Phosphorothioate DNA (PSDNA) and / or phosphodiester DNA (PODNA) and / or other materials to treat cancer and / or neurodegenerative disorders

Abstract

A method of cancer treatment, comprising: delivering a physiologically effective amount of phosphorothioate DNA (PSDNA) and / or phosphodiester DNA (PODNA)to cancer cells. Related compositions are also disclosed. Also disclosed are methods of treating neurodegenerative disorders using other compounds and related compositions.

Description

[0001] TITLE: PHOSPHOROTHIOATE DNA (PSDNA) AND / OR PHOSPHODIESTER DNA (PODNA) AND / OR OTHER MATERIALS TO TREAT CANCER AND / OR NEURODEGENERATIVE DISORDERS

[0002] Related applications:

[0003] This PCT application claims priority under 35 U.S.C §119(a) from Israeli application 307313 filed on September 27 , 2023 by the same Applicant and entitled "Use of PSDNA to Reduce Prion Formation to Thereby Prevent and Reverse Growth of Cancer and Use of Specific Compounds and Microgravity for Affecting Amyloid Formation to Enable Study, Treatment, and Diagnosis of Neurodegenerative Disorders";

[0004] This PCT application also claims priority under 35 U.S.C §119(e) from US provisional application 63 / 698,573 filed on September 25, 2024 by the same Applicant and entitled "PHOSPHOROTHIOATE DNA (PSDNA) AND / OR PHOSPHODIESTER DNA (PODNA) AND / OR OTHER MATERIALS TO TREAT CANCER AND / OR NEURODEGENERATIVE DISORDERS";

[0005] Each of these earlier applications is fully incorporated herein by reference for all that it contains including the color content of the figures.

[0006] FIELD OF THE INVENTION

[0007] The invention is in the field of pharmaceuticals and medical treatment, more specifically treatment of cancer and / or neurodegenerative disorders.

[0008] BACKGROUND OF THE INVENTION

[0009] PSDNA and Prion Formation

[0010] DNA binding proteins are a diverse group of proteins that interact with DNA and modulate its structure, accessibility, and function. They play essential roles in various cellular processes, including transcription, replication, and repair. Specific DNA binding proteins can recognize and bind to short DNA fragments, such as transcription factors binding to regulatory regions of genes, or repair proteins recognizing damaged DNA sites.

[0011] Several extracellular membrane proteins have been reported to bind to DNA. Toll-like receptor 9 (TLR9) is a pattern recognition receptor primarily expressed in immune cells that recognizes unmethylated CpG motifs in DNA, playing a crucial role in innate immune responses and triggering inflammatory reactions upon DNA activation. High Mobility Group Box 1 (HMGB1), a non-histone chromosomal protein released by damaged cells, binds to DNA and facilitates its internalization by cells. This mediates inflammatory responses and immune cell activation. The DNA binding site of HMGl protein is composed of two similar segments (HMG boxes), both of which have counterparts in other eukaryotic regulatory proteins.

[0012] Pentraxin 3 (PTX3), an extracellular pattern recognition receptor involved in innate immunity, has been shown to bind to DNA and contribute to immune responses. Spermine oxidase (SMOX), an enzyme involved in polyamine metabolism, interacts with DNA and modulates DNA-binding protein activity. Thrombospondin-1 (TSP-1), a matricellular protein, binds to DNA and influences cell adhesion, migration, and angiogenesis.

[0013] Prion is composed solely of the disease-causing prion protein (PrPSc) that is formed from the cellular isoform PrPC by a posttranslational process.

[0014] It has been previously demonstrated that short Phosphorothioate DNA (PS-DNA) oligonucleotides diminished the levels of both PrPC and PrPSc in prion-infected neuroblastoma (ScN2a) cells. The effect of PS-DNA on PrP levels was independent of the nucleotide sequence. The effective concentration (EC50) of PS-DNA required to achieve half-maximal diminution of PrPSc was ~70 nM, whereas the EC50 of PS-DNA for PrPC was more than 50-fold greater. This finding indicated that diminished levels of PrPSc after exposure to PS-DNA are unlikely to be due to decreased PrPC levels. Bioassays in transgenic mice demonstrated a substantial diminution in the prion infectivity after ScN2a cells were exposed to PS-DNAs. Whether PS- DNA will be useful in the treatment of prion disease in people or livestock remains to be seen.

[0015] The DNA binding site that caused the PrPc to be downregulated following its specific binding to the PSDNA was subsequently identified. These findings highlight the diverse roles of extracellular membrane proteins in DNA binding and their potential impact on immune responses, cellular function, and disease processes.

[0016] In the context of cancer cell death due to exposure to short DNA fragments, several mechanisms can come into play. Short DNA fragments can be generated through various processes, including DNA damage, degradation, or fragmentation during cellular stress or apoptotic events. These fragments can potentially activate p53 and other DNA-binding proteins, leading to downstream signaling events that ultimately result in cancer cell death.

[0017] The protein p53 is a transcription factor that plays a crucial role in regulating cell division and preventing the development of cancer. It functions primarily as a tumor suppressor by controlling the expression of genes involved in cell cycle arrest, DNA repair, and apoptosis (programmed cell death). When DNA is damaged or there are abnormalities in the cell, p53 is activated to trigger appropriate cellular responses, including repair mechanisms or inducing apoptosis to eliminate the damaged cells. Interestingly it was demonstrated that p53 could be redirected out of the cells.

[0018] One possible mechanism is that short DNA fragments may directly bind to p53, promoting its activation and subsequent transcriptional activation of pro-apoptotic genes. This activation can lead to the induction of apoptosis in cancer cells, effectively eliminating them.

[0019] Additionally, short DNA fragments can also be recognized by other DNA binding proteins involved in DNA repair pathways, such as DNA damage response proteins. Activation of these proteins by short DNA fragments can trigger DNA repair processes, which, if overwhelmed or prolonged, can lead to cell death.

[0020] It is important to note that the effectiveness of cancer cell death due to exposure to short DNA fragments may vary depending on various factors, including the nature and extent of DNA damage, the specific DNA binding proteins involved, the cellular context, and the overall health of the cell.

[0021] The prion protein, commonly known for its association with neurodegenerative diseases, has also been implicated in cancer. The prion protein, encoded by the PRNP gene, is primarily expressed in various cell types, including neurons, immune cells, and cancer cells.

[0022] Emerging evidence suggests that the prion protein, particularly its isoform PrPC, plays a role in tumorigenesis and tumor progression. PrPC has been shown to interact with multiple signaling pathways involved in cell survival, proliferation, and metastasis, including those mediated by growth factors, cell adhesion molecules, and matrix metalloproteinases.

[0023] Studies have demonstrated that the expression of the prion protein is dysregulated in several types of cancer, and its overexpression has been associated with increased cell proliferation, survival, and resistance to chemotherapy. Additionally, the prion protein has been implicated in promoting cancer cell migration, invasion, and angiogenesis. The precise mechanisms underlying the contribution of the prion protein to cancer development are still not fully understood.

[0024] In a previous study, it was shown that shorter Phosphorothioate oligonucleotides (PSDNA) ranging from 18 to 44 nucleotides in length, exhibit greater nuclease resistance compared to phosphodiester DNA (PODNA) oligonucleotides, resulting in the downregulation of the prion protein in neuronal cell lines. Notably, this downregulation was achieved through lysosomal degradation and did not induce cellular death.

[0025] Amyloids and Neurodegenerative Disorders

[0026] Neurodegenerative diseases, ranging from Alzheimer's dementia to Parkinson's Disease pose a deep challenge to medical sciences, as well as a burden on those who are afflicted and their families. The societal costs are enormous.

[0027] Neurodegenerative diseases are caused by the formation of protein precipitates called amyloids. Amyloids are protein aggregates that form insoluble structures resistant to degradation. They tend to accumulate in the brain, forming clumps known as amyloid plaques. These plaques accumulate and grow, leading to the loss of connections among nerve cells and ultimately causing their death. Since nerve cells do not regenerate, this damage is irreversible. Amyloids are abnormal proteins found in the brain, resulting from genetic inheritance or abnormal formation, often manifesting in later ages. The accumulation of different types of amyloids in the brain causes various diseases.

[0028] Various environmental factors such as temperature and pH affect amyloid formation. However, very little is known regarding the impact of gravity on these aggregates. In addition, very little is known regarding the impact of particular pharmaceuticals on the growth of amyloids, including both the promotion of growth of such amyloids, which may be useful for in vitro studies, and the reversal of growth of such amyloids.

[0029] SUMMARY OF THE INVENTION

[0030] According to one aspect of some embodiments of the invention exposure of cancer cells to phosphorothioate DNA (PSDNA) and / or phosphodiester DNA (PODNA) contributes to downregulation of the PrP protein and / or contributes to decreased cell proliferation (using the MTT assay). Alternatively or additionally, in some embodiments exposure to PSDNA contributes to overcoming cancer mutations and / or to cell death. According to this aspect, PSDNA is useful in combination with, or as a replacement for or conventional therapy such as chemotherapy, immunotherapy and irradiation. In some embodiments the effects of PSDNA on the PrP protein contribute to its therapeutic efficacy. Specifically, PrP expression has a negative effect on chemotherapy, so PSDNA treatment appears useful to enhance the efficacy of a wide range of drugs in combination either before or in combination of the drug.

[0031] According to another aspect of some embodiments of the invention one or more members of the group consisting of a fungal extract, OMEGA 3, Dorminol, Valerian root extract, curcumin, Lamodex, Clonozapame, Frisium, Rispond, Statin(s), Tardiferol, Vimpat, Colonzapam, Bisoprolol fumarate, inovamed, Olanzapine, Tadam, Sinemet, valproate, sulpride, clomipramine, and Topiramate exert a negative influence on amyloid formation and / or the effect of microgravity on amyloid formation. According to various exemplary embodiments of the invention the drugs themselves reduce amyloid formation or enhance the amyloid inhibiting ability of other proteins.

[0032] According to a further aspect of some embodiments of the invention relates to using the differential effect of these compounds on different proteins in the assay as an early diagnosis tool to distinguish one patient from another.

[0033] According to various exemplary embodiments of the invention, the effect of various drugs is tested using one or more of three methods. The first method, Thioflavin T (ThT) fluorescence assay, uses a fluorescent molecule capable of forming chemical bonds with amyloids to quantify amyloid plaques. The second method, Fourier Transform Infrared Spectroscopy (FTIR), is employed to characterize chemical compounds. The FTIR spectrum in the infrared wavelength range (750 nm to millimeter), serves as a fingerprint for the material, facilitating its unequivocal identification. The third method, Atomic Force Microscopy (AFM), images surface topography at atomic-level resolutions.

[0034] Collagen is a central building block of the extracellular matrix, providing structural support and substrate for tissue growth and regeneration. The collagen is produced in the tissues according to the need when they are built or regenerated.

[0035] Hyaluronic acid is a carbohydrate polymer found throughout the body, contributing to synovial fluid thickness, articular cartilage structure, and skin repair. Hyaluronic acid also greatly contributes to cell proliferation and migration and participates in intercellular signaling. The molecular mass of hyaluronan decreases with age.

[0036] CLONEX™ is a benzodiazepine sedative, that mainly treats anxiety attacks and states of anxiety, as well as treating manic depression. CLONEX acts on GABA receptors and chloride channels in nerve cells inside the brain, reducing cellular activity and inducing relaxation.

