concentration of monomers compared to samples without external field.

Figure 23 depicts estimation of monomer percentage at 280 nm (tyrosine estimation) by measuring the intensity of 100 μΜ Αβΐ-42 peptide in solution. Absorbance at 280 nm is measured with strength of external electric field as the only variable at 0th and 24th h of incubation. The higher intensity of the 0th h sample (black) signifies large population of monomers.

After 24 h, the supernatant obtained after ultracentrifugation of the control sample shows significant reduction in the monomer concentration, thus, suggesting formation of large aggregates with large molecular weight and sedimentation co-efficient corresponding to its size. Supernatant of both the field exposed samples, however, has noticeably higher absorbance than the control. Sample exposed to 150 Vcm^"1 in particular, appears to be more effective in retarding the aggregation of monomers, confirming the inferences of ThT assay and Static light scattering experiments.

Stability assay: Stability of the non β-conformations, formed under the influence of external electric field by Αβ peptide, holds the key in consolidating field based treatment strategy. We have tested the stability of structures formed under 150 Vcm^"1 and 300 Vcm^"1 (DC) by removing the applied field and recording the ThT fluorescence intensity at regular intervals of time for the next 24 h.

Figure 24 depicts stability of the field exposed Αβΐ-42 samples. Different color codes are used for different external field strengths at the 0th, 12th and 24th hour after removing from the field.

The stability assay data shows at 0th hour after removing the field, electric field exposed samples, in general, show reduced ThT fluorescence confirming its retarding effect on the aggregation process. Sample exposed to 300 Vcm^"1 show gradual increase in ThT fluorescence intensity and a 100% increase is observed after 24h when compared to 0th hour. Samples in 150 Vcm^"1, however, has only a marginal increase in the ThT fluorescence intensity after 24h.

Claims

CLAIMS We claim:

1. A portable, wearable, non-invasive, electric field device for the treatment of Alzheimer's disease comprising of:

a) the power supply/charging circuit for battery inside,

b) oscillator/transistor stage,

c) the voltage booster stage.

d) and two parallel aluminum electrodes separated by a distance of 17 cm, anchored to the helmet like plastic case.

2. The device of claim 1 wherein the said assembly is used to deliver appropriate electric field for medical treatment.

3. The device of claim 1 wherein a voltage booster circuit is employed to producing the said electric field for medical treatment.

4. The device of claim 1 wherein a parallel insulated electrode system is employed to achieve directional electric field across the brain of the patient electric field for medical treatment.

5. The device of claim 1 wherein a rechargeable battery is employed as a power source.

6. The device of claim 1 wherein a charging system is connected to the battery.

7. The device of claim 1 capable of producing AC (alternating current) and DC (direct current) field for medical treatment of Alzheimer's disease, dementia and memory-loss disorders in a patient.

8. A method for medical treatment of Alzheimer's disease using a wearable portable head worn device by transmitting electric field to the brain of the wearer.

9. A device as in claim 8 wherein said transmitting means a mode for directing the electric field signal to a predetermined part of the brain.

10. A method for medical treatment of Alzheimer's disease, dementia and memory- loss disorders in a patient where in the stimulus is an electric field.