Methods for inhibiting degradative protease activity in aging - Patents.com

Abstract

Provided herein is a method of reversing serine protease accumulation, reversing cellular damage, and / or preserving extracellular matrix in an organ of a subject, comprising the steps of selecting a subject at risk of damage to the organ and administering a therapeutically effective amount of a serine protease inhibitor. The serine protease inhibitor can be an orally administered competitive inhibitor.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 300,409, filed January 18, 2022. The disclosure of the prior application is considered part of the disclosure of this application and is incorporated by reference herein in its entirety. [Background technology]

[0002] Serine proteases (e.g., trypsin) activate secondary proenzymes (e.g., members of the matrix metalloproteinase, kallikrein, cathepsin family, etc.) that are involved in tissue degradation and loss of cellular function due to cleavage of extracellular matrix proteins (collagenolysis) and membrane receptor cleavage (e.g., insulin receptor cleavage, loss of insulin receptor binding sites, and "insulin resistance"). Chronic matrix metalloproteinase (MMP) inhibition (e.g., doxycycline, grape seed extract, resveratrol, etc.) has also been shown to reduce symptoms of metabolic syndrome in experimental models and short-term human trials. Although multiple genes associated with longevity have been identified (e.g., by genome-wide association studies) in specific aging human populations and animal models, they do not identify a common mechanism for aging in different species. Summary of the Invention

[0003] Provided herein is a method for reversing accumulation of a serine protease in an organ of a subject, the method comprising: (a) selecting a subject having or at risk of accumulation of a serine protease in the organ; and (b) administering a therapeutically effective amount of a serine protease inhibitor, thereby reversing accumulation of a serine protease in the organ of the subject.

[0004] Further provided herein is a method of reversing cellular damage in an organ of a subject, the method comprising: (a) selecting a subject having or at risk of cellular damage to the organ; and (b) administering a therapeutically effective amount of a serine protease inhibitor, thereby reversing cellular damage in the organ of the subject.

[0005] Further provided herein is a method of preserving extracellular matrix in an organ of a subject, the method comprising the steps of: (a) selecting a subject having or at risk of losing extracellular matrix in the organ; and (b) administering a therapeutically effective amount of a serine protease inhibitor, thereby preserving extracellular matrix in the organ of the subject.