[0037] Valerian is a wild flowering plant, there is a medicinal use of it mainly in its roots for sleep problems and stress. Most of the medical use relies on traditional use, and not enough clinical studies have been done to prove its effectiveness. The way valerian root remedies work is not fully known, but from some experiments, it is thought to have effects on the brain that lead to relaxation and sleepiness. Omega-3 Fatty acids, especially eicosatetraenoic acid (EPA) and docosahexaenoic acid (DHA), have received attention for their potential neuroprotective effects in neurodegenerative diseases. Epidemiological studies have reported inverse relationships between fish consumption, rich in omega-3 fatty acids, and the risk of cognitive decline and neurodegenerative diseases. Mechanistically, omega-3 fatty acids modulate inflammatory pathways, enhance synaptic plasticity, and promote neuronal survival and repair. Furthermore, DHA plays a crucial role in maintaining membrane integrity, ion channel function, and neurotransmitter release in the brain.

[0038] Lysozyme was used as a model protein known to form amyloids and is suitable for future FTIR analysis, which requires a high concentration of amyloid for detection. Lysozyme is an enzyme that breaks down bacterial cell wall crosslinks and is found in human tears. Additionally, it serves as a model protein for studying amyloid aggregation in neurodegenerative disease research.

[0039] Experiments set forth hereinbelow using the Thioflavin T (ThT) fluorescence method, evaluated the impact of several substances (valerian, omega-3, CLONEX, hyaluronic acid + collagen mixture) on amyloid aggregation for peptides such as polyglutamine (Q.10), tau, prion, alpha-synuclein, and amyloid beta. The experiments included initial runs at 55°C and pH 7.4, with a follow-up study at 37°C for validation and comparison. Short and long incubation times were employed.

[0040] Results are summarized in Table 1.

[0041] Table 1. Summary of the results from the two experiments from the Thioflavin T method. NC=No change.

[0042] The left column indicates the name of the amyloid peptide, the right column describes the experimental conditions with an emphasis on the difference between the experiments, and the first row describes the tested substance. Each cell under the tested substance describes how the substance affected the peptide and in which experiment. In case there are two modes of influence the primary influence is listed first.

[0043] For purposes of this specification and the accompanying claims, the term "microgravity" indicates conditions on Earth that mimic zero gravity conditions. In some embodiments, microgravity is achieved using a well-characterized rotating machine that creates these conditions. Anything placed on the platform of the machine is subject to zero gravity.

[0044] Various exemplary embodiments of the invention relate to one, two, or all three of the aspects set forth above.

[0045] It will be appreciated that the various aspects described above relate to solution of technical problems associated with inhibition of formation of prion protein (PrP) in cells diminishing deposits of PrP after they are formed.

[0046] Alternatively or additionally, it will be appreciated that the various aspects described above relate to solution of technical problems related to associated with inhibition formation of amyloid plaques in cells diminishing deposits of amyloid plaques, after they are formed.

[0047] In some exemplary embodiments of the invention there is provided a method of cancer treatment, including: delivering a physiologically effective amount of phosphorothioate DNA (PSDNA) to cancer cells. In some embodiments the physiologically effective amount is at least 1 mM. Alternatively or additionally, in some embodiments the physiologically effective amount does not exceed 10 mM. Alternatively or additionally, in some embodiments the PSDNA includes a mixture of random oligonucleotides. Alternatively or additionally, in some embodiments the delivering includes systemic delivery to a patient. Alternatively or additionally, in some embodiments the delivering includes direct delivery to a tumor.

[0048] In some exemplary embodiments of the invention there is provided a method of cancer treatment, including: delivering a physiologically effective amount of phosphodiester DNA (PODNA) to cancer cells. In some embodiments the physiologically effective amount is at least 10 pM. Alternatively or additionally, in some embodiments the physiologically effective amount does not exceed 100 pM. Alternatively or additionally, in some embodiments the PODNA includes a mixture of random oligonucleotides. Alternatively or additionally, in some embodiments the delivering includes systemic delivery to a patient. Alternatively or additionally, in some embodiments the delivering includes direct delivery to a tumor.

[0049] In some exemplary embodiments of the invention there is provided a physiologically effective amount of phosphorothioate DNA (PSDNA) for use in the treatment of cancer. In some embodiments the physiologically effective amount is at least lpM. Alternatively or additionally, in some embodiments the physiologically effective amount does not exceed 10 pM. Alternatively or additionally, in some embodiments the PSDNA includes a mixture of random oligonucleotides. Alternatively or additionally, in some embodiments the compound is formulated for systemic delivery to a patient. Alternatively or additionally, in some embodiments the compound is formulated for direct delivery to a tumor.

[0050] In some exemplary embodiments of the invention there is provided a physiologically effective amount of phosphodiester DNA (PODNA) for use in the treatment of cancer. In some embodiments the physiologically effective amount is at least 10 pM. Alternatively or additionally, in some embodiments the physiologically effective amount does not exceed 100 pM. Alternatively or additionally, in some embodiments the PODNA includes a mixture of random oligonucleotides. Alternatively or additionally, in some embodiments the compound is formulated for systemic delivery to a patient. Alternatively or additionally, in some embodiments the compound is formulated for direct delivery to a tumor.

[0051] In some exemplary embodiments of the invention there is provided pharmaceutical composition including: a physiologically effective amount of phosphorothioate DNA (PSDNA) as an active ingredient; and diluents and / or excipients; wherein the physiologic effect is retardation or reversal to tumor growth. In some embodiments the physiologically effective amount for a single oral dosage form is at least 5 mg. Alternatively or additionally, in some embodiments the physiologically effective amount for a single oral dosage form does not exceed 50 mg. Alternatively or additionally, in some embodiments the physiologically effective amount for a single systemic injectable dosage form is at least 1 mg. Alternatively or additionally, in some embodiments the physiologically effective amount for a single systemic injectable dosage form does not exceed 10 mg. Alternatively or additionally, in some embodiments the physiologically effective amount for a single locally injectable dosage form is at least 0.5 mg. Alternatively or additionally, in some embodiments the physiologically effective amount for a single locally injectable dosage form does not exceed 5 mg. Alternatively or additionally, in some embodiments the PSDNA includes a mixture of random oligonucleotides.

[0052] In some exemplary embodiments of the invention there is provided a pharmaceutical composition including: a physiologically effective amount of phosphodiester DNA (PODNA) as an active ingredient; and diluents and / or excipients; wherein the physiologic effect is retardation or reversal to tumor growth. In some embodiments the physiologically effective amount for a single oral dosage form is at least 10 mg. Alternatively or additionally, in some embodiments the physiologically effective amount for a single oral dosage form does not exceed 100 mg. Alternatively or additionally, in some embodiments the physiologically effective amount for a single systemic injectable dosage form is at least 5 mg. Alternatively or additionally, in some embodiments the physiologically effective amount for a single systemic injectable dosage form does not exceed 50 mg. Alternatively or additionally, in some embodiments the physiologically effective amount for a single locally injectable dosage form is at least 2 mg. Alternatively or additionally, in some embodiments the physiologically effective amount for a single locally injectable dosage form does not exceed 20 mg. Alternatively or additionally, in some embodiments the PODNA includes a mixture of random oligonucleotides.

[0053] In some exemplary embodiments of the invention there is provided a method of preventing or reversing amyloid formation, including: delivering a physiologically effective concentration of at least one member of the group of active ingredients consisting of OMEGA 3, Dorminol, and Valerian root extract, curcumin, Lamodex, Clonazepam, Frisium, Rispond, Statin, Tardiferol, Vimpat, Colonzapam, Bisoprolol fumarate inovamed, Olanzapine, Tadam, Sinemet, valproate, sulpride, clomipramine, Topiramate, and a combination of collagen and hyaluronic acid to cells producing, or in danger of producing, amyloid plaques. In some embodiments the method is performed under microgravity conditions. Alternatively or additionally, in some embodiments the active ingredient includes a combination of collagen and hyaluronic acid. Alternatively or additionally, in some embodiments the active ingredient includes Clonazepam. Alternatively or additionally, in some embodiments the active ingredient includes Valerian root extract. Alternatively or additionally, in some embodiments the active ingredient includes OMEGA 3. Alternatively or additionally, in some embodiments the cells are brain cells. Alternatively or additionally, in some embodiments the cells are cancer cells.

[0054] In some exemplary embodiments of the invention there is provided at least one member of the group of active ingredients consisting of OMEGA 3, Dorminol, and Valerian root extract, curcumin, Lamodex, Clonazepam, Frisium, Rispond, Statin, Tardiferol, Vimpat, Colonzapam, Bisoprolol fumarate inovamed, Olanzapine, Tadam, Sinemet, valproate, sulpride, clomipramine, Topiramate, and a combination of collagen and hyaluronic acid for use in a composition for preventing, retarding or reversing formation of amyloid plaques in or on cells. In some embodiments Alternatively or additionally, in some embodiments the composition is deployed under microgravity conditions. Alternatively or additionally, in some embodiments the active ingredient includes a combination of collagen and hyaluronic acid. Alternatively or additionally, in some embodiments the active ingredient includes Clonazepam. Alternatively or additionally, in some embodiments the active ingredient includes Valerian root extract. Alternatively or additionally, in some embodiments the active ingredient includes OMEGA 3. Alternatively or additionally, in some embodiments the cells are brain cells or nerve cells. Alternatively or additionally, in some embodiments the cells are cancer cells.

[0055] In some exemplary embodiments of the invention there is provided a pharmaceutical composition including: at least one member of the group of active ingredients consisting of OMEGA 3, Dorminol, and Valerian root extract, curcumin, Lamodex, Clonazepam, Frisium, Rispond, Statin, Tardiferol, Vimpat, Colonzapam, Bisoprolol fumarate inovamed, Olanzapine, Tadam, Sinemet, valproate, sulpride, clomipramine, Topiramate, and a combination of collagen and hyaluronic acid; and diluents and / or excipients; wherein the active ingredient is present in an amount sufficient to prevent, retard, or reverse formation of amyloid plaques in or on cells. In some embodiments the active ingredient includes a combination of collagen and hyaluronic acid. Alternatively or additionally, in some embodiments the active ingredient includes Clonazepam. Alternatively or additionally, in some embodiments the active ingredient includes Valerian root extract. Alternatively or additionally, in some embodiments the active ingredient includes OMEGA 3. Alternatively or additionally, in some embodiments the composition is formulated for activity on brain cells or nerve cells. Alternatively or additionally, in some embodiments the composition is formulated for activity on cancer cells.

[0056] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although suitable methods and materials are described below, methods and materials similar or equivalent to those described herein can be used in the practice of the present invention. In case of conflict, the patent specification, including definitions, will control. All materials, methods, and examples are illustrative only and are not intended to be limiting. As used herein, the terms "comprising" and "including", or grammatical variants thereof are to be taken as specifying the inclusion of the stated features, integers, actions or components without precluding the addition of one or more additional features, integers, actions, components or groups thereof. This term is broader than and includes the terms "consisting of" and "consisting essentially of" as defined by the Manual of Patent Examination Procedure of the United States Patent and Trademark Office. Thus, any recitation that an embodiment "includes" or "comprises" a feature is a specific statement that sub embodiments "consist essentially of" and / or "consist of" the recited feature.

[0057] The phrase "consisting essentially of" or grammatical variants thereof when used herein are to be taken as specifying the stated features, integers, steps or components but do not preclude the addition of one or more additional features, integers, steps, components or groups thereof but only if the additional features, integers, steps, components or groups thereof do not materially alter the basic and novel characteristics of the claimed composition, device or method.

[0058] The phrase "adapted to" as used in this specification and the accompanying claims imposes additional structural limitations on a previously recited component.

[0059] The term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of architecture and / or computer science.

[0060] Percentages (%) are W / V (weight per volume) unless otherwise indicated.

