None.
The present invention relates in general to the field of prevention and/or treatment of Alzheimer's disease and related disorders, and more particularly, to the use of BCAA-lowering compounds for prevention and/or treatment of Alzheimer's disease and related disorders.
None.
Without limiting the scope of the invention, its background is described in connection with Alzheimer's disease.
Alzheimer's disease (AD) is an irreversible neurodegenerative disorder ranking as the sixth leading cause of mortality in the US although more recent estimates suggest that the number is underestimated and it actually comes at third just after heart disease and cancer21,22. Unfortunately, no cure or effective treatments exist currently that slow down the progression of AD. Without a specific biomarker that can predict the early onset of AD today we are mainly relying on several diagnostic tools to detect AD. NMR techniques such as PET imaging has been widely used to detect amyloid beta (AB) aggregates tau deposits in the brain (CNS) for AD diagnosis24,25. However, injection of radioactive tracers, the need of trained physicians/technicians, uncomfortable procedure, and expensive cost remain as important limitations. Several diagnostic biomarkers can be obtained from the cerebrospinal fluid (CSF) that are both highly sensitive and specific25,28, however the procedure involves an invasive lumbar puncture hat can cause nausea and severe backache in older patients. Clearly, ideal biomarkers would have not only high validation of diagnosis/prediction, but also only require a minimally invasive and risky technique such as those that can be obtained from blood. Several plasma/serum inflammatory cytokines29-32 Aβ peptides28,33, and mi-RNAs34-36 have shown promising results for detection of AD, although inconsistent findings and the lack of reproducibility far limit the applicability of these potential biomarkers. Collectively, identification of reliable circulating biomarkers that are minimally invasive and patient-friendly remain as a great interest and priority in the field.
Alzheimer's disease (AD) affects over 5 million individuals in the US without any cure or effective treatments. Further, there is no reliable biomarker today that can help predict individual risk for AD, thus raising the national urgency to bridge the major gap in strategies to prevent and/or treat AD.
Thus, what is needed are novel methods for preventing and treating AD.
In one embodiment, the present invention includes a method of treating CNS-related conditions, comprising: administering an effective amount of a compound that lowers the levels of one or more branched-chain amino acids (BCAAs) or metabolites thereof, or any pharmaceutically acceptable salt thereof, wherein the CNS-related conditions is selected from the group consisting of Alzheimer's disease and dementia selected from the group consisting of vascular dementia, Lewy body dementia, frontotemporal dementia, Creutzfeldt-Jacob disease, Wernicke-Korsakoff disease, and Huntington's disease, Multiple sclerosis, Parkinson's disease, autism, Amyotrophic lateral sclerosis (ALS), Hereditary diffuse leukoencephalopathy with spheroids (HDLS) and epilepsy, neuropsychiatric disorders such as all anxiety disorders including, but not limited to, generalized anxiety disorder, social anxiety disorder, specific phobias and separation anxiety disorder, and a collection of depression disorders including, but not limited to, clinical depression (major depression), bipolar depression, persistent depressive disorder (dysthymia), seasonal affective disorder, atypical depression, treatment-resistant depression, psychotic depression, postpartum depression, premenstrual dysphoric disorder, and situational depression (stress response syndrome). In one aspect, the compound is a (S)-α-chloro-phenylpropionic acid and is administered in an amount within a range of from 10-200 mg/kg and is provided daily. In another aspect, the compound is a BT2 compound and is provided at administered in an amount within a range of from 10-100 mg/kg/day and is provided daily. In another aspect, the BT2 compounds is selected from at least one of: BT2 (3,6-dichlorobenzo[b]thiophene-2-carboxylic acid); BT2F (3-chloro-6-fluorobenzo[b]thiophene-2-carboxylic acid); or BT3 (N-(4-acetamido-1,2,5-oxadiazol-3-yl)-3,6-dichlorobenzo[b]thiophene-2-carboxamide). In another aspect, the compound is a benzothiopene-2-carboxylic acid (BT2) compound and is provided at administered in an amount within a range of from 10-100 mg/kg/day and is provided daily. In another aspect, the compound is administered using a method selected from the group consisting of oral, intravenous, subcutaneous, intranasal, pulmonary, intracranial, intracerebroventricular, topical, enteral, parenteral, or rectal. In another aspect, the compound is administered in a formulation comprising a carrier, the carrier being selected from the group consisting of: lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starches, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl benzoate, propyl benzoate, talc, magnesium stearate, and mineral oil. In another aspect, the CNS-related condition treated by the compound is Alzheimer's Disease. In another aspect, the method further comprises obtaining a biological sample from the patient with the CNS-related condition and determining if the biological sample has an increase in BCAAs or metabolites thereof when compared to a sample from a subject without the CNS-related condition.