[0006] In some embodiments, the subject is at least 40 years old. In some embodiments, the subject is at least 50 years old. In some embodiments, the subject is at least 60 years old. In some embodiments, the subject is not at risk of developing shock and / or septic shock. In some embodiments, the subject is HIV-free. In some embodiments, the organ is selected from the group consisting of brain, spinal cord, heart, kidney, muscle, liver, and lung. In some embodiments, the organ is selected from the group consisting of brain, heart, and muscle. In some embodiments, the organ is brain. In some embodiments, the selecting step comprises selecting a subject with a brain disease or brain pathology. In some embodiments, the brain disease or brain pathology is selected from the group consisting of Alzheimer's disease, dementia including frontotemporal dementia, epilepsy or other seizure disorder, psychiatric disorder, multiple sclerosis, Huntington's disease, Parkinson's disease, amyotrophic lateral sclerosis, meningitis, encephalitis, brain tumor, Creutzfeldt-Jakob disease, chronic traumatic encephalopathy, long COVID associated dementia, and stroke. In some embodiments, the organ is a heart. In some embodiments, the step of selecting the subject comprises selecting a subject having a heart disease or a heart condition. In some embodiments, the heart disease or a heart condition is selected from the group consisting of coronary heart disease, angina, unstable angina, heart failure, cardiac arrhythmia, valvular disease, hypertension, cardiac arrhythmia, endocarditis, pericardial disease, and cardiomyopathy. In some embodiments, the organ is a muscle. In some embodiments, the step of selecting the subject comprises selecting a subject having a muscle disease or a muscle condition. In some embodiments, the muscle disease or a muscle condition is selected from the group consisting of fibromyalgia, myositis, including polymyositis and dermatomyositis, muscular dystrophy, myasthenia gravis, amyotrophic lateral sclerosis, rhabdomyolysis, cardiomyopathy, sarcopenia, Charcot-Marie-Tooth disease, multiple sclerosis, myopathy, peripheral neuropathy, and spinal muscular atrophy. In some embodiments, the organ is a kidney. In some embodiments, selecting an at-risk subject comprises selecting a subject with a renal disease or condition.In some embodiments, the kidney disease or condition is selected from the group consisting of chronic kidney disease, diabetic kidney disease, acute kidney injury, kidney stones, kidney infections including pyelonephritis, kidney cysts, and kidney cancer. In some embodiments, the organ is the liver. In some embodiments, the step of selecting the subject comprises the step of selecting a subject having a liver disease or condition. In some embodiments, the liver disease or condition is selected from the group consisting of hepatitis A, hepatitis B, hepatitis C, autoimmune hepatitis, primary biliary cholangitis, primary sclerosing cholangitis, hemochromatosis, Wilson's disease, alpha-1 antitrypsin deficiency, hepatoma, cholangiocarcinoma, hepatic adenoma, nonalcoholic fatty liver disease, and nonalcoholic steatohepatitis. In some embodiments, the serine protease comprises at least one of trypsin, subtilisin, or a combination thereof. In some embodiments, the serine protease comprises at least one of trypsin, elastase, chymotrypsin, or a combination thereof. In some embodiments, the serine protease comprises trypsin. In some embodiments, the serine protease inhibitor is a competitive inhibitor. In some embodiments, the serine protease inhibitor is selected from the group consisting of nafamostat mesilate (Futhan), camostat mesilate (FOY 305), gabexate mesilate (FOY) or derivatives, serine protease inhibitor Kazal-1 type (SPINK1), aprotinin, tranexamic acid, urinastatin, granzyme A, granzyme B, UAMC-00050, 4-(2 aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF), soybean trypsin inhibitor, meprin inhibitor, setomelanotide, alpha-1-antitrypsin, and serpin. In some embodiments, the serine protease inhibitor comprises tranexamic acid. In some embodiments, the therapeutically effective amount of the serine protease inhibitor is less than 10% of the subject's digestive enzyme activity. In some embodiments, the therapeutically effective amount of the serine protease inhibitor is less than 10 μM. In some embodiments, the therapeutically effective amount of the serine protease inhibitor is less than 5 μM.In some embodiments, the serine protease inhibitor is administered enterally, intraperitoneally, intravenously, intramuscularly, subcutaneously, intradermally, orally, intranasally, pulmonary, rectally, or by an external or implanted infusion pump controlled by telemetry. In some embodiments, the serine protease inhibitor is administered orally. In some embodiments, the serine protease inhibitor is administered by a telemetry-controlled infusion pump. In some embodiments, the serine protease inhibitor is administered as a liposomal composition or as a nanoparticle encapsulation. In some embodiments, the serine protease inhibitor is administered as an eye drop. In some embodiments, the telemetry-controlled infusion pump is directed to an organ. In some embodiments, the serine protease inhibitor is administered for more than one week. In some embodiments, the serine protease inhibitor is administered for more than two weeks. In some embodiments, the serine protease inhibitor is administered for more than four weeks.