[0061] BRIEF DESCRIPTION OF THE DRAWINGS

[0062] In order to understand the invention and to see how it may be carried out in practice, embodiments will now be described, by way of non-limiting example only, with reference to the accompanying figures. In the figures, identical and similar structures, elements or parts thereof that appear in more than one figure are generally labeled with the same or similar references in the figures in which they appear. Dimensions of components and features shown in the figures are chosen primarily for convenience and clarity of presentation and are not necessarily to scale. The attached figures are:

[0063] FIG. 1 is a Western blot analysis of the PrP protein in various cell lines and representative proliferation assay following exposure of cells to PSDNA demonstrating specific downregulation of PrP in cancer cell lines and its specific inhibition of cancer cell growth according to some embodiments of the invention; FIG. 2 illustrates an MTT assay and live images showing how PSDNA specifically inhibits the growth of cancer cells and is associated with the expression of PrP according to some embodiments of the invention;

[0064] FIG. 3 illustrates fluorescent images and Western blot analysis of human PrP siRNA showing specific inhibition of human PrP using siRNA in fluorescently labeled human MDA-MB-231 cell lines leads to cell death, as observed with non-specific siRNA, using a fluorescent microscope according to some embodiments of the invention;

[0065] FIG. 4 illustrates Transcriptomic analysis of mouse triple negative cancer cell line 4T1 according to some embodiments of the invention;

[0066] FIG. 5 illustrates how PSDNA treatment reduces or eliminates mice and human triple negative cancer according to some embodiments of the invention;

[0067] FIG. 6 illustrates an Impact of PSDNA treatment on PrP levels in primary tumors and its effect on tumor migration according to some embodiments of the invention;

[0068] Fig. 7 illustrates testing for amyloids using ThT staining according to some embodiments of the invention;

[0069] Fig. 8A, Fig. 8B, Fig. 8C, and Fig. 8D illustrate results of assays regarding the effect of different compounds on amyloid formation according to some embodiments of the invention;

[0070] Fig. 9A, Fig. 9B and Fig. 9C illustrate experimental results regarding the effect of microgravity conditions on amyloid formation according to some embodiments of the invention;

[0071] Fig. 10 illustrates that PolyQ Amyloidogenic aggregates specifically dissolve following incubation with Valerian and Omega 3; Poly Q.10 incubation with: (A) Valerian=Val, (B) Omega 3=03, (C) Hyaluronic acid=Hia+ Collagen=Col, and (D) Clonozapam=Clo; Yellow lines represent the peptide in the presence of the tested drug and the blue line represents the control peptide incubated with water alone (DDW);Lower Error Bar (LB) and Upper Error Bars (UB) are included to evaluate the significance of the results;

[0072] Fig. 11 illustrates that PolyQ Amyloidogenic aggregates specifically dissolve following incubation with Valerian and Omega 3 at 37 °C; Poly Q10 incubation with: (A) Valerian=Val, (B) Omega 3=03. Yellow lines represent the peptide in the presence of the tested drug and the blue line represents the control peptide incubated with water alone (DDW); Lower Error Bar (LB) and Upper Error Bars (UB) are included for measurement;

[0073] Fig. 12 illustrates that Tau Amyloidogenic aggregates specifically dissolve following incubation with Valerian and Omega 3;Tau peptide incubation with: (A) Valerian=Val, (B) Omega 3=03, (C) Hyaluronic acid=Hia+ Collagen=Col, and (D) Clonozapam=Clo; Yellow lines represent the peptide in the presence of the tested drug and the blue line represents the control peptide incubated with water alone (DDW); Lower Error Bar (LB) and Upper Error Bars (UB) are included for measurement;

[0074] Fig. 13 illustrates that Tau Amyloidogenic aggregates specifically dissolve following incubation with Valerian and Omega 3 in 37 °C; Tau peptide incubation with: (A) Valerian=Val,

[0075] (B) Omega 3=03; Yellow lines represent the peptide in the presence of the tested drug and the blue line represents the control peptide incubated with water alone (DDW); Lower Error Bar (LB) and Upper Error Bars (UB) are included for measurement;

[0076] Fig. 14 illustrates that Alpha-synuclein and A Beta Amyloidogenic aggregates specifically dissolve following incubation with Valerian and Omega 3 in different solvents. Alpha- synuclein incubation with (A) Valerian=Val, (B) Alpha-synuclein incubation with Omega 3=03.

[0077] (C) A Beta-amyloid without DMSO with valerian. (D) A Beta-amyloid with DMSO with Valerian (E) A Beta-amyloid without DMSO with omega 3. (F) A beta with DMSO with Omega 3;

[0078] Fig. 15 illustrates that AFM analysis demonstrates the anti-amyloid effect of omega 3 and valerian in polyglutamine and A beta amyloid structures. (A-C) The polyglutamine peptide or A beta peptide (D-F) with DDW (A, D), Omega 3 (B, E), and Valerian (C, F). J is the zoom-in image of A and H is the zoom-in of H;

[0079] Fig. 16 illustrates autofluorescence of tested compounds and their ability to form amyloid; (A) Valerian=Val, (B) Omega 3=03, (C) Hyaluronic acid=Hia+ Collagen=Col, and (D) Clonozapam=Clo; Orange line represents the substance in the presence of ThT and the blue line represents the control sample with water and ThT alone (DDW); Green line represents only the substance;

[0080] Fig. 17 illustrates that Lysozyme Amyloidogenic aggregates specifically dissolve following incubation with Valerian and Omega 3; Lysozyme incubation with: (A) Valerian=Val, (B) Omega 3=03, (C) Hyaluronic acid=Hia+ Collagen=Col, and (D) Clonozapam=Clo; Yellow lines represent the peptide in the presence of the tested drug and the blue line represents the control peptide incubated with water alone (DDW); Lower Error Bar (LB) and Upper Error Bars (UB) are included for measurement.

[0081] Fig. 18 illustrates results of a western blot: upper panel downregulation of PrP (stained using the D18 antibody) by PSDNA (PS) relative to a negative control, normalized to GAPDH in 4T1 cells; lower panel downregulation of PrP by PSDNA (PS) relative to a negative control, normalized to Vinculin in LRK1O and LRK 14 cells;

[0082] Fig. 19 illustrates that mouse and human cancer cell growth is inhibited by cellular aggregation following specific exposure to PSDNA, and this effect is sequence-independent; Fig. 20 illustrates the results of a western blot indicating that downregulation of PrP by PSDNA (PS) is time dependent and sequence dependent; C blank negative control, PO indicates PODNA as an additional negative control; normalized to Vinculin;

[0083] Fig. 21 illustrates that PSDNA inhibits aggressive cancer cell growth in mice and humans in breast and lung adenocarcinoma which is bears specific genetic mutations in a dosedependent manner and specific to cancer cell lines (no significant inhibition of the cell proliferation of the noncancer HEK cell line) with an IC50 of approximately 0.01 micromolar; Fig. 22A is a western blot of 4T1 breast cancer mouse cell lines exposed to PSDNA for 3 hours, 2 days, and 5 days illustrating increased STING phosphorylation (Left lower panel), and PARP cleavage (Right) correlates with PrP downregulation (left upper panel Vinculin is used to demonstrate equal amount of total protein loaded on each well;

[0084] Fig. 22B is a western blot of 4T1 breast cancer mouse cell lines exposed to PSDNA(PS) PODNA(PO) or a blank (C), for 3 or 24 hours, illustrating PARP and pSTING levels (supporting findings in figure 22A); Silver staining of the gel was used to indicate equal amount of total protein loaded for each time point;

[0085] Fig. 22C is a western blot of 4T1 breast cancer mouse cell lines exposed to PSDNA (PS) for 3 hours, 2 days or 5 illustrating MONDO A cleavage; Ponceau S staining was used to demonstrate equal loading of total protein in each well;

[0086] Fig. 22D is a western blot of HEK cells exposed to PSDNA(PS) PODNA(PO) or a blank (C), for 3 or 24 hours, illustrating PARP levels; Silver staining of the gel was used to indicate equal amount of total protein loaded for each time point;

[0087] Fig. 23 is a series of bar graphs illustrating that PSDNA treatment has a synergistic effect with Osimertinibe in KRAS mutant B901L Human lung cancer cells; and

[0088] Fig. 24 is a series of photographs illustrating cell aggregation resulting from treatment with lpM PSDNA; Mouse mammary breast cancer cell lines 4T1 and mouse lung carcinoma cell lines B901L form cell aggregates following exposure to ImM PSDNA for 24 hours; HEK cells serve as a non-cancer control and were not affected by PSDNA in the media.

[0089] DETAILED DESCRIPTION OF EMBODIMENTS The principles and operation of a pharmaceutical composition and / or method and / or compound for use according to exemplary embodiments of the invention may be better understood with reference to the drawings and accompanying descriptions.

[0090] Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting.

[0091] Embodiments of the invention relate to pharmaceutical compositions and treatment methods that ameliorate cancer and / or neurodegenerative disorders.

[0092] Specifically, some embodiments of the invention relate to use of phosphorothioate DNA (PSDNA) and / or phosphodiester DNA (PODNA) to retard or prevent tumor formation.

[0093] Alternatively or additionally, some embodiments of the invention relate to use of a fungal extract, and / or OMEGA 3, and / or Dorminol, and / or Valerian root extract, and / or curcumin, and / or Lamodex, and / or Clonozapame, and / or Frisium, and / or Rispond, and / or Statin, and / or Tardiferol, and / or Vimpat, and / or Colonzapam, and / or Bisoprolol fumarate and / or inovamed, and / or Olanzapine, and / or Tadam, and / or Sinemet, and / or valproate, and / or sulpride, and / or clomipramine, and / or Topiramate to retard or reverse amyloid formation.

[0094] Exemplary PSDNA treatment methods

[0095] In some exemplary embodiments of the invention there is provided a method of cancer treatment, including delivering a physiologically effective amount of phosphorothioate DNA (PSDNA) to cancer cells. In some embodiments the physiologically effective amount is at least 1 pM. Alternatively or additionally, in some embodiments the physiologically effective amount does not exceed 10 pM. In some exemplary embodiments of the invention, the PSDNA comprises a mixture of random oligonucleotides. According to these embodiments, the use of random oligonucleotides eliminates any antisense effect.

[0096] In some exemplary embodiments of the invention, the delivering includes systemic delivery to a patient. Systemic delivery includes but is not limited to oral (or buccal) administration, intravenous injection, intraperitoneal administration and other injection routes.

[0097] In other exemplary embodiments of the invention, the delivering includes direct delivery to a tumor e.g. by injection into a tumor or lavage of the tumor. In some exemplary embodiments of the invention there is provided method of cancer treatment, including delivering a physiologically effective amount of phosphodiester DNA (PODNA) to cancer cells. In some embodiments the physiologically effective amount is at least 10 pM. Alternatively or additionally, in some embodiments the physiologically effective amount does not exceed 100 pM.

[0098] In some exemplary embodiments of the invention, the PODNA comprises a mixture of random oligonucleotides (which eliminates an antisense effect as described above).

[0099] In some exemplary embodiments of the invention, the delivering includes systemic delivery to a patient. Systemic delivery includes, but is not limited to, oral administration, intravenous injection, intraperitoneal administration, and other injection routes.

[0100] In other exemplary embodiments of the invention, the delivering includes direct delivery to a tumor (e.g. by injection into a tumor or lavage of the tumor).

[0101] Exemplary PSDNA compounds for use

[0102] In some exemplary embodiments of the invention there is provided a physiologically effective amount of phosphorothioate DNA (PSDNA) for use in the treatment of cancer.

[0103] In some exemplary embodiments of the invention, the physiologically effective amount is at least lpM. Alternatively or additionally, in some embodiments the physiologically effective amount does not exceed 10 pM.

[0104] In some exemplary embodiments of the invention, the PSDNA comprises a mixture of random oligonucleotides.

[0105] In some exemplary embodiments of the invention, the compound for use is formulated for systemic delivery to a patient (e.g. as an oral dosage form or an injectable dosage form).

[0106] In other exemplary embodiments of the invention, the compound for use is formulated for direct delivery to a tumor (e.g. for injection into a tumor or lavage of the tumor).

[0107] Exemplary PODNA compounds for use

[0108] In some exemplary embodiments of the invention there is provided a physiologically effective amount of phosphodiester DNA (PODNA) for use in the treatment of cancer. In some exemplary embodiments of the invention, the physiologically effective amount is at least 10 pM. Alternatively or additionally, in some embodiments the physiologically effective amount does not exceed 100 pM.

[0109] In some exemplary embodiments of the invention, the PODNA includes a mixture of random oligonucleotides (which eliminates any antisense effect). In some exemplary embodiments of the invention, the compound for use is formulated for systemic delivery to a patient (e.g. as an oral dosage form or an injectable dosage form). In other exemplary embodiments of the invention, the compound for use is formulated for direct delivery to a tumor (e.g. injection into tumor or lavage of the tumor).

[0110] Exemplary PSDNA pharmaceutical composition

[0111] In some exemplary embodiments of the invention there is provided pharmaceutical composition comprising: a physiologically effective amount of phosphorothioate DNA (PSDNA) as an active ingredient with diluents and / or excipients; wherein the physiologic effect is retardation or reversal to tumor growth. In some exemplary embodiments of the invention, physiologically effective amount for a single oral dosage form is at least 5 mg. Alternatively or additionally, in some embodiments the physiologically effective amount for a single oral dosage form does not exceed 50 mg.

[0112] In other exemplary embodiments of the invention, the physiologically effective amount for a single systemic injectable dosage form is at least 1 mg. Alternatively or additionally, in some embodiments the physiologically effective amount for a single systemic injectable dosage form does not exceed 10 mg.

[0113] In other exemplary embodiments of the invention, the physiologically effective amount for a single locally injectable dosage form is at least 0.5 mg. Alternatively or additionally, in some embodiments the physiologically effective amount for a single systemic locally injectable dosage form does not exceed 5 mg. Local injection indicates direct injection into a tumor.

[0114] In some exemplary embodiments of the invention, the PSDNA includes a mixture of random oligonucleotides, which eliminates an antisense effect.

[0115] Exemplary PODNA pharmaceutical composition

[0116] In some exemplary embodiments of the invention there is provided a pharmaceutical composition including a physiologically effective amount of phosphodiester DNA (PODNA) as an active ingredient with diluents and / or excipients; wherein the physiologic effect is retardation or reversal to tumor growth. In some exemplary embodiments of the invention, the physiologically effective amount for a single oral dosage form is at least 10 mg. Alternatively or additionally, in some embodiments the physiologically effective amount for a single oral dosage form does not exceed 100 mg.

[0117] In other exemplary embodiments of the invention, the physiologically effective amount for a single systemic injectable dosage form is at least 5 mg. Alternatively or additionally, in some embodiments the physiologically effective amount for a single systemic injectable dosage form does not exceed 50 mg.

[0118] In other exemplary embodiments of the invention, the physiologically effective amount for a single locally injectable dosage form is at least 2 mg. Alternatively or additionally, in some embodiments the physiologically effective amount for a single locally injectable dosage form does not exceed 20 mg. Local injection indicates directly into a tumor.

[0119] Alternatively or additionally, in some embodiments PODNA includes a mixture of random oligonucleotides, which eliminates any antisense effect.

[0120] In some exemplary embodiments of the invention there is provided method of preventing or reversing amyloid formation, including: delivering a physiologically effective concentration of at least one member of the group of active ingredients consisting of OMEGA 3, Dorminol, and Valerian root extract, curcumin, Lamodex, Clonazepam, Frisium, Rispond, Statin, Tardiferol, Vimpat, Colonzapam, Bisoprolol fumarate inovamed, Olanzapine, Tadam, Sinemet, valproate, sulpride, clomipramine, Topiramate, and a combination of collagen and hyaluronic acid to cells producing, or in danger of producing, amyloid plaques. In some exemplary embodiments of the invention, the method is performed under microgravity conditions.

[0121] In some exemplary embodiments of the invention, the active ingredient includes a combination of collagen and hyaluronic acid.

[0122] In one exemplary embodiment of the invention a combination of collagen and hyaluronic acid is prepared as follows:

[0123] One pill (containing 1200 mg type II collagen, 720 mg hydrolyzed collagen type II, chondroitin sulfate 192 mg, hyaluronic acid 120 mg, 100 mg vitamin C as calcium ascorbate) is dissolved in 10ml DDW, centrifuged at 3000 RPM and 5 microliters of the supernatant was diluted to a total volume of 80 microliters.

[0124] The final concentrations are:

[0125] Type II collagen: 7.5 pg / pL;

[0126] Hydrolysed collagen type II: 4.5 pg / pL;

[0127] Chondroitin sulfate: 1.2 pg / pL;

[0128] Hyaluronic acid: 0.75 pg / pL;

[0129] Vitamin C (calcium ascorbate): 0.625 pg / pL;

[0130] In some exemplary embodiments of the invention, the active ingredient includes Clonazepam. In some exemplary embodiments of the invention, the concentration of clonazepam is at least 0.02 pg / pL and / or not more than 0.08 pg / pL. In some embodiments about 0.03125 pg / pL.

[0131] Alternatively or additionally, in some embodiments the active ingredient includes Valerian root extract. According to various exemplary embodiments of the invention the physiologically effective concentration of Valerian is at least 2.5 pg / pL and / or not more than 25 pg / pL. In some embodiments the physiologically effective concentration is about 12.5 pg / pL

[0132] In some embodiments the active ingredient includes OMEGA 3. According to various exemplary embodiments of the invention the physiologically effective concentration of OMEGA 3 is at least 2.5 pg / pL and / or not more than 25 pg / pL. In some embodiments the physiologically effective concentration is about 12.5 pg / pL

[0133] According to various exemplary embodiments of the invention cells include brain cells, and / or cells are cancer cells.

[0134] Exemplary compounds for use against amyloid plaques

[0135] In some exemplary embodiments of the invention there is provided at least one member of the group of active ingredients consisting of OMEGA 3, Dorminol, and Valerian root extract, curcumin, Lamodex, Clonazepam, Frisium, Rispond, Statin, Tardiferol, Vimpat, Colonzapam, Bisoprolol fumarate inovamed, Olanzapine, Tadam, Sinemet, valproate, sulpride, clomipramine, Topiramate, and a combination of collagen and hyaluronic acid for use in preventing, retarding or reversing formation of amyloid plaques in or on cells. In some exemplary embodiments of the invention, the composition for use is deployed under microgravity conditions.

[0136] Alternatively or additionally, in some embodiments the active ingredient includes a combination of collagen and hyaluronic acid. In some exemplary embodiments of the invention, the combination of collagen and hyaluronic acid is as disclosed hereinabove.

[0137] Alternatively or additionally, in some embodiments the active ingredient includes Clonazepam. In some exemplary embodiments of the invention, the concentration of clonazepam is at least 0.02 pg / pL and / or does not exceed 0.08 pg / pL. In some embodiments the concentration is about 0.03125 pg / pL.

[0138] Alternatively or additionally, in some embodiments the active ingredient includes Valerian root extract. In some exemplary embodiments of the invention, there is at least 10 pM and / or not more than 50 pM of Valerian root extract.

[0139] Alternatively or additionally, in some embodiments the active ingredient includes OMEGA 3. In some exemplary embodiments of the invention, the Omega-3 (DHA / EPA) is present at an amount of at least 10 pM and / or not more than 100 pM.

[0140] According to various exemplary embodiments of the cells include brain cells and / or nerve cells and / or cancer cells.

[0141] Exemplary pharmaceutical composition for amyloid plaques

[0142] In some exemplary embodiments of the invention there is provided a pharmaceutical composition including: at least one member of the group of active ingredients consisting of OMEGA 3, Dorminol, and Valerian root extract, curcumin, Lamodex, Clonazepam, Frisium, Rispond, Statin, Tardiferol, Vimpat, Colonzapam, Bisoprolol fumarate inovamed, Olanzapine, Tadam, Sinemet, valproate, sulpride, clomipramine, Topiramate, and a combination of collagen and hyaluronic acid; and diluents and / or excipients; wherein the active ingredient is present in an amount sufficient to prevent, retard, or reverse formation of amyloid plaques in or on cells.

[0143] In some exemplary embodiments of the invention, the active ingredient includes a combination of collagen and hyaluronic acid. In some embodiments the combination of collagen and hyaluronic acid is as set forth hereinabove. In some exemplary embodiments of the invention, \hyaluronic acid is used in concentrations of at least 10 pg / mL and / or not more than 100 pg / mL. Alternatively or additionally, in some embodiments collagen is used in a concentrations of at least 1 pg / mL and or not more than 100 pg / mL.

[0144] In some exemplary embodiments of the invention, the active ingredient includes Clonazepam. In some embodiments the clonazepam is present at a concentration of 0.1 pM and / or at a concentration not exceeding 10 pM.

[0145] In some embodiments the active ingredient comprises Valerian root extract (e.g. at concentrations as set forth hereinabove).

[0146] In some exemplary embodiments of the invention, the active ingredient includes OMEGA 3 (concentrations as set forth hereinabove).

[0147] According to various exemplary embodiments of the invention the pharmaceutical composition is formulated for activity on brain cells and / or nerve cells and / or cancer cells.

[0148] In some exemplary embodiments of the invention, exposure of cancer cells for 3-15h to PSDNA led to the downregulation of the PrP protein. A 24-hour exposure of cancer cells to the PSDNA decreased cell proliferation as demonstrated by the MTT assay.

[0149] Referring to FIG. 1, Western blot analysis using the anti-PrP antibody D18 revealed different glycosylation patterns (A and B). An antibody against GAPDH was used as a normalizing control to demonstrate an equal amount of total protein loaded for each cell line. MTT assay was conducted for two cell lines that express PrP and cell lines and are affected by PrP (A9 and ID8 panel C) compared to a cell line that is not affected by PSDNA (RAW panel C). In addition, PrP in the mouse Neuroblastoma NR67, human Glioblastoma LN299, mouse Fibrosarcoma WEHI164, and human MBA-MB-231-WT cell lines is downregulated following exposure to PSDNA for 24h. MBA-MB-231-PrPKO needs to be retested since the tubulin was negative (panel D). Ponceau S staining indicates the total protein loaded in each well.

[0150] In order to test the correlation between PrP expression, cell proliferation, and PSDNA exposure in two cancer cell lines that represent uncurable cancer patients the triple negative cancer cell line (MDA-MB-231) and the pancreatic cancer cell line (KPC) were employed. Equal numbers of cells were seeded onto a 96 well plate and cell growth was measured after 24, 48 and 72 hours with and without exposure to the PSDNA. An MDA-MB-231 cell line with reduced PrP expression level was generated using the CRISPR / CAS9 PrPKO method to test the effect of PrP expression. An additional control was the HEK 293Tcell line, which is a non-cancer cell line which does not express PrP (Fig. 2). Results presented in Fig. 2 indicate that KPC (which has the highest levels of PrP) proliferates significantly faster than the MDA-MB-231 cell line. The two cell lines that have no PrP (HEK293T) or low PrP levels (MDA-MB-231 PrPKO) were slower in growth and were not affected by the exposure to PSDNA.

[0151] FIG. 2 demonstrates that PSDNA specifically inhibits the growth of cancer cells and is associated with the expression of PrP. The rate of cell proliferation is correlated with PrP expression, and the inhibitory effect of PSDNA on cell proliferation is evident in the MTT assay (Panel A) and live images taken after 24 hours (Panel B).

[0152] To further demonstrate that PrP downregulation leads to either cell death or reduced proliferation the PrP protein was targeted using the SiRNA method. A human specific SiRNA guide was transfected into the triple negative cancer cell line MDA-MB-231 labelled with RFP. Fig. 3 demonstrates that depletion of the prion protein specifically in the human MDA cell line leads to cell death in these cells. The mouse KPC cells and Human HEK293T cells served as negative controls. No cell death was observed in either cell line (data not shown) nor PrP downregulation in the KPC cells as can be observed in Fig. 3, lower panel.

[0153] Fig. 3 illustrates fluorescent images and Western blot analysis of human PrP siRNA. MDA-MB-231-GFP cell lines were exposed to control siRNA (left upper panel) or PrP SiRNA (right upper panel). Western blot analysis using the anti-PrP antibody D18 revealed the specific elimination of the PrP expression in the MDA human cancer cell line. PrP levels are not affected in the mouse KPC cell line. HEK cell line was used as a negative control. An antibody for betaactin on the same samples demonstrates equal loading of total protein (lower right panel).

[0154] In order to further understand the mechanism of action and the pathways that might be Involved in this process, a transcriptomic analysis assay of the mouse triple negative cancer cell line model 4T1 following exposure of the active PSDNA or the inactive PODNA. Two independent cell samples and two independent sequencing (set 1 and set 2) were performed on these two groups. According to the pre-specified criteria (false discovery rate (FDR) < 0.05 and two-fold difference in expression), in combination with the VIB / UGENT analysis tool (Draw Venn Diagram. https: / / bioinformatics.psb.u ent.be / webtools / Venn / ) expression was lower for 121 genes and upregulated for 195 in the PSDNA group as compared to the PODNA group (Fig. 4 panel A). Thereafter, the most significantly upregulated and downregulated genes were placed separately into the Metascape pathway analysis tool to identify the pathways that are involved following PSDNA exposure (Figure 4 panel B). The highest impact for downregulated genes (Log(q-value) of -3.76) was on the defense response to virus (G0:0051607) represented by the following genes: Ifitl, Ifit3, Mx2, Statl, Oaslg, Oaslb, Oasl2, Zbpl, Bst2, Trim30d, Ddx60, Isgl5, Tgtpl, Masp2, GbplO, Ccnd2, Gadd45b, Gadd45g, TnfsflO. The second pathway that was significantly impacted (Log(q-value) of -2.040) was the response to interferon-beta pathway (G0:0035456). The highest impact for upregulated genes (Log(q-value) of -23.03) was on the Cholesterol metabolism with Bloch and Kandutsch-Russell pathways (WikiPathways, WP4346) represented by the following genes: Cyp51, Fasn, Fdftl, Hsdl7b7, Lss, Nsdhl, Scdl, Scd2, Sqle, Srebfl, Srebf2, Fads2, Msmol, Pmvk, Tm7sf2, Dhcr24, Fdps, Acat2, Hmgcsl, Idil, Insigl, Cyp39al, Abcc3, Stard4, Idhl, Ldlr, Apobr, Cish, Gstml, Gstm2, St3gall, Pcyt2, Elovl6, Acsl5, Rdhll, Slc5a3, Dio2, Slcl6a2, Slco4al, Dbi, Aldoc. The second pathway that was significantly impacted (Log(q-value) of -2.040) was the response to interferon-beta pathway (G0:0035456). Additional pathways include the sterol biosynthetic process, Metabolism of steroids, Cholesterol biosynthesis, secondary alcohol metabolic process, cholesterol biosynthetic process, secondary alcohol biosynthetic process, cholesterol metabolic process, cholesterol metabolic process, sterol metabolic process, steroid biosynthetic process, lipid biosynthetic process, alcohol metabolic process, organic hydroxy compound metabolic process, Metabolism of lipids, organic hydroxy compound biosynthetic process, steroid metabolic process. All are with a significant impact between (Log(q-value) of -21.604 to -7.345). Moreover, there is a list of pathways correlating directly to the cell cycle with a significant impact of Log(q-value) of -4.575 to -2.835, such as the nuclear chromosome segregation genes (Aspm, Ccnb2, Plkl, Aurka, Pttgl, Psrcl, Spc25, Fam83d, Cdc20, Cenpf, Nusapl, Kif22, Cenpe, Kifl4, Ccnf, Cenpa, Ier3, Mucl, Pim2, Kif2Oa, Fbxll5, Clip4, Gas2l3, Ccdc68, Mki67, Ccl2, Pifl, S100a8, Trib2, Arhgap6, Ckb, Dockll, Depdclb, Arhgef39, Cyp51, Ldlr, Serpine2), meiotic spindle assembly(Aspm, Ccnb2, Aurka, Cenpe), spindle assembly involved in female meiosis(Ccnb2, Aurka, Cenpe), cell division (Aspm, Ccnb2, Ccnf, Cenpa, Plkl, Kif2Oa, Aurka, Pttgl, Psrcl, Spc25, Fam83d, Cdc20, Nusapl, Cenpe, Kif 14), The Peroxisome proliferator- activated receptor (PPAR) signaling pathway (Dbi, Scdl, Scd2, Fads2, Mmplb, Hmgcsl, Acsl5), regulation of cell migration (Add2, Slprl, Cxcll, S100a8, Ccl2, Cxcl5, Sele, Selp, Spn, Cxcll6, Itgal) and cellular extravasation (Add2, Ccl2, Sele, Selp, Spn, Itgal, Cdl4, Cxcll, Ldlr, Mmp3, Tlr4, Mmplb, Csf3, S100a8, Egrl, Angpt2, Tmsb4x, Kif2Oa, Serpine2, P2ryl2, Dockll, Kif22, Cenpe, Itga8, Vtcnl, Tigit).

[0155] These findings suggest a connection between novel biochemical pathways and the downregulation of PrP, highlighting the potential of PSDNA as a promising anti-cancer drug. This is particularly significant for somatic cancers that currently lack effective treatments.

[0156] FIG. 4 illustrates transcriptomic analysis of mouse triple negative cancer cell line 4T1. Two independent 4T1 cancer cell lines were exposed for 3h to the active PSDNA or to the inactive PODNA. These samples were sequenced twice (setl and set2) and the number of common upregulated and downregulated genes (A) were uploaded onto the metascape analysis to identify pathways that are significantly represented among these genes (B). Volcano plot analysis identified the most significantly downregulated (blue) and upregulated (red) genes (C).

[0157] The next step was to inject PSDNA directly into the tumor of a mouse and a human mammary fat pad cancer model. Using the independent student 2 tailed distribution with different standard deviation between groups t test, demonstrates that PSDNA effectively reduced or eliminated tumor growth, as shown in FIG. 5. Interestingly the effect of the PSDNA was prominent in the pretreatment in both models as could be seen from the early significant difference in tumor growth (Fig. 5 upper panel A).

[0158] FIG. 5 illustrates how PSDNA treatment reduces or eliminates mice and human triple negative cancer. Mouse 4T1 and human MDA-MB-231 triple negative cancer cell lines were either exposed for 3h to the PSDNA or the PODNA before their inoculation into the mammary fat pad (pre) or directly after the tumor induction at day 10 (post). Western blot analysis revealed a decrease in prion protein levels in the remaining tumors, as shown in FIG. 6.

[0159] FIG. 6 illustrates the Impact of PSDNA treatment on PrP levels in primary tumors and its effect on tumor migration. Furthermore, treatment of the tumors with PSDNA resulted in the inhibition or significant reduction of metastasis, as shown in FIG. 6, panels c and d.

[0160] Further research may provide additional information regarding the mechanism of action of PSDNA on PrP levels, and help evaluate the therapeutic potential of PSDNA as an anti-cancer treatment.

[0161] One specific aim is to explore the in-vivo therapeutic potential of PSDNA in various animal models.

[0162] Triple negative breast cancer (TNBC), fibrosarcoma, pancreatic cancer, and small cell lung carcinoma (SCLC) are four types of aggressive and challenging cancers that currently lack effective curative treatments. Triple-negative breast cancer is characterized by the absence of the estrogen receptor, the progesterone receptor, and the human epidermal growth factor receptor 2 (HER2). TNBC tends to be more aggressive and has limited targeted therapy options compared to other breast cancer subtypes. Fibrosarcoma is a rare type of soft tissue sarcoma that arises from fibrous connective tissue. It often presents as an aggressive and invasive tumor with a high likelihood of metastasis. Pancreatic cancer, notorious for its high mortality rate, is challenging to treat due to its late-stage diagnosis and resistance to many conventional therapies. Limited therapeutic options and the rapid progression of the disease contribute to its poor prognosis. Small cell lung carcinoma, specifically Louis lung carcinoma (LLC), is an aggressive and highly malignant form of lung cancer. It is known for its rapid growth, early metastasis, and poor response to standard treatments, resulting in a significant need for novel therapeutic strategies. Addressing the unique characteristics and underlying mechanisms of these incurable cancers is crucial to develop innovative treatments and improve patient outcome.

[0163] Cell line models have been established for studying various types of cancer, including triple negative breast cancer (TNBC), fibrosarcoma, pancreatic cancer, and small cell lung carcinoma (SCLC). These cell lines provide valuable tools for investigating the underlying biology of these cancers and testing potential therapeutic approaches. Our previous studies have shown that phosphorothioate oligonucleotides (PSDNA) can downregulate the expression of the prion protein (PrP) in multiple cell lines representing three of these cancer types. For TNBC, we utilized the MDA-MB-231 cell line, derived from metastatic breast adenocarcinoma. The 4T1 cell line, derived from a spontaneous mammary tumor in mice, serves as a valuable model for investigating TNBC metastasis. In the case of pancreatic cancer, the KPC cell line, with mutations in Kras and Trp53 genes, provides insights into pancreatic ductal adenocarcinoma (PDAC). The Kras mutation G12D leads to the activation of the Kras oncogene, a key driver of pancreatic cancer initiation and progression. The Trp53 mutation R172H results in the loss of normal Trp53 tumor suppressor function, contributing to the aggressive nature of PDAC. Additionally, the D122 cell line, also known as 3LL-D122 or 3LL, derived from a mouse lung tumor, is widely studied in the context of lung cancer. Our In vivo experiments demonstrated that direct injection of PSDNA into MDA-MB-231 or 4T1 cell-derived tumors in mice significantly reduced or eliminated both the primary tumors and metastasis. To further explore the effect of PSDNA, we will test if our WEHI-164 murine fibrosarcoma cell line express PrP and if PSDNA attenuates its proliferation and lead to cell death. A positive result will lead us to establish an RFP expressing cell line to follow the ability of PSDNA to inhibit cancer metastasis. We will acquire the HT1080 fibrosarcoma cell line derived from a human adult fibrosarcoma tumor, as well as the HT- 1080GFP, a genetically modified HT1080 cell line expressing green fluorescent protein (GFP) for tracking tumor cells. These additional cell lines will enable us to investigate the impact of PSDNA on fibrosarcoma. Thereafter, these cell lines will be injected into the adequate mouse host and the effect of direct injection of PSDNA will be tested.

[0164] Effect of Molecules and Microgravity on Amyloid Formation

[0165] Referring to Fig. 7, Fig. 8A, Fig. 8B, Fig. 8C, Fig. 8D, Fig. 9A, Fig. 9B, and Fig. 9C, neurodegenerative diseases, ranging from Alzheimer's dementia to Parkinson's Disease pose a deep challenge to medical sciences, as well as a burden on those who are afflicted and their families. The societal costs are enormous.

[0166] Protein misfolding and aggregation is a hallmark of these diseases. Various environmental factors such as temperature and pH affect amyloid formation.

[0167] Aggregation and misfolding of proteins are associated with tissue deposition of amyloid fibrils or plaques in neurodegenerative diseases (NDS) like Alzheimer's disease (AD), Parkinson's disease (PD), Prion disease (PrD), Huntington's disease (HD), and Amyotrophic Lateral Sclerosis (ALS). In each disorder, a different fraction of a specific peptide within a particular protein forms various types of aggregates in brain tissue, which have been associated with neurodegeneration. Amyloid aggregates are now considered a hallmark of those diseases and their formation is monitored in patients by in vivo imaging, as a disease reporter. Moreover, in-vitro and in-vivo studies indicate that understanding the conditions that enhance or prevent the formation of such aggregates might be the key approach to identify future therapies.

[0168] Recent evidence suggests that amyloid formation and protein crystallization share phenomenological similarities during molecular assembly processes. Both protein crystals and amyloid fibrils can form in a supersaturated solution via initial nucleation and subsequent growth despite their morphological differences. In the case of protein crystallization, gravity notably influences the assembly process, as it causes sedimentation and convection flow, thereby perturbing microenvironments surrounding the crystal. The present disclosure identifies certain drugs that affect amyloid formation. This includes drugs that are known to penetrate the BBB as well as Nutraceuticals that could be approved quickly if found to be effective.

[0169] Fig. 7, Fig. 8A, fig. 8B, fig. 8C, Fig. 8D, Fig. 9A, Fig. 9B, and Fig. 9C include results from studies performed using various compounds, in which the compounds are identified by number. A key to the numbering appears in Table 2 below.

[0170] Table 2 - Compounds Studied in Connection with Amyloid Formation

[0171] In order to explore the possible future effect of commonly used drugs the Thioflavin T (ThT) amyloidogenic specific fluorescence staining assay was established using a conventional 96 well plate. Data indicates that this assay provides amyloid specific staining. For example, FIG. 8B illustrates that the staining of Thioflavin T (ThT) is amyloid specific. PBS (shown in lower graph (a)) and IgG peptide (shown in lower graph (b)) led to insignificant fluorescence. ThT is excited at 440 and emission is at 485 nm, as shown in FIG. 7.

[0172] FIG. 8B further illustrates the specific effect of pH on amyloid formation. Significant and specific fluorescence of ThT Incubation occurs with two peptides representing the Huntingtin and the Tau protein, respectively. The aggregation of the Q.7 peptide is pH independent (upper graph (a)) while the aggregation of the Tau peptide, as a representative of the previously tested hexameric peptides, is significantly lower at pH 3 and at pH 12 it is significantly higher than physiological pH (upper graph (b)).

[0173] These tests have been performed with respect to amyloid formation in animal models, including in particular mouse cells, among other testing targets.

[0174] Using the amyloid formation assay, it was then possible to study the effect of different compounds on the amyloid formation.

[0175] Neurodegenerative disorders are incurable and most of the patients are diagnosed when it is too late. Therefore, prevention of the formation of amyloid prior to onset of clinical signs can be a key to treatment. The FDA approved drugs listed in table 2 which are orally taken for other neuronal related conditions such as depression, headache, anxiety and epilepsy, are excellent candidates for repurposing. Nutraceuticals could be an additional avenue since these drugs could be sold without prescription.

[0176] When a drug is identified as an anti-amyloidogenic drug in the ThT amyloidogenic specific fluorescence staining assay, the active compound can be tested in an in vitro model, and in parallel the effect of this drug can be tested directly in an animal model. Synthetic polyQ.10 peptide was chosen for an initial study since the HD model is well established and the model is tested over a short time (12 weeks compared to several months to years in AD or Prion models). All assays were conducted in physiological pH since amyloidogenic peptides form amyloid in these conditions. The results indicate that the FDA approved drug C20 inhibits the formation of amyloid and even could potentially reverse existing amyloid aggregates. Moreover, two Nutraceuticals C9 and C24 had similar effect. It was demonstrated that the formation of amyloid in the gut might influence the formation of these aggregates in the brain.

[0177] This methodology was used to identify various drugs that reverse existing amyloid plaques as well as inhibiting the formation of new amyloid plaques. These drugs include a fungal extract, OMEGA 3, Dorminol, and Valerian root extract, curcumin, Lamodex, Clonozapame, Frisium, Rispond, Statin, Tardiferol, Vimpat, Colonzapam, Bisoprolol fumarate inovamed, Olanzapine, Tadam, Sinemet, valproate, sulpride, clomipramine, Topiramate. We are testing Ritalin and Resital. These compounds are promising candidates for both prophylaxis and treatment of disorders characterized by formation of amyloid plaques.

[0178] As shown in FIG. 8D, compound 3 and compound 9 are two prescribed and over the counter drugs, respectively that clearly inhibit the formation of amyloid aggregates of the poly Q.10 (a) and the prion peptide (b). The lower graph illustrates HTT and ThT, while the upper graph illustrates Prion hexamer and ThT.

[0179] In addition, two drugs that are prescribed and consumed for long periods of time were demonstrated to induce the formation of amyloid aggregates, a combination of collagen and hyaluronic acid, and an over-the-counter compound Grana Gard (there is also preliminary data with Lorivan). In the data, two FDA approved drugs C31 and C27 significantly enhanced amyloid formation and the Nutraceutical C29 had the same effect. It is important to further investigate the effect of these drugs since they are taken for long periods of time by patients and could be the source for amyloid formation in those patients. These compounds can be used to accelerate amyloid plaque formation in all existing models and shorten the screening time in the field of amyloid formation. The effect of some of these drugs was enhanced under microgravity conditions.

[0180] The amyloid-enhancing products may be used as accelerators for detection assays and models for future high throughput screening. Such screening with respect to amyloid formation may be useful for providing the first "safety" stamp for over-the-counter drugs and compounds FDA-approved for chronic use to assure patients that specific drugs are not harmful in the long term (at least with regard to amyloid plaque formation).

[0181] The assay described above is potentially important as part of a premarket approval process, in order to ensure that toxic compounds are not approved. Another possible use is to use the differential effect of compounds on one type of amyloid compared to the other as a possible early diagnostic tool between neurodegenerative diseases. In addition, the methods described herein may be used for early diagnosis of amyloid formation and neurodegenerative diseases through the specific effect of compounds.

[0182] Microgravity substantially improves the growth of protein crystals. This is because, in the absence of buoyancy-induced convection, the movement of protein molecules in microgravity is driven only by random diffusion and is therefore much slower than on Earth. Furthermore, when gravity as a masking factor is eliminated, other interactions can become prevalent. Consequently, other crystalline structures (polymorphs) may arise, even though they are very rare on Earth. It might even be possible to crystallize materials which were not successfully crystallized at normal terrestrial g force. For example, some of the proteins involved in neurodegenerative diseases crystallize on Earth but not with enough quality and uniformity to determine their structures.

[0183] Studies with hexameric peptides derived from specific proteins known to form amyloid fibrils associated with specific diseases have demonstrated identical characteristics in-vitro. Moreover, their aggregation into self-assembled amyloid structures and the kinetics of this process has been demonstrated to be dependent on various environmental factors such as temperature, pH, and salt concentration. However, very little is known regarding the impact of gravity on these aggregates.

[0184] To simulate the effect of zero gravity in space, identical 96 well plates were mounted onto Random Positioning Machines (RPMs) as a ground-based model to simulate microgravity. This model is well-established and it is used to predict the influence of zero gravity. The Random Positioning Machine (RPMs) were set up at room temperature to conditions that RPM's rotation was faster than the biological process of amyloid formation, but not so fast that undesired side effects appear. Therefore, the inner frame was set to 25 RPM and the outer frame was set to 40RPM. Control plates were positioned at the same temperature on a horizontal shaker. The initial experiment focused on the polyQlO peptide which forms amyloidogenic compounds and screened selected drugs which are chronically used such as Nutraceuticals or FDA approved drugs. Amyloid formation, within the 96 well plate under gravity conditions, was continuously measured and was set up for the identical "shaking" conditions as the 96 well plate that was mounted on the RPM. Experiments comparing the end point to the initial levels of amyloid, both plates were set up simultaneously, measured in the Cytoscan 5 plate reader and the identical incubation time was given to both plates under gravity and microgravity conditions. One first environmental parameter to be tested is the effect of pH on amyloid formation, in gravity and microgravity conditions.

[0185] Fig. 9A, Fig. 9B and Fig. 9C demonstrate the effect of microgravity on amyloid formation in the presence and absence of potential anti-amyloidogenic drugs.

[0186] In addition to the potential therapeutic impact of these findings, they are significant since when traveling to space it might be important to utilize this screening method to test the potential harmful effect of the combinatorial impact of microgravity and certain drugs. On the other hand, the disruptive microgravity environment allows for additional accelerated biophysical screening. The opportunity to study drugs and cells in microgravity permits exploration into chemical and biological properties that might not be present under usual conditions due to the masking effects of gravity forces. Such explorations under extreme conditions are an elegant tool for drug discovery of new therapeutic treatments for neurodegenerative diseases.

[0187] This application suggests, for the first time, testing compounds for these parameters as a condition for regulatory approval (e.g. by the FDA).

[0188] Inhibition of amyloid plaque formation

[0189] Valerian exhibits a potent inhibitory effect on polyglutamine (polyQ) amyloid structures immediately upon incubation, as shown in Fig. 10(panel a) and Fig. 11 (panel a). This effect intensifies over time, eventually leading to a near-complete reduction of polyQ. amyloids (Fig. 10 panel a). A similar pattern is observed with tau amyloidogenic aggregates: valerian's effect is immediate, reducing the presence of tau aggregates significantly (Fig. 13 panel a) and preventing the formation of new amyloids (Fig. 12 panel a and Fig. 13 panel a). Over time, the tau aggregates diminish completely with extended incubation (Fig. 13 panel a). Valerian's inhibitory effect also extends to alpha-synuclein aggregates (Fig. 14 panel a) and amyloid beta (A ) peptides, both in DDW (Fig. 14 panel c) and DMSO (Fig 14 panel d). In addition to inhibiting the formation of new alpha-synuclein aggregates, valerian disrupts existing ones. The effect of valerian on polyQ and AP amyloids was confirmed through AFM analysis comparing untreated samples in DDW (Fig. 15 panel a) to those treated with valerian (Fig. 16 panel c).

[0190] Omega-3 also shows a strong, immediate effect on polyQ. amyloids (Fig. 10 panel b and Fig 11 panel b), though its efficacy declines after 66 to 166 hours, potentially due to the compound's instability. However, the long-term inhibition of both existing and newly formed A amyloid aggregates (Fig 12 panel b and Fig 13 panel b) suggests that omega-3 may interact with tau peptide in a way that stabilizes its inhibitory effects. This theory is further supported by the immediate and sustained action of omega-3 on alpha-synuclein aggregates (Fig. 14 panel b) and AP amyloids (Fig. 14 panel e and Fig. 14 panel f), maintaining its potency even after 90 hours of incubation. AFM analysis also confirmed omega-3's inhibitory effect on polyQ. aggregates (Fig. 15 panel a vs. Fig. 15 panel b) and AP amyloids (Fig. 15 panel b vs. Fig. 15 panel d).

[0191] Interestingly, clonazepam (Clonex) exhibited a specific inhibitory effect on tau amyloid aggregates, particularly after extended incubation times, suggesting a potential for selective amyloid inhibition.

[0192] The inhibitory effects of valerian, omega-3, and clonazepam on newly formed amyloidogenic structures were also observed with lysozyme amyloid aggregates, as demonstrated by the ThT assay (Fig. 17). Notably, the combination of hyaluronic acid and collagen inhibited lysozyme aggregation, highlighting the specificity of this compound mix. This is particularly interesting, as in previous experiments, the same combination was shown to enhance polyQ amyloid formation, suggesting distinct interactions depending on the amyloid type.

[0193] AFM Results

[0194] AFM analysis presented in Fig. 15 demonstrates the anti-amyloid effect of omega 3 and valerian in polyglutamine and A beta amyloid structures. Fig. 15 panel A and Fig. 15 panel B represent existing polyQ peptide amyloidogenic constructs in DDW (blue line) which are dissolved in the presence of valerian and Omega 3 respectively (yellow line). Figure 15 panel C and Fig. 15 panel D represent a beta existing amyloidogenic peptide construct diluted in DDW or DMSO respectively, and dissolved with Valerian. Fig. 15 panel E and Fig. 15 panel F represent a beta existing amyloidogenic peptide construct diluted in DDW or DMSO respectively, and incubated with omega3. Panel B of Fig. 15 shows that the polyglutamine peptide with omega-3 formed big amyloid structures which were less dense than the polyglutamine peptide in DDW alone.

[0195] Panel C of Fig. 15 shows polyglutamine peptide in the presence of valerian did not form amyloid. These protrusions are not amyloids as seen in DDW and omega-3 samples.

[0196] Panel D of Fig. 15 shows the amyloid beta with DDW formed large amyloids and were different from poly Q amyloids.

[0197] Panel E of Fig. 15 shows the amyloid beta with omega-3 formed amyloids in low density which compared to DDW.

[0198] Panel F of Fig. 15 shows the amyloid beta with valerian did not form amyloid.

[0199] Panel G of Fig. 15 is a higher magnification of the amyloids in panel A.

[0200] Panel H of Fig. 15 is a higher magnification of Panel B showing the difference between the structures of the amyloids with omaga-3 and DDW.

[0201] Panel I of Fig. 15 is a higher magnification of Panel E which shows the amyloids are not significant.

[0202] Panel J of Fig. 15 is an example of the sizes of the image in Panel G.

[0203] Panel K of Fig. 15 is a surface control without peptides and substances.

[0204] Fig. 16 illustrates the autofluorescence of the tested compounds and their ability to form amyloid. (A) Valerian=Val, (B) Omega 3=03, (C) Hyaluronic acid=Hia+ Collagen=Col, and (D) Clonozapam=Clo. The orange line represents the substance in the presence of ThT and the blue line represents the control sample with water and ThT alone (DDW). The green line represents only the substance.

[0205] Fig. 16 Panel A, Fig. 16 Panel B and Fig. 16 panel C show that the fluorescence intensity of valerian, omega-3, hyaluronic acid, and collagen is higher than that of water, indicating that these substances exhibit autofluorescence. In contrast, Fig. 16 panel D shows the Clonex graph either overlapping with or slightly above the water graph, suggesting that Clonex does not exhibit significant autofluorescence. None of these substances were observed to form amyloids to a significant degree.

[0206] Fig. 17 illustrates Lysozyme Amyloidogenic aggregates specifically dissolve following incubation with Valerian and Omega 3. Lysozyme incubation with: (A) Valerian=Val, (B) Omega 3=03, (C) Hyaluronic acid=Hia+ Collagen=Col, and (D) Clonozapam=Clo. The yellow lines represent the peptide in the presence of the tested drug and the blue line represents the control peptide incubated with water alone (DDW). Lower Error Bar (LB) and Upper Error Bars (UB) are included for measurement.

[0207] Fig. 18 demonstrates that PSDNA inhibits cell proliferation and that downregulation of PrP in 4T1 cells leasing to cell death using siRNA. Fig. 19 shows that 4T1 cells form aggregates when exposed to PSDNA. This phenomenon was observed in three cancer cell lines (4T1, LRK1O, and LRK14) using two different 22-mer PSDNA sequences (Fig. 19 PSI and PS2). Interestingly, this effect is apparently not due solely to the negative charge of the PSDNA, as the 22-mer PO- DNA, which has an identical sequence to PS2 and was used as a negative control (Fig. 19 PO), did not induce cell aggregation or PrP downregulation, even after extended incubation times.

[0208] Additionally, this effect appears to be cell-specific, as no aggregation was observed in HEK cells (See Fig. 24). Data presented herein indicates that PARP is involved in the PSDNA- mediated cell death mechanism.

[0209] Fig. 19illustrates that mouse and human cancer cell growth is inhibited by cellular aggregation following specific exposure to PSDNA, and this effect is sequence-independent.

[0210] In addition, the PrP downregulation is specific and time-dependent as illustrated by Fig. 20.

[0211] The downregulation of PrP following PSDNA exposure (Indicated as PS in Fig. 20) suggests that PrP plays a role in maintaining cellular integrity or proliferation in cancer cells. More specifically, the results suggest that reduction of PrP contributes to cell aggregation or other cytotoxic effects. Since PrP is involved in cell signaling and cell adhesion, its loss could also drive cell detachment and aggregation, leading to inhibited growth.

[0212] An MTT assay was performed to assess the effect of PSDNA on cellular viability. Various concentrations of PSDNA were in vitro to determine the IC50 of the PSDNA. Cells were incubated for 72 hours in the case of slower-growing cells (HEK) orthose seeded at low confluency (LRK10), and for 24 hours for faster-growing cells or those seeded at high confluency (4T1, B901K).

[0213] The results indicate that PSDNA specifically inhibits or induces cancer cell death in a targeted manner. This effect is observed in the mouse mammary cancer cell line 4T1, as well as in other cancer cell types, with an IC50 of approximately 0.01 micromolar for most cell lines (Fig. 21 Panels A (4T1); B (B901L); C (HEK); and D (10LRK)). This effect is not strain-specific, as it occurs in both human and mouse cell lines. These results are consistent with previous findings. Fig. 42 illustrates that PSDNA inhibits cancer cell growth in mice and humans.

[0214] In a subsequent experiment 4T1 breast cancer mouse cell lines were exposed to PSDNA for 3 hours, 2 days, and 5 days. The STING pathway is activated, as shown by increased STING phosphorylation (Fig. 22A and Fig. 22B). In addition PARP cleavage was detected (Fig. 22 A and Fig. 22B), indicating its inactivation, while Mondo A levels decreased over time to its expected size of 95 kDa. However, there was an accumulation of cleaved Mondo A (Fig. 22C), a phenomenon that has not been previously described in the literature.

[0215] Interestingly, PARP cleavage mediated by PSDNA is cell-specific, as no PARP cleavage was observed in HEK cells following PSDNA exposure (Fig. 22D). To ensure equal loading of total protein across control and experimental samples, we used vinculin staining (Fig. 22A), total protein on the Bio-Rad Mini-Protean TGX stain-free gel (Fig. 22B and 22D), or Ponceau S staining (Fig. 22C). Furthermore, there was a reduction in total STING expression after prolonged PSDNA exposure, making the increase in pSTING even more significant (Fig. 22A).

[0216] These findings suggest a multi-faceted response in the 4T1 breast cancer cell line when exposed to PSDNA, involving both immune signaling and metabolic regulatory pathways. STING phosphorylation indicates activation of the cGAS-STING pathway, a key innate immune response to cytosolic DNA. This pathway typically responds to double-stranded DNA (dsDNA) from pathogens or cellular damage, leading to interferon production and inflammatory signaling. The decrease in total STING over time after PSDNA exposure, alongside increased phosphorylated STING (pSTING), suggests a tightly regulated response where the activated form of STING is upregulated, but overall STING protein levels are reduced as part of a negative feedback loop to avoid excessive immune activation. This may signal the cell's attempt to modulate immune signaling to prevent chronic inflammation or apoptosis. PARP cleavage is a hallmark of apoptosis. Cleaved PARP indicates its inactivation, which prevents the repair of damaged DNA, thus pushing the cell toward programmed cell death. The absence of PARP cleavage in HEK cells upon PSDNA exposure shows that the apoptosis induction by PSDNA is cell-specific, potentially due to differences in the DNA damage response, immune signaling, or cell cycle regulation between cancer and non-cancer cells. PARP inactivation is particularly important because it suggests that PSDNA induces not just immune activation, but also cell death directly via apoptosis in 4T1 cells.

[0217] Mondo A is a transcription factor involved in glucose and metabolic regulation, particularly in response to intracellular glucose levels. The accumulation of cleaved Mondo A is an intriguing finding, as this has not been well documented. The cleavage of Mondo A may represent a novel regulatory mechanism linked to metabolic stress or DNA damage responses in cancer cells. PSDNA might be causing Mondo A cleavage either as part of a stress response or as a consequence of disrupted cellular metabolism due to immune signaling activation (e.g., STING) or apoptosis (e.g., PARP cleavage). Further exploration of the function of cleaved Mondo A could uncover new insights into cancer cell metabolism and survival pathways.

[0218] The stronger response in syngeneic mice compared to PDX models suggests that the immune system plays a key role in the anti-cancer effect of PSDNA. Syngeneic models maintain the host immune system, while PDX models (human tumors in immunodeficient mice) lack a fully functional immune response. This highlights the importance of immune-mediated mechanisms— possibly through the activation of the STING pathway— in the anti-tumor effects observed in your in vivo studies.

[0219] In conclusion, the exposure of 4T1 cells to PSDNA triggers the activation of the cGAS- STING pathway (innate immune activation), apoptosis via PARP cleavage, and possibly a novel metabolic response via Mondo A cleavage. The absence of PARP cleavage in HEK cells underscores the specificity of this response to cancer cells. These findings suggest a dual mechanism of immune activation and metabolic disruption contributing to cell death and may open up new therapeutic insights, especially about Mondo A's role in cancer metabolism.

[0220] Fig. 22A, Fig. 22 B, Fig. 22C, and Fig. 22D are western blot analyses of 4T1 cells incubated with PSDNA suggesting that PSDNA induces cell death through the activation of multiple pathways.

[0221] Fig. 23 Illustrates that PSDNA treatment has a synergistic effect with Osimertinibe in KRAS mutant B901L Human lung cancer cells.

[0222] Fig. 24 illustrates that treatment of cells with ImM PSDNA forms specific cancer cell aggregates. Mouse mammary breast cancer cell lines 4T1 and mouse lung carcinoma cell lines B901L form cell aggregates following exposure to PSDNA for 24 hours. Human embryonic kidney cells serve as a non-cancer control cell line and were not affected by the presence of PSDNA in the media.

[0223] Exemplary considerations regarding the blood brain barrier

[0224] When contemplating treatment of brain cells, the ability of compounds to cross the blood-brain barrier (BBB) can be an important consideration. Clonazepam and DHA (from omega-3 fatty acids) are known to penetrate the BBB efficiently.

[0225] Clonazepam, as a benzodiazepine, already plays a role in neurological treatments and could be considered for managing symptoms like seizures or neuroinflammation.

[0226] DHA (docosahexaenoic acid; a component of omega-3 fatty acids), an essential component of brain phospholipids, has shown neuroprotective effects and might be explored as part of a long-term therapy to slow neurodegenerative progression.

[0227] For valerian root, active compounds such as valerian may offer anxiolytic and neuroprotective properties, and their ability to cross the BBB could support managing symptoms like anxiety and sleep disturbances associated with neurodegenerative diseases. Combination of Valerian with other compounds has the potential to enhance its therapeutic potential.

[0228] The delivery of compounds that cross the BBB would likely be most effective via oral administration or intranasal routes. In cases where systemic delivery is insufficient, advanced drug delivery systems such as liposomal formulations or nanocarriers are available to enhance BBB penetration This may be especially relevant for compounds hyaluronic acid and / or collagen, which have limited BBB permeability in their native forms.

[0229] Some forms of low-molecular-weight hyaluronic acid have been investigated for delivery to the brain.

[0230] The ThT assay

[0231] The relative amount of amyloid present in each solution is measured by combining solutions of the peptides (100 micrograms in 25 microliter) with 45microloter of 100 mM MES (pH 7.4) and 5 microliters of compound at the appropriate concentration incubated at 37°C in a black 96-well microtiter plate. ThT (5 microliters of 10 mM solution) is added, and the resultant fluorescence at 485 nm from excitation at 440 nm is measured with a Cytation 5 fluorescence microtiter plate reader.

[0232] Exemplary dosage information

[0233] The final concentration of Ritalin in solution is 0.25 mg / ml after dissolving a 40 mg pill in 10 ml, taking 5 microliters of this solution, and diluting it in a total volume of 80 microliters.

[0234] Ritalin (methylphenidate) is typically used in a range of 1 pM to 100 pM in various cell studies involving neuroactive drugs

[0235] Common Doses of Ritalin in Mice Experiments:

[0236] 1. Oral administration (via drinking water or food): o Typical doses range from 5 mg / kg to 50 mg / kg.

[0237] 2. Intraperitoneal (IP) injection: o Doses often range from 0.5 mg / kg to 10 mg / kg.

[0238] 3. Intravenous (IV) administration: o Lower doses, such as 0.1 mg / kg to 5 mg / kg, are generally used.

[0239] Final estimated concentration in the dorminol active ingredients in the ThT exp: Final Concentrations in the 80 pL Reaction:

[0240] • Valerian Root Extract: 1.875 mg / mL

[0241] • Hops Extract: 0.9375 mg / mL

[0242] • Passionflower Extract: 0.625 mg / mL

[0243] • Melatonin: 0.0125 mg / mL

[0244] Typical Concentrations for In Vitro Use:

[0245] 1. Valerian Root Extract: o Typical Range: 10-500 pg / mL o Purpose: Investigating anti-proliferative, anti-inflammatory, or sedative effects.

[0246] 2. Hops Extract: o Typical Range: 10-200 pg / mL o Purpose: Testing for antioxidant, anti-cancer, or anti-inflammatory properties.

[0247] 3. Passionflower Extract: o Typical Range: 10-200 pg / mL o Purpose: Known for anxiolytic and sedative properties, it's usually tested for anti-anxiety or neuroprotective effects.

[0248] 4. Melatonin: o Typical Range: 0.1-100 pM (which corresponds to 0.023-23 pg / mL) o Purpose: Melatonin is commonly tested for its role in circadian rhythm, antioxidant properties, and protective effects on neuronal cells.

[0249] It is expected that during the life of this patent many new cancer varieties and new neurodegenerative disorders will be characterized and the scope of the invention is intended to include all such new technologies a priori.

[0250] As used herein the term "about" refers to ± 10 %. Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

[0251] Specifically, a variety of numerical indicators have been utilized. It should be understood that these numerical indicators could vary even further based upon a variety of engineering principles, materials, intended use and designs incorporated into the various embodiments of the invention. Additionally, components and / or actions ascribed to exemplary embodiments of the invention and depicted as a single unit may be divided into subunits. Conversely, components and / or actions ascribed to exemplary embodiments of the invention and depicted as sub-units / individual actions may be combined into a single unit / action with the described / depicted function.

[0252] Alternatively, or additionally, features used to describe a method can be used to characterize a pharmaceutical composition or composition for use and features used to describe a pharmaceutical composition or composition for use can be used to characterize a method.

[0253] It should be further understood that the individual features described hereinabove can be combined in all possible combinations and sub-combinations to produce additional embodiments of the invention. The examples given above are exemplary in nature and are not intended to limit the scope of the invention which is defined solely by the following claims.

[0254] Each recitation of an embodiment of the invention that includes a specific feature, part, component, module or process is an explicit statement that additional embodiments of the invention not including the recited feature, part, component, module or process exist.

[0255] Alternatively or additionally, various exemplary embodiments of the invention exclude any specific feature, part, component, module, process or element which is not specifically disclosed herein.

[0256] Specifically, the invention has been described in the context of cancer and neurodegenerative disorders but might also be used in the context of other diseases.

[0257] All publications, references, patents and patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention.

[0258] The terms "include", and "have" and their conjugates as used herein mean "including but not necessarily limited to".

Claims

CLAIMS:

1. A method of cancer treatment, comprising: delivering a physiologically effective amount of phosphorothioate DNA (PSDNA) to cancer cells.

2. The method of claim 1, wherein said physiologically effective amount is at least 1 pM.

3. The method of claim 1, wherein said physiologically effective amount does not exceed 10 pM.

4. The method of claim 1, wherein said PSDNA comprises a mixture of random oligonucleotides.

5. The method of claim 1, wherein said delivering comprises systemic delivery to a patient.

6. The method of claim 1, wherein said delivering comprises direct delivery to a tumor.

7. A method of cancer treatment, comprising: delivering a physiologically effective amount of phosphodiester DNA (PODNA) to cancer cells.

8. The method of claim 7, wherein said physiologically effective amount is at least 10 pM.

9. The method of claim 7, wherein said physiologically effective amount does not exceed 100 pM.

10. The method of claim 7, wherein said PODNA comprises a mixture of random oligonucleotides.

11. The method of claim 7, wherein said delivering comprises systemic delivery to a patient.

12. The method of claim 7, wherein said delivering comprises direct delivery to a tumor.

13. A physiologically effective amount of phosphorothioate DNA (PSDNA) for use in the treatment of cancer.

14. The compound for use of claim 13, wherein said physiologically effective amount is at least lpM.

15. The compound for use of claim 13, wherein said physiologically effective amount does not exceed 10 pM.

16. The compound for use of claim 13, wherein said PSDNA comprises a mixture of random oligonucleotides.

17. The compound for use of claim 13, formulated for systemic delivery to a patient.

18. The compound for use of claim 13, formulated for direct delivery to a tumor.

19. A physiologically effective amount of phosphodiester DNA (PODNA) for use in the treatment of cancer.

20. The compound for use of claim 19, wherein said physiologically effective amount is at least 10 pM.

21. The compound for use of claim 19, wherein said physiologically effective amount does not exceed 100 pM.

22. The compound for use of claim 19, wherein said PODNA comprises a mixture of random oligonucleotides.

23. The compound for use of claim 19, formulated for systemic delivery to a patient.

24. The compound for use of claim 19, formulated for direct delivery to a tumor.

25. A pharmaceutical composition comprising: a physiologically effective amount of phosphorothioate DNA (PSDNA) as an active ingredient; and diluents and / or excipients; wherein the physiologic effect is retardation or reversal to tumor growth.

26. The pharmaceutical composition of claim 25, wherein said physiologically effective amount for a single oral dosage form is at least 5 mg.

27. The pharmaceutical composition of claim 25, wherein said physiologically effective amount for a single oral dosage form does not exceed 50 mg.

28. The pharmaceutical composition of claim 25, wherein said physiologically effective amount for a single systemic injectable dosage form is at least 1 mg.

29. The pharmaceutical composition of claim 25, wherein said physiologically effective amount for a single systemic injectable dosage form does not exceed 10 mg.

30. The pharmaceutical composition of claim 25, wherein said physiologically effective amount for a single locally injectable dosage form is at least 0.5 mg.

31. The pharmaceutical composition of claim 25, wherein said physiologically effective amount for a single locally injectable dosage form does not exceed 5 mg.

32. The pharmaceutical composition of claim 25, wherein said PSDNA comprises a mixture of random oligonucleotides.

33. A pharmaceutical composition comprising: a physiologically effective amount of phosphodiester DNA (PODNA) as an active ingredient; and diluents and / or excipients;wherein the physiologic effect is retardation or reversal to tumor growth.

34. The pharmaceutical composition of claim 33, wherein said physiologically effective amount for a single oral dosage form is at least 10 mg.

35. The pharmaceutical composition of claim 33, wherein said physiologically effective amount for a single oral dosage form does not exceed 100 mg36. The pharmaceutical composition of claim 33, wherein said physiologically effective amount for a single systemic injectable dosage form is at least 5 mg.

37. The pharmaceutical composition of claim 33, wherein said physiologically effective amount for a single systemic injectable dosage form does not exceed 50 mg.

38. The pharmaceutical composition of claim 33, wherein said physiologically effective amount for a single locally injectable dosage form is at least 2 mg.

39. The pharmaceutical composition of claim 33, wherein said physiologically effective amount for a single locally injectable dosage form does not exceed 20 mg.

40. The pharmaceutical composition of claim 33, wherein said PODNA comprises a mixture of random oligonucleotides.

41. A method of preventing or reversing amyloid formation, comprising: delivering a physiologically effective concentration of at least one member of the group of active ingredients consisting of OMEGA 3, Dorminol, and Valerian root extract, curcumin, Lamodex, Clonazepam, Frisium, Rispond, Statin, Tardiferol, Vimpat, Colonzapam, Bisoprolol fumarate inovamed, Olanzapine, Tadam, Sinemet, valproate, sulpride, clomipramine, Topiramate, and a combination of collagen and hyaluronic acid to cells producing, or in danger of producing, amyloid plaques.

42. The method of claim 41, performed under microgravity conditions.

43. The method of claim 41, wherein said active ingredient comprises a combination of collagen and hyaluronic acid.

44. The method of claim 41, wherein said active ingredient comprises Clonazepam.

45. The method of claim 41, wherein said active ingredient comprises Valerian root extract.

46. The method of claim 41, wherein said active ingredient comprises OMEGA 3.

47. The method of claim 41, wherein said cells are brain cells.

48. The method of claim 41, wherein said cells are cancer cells.

49. At least one member of the group of active ingredients consisting of OMEGA 3, Dorminol, and Valerian root extract, curcumin, Lamodex, Clonazepam, Frisium, Rispond, Statin, Tardiferol, Vimpat, Colonzapam, Bisoprolol fumarate inovamed, Olanzapine, Tadam, Sinemet, valproate, sulpride, clomipramine, Topiramate, and a combination of collagen and hyaluronic acid for use in a composition for preventing, retarding or reversing formation of amyloid plaques in or on cells.

50. The composition for use of claim 49, deployed under microgravity conditions.

51. The composition for use of claim 49, wherein said active ingredient comprises a combination of collagen and hyaluronic acid.

52. The composition for use of claim 49, wherein said active ingredient comprises Clonazepam.

53. The composition for use of claim 49, wherein said active ingredient comprises Valerian root extract.

54. The composition for use of claim 49, wherein said active ingredient comprises OMEGA 3.

55. The composition for use of claim 49, wherein said cells are brain cells or nerve cells.

56. The composition for use of claim 49, wherein said cells are cancer cells.

57. A pharmaceutical composition comprising: at least one member of the group of active ingredients consisting of OMEGA 3, Dorminol, and Valerian root extract, curcumin, Lamodex, Clonazepam, Frisium, Rispond, Statin, Tardiferol, Vimpat, Colonzapam, Bisoprolol fumarate inovamed, Olanzapine, Tadam, Sinemet, valproate, sulpride, clomipramine, Topiramate, and a combination of collagen and hyaluronic acid; and diluents and / or excipients; wherein said active ingredient is present in an amount sufficient to prevent, retard, or reverse formation of amyloid plaques in or on cells.

58. The pharmaceutical composition of claim 57, wherein said active ingredient comprises a combination of collagen and hyaluronic acid.

59. The pharmaceutical composition of claim 57, wherein said active ingredient comprises Clonazepam.

60. The pharmaceutical composition of claim 57, wherein said active ingredient comprises Valerian root extract.

61. The pharmaceutical composition of claim 57, wherein said active ingredient comprises OMEGA 3.

62. The pharmaceutical composition of claim 57, formulated for activity on brain cells or nerve cells.

63. The pharmaceutical composition of claim 57, formulated for activity on cancer cells.

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