In another embodiment, the present invention includes a method of identifying and treating CNS-related conditions, comprising: obtaining a biological sample from the patient with the CNS-related condition and determining if the biological sample has an increase in one or more branched-chain amino acids (BCAAs) or metabolites thereof, when compared to a sample from a subject without the CNS-related condition; and administering an effective amount of a compound that lowers the levels of one or more branched-chain amino acids (BCAAs) or metabolites thereof, or any pharmaceutically acceptable salt thereof, wherein the CNS-related conditions is selected from the group consisting of Alzheimer's disease and dementia selected from the group consisting of vascular dementia, Lewy body dementia, frontotemporal dementia, Creutzfeldt-Jacob disease, Wernicke-Korsakoff disease, and Huntington's disease, Multiple sclerosis, Parkinson's disease, autism, Amyotrophic lateral sclerosis (ALS), Hereditary diffuse leukoencephalopathy with spheroids (HDLS) and epilepsy, neuropsychiatric disorders such as all anxiety disorders including, but not limited to, generalized anxiety disorder, social anxiety disorder, specific phobias and separation anxiety disorder, and a collection of depression disorders including, but not limited to, clinical depression (major depression), bipolar depression, persistent depressive disorder (dysthymia), seasonal affective disorder, atypical depression, treatment-resistant depression, psychotic depression, postpartum depression, premenstrual dysphoric disorder, and situational depression (stress response syndrome). In one aspect, the compound is a (S)-α-chloro-phenylpropionic acid and is administered in an amount within a range of from 10-200 mg/kg and is provided daily. In another aspect, the compound is a benzothiopene-2-carboxylic acid (BT2) compound and is provided at administered in an amount within a range of from 10-100 mg/kg/day and is provided daily. In another aspect, the BT2 compounds is selected from at least one of: BT2 (3,6-dichlorobenzo[b]thiophene-2-carboxylic acid); BT2F (3-chloro-6-fluorobenzo[b]thiophene-2-carboxylic acid); or BT3 (N-(4-acetamido-1,2,5-oxadiazol-3-yl)-3,6-dichlorobenzo[b]thiophene-2-carboxamide). In another aspect, the compound is a BT2 compound and is provided at administered in an amount within a range of from 10-100 mg/kg/day and is provided daily. In another aspect, the compound is administered using a method selected from the group consisting of oral, intravenous, subcutaneous, intranasal, pulmonary, intracranial, intracerebroventricular, topical, enteral, parenteral, or rectal. In another aspect, the compound is administered in a formulation comprising a carrier, the carrier being selected from the group consisting of: lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starches, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl benzoate, propyl benzoate, talc, magnesium stearate, and mineral oil. In another aspect, the CNS-related condition treated by the compound is Alzheimer's Disease. In another aspect, the method further comprises obtaining one or more additional biological samples at a different time from the patient with the CNS-related condition after treatment with the composition that lowers one or more BCAAs or metabolites thereof, and determining if the biological sample has an decrease in BCAAs when compared to a prior sample from the subject with the CNS-related condition.
In another embodiment, the present invention includes a method for treating a patient with a CNS-related condition that comprises an increase in one or more branched-chain amino acids (BCAAs) or metabolites thereof, the method comprising the steps of: performing or having performed an assay from a biological sample from the patient with the CNS-related condition to determine if the biological sample has an increase in one or more branched-chain amino acids (BCAAs) or metabolites thereof, when compared to a sample from a subject without the CNS-related condition; and if the biological sample from the patient with the CNS-related condition has an increase in the one or more BCAAs or metabolites thereof, then: treating the patient with an effective amount of a compound that lowers the levels of one or more branched-chain amino acids (BCAAs) or metabolites thereof, or any pharmaceutically acceptable salt thereof, wherein the CNS-related conditions is selected from the group consisting of Alzheimer's disease and dementia selected from the group consisting of vascular dementia, Lewy body dementia, frontotemporal dementia, Creutzfeldt-Jacob disease, Wernicke-Korsakoff disease, and Huntington's disease, Multiple sclerosis, Parkinson's disease, autism, Amyotrophic lateral sclerosis (ALS), Hereditary diffuse leukoencephalopathy with spheroids (HDLS) and epilepsy, neuropsychiatric disorders such as all anxiety disorders including, but not limited to, generalized anxiety disorder, social anxiety disorder, specific phobias and separation anxiety disorder, and a collection of depression disorders including, but not limited to, clinical depression (major depression), bipolar depression, persistent depressive disorder (dysthymia), seasonal affective disorder, atypical depression, treatment-resistant depression, psychotic depression, postpartum depression, premenstrual dysphoric disorder, and situational depression (stress response syndrome). In one aspect, the compound is a (S)-α-chloro-phenylpropionic acid and is administered in an amount within a range of from 10-200 mg/kg and is provided daily. In another aspect, the compound is a benzothiopene-2-carboxylic acid (BT2) compound and is provided at administered in an amount within a range of from 10-100 mg/kg/day and is provided daily. In another aspect, the BT2 compounds is selected from at least one of: BT2 (3,6-dichlorobenzo[b]thiophene-2-carboxylic acid); BT2F (3-chloro-6-fluorobenzo[b]thiophene-2-carboxylic acid); or BT3 (N-(4-acetamido-1,2,5-oxadiazol-3-yl)-3,6-dichlorobenzo[b]thiophene-2-carboxamide). In another aspect, the compound is a BT2 compound and is provided at administered in an amount within a range of from 10-100 mg/kg/day and is provided daily. In another aspect, the compound is administered using a method selected from the group consisting of oral, intravenous, subcutaneous, intranasal, pulmonary, intracranial, intracerebroventricular, topical, enteral, parenteral, or rectal.
In another embodiment, the present invention includes a method for diagnosing a patient with a CNS-related condition related to increases in branched-chain amino acids (BCAAs) or metabolites thereof, the method comprising: performing or having performed an assay from a biological sample from the patient with the CNS-related condition to determine if the biological sample has an increase in one or more branched-chain amino acids (BCAAs) or metabolites thereof, when compared to a sample from a subject without the CNS-related condition, wherein the BCAA is selected from at least one of valine, leucine, and isoleucine, or metabolites thereof. In one aspect, the biological sample is selected from blood, plasma, serum, tear, sweat, or sputum. In another aspect, the CNS-related condition is selected from at least one of: Alzheimer's disease and dementia selected from the group consisting of vascular dementia, Lewy body dementia, frontotemporal dementia, Creutzfeldt-Jacob disease, Wernicke-Korsakoff disease, and Huntington's disease, Multiple sclerosis, Parkinson's disease, autism, Amyotrophic lateral sclerosis (ALS), Hereditary diffuse leukoencephalopathy with spheroids (HDLS) and epilepsy, neuropsychiatric disorders such as all anxiety disorders including, but not limited to, generalized anxiety disorder, social anxiety disorder, specific phobias and separation anxiety disorder, and a collection of depression disorders including, but not limited to, clinical depression (major depression), bipolar depression, persistent depressive disorder (dysthymia), seasonal affective disorder, atypical depression, treatment-resistant depression, psychotic depression, postpartum depression, premenstrual dysphoric disorder, and situational depression (stress response syndrome).
For a more complete understanding of the features and advantages of the present invention, reference is now made to the detailed description of the invention along with the accompanying figures and in which:
While the making and using of various embodiments of the present invention are discussed in detail below, it should be appreciated that the present invention provides many applicable inventive concepts that can be embodied in a wide variety of specific contexts. The specific embodiments discussed herein are merely illustrative of specific ways to make and use the invention and do not delimit the scope of the invention.
To facilitate the understanding of this invention, a number of terms are defined below. Terms defined herein have meanings as commonly understood by a person of ordinary skill in the areas relevant to the present invention. Terms such as “a”, “an” and “the” are not intended to refer to only a singular entity, but include the general class of which a specific example may be used for illustration. The terminology herein is used to describe specific embodiments of the invention, but their usage does not delimit the invention, except as outlined in the claims.
The present inventors identified a class of existing compounds to prevent or delay the progression of Alzheimer's disease (AD). There are several important aspects of the present invention. (1) These compounds' mechanism of action has been already tested and validated by many laboratories and are easily available. Since current drugs are not effective at treating AD, this is a huge advantage because pharmaceutical companies do not need to spend millions of dollars just to develop an effective compound, and instead can take advantage of these validated compounds providing an immediate benefit to patients. (2) Another innovative element lies in the process for identifying novel uses for these compounds. These compounds have recently been shown to lower branched-chain amino acids (BCAAs) in rodents and alleviate insulin resistance and restore heart remodeling. The present inventor shows herein that circulating BCAAs are higher in both individuals and animal model of AD, and BCAA supplementation induces features of AD pathology, suggesting that the BCAA-lowering compounds can potentially ameliorate AD pathology and symptoms. (3) Development/refining of these compounds to test its efficacy and safety can be conducted using well-known neuroscience and biochemistry methods and models. Rapid, successful testing in both animals and humans allows these compounds to immediately benefit patients.
Alzheimer's disease (AD) is the third leading cause of mortality in the US and the number of cases is expected to exponentially increase highlighting the need for developing an effective treatment. The annual healthcare expenditure is nearly $300 billion and is a huge burden on the nation's healthcare system. It is estimated that the number of people with AD and other forms of dementia will grow to 13.9 million by 2060. The quality of life of AD patients is severely impacted by memory loss; they are unable to care for themselves and rely on assisted living and the support of unpaid caregivers. It is also important to tend to the well-being and needs of the caregivers. The development of drugs that improve the control of symptoms will be very beneficial for at-risk patients and caregivers to plan for specialized professional care. Moreover, given that AD shares similar pathophysiological abnormalities with other neurodegenerative diseases such as Parkinson's disease, Huntington's disease, autism, and other prevalent chronic disorders such as obesity, Type 2 diabetes and cardiovascular diseases, the proposed compounds may potentially be broadly used for treating these different disorders alone or in conjunction with current existing therapies. The present invention can be used to treat Alzheimer's disease and dementia selected from the group consisting of vascular dementia, Lewy body dementia, frontotemporal dementia, Creutzfeldt-Jacob disease, Wernicke-Korsakoff disease, and Huntington's disease, Multiple sclerosis, Parkinson's disease, autism, Amyotrophic lateral sclerosis (ALS), Hereditary diffuse leukoencephalopathy with spheroids (HDLS) and epilepsy, neuropsychiatric disorders, generalized anxiety disorder, social anxiety disorder, specific phobias and separation anxiety disorder, depression disorders, clinical depression (major depression), bipolar depression, persistent depressive disorder (dysthymia), seasonal affective disorder, atypical depression, treatment-resistant depression, psychotic depression, postpartum depression, premenstrual dysphoric disorder, and situational depression (stress response syndrome).
A dosage unit for use of the compounds or agents that reduce a level of branched-chain amino acids (BCAAs) of the present invention, may be a single compound or mixtures thereof with other compounds. The compounds may be mixed together, form ionic or even covalent bonds. The BCAA-reducing agents of the present invention may be administered in oral, intravenous (bolus or infusion), intraperitoneal, subcutaneous, or intramuscular form, all using dosage forms well known to those of ordinary skill in the pharmaceutical arts. Depending on the particular location or method of delivery, different dosage forms, e.g., tablets, capsules, pills, powders, granules, elixirs, tinctures, suspensions, syrups, and emulsions may be used to provide the reduction in the levels of BCAAs of the present invention to a patient in need of therapy for CNS-related conditions, such as Alzheimer's Disease and other neurological conditions caused by an increase in BCAAs. The BCAA reducing agents may also be administered as any one of known salt forms.
BCAA-reducing agents re typically administered in admixture with suitable pharmaceutical salts, buffers, diluents, extenders, excipients and/or carriers (collectively referred to herein as a pharmaceutically acceptable carrier or carrier materials) selected based on the intended form of administration and as consistent with conventional pharmaceutical practices. Depending on the best location for administration, the BCAA reducing agents may be formulated to provide, e.g., maximum and/or consistent dosing for the particular form for oral, rectal, topical, intravenous injection or parenteral administration. While each of the BCAA reducing agents may be administered alone, it will generally be provided in a stable salt form mixed with a pharmaceutically acceptable carrier. The carrier may be solid or liquid, depending on the type and/or location of administration selected.
The following are non-limiting examples of lower BCAA lowering agents for use with the present invention. (S)-CPP: (S)-α-chloro-phenylpropionic acid. All in vitro pharmacokinetics (ex. affinity binding, binding site, functional activity) examined and demonstrated (Tso S C et al., PNAS, 2013). Half-life of ˜7 hours in hepatocytes, and 0.113 ml/min clearance rate in CD-1 mice during in vivo pharmacokinetics study (meaning it stays long enough in the body). Shown to effectively inhibit BCKDH kinase (BCKDH suppressor), thereby activating BCKDH to break down BCAAs in liver, muscle, kidney, and heart, and thus lowering plasma BCAAs in mice (Tso S C et al., PNAS, 2013). A commonly used dose is: 40-160 mg/kg/day intraperitoneal (ip) injection for 3-8 weeks, but doses of 1-250 mg can be used, e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 125, 130, 140, 150, 160, 170, 175, 180, 190 or 200 mg can be used. Dose and/or duration are tested for safety and efficacy in Alzheimer's mice and then humans. BT2 compounds: BT2 (3,6-dichlorobenzo[b]thiophene-2-carboxylic acid); BT2F (3-chloro-6-fluorobenzo[b]thiophene-2-carboxylic acid); BT3 (N-(4-acetamido-1,2,5-oxadiazol-3-yl)-3,6-dichlorobenzo[b]thiophene-2-carboxamide). All in vitro pharmacokinetics (ex. affinity binding, binding site, functional activity) examined and demonstrated (Tso S C et al. J Biol Chem, 2014). Half-life of >4 hours in vitro and ˜12 hours when injected ip in CD-1 mice during in vivo pharmacokinetics study (meaning it stays long enough in the body). Shown to effectively inhibit BCKDH kinase (BCKDH suppressor), thereby activating BCKDH to break down BCAAs in liver, muscle, kidney, and heart, and thus lowering plasma BCAAs in mice (Tso S C et al., J Biol Chem, 2014). A commonly used dose is: 20-40 mg/kg/day ip injection or oral gavage for 3-8 week, but doses of 1-100 mg can be used, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 25, 30, 40, 50, 60, 70, 75, 80, 90 or 100 mg can be used. Dose and/or duration are tested for safety and efficacy in Alzheimer's mice and then humans. BT2 is used for Lowering plasma BCAAs and their metabolites to alleviate obesity-related insulin resistance. Lowering heart BCAAs and their metabolites to restore heart function or remodeling after experimental heart failure.
Techniques and compositions for making useful dosage forms using the present invention are described in one or more of the following references: Anderson, Philip O.; Knoben, James E.; Troutman, William G, eds., Handbook of Clinical Drug Data, Tenth Edition, McGraw-Hill, 2002; Pratt and Taylor, eds., Principles of Drug Action, Third Edition, Churchill Livingston, New York, 1990; Katzung, ed., Basic and Clinical Pharmacology, Ninth Edition, McGraw Hill, 2007; Goodman and Gilman, eds., The Pharmacological Basis of Therapeutics, Tenth Edition, McGraw Hill, 2001; Remington's Pharmaceutical Sciences, 20th Ed., Lippincott Williams & Wilkins, 2000, and updates thereto; Martindale, The Extra Pharmacopoeia, Thirty-Second Edition (The Pharmaceutical Press, London, 1999); relevant portions incorporated herein by reference.
For example, the BCAA-reducing agents may be included in a tablet. Tablets may contain, e.g., suitable binders, lubricants, disintegrating agents, coloring agents, flavoring agents, flow-inducing agents and/or melting agents. For example, oral administration may be in a dosage unit form of a tablet, gelcap, caplet or capsule, the active drug component being combined with an non-toxic, pharmaceutically acceptable, inert carrier such as lactose, gelatin, agar, starch, sucrose, glucose, methyl cellulose, magnesium stearate, dicalcium phosphate, calcium sulfate, mannitol, sorbitol, mixtures thereof, and the like. Suitable binders for use with the present invention include: starch, gelatin, natural sugars (e.g., glucose or beta-lactose), corn sweeteners, natural and synthetic gums (e.g., acacia, tragacanth or sodium alginate), carboxymethylcellulose, polyethylene glycol, waxes, and the like. Lubricants for use with the invention may include: sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, mixtures thereof, and the like. Disintegrators may include: starch, methyl cellulose, agar, bentonite, xanthan gum, mixtures thereof, and the like.
BCAA-reducing agents may be administered in the form of liposome delivery systems, e.g., small unilamellar vesicles, large unilamellar vesicles, and multilamellar vesicles, whether charged or uncharged. Liposomes may include one or more: phospholipids (e.g., cholesterol), stearylamine and/or phosphatidylcholines, mixtures thereof, and the like.
BCAA-reducing agents may also be coupled to one or more soluble, biodegradable, bioacceptable polymers as drug carriers or as a prodrug. Such polymers may include: polyvinylpyrrolidone, pyran copolymer, polyhydroxylpropylmethacrylamide-phenol, polyhydroxyethylasparta-midephenol, or polyethyleneoxide-polylysine substituted with palmitoyl residues, mixtures thereof, and the like. Furthermore, the BCAA reducing agents may be coupled one or more biodegradable polymers to achieve controlled release of the BCAA reducing agents, biodegradable polymers for use with the present invention include: polylactic acid, polyglycolic acid, copolymers of polylactic and polyglycolic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacylates, and crosslinked or amphipathic block copolymers of hydrogels, mixtures thereof, and the like.
In one embodiment, gelatin capsules (gelcaps) may include the BCAA-reducing agents and powdered carriers, such as lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like. Like diluents may be used to make compressed tablets. Both tablets and capsules may be manufactured as immediate-release, mixed-release or sustained-release formulations to provide for a range of release of medication over a period of minutes to hours. Compressed tablets may be sugar coated or film coated to mask any unpleasant taste and protect the tablet from the atmosphere. An enteric coating may be used to provide selective disintegration in, e.g., the gastrointestinal tract.
For oral administration in a liquid dosage form, the oral drug components may be combined with any oral, non-toxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like. Examples of suitable liquid dosage forms include solutions or suspensions in water, pharmaceutically acceptable fats and oils, alcohols or other organic solvents, including esters, emulsions, syrups or elixirs, suspensions, solutions and/or suspensions reconstituted from non-effervescent granules and effervescent preparations reconstituted from effervescent granules. Such liquid dosage forms may contain, for example, suitable solvents, preservatives, emulsifying agents, suspending agents, diluents, sweeteners, thickeners, and melting agents, mixtures thereof, and the like.
Liquid dosage forms for oral administration may also include coloring and flavoring agents that increase patient acceptance and therefore compliance with a dosing regimen. In general, water, a suitable oil, saline, aqueous dextrose (e.g., glucose, lactose and related sugar solutions) and glycols (e.g., propylene glycol or polyethylene glycols) may be used as suitable carriers for parenteral solutions. Solutions for parenteral administration include generally, a water soluble salt of the active ingredient, suitable stabilizing agents, and if necessary, buffering salts. Antioxidizing agents such as sodium bisulfite, sodium sulfite and/or ascorbic acid, either alone or in combination, are suitable stabilizing agents. Citric acid and its salts and sodium EDTA may also be included to increase stability. In addition, parenteral solutions may include pharmaceutically acceptable preservatives, e.g., benzalkonium chloride, methyl- or propyl-paraben, and/or chlorobutanol. Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, Mack Publishing Company, a standard reference text in this field, relevant portions incorporated herein by reference.
For direct delivery to the nasal passages, sinuses, mouth, throat, esophagus, trachea, lungs and alveoli, the BCAA reducing agents may also be delivered as an intranasal form via use of a suitable intranasal vehicle. For dermal and transdermal delivery, the BCAA reducing agents may be delivered using lotions, creams, oils, elixirs, serums, transdermal skin patches and the like, as are well known to those of ordinary skill in that art. Parenteral and intravenous forms may also include pharmaceutically acceptable salts and/or minerals and other materials to make them compatible with the type of injection or delivery system chosen, e.g., a buffered, isotonic solution. Examples of useful pharmaceutical dosage forms for administration of BCAA reducing agents may include the following forms.
Capsules. Capsules may be prepared by filling standard two-piece hard gelatin capsules each with 1 to 500 milligrams of powdered active ingredient, 5 to 150 milligrams of lactose, 5 to 50 milligrams of cellulose and 6 milligrams magnesium stearate.
Soft Gelatin Capsules. A mixture of active ingredient is dissolved in a digestible oil such as soybean oil, cottonseed oil or olive oil. The active ingredient is prepared and injected by using a positive displacement pump into gelatin to form soft gelatin capsules containing, e.g., 100-500 milligrams of the active ingredient. The capsules are washed and dried.
Tablets. A large number of tablets are prepared by conventional procedures so that the dosage unit was 1 to 500 milligrams of active ingredient, 0.2 milligrams of colloidal silicon dioxide, 5 milligrams of magnesium stearate, 50-275 milligrams of microcrystalline cellulose, 11 milligrams of starch and 98.8 milligrams of lactose. Appropriate coatings may be applied to increase palatability or delay absorption.
To provide an effervescent tablet appropriate amounts of, e.g., monosodium citrate and sodium bicarbonate, are blended together and then roller compacted, in the absence of water, to form flakes that are then crushed to give granulates. The granulates are then combined with the active ingredient, drug and/or salt thereof, conventional beading or filling agents and, optionally, sweeteners, flavors and lubricants.
Injectable solution. A parenteral composition suitable for administration by injection is prepared by stirring 1.5% by weight of active ingredient in deionized water and mixed with, e.g., up to 10% by volume propylene glycol and water. The solution is made isotonic with sodium chloride and sterilized using, e.g., ultrafiltration.
Suspension. An aqueous suspension is prepared for oral administration so that each 5 ml contain 1 to 500 mg of finely divided active ingredient, 200 mg of sodium carboxymethyl cellulose, 5 mg of sodium benzoate, 1.0 g of sorbitol solution, U.S.P., and 0.025 ml of vanillin.
For mini-tablets, the active ingredient is compressed into a hardness in the range 6 to 12 Kp. The hardness of the final tablets is influenced by the linear roller compaction strength used in preparing the granulates, which are influenced by the particle size of, e.g., the monosodium hydrogen carbonate and sodium hydrogen carbonate. For smaller particle sizes, a linear roller compaction strength of about 15 to 20 KN/cm may be used.
Kits. The present invention also includes pharmaceutical kits useful, for example, for the treatment of cancer, which comprise one or more containers containing a pharmaceutical composition comprising a therapeutically effective amount of BCAA-reducing agents. Such kits may further include, if desired, one or more of various conventional pharmaceutical kit components, such as, for example, containers with one or more pharmaceutically acceptable carriers, additional containers, etc., as will be readily apparent to those skilled in the art. Printed instructions, either as inserts or as labels, indicating quantities of the components to be administered, guidelines for administration, and/or guidelines for mixing the components, may also be included in the kit. It should be understood that although the specified materials and conditions are important in practicing the invention, unspecified materials and conditions are not excluded so long as they do not prevent the benefits of the invention from being realized.
Examples of suitable liquid dosage forms include solutions or suspensions in water, pharmaceutically acceptable fats and oils, alcohols or other organic solvents, including esters, emulsions, syrups or elixirs, suspensions, solutions and/or suspensions reconstituted from non-effervescent granules and effervescent preparations reconstituted from effervescent granules. Such liquid dosage forms may contain, for example, suitable solvents, preservatives, emulsifying agents, suspending agents, diluents, sweeteners, thickeners, and melting agents. Oral dosage forms optionally contain flavorants and coloring agents. Parenteral and intravenous forms may also include minerals and other materials to make them compatible with the type of injection or delivery system chosen.
As used herein, the term “chewable” refers to semi-soft, palatable and stable chewable treat without addition of water. It should be appreciated to the skilled artisan that a chewable composition will be stable and palatable, fast disintegrating, semi-soft medicated chewable tablets (treats) by extrusion without the addition of extraneous water. A soft, chewable tablet does not harden on storage and are resistant to microbial contamination. A semi-soft chewable contain a blend of any one or more of binders, flavours, palatability enhancers, humectants, disintegrating agents, non-aqueous solvents, and diluents that are plasticized with liquid plasticizers, such as glycols and polyols to make them ductile and extrudable. The chewable can be made by extrusion, e.g., including fats or lipids as plasticizers and binding agents, is manufactured in the absence of added water, uses plasticizers to replace water in extrudable matrices, contains humectants to maintain the extrudable chew in a pliant and soft state during its shelf life, or any combination thereof. The chewable form may be provided in conjunction with one or more flavorants and/or taste masking agents that improve the taste of the formulation greater than 10, 20, 30, 40, 50, 60, 70, 80, or 90%. The chewable can include the active agent and the ion exchange resin to enhance taste masking.
For topical administration, the composition can be incorporated into creams, ointments, gels, transdermal patches and the like. The composition can also be incorporated into medical dressings, for example wound dressings, e.g., woven (e.g., fabric) dressings or non-woven dressings (e.g., gels or dressings with a gel component). The use of alginate polymers in dressings is known, and such dressings, or indeed any dressings, may further incorporate the alginate oligomers of the invention.