[0007] Provided herein is a pharmaceutical composition for the treatment of aging or age-related pathologies, comprising a serine protease inhibitor. In some embodiments, the pharmaceutical composition treats age-related pathologies that affect organs selected from the group consisting of the brain, spinal cord, heart, kidney, muscle, liver, and lung. In some embodiments, the age-related pathologies affect organs selected from the group consisting of the brain, heart, and muscle. In some embodiments, the organ is the brain. In some embodiments, the age-related pathologies are selected from the group consisting of Alzheimer's disease, dementia, including frontotemporal dementia, memory, balance, sensation, pain, including but not limited to back pain, age-related loss of neuronal function, including but not limited to epilepsy or other seizure disorders, psychiatric disorders, multiple sclerosis, Huntington's disease, Parkinson's disease, amyotrophic lateral sclerosis, meningitis, encephalitis, brain tumors, and transient ischemic attacks. In some embodiments, the organ is the heart. In some embodiments, the age-related condition is selected from the group consisting of coronary heart disease, angina, unstable angina, heart failure, valvular disease, hypertension, cardiac arrhythmia, endocarditis, pericardial disease, and cardiomyopathy. In some embodiments, the organ is muscle. In some embodiments, the age-related condition is selected from the group consisting of fibromyalgia, myositis, including polymyositis and dermatomyositis, muscular dystrophy, myasthenia gravis, amyotrophic lateral sclerosis, rhabdomyolysis, cardiomyopathy, sarcopenia, Charcot-Marie-Tooth disease, multiple sclerosis, myopathy, peripheral neuropathy, and spinal muscular atrophy. In some embodiments, the organ is kidney. In some embodiments, the age-related condition is selected from the group consisting of acute kidney injury, kidney stones, kidney infections, including pyelonephritis, kidney cysts, subjects requiring kidney dialysis, and kidney cancer. In some embodiments, the organ is liver. In some embodiments, the age-related condition is selected from the group consisting of hepatitis A, hepatitis B, hepatitis C, autoimmune hepatitis, primary biliary cholangitis, primary sclerosing cholangitis, hemochromatosis, Wilson's disease, alpha-1 antitrypsin deficiency, hepatocarcinoma, cholangiocarcinoma, hepatic adenoma, nonalcoholic fatty liver disease, and nonalcoholic steatohepatitis. In some embodiments, the serine protease inhibitor is a competitive inhibitor.In some embodiments, the serine protease inhibitor is selected from the group consisting of nafamostat mesylate (Futhan), camostat mesylate (FOY 305), gabexate mesylate (FOY) or derivatives, serine protease inhibitor Kazal-1 type (SPINK1), aprotinin, tranexamic acid, urinastatin, granzyme A, granzyme B, UAMC-00050, 4-(2 aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF), soybean trypsin inhibitor, meprin inhibitor, setomelanotide, alpha-1-antitrypsin, and serpin. In some embodiments, the serine protease inhibitor comprises tranexamic acid. In some embodiments, the serine protease inhibitor is administered at less than 10% of the subject's digestive enzyme activity. In some embodiments, the serine protease inhibitor is less than 10 μM. In some embodiments, the serine protease inhibitor is less than 5 μM. In some embodiments, the serine protease inhibitor is administered enterally, intraperitoneally, intravenously, intramuscularly, subcutaneously, intradermally, orally, intranasally, pulmonary, rectally, or by an external or implanted infusion pump controlled by telemetry. In some embodiments, the serine protease inhibitor is administered orally. In some embodiments, the serine protease inhibitor is administered by a telemetry-controlled infusion pump. In some embodiments, the serine protease inhibitor is administered as a liposomal composition or nanoparticles. In some embodiments, the serine protease inhibitor is administered as an eye drop. In some embodiments, the telemetry-controlled infusion pump is directed to an organ. In some embodiments, the serine protease inhibitor is administered for more than one week. In some embodiments, the serine protease inhibitor is administered for more than two weeks. In some embodiments, the serine protease inhibitor is administered for more than four weeks.

[0008] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Methods and materials are described herein for use in the present invention, and other suitable methods and materials known in the art can also be used. Materials, methods, and examples are merely illustrative and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0009] Other features and advantages of the invention will become apparent from the following detailed description and drawings, and from the claims. [Brief description of the drawings]