Branched-chain amino acids (BCAAs) are essential amino acids that need to be obtained from food. They compete for same transporter with other aromatic amino acids that serve as the precursors of several critical neurotransmitters in the brain such as dopamine, serotonin, and norepinephrine1, hence excess BCAAs may lead to profound imbalance of these key neurotransmitters. Moreover, BCAA overload has been shown to induce neural oxidative stress and apoptosis4-9, and mTOR hyperactivation commonly induced by BCAAs lead to insulin resistance in the brain10-11. Importantly, all of these abnormalities represent the pathophysiological hallmarks of AD, making BCAAs a potentially significant contributor to the development of AD. Interestingly, BCAAs are recently implicated in insulin resistance/Type 2 diabetes (T2D)12-17. The present inventors determined if defective BCAA metabolism drives development or exacerbation of AD, however the role of BCAAs in AD pathogenesis is poorly understood.
Time-course study to determine plasma and brain BCAA profiles are associated with AD. Pathological features of AD such as phosphorylated Tau protein and AB aggregates in the neocortex and hippocampus and cognitive deficits will be assessed in a well-established APP/PS1 mouse model of AD.
Test if high plasma BCAAs and/or their metabolites in AD are due to impaired hepatic BCAA catabolism. He inventor has previously shown that insulin regulates plasma BCAAs by inducing hepatic BCAA breakdown. Therefore, BCAA catabolic control in response to insulin will be interrogated in AD mice. Hyperinsulinemic euglycemic clamps will be performed based on the inventors prior findings.
Cognitive functions and Ap deposits and pTau protein in AD mice can be examined following dietary BCAA restriction or supplementation for months. The genes involved in neuronal/cellular functions in the hippocampus and neocortex will be assessed for potential mechanisms.
Branched-chain amino acids and the CNS; BCAAs (i.e., leucine, isoleucine, and valine) are essential amino are not produced in our body, hence the need to obtain them from food. While BCAA acids that supplementation is widely used for improving physical fitness37-38 and for preventing muscle wasting in patients40-49 with cirrhosis, kidney failure, cancer, or sepsis mounting evidence suggests that BCAAs or their derived metabolites can induce neural oxidative stress4,5,8,9, mitochondrial dysfunction50,51 and apoptosis6,7. In support of these findings, individuals with maple syrup urine disease, a rare genetic disease that causes a mutation in genes critical BCAA-degrading enzyme complex, have nearly 10-fold higher BCAAs and their toxic keto-acids in the plasma compared to healthy controls52,53 leading to serious neurological impairments.
Further evidence correlating BCAAs to brain function comes from the observation that the enzyme branched-chain aminotransferase (BCAT) converts BCAAs to glutamate, an excitatory neurotransmitter. Moreover, BCAAs compete for the same large amino acid transporter (LAT) with aromatic amino acids like phenylalanine, tyrosine, and tryptophan—the precursors of neurotransmitters like dopamine, norepinephrine, and serotonin. Thus, excess circulating BCAAs may lead production of too much glutamate that contribute excitotoxicity, as well as trigger a significant imbalance and reduction some key neurotransmitters in the brain.
The inventor recognized that such metabolic diseases share a number of abnormal features with AD. These findings further underscore that BCAA control is similarly impaired in AD, however prior to the present invention the role of BCAAs and their metabolism in the onset of AD, or the CNS health in general was unknown.
Recent clinical studies have tried to address this and examine the association between BCAAs and AD, but the findings are inconsistent50-62. Thus, the present invention looked at fasting plasma BCAAs and/or their metabolites are increased in AD patients (
The present invention provides significant and relevant information related to human health because it enabled the determination of circulating BCAAs and/or their metabolites as a predictive/diagnostic biomarker for AD. Individuals may be easily tested for their susceptibility to or the onset/progression of AD by measuring blood levels of BCAAs and their metabolites in conjunction with existing markers, with minimal risks and invasiveness. In addition, the study will provide a novel dietary interventional strategy that can possibly delay further progression of AD symptoms, which evidently has significant implications in the financial burden and the quality of life for AD patients and their families.
Previously unidentified role of BCAAs in the CNS function neurodegenerative diseases such as AD in vivo. The present invention provides a novel biomarker that can be used to either predict or diagnose AD. The detection of circulating biomarkers is ideal compared to neuroimaging techniques or CSF analysis because it is simpler, less invasive, cost-effective, and patient-friendly. As a novel biomarker, BCAAs significantly improve the clinical utility and predictive power of existing methods of AD assessment.
Test the efficacy of dietary BCAA intake on AD progression using iso-caloric and isonitrogenous diet. A multi-dimensional approach (transgenic, integrative physiology, molecular biology, and behavioral) is used to understand the impact of BCAAs and their metabolism in the development of AD.
Few earlier rodent studies have demonstrated sex differences in diurnal regulation of BCAA metabolism53,84. The inventor has shown that hepatic BCAA-degrading enzyme is lower in obese and diabetic men compared to lean men, but the difference disappears in women, suggesting potential sex/hormonal differences in the control of BCAA metabolism in humans well. Thus, studying both sexes is deemed necessary.
Examining the levels of BCAAs in both sexes is important because, in addition to its catabolic capacity, circulating amino acids (AA) like BCAAs closely reflect immediate dietary BCAA intake. This is due to the lack of the body's AA-storing mechanism for later use, unlike in the case of excess carbohydrate or fat. It is shown herein that: (1) elevated plasma BCAAs and their metabolites in both AD men and mice, (2) even higher levels in patients with AD+T2D vs. T2D alone, and (3) BCAAs as a significant composite predictor AD (
Therefore, will conduct a time-course study using double-transgenic APP/PS1 AD mice69,71 (
A transgenic APP/PS1 mouse model of AD is used because exhibit cognitive impairment at the age of 7-8 months. Thus, two month-old WT littermates and APP/PS1 (AD) mice can be divided into two cohorts with one cohort subjected to cognitive behavioral tests when they are 3 months old (intact cognition), and the other when they are 8 months old (4 groups total for each sex; n=13/group; 3 month-old WT or APP/PS1 mice; 8 month-old WT or APP/PS1 mice). These animals are readily available since they were breed the transgenic mice at the Texas Tech animal facility. Animals throughout the proposal will be placed on ad libitum chow diet and water unless otherwise noted. They will be group-housed and their weekly body weight and food intake will be monitored. To assess cognitive function at both 3 and months of age, the mice will undergo widely used behavioral tests: Y-maze with spontaneous alternation and Morris Water Maze. Y-maze is often used to assess working memory in animals. This method takes the advantage of rodent's tendency to explore a new arm of the maze rather than a familiar arm, and this executive action requires multiple brain regions like basal forebrain, hippocampus, and prefrontal cortex. Morris Water Maze (MWM) is another widely used behavioral test to study spatial learning and memory in rodents. Details of these behavioral assays are described by the inventor.75 One week of rest will be given between each behavior test. Four days after the last behavioral test trial, overnight-fasted animals can be sacrificed and blood through cardiac puncture can be collected for plasma isolation. Peripheral tissues like liver, muscle, and white adipose tissue (WAT) will be harvested, snap frozen, and stored at −80° C. Brains will be also harvested and the neocortex and hippocampus will be dissected and immediately frozen. The P1 is highly experienced in rodent neuroanatomy and microdissection19,75,78. Total plasma, cortical, and hippocampal BCAAs will be measured by spectrophotometric assay that measures NADH generated from BCAA oxidation's. Individual BCAAs, their derived keto acids and acylcarnitines can be analyzed by LC-MS. From the neocortex and hippocampus, branched-chain aminotransferase (BCATc)—the first enzyme in the BCAA degradation pathway found in neurons, branched-chain a-keto acid dehydrogenase (BCKDH) the rate-limiting enzyme, BCKDH phosphatase and kinase-BCKDH activating and inhibiting enzyme, respectively, the phosphorylated (inactive) state of the enzyme BCKDH/BCKDH (inactivation index)19,80 and KIV oxidation (i.e., functional BCKDH activity readout)19 will be determined altogether in order to comprehensively assess BCAA catabolism. The cortical and hippocampal aggregates of insoluble AB (1-40; 1-42), soluble Ap oligomers, and phosphorylated Tau (pTau) the protein deposits thought to be responsible for cognitive deficits seen in AD patients, will be measured by ELISA and/or western blots. Thus, a correlation between BCAA metabolism to AD-related brain pathology and memory deficits is obtained as a function of time.
It can be expected that plasma BCAAs and their metabolites will be higher in 8 month-old AD mice compared to WT mice, which will be associated with their impaired cognitive/memory functions as serve assessed by the behavioral tests, indicating that plasma BCAAs and their metabolites may surrogate marker for AD-like symptoms. In the neocortex and hippocampus of AD mice, increased Af aggregates and pTau can be observed, as well as elevated BCAAs/their metabolites that are associated with impaired BCAA catabolism.
High plasma BCAAs and their metabolites in AD mice at months of age, when there is no visible AD-related cognitive dysfunction can be determined to indicate a predictive role in the onset of AD, in keeping with the inventor's findings in 5 month-old AD mice. If these changes are absent in female mice, then a role of estrogen in plasma BCAAs in AD is determined by performing an ovariectomy. However, women usually develop AD at 65 years of age or greater, at which they are well within the postmenopausal, low-estrogen stage, so estrogen may not critical mediator. Interestingly, during postmenopause there compensatory surge of follicular-stimulating hormone (FSH) from the anterior pituitary in circulation, and, thus, may have an important role other than stimulating ovarian functions. The role of FSH in the AD female mice is tested using FSH-specific antibody. While APP/PS1 mouse model was chosen here mainly because of the strong and persistent transgene expressions in the brain unlike other models, in which they are temporally induced by doxycycline (e.g., AAV-mediated transfection, to extend these findings to other AD mouse models for reproducibility).
The findings above indicate defective BCAA degradation in liver, an organ with high BCAA catabolic activity from transgenic AD mice at both protein and gene levels (
Design and Analysis: Observe sex differences studying females and males. 8 month-old WT or transgenic AD (APP/PS1) mice (4 groups total for each sex; n=13/group; WT or APP/PS1 mice+ or 4mU clamps) will be implanted with jugular and arterial catheters. After recovery (usually 4-5 days), the animals will undergo hyperinsulinemic euglycemic clamps (
WT mice will have lower plasma BCAAs and their derived metabolites during hyperinsulinemic clamps compared to 1 mU basal clamps. WT mice will also display enhanced hepatic BCAA catabolism increased BCKDH protein and activity during hyperinsulinemic clamps. On other hand, the ability insulin to increase hepatic BCAA degradation will be diminished in APP/PS1 mice, resulting in higher plasma BCAAs and their metabolites. These outcomes would show that APP/PS1 mice indeed have impaired hepatic BCAA metabolism. BCAA catabolism in other peripheral insulin-sensitive tissues like white adipose tissue and muscle may be impaired as well in APP/PS1 mice, however, this may be unlikely as these organs play a minor role regulating BCAAs. Protein turnover can be assessed to test if protein catabolism is a major contributor of elevated plasma BCAAs in AD mice.
Dietary intervention has been shown to lower the susceptibility to age-related cognitive impairment or AD. A nutritional strategy aimed at decreasing AD pathology is used. Others have shown that protein restriction improves cognitive performance in AD mice88 although the role of BCAAs in this limited protein intake is unknown. A body of literature points to the deleterious effects of BCAA overload on neuronal functions and neurotransmitter balance in the brain149.
To first determine a possible link between circulating BCAA levels and AD, serum samples from male individuals with AD or age-matched healthy individuals were analyzed through untargeted metabolomics by LC-MS. From 95 metabolites that are found to be differentially regulated, Compound Discoverer database identified BCAA metabolism as one of the most affected biological pathways in AD patients. As shown in
Transgenic AD mice reveal (share) remarkable similarities with humans in regards to the formation of amyloid plaques, neurofibrillary tangles, and related cognitive decline, thus serving as an excellent model to study the disease pathophysiology34. APPswe mouse (also known as Tg2576) is one of the most widely used mouse models of AD that overexpresses human amyloid precursor protein (APP) containing Swedish mutation and shows a cognitive impairment as early as 6 months of age35. The inventors measured plasma BCAA levels from 8-month-old APPswe mice to determine if a relationship similar to what the inventors observed in humans exists in these mice. Indeed, plasma BCAAs were significantly elevated in APPswe mice compared to WT controls (
Elevated systemic levels of BCAAs and their metabolites in AD raise a possibility that BCAAs and AD are causally linked. To test this, 0, 1, 5, or 10 mM of a mixture of BCAAs (leucine, isoleucine, valine; 1:2:1 ratio) was supplemented to differentiated HT-22 hippocampal neurons for 24 h. The in vitro results showed that BCAA exposure dose-dependently downregulates mRNA of neuronal health markers such as LC3A (autophagy), NRF1 (mitochondrial biogenesis), PSD 95 (synapse formation), as well as OPA1, Mfn1, and Mfn2 (mitochondrial fusion;
Given the strong association between circulating BCAAs/metabolites and AD (
The inventors predicted that if BCAAs are indeed a significant contributor to AD pathogenesis, then lowering BCAA intake would at least partly slow down the disease progression. APP/PS1 mice displayed higher plasma BCAA levels compared to WTs before dietary treatment (
To understand possible mechanisms by which limiting BCAA intake slows down AD development, the inventors focused on examining key markers of AD-related brain pathology and neurotransmitter levels in the hippocampus and cortex as an indicator of neuronal damage and health. BCAA-restricted APP/PS1 mice had a significantly lower ratio of pBCKDH to BCKDH in liver compared to regular chow-fed APP/PS1 mice (
As a complementary strategy to dietary BCAA restriction, the inventors used a pharmacological approach to lower circulating BCAAs and examine the beneficial effects in AD mice. BT2 is an allosteric inhibitor of BCKDH kinase that suppresses BCKDH activity, leading to increased BCAA catabolism and lower plasma BCAA levels. For one month, BT2 (40 mg/kg ip) was daily injected to 5xFAD mice, another widely used AD model that overexpresses three APP and two presenilin (PSEN1) mutations. These transgenic mice develop amyloid plaques and neuroinflammation at as early as 1.5-2 months of age7, thus allowing the inventors to investigate the role of BCAAs in AD-related brain pathology when they are young. Body weight (
It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method, kit, reagent, or composition of the invention, and vice versa. Furthermore, compositions of the invention can be used to achieve methods of the invention.
It will be understood that particular embodiments described herein are shown by way of illustration and not as limitations of the invention. The principal features of this invention can be employed in various embodiments without departing from the scope of the invention. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of this invention and are covered by the claims.
All publications and patent applications mentioned in the specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and/or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” The use of the term “of” in the claims is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and/or.” Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects.
As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. In embodiments of any of the compositions and methods provided herein, “comprising” may be replaced with “consisting essentially of” or “consisting of”. As used herein, the phrase “consisting essentially of” requires the specified integer(s) or steps as well as those that do not materially affect the character or function of the claimed invention. As used herein, the term “consisting” is used to indicate the presence of the recited integer (e.g., a feature, an element, a characteristic, a property, a method/process step or a limitation) or group of integers (e.g., feature(s), element(s), characteristic(s), propertie(s), method/process steps or limitation(s)) only.
The term “or combinations thereof” as used herein refers to all permutations and combinations of the listed items preceding the term. For example, “A, B, C, or combinations thereof” is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, AB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context.
As used herein, words of approximation such as, without limitation, “about”, “substantial” or “substantially” refers to a condition that when so modified is understood to not necessarily be absolute or perfect but would be considered close enough to those of ordinary skill in the art to warrant designating the condition as being present. The extent to which the description may vary will depend on how great a change can be instituted and still have one of ordinary skilled in the art recognize the modified feature as still having the required characteristics and capabilities of the unmodified feature. In general, but subject to the preceding discussion, a numerical value herein that is modified by a word of approximation such as “about” may vary from the stated value by at least ±1, 2, 3, 4, 5, 6, 7, 10, 12 or 15%.
Additionally, the section headings herein are provided for consistency with the suggestions under 37 CFR 1.77 or otherwise to provide organizational cues. These headings shall not limit or characterize the invention(s) set out in any claims that may issue from this disclosure. Specifically and by way of example, although the headings refer to a “Field of Invention,” such claims should not be limited by the language under this heading to describe the so-called technical field. Further, a description of technology in the “Background of the Invention” section is not to be construed as an admission that technology is prior art to any invention(s) in this disclosure. Neither is the “Summary” to be considered a characterization of the invention(s) set forth in issued claims. Furthermore, any reference in this disclosure to “invention” in the singular should not be used to argue that there is only a single point of novelty in this disclosure. Multiple inventions may be set forth according to the limitations of the multiple claims issuing from this disclosure, and such claims accordingly define the invention(s), and their equivalents, that are protected thereby. In all instances, the scope of such claims shall be considered on their own merits in light of this disclosure, but should not be constrained by the headings set forth herein.
All of the compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
To aid the Patent Office, and any readers of any patent issued on this application in interpreting the claims appended hereto, applicants wish to note that they do not intend any of the appended claims to invoke paragraph 6 of 35 U.S.C. § 112, U.S.C. § 112 paragraph (f), or equivalent, as it exists on the date of filing hereof unless the words “means for” or “step for” are explicitly used in the particular claim.
For each of the claims, each dependent claim can depend both from the independent claim and from each of the prior dependent claims for each and every claim so long as the prior claim provides a proper antecedent basis for a claim term or element.
This invention was made with government support under 1R21AG069140-01 awarded by the National Institutes of Health/NSF/DARPA. The government has certain rights in the invention.
Filing Document | Filing Date | Country | Kind |
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PCT/US2021/060448 | 11/23/2021 | WO |
Number | Date | Country | |
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63123886 | Dec 2020 | US |