[0010] [Figure 1-1] Figure 1A: Images showing pancreatic trypsin accumulation in rat myocardium by immunohistochemistry with a monoclonal antibody against pancreatic trypsin in young (16 week old) rats, old (100 week old) rats, and old treated rats (100 week old, blockade of pancreatic trypsin with oral tranexamic acid (TXA) for 2 weeks). The figure shows the same images in the original bright field and after digital color extraction of the IHC labeling. [Figure 1-2] FIG 1B: Images showing rat myocardial collagen fragmentation in young (16 week old), old (100 week old), and old treated rats (100 week old, blockade of pancreatic trypsin with oral tranexamic acid for 2 weeks). The left panel shows bright field images, and the right panel shows collagen fragmentation after color extraction. [Figure 2-1]Figure 2A: Images showing pancreatic trypsin accumulation in rat brain sections by immunohistochemistry with a monoclonal antibody against pancreatic trypsin in young (16 week old) rats, old (100 week old) rats, and old treated rats (100 week old, blockade of pancreatic trypsin with oral tranexamic acid for 2 weeks). The figure shows the same images in the original bright field and after digital color extraction of the IHC labeling. [Figure 2-2] Figure 2B: Images showing collagen degradation in brain sections from young (16 weeks old), old (100 weeks old), and old treated rats (100 weeks old, blockade of pancreatic trypsin with oral tranexamic acid for 2 weeks) by labeling with a peptide that binds to fragmented collagen fibers. The figure shows the original bright field and the same image after digital color extraction of the collagen-binding peptide label. [Figure 3-1] Figure 3A: Images showing pancreatic trypsin accumulation in rat intestine by immunohistochemistry using a monoclonal antibody against pancreatic trypsin in young (16 week old) rats, old (100 week old) rats, and old treated rats (100 week old, 2 week blockade of pancreatic trypsin with oral tranexamic acid (TXA)). The figure shows the same images in the original bright field and after digital color extraction of the IHC labeling. [Figure 3-2] FIG. 3B: Images showing mucin-containing mucus layer on epithelial cells of small intestine stained with Alcian blue (pH 2.5) in young (16-week-old), old (100-week-old), and old treated rats (100-week-old, 2-week blockade of pancreatic trypsin with oral tranexamic acid (TXA)). The figure shows the original brightfield image with the corresponding color-extracted image below. In addition, the top brightfield image shows that epithelial cells of small intestine were stained with amylase (brown) in young (16-week-old), old (100-week-old), and old treated rats (100-week-old, 2-week blockade of pancreatic trypsin with oral tranexamic acid (TXA)). An increase in amylase in small intestine was observed in old rats compared to young rats, and this increase was attenuated by administration of oral tranexamic acid for 2 weeks. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] The present disclosure describes methods of inhibiting serine proteases and reducing serine protease activity outside the gastrointestinal (GI) tract in a subject.

[0012] Various non-limiting aspects of these methods are described herein and may be used in any combination without limitation. Further aspects of the various components of the methods described herein are known in the art.

[0013] It must be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0014] As used herein, the term "about" refers to a value that is comparable in the context of the referenced value when used herein with reference to a value. In general, a person skilled in the art familiar with the context will understand the reasonable degree of variation that is encompassed by "about" in that context. For example, in some embodiments, the term "about" can encompass a range of values ​​that are within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the referenced value.

[0015] As used herein, "cell" can refer to either a prokaryotic or eukaryotic cell, obtained from the subject or from a commercial source, as appropriate.

[0016] The Aging Process As used herein, the term "aging" refers to the processes associated with growing older. The term specifically refers to humans, many animals, and fungi, but in a broader sense, aging can also refer to single cells within an organism that stop dividing (cellular senescence) and exhibit reduced cellular function (e.g., response to growth hormone, insulin) and gene expression. In humans, aging represents an accumulation of changes over time, including physical and psychological changes. For example, aging involves the loss of cellular and tissue function, clinically developing comorbidities with increased susceptibility to disease, and ultimately complete organ failure. A range of biological processes (e.g., cellular and mitochondrial function, stem cell proliferation and differentiation, genetic lesions, histones, DNA repair mechanisms, epigenetics, protein folding, intracellular and intercellular signaling, and nutrient utilization) become dysregulated, unstable, and exhausted. Pathophysiological mechanisms in aging may include impaired resistance to molecular stressors, chronic low-grade inflammation, genomic instability, telomere shortening and cellular senescence, epigenetic changes, loss of protein homeostasis, dysregulated nutrient sensing, stem cell depletion, and / or altered intercellular communication. Vascular and immunological cell functions become compromised with pathological remodeling and the development of age-related risk factors and diseases, while multiple different tissues share molecular and cellular mechanisms for microvascular and macrovascular pathology in aging. Aging is also accompanied by chronic low-grade inflammation, and since the inflammatory cascade essentially serves tissue repair, there may be chronic mechanisms in aging that cause tissue damage. In all organs, cells and extracellular matrix are known to deteriorate, and the mechanisms proposed are due to reactive oxygen species, radiation exposure, and repeated small damage.

[0017] Aging is one of the biggest known risk factors for most human diseases, and of the approximately 150,000 people who die every day worldwide, about two-thirds die from age-related causes. Aging involves dynamic biological, physiological, environmental, psychological, behavioral, and social process changes. As used herein, "symptoms of biological aging" can refer to the general signs and symptoms of aging, which may include, but are not limited to, deterioration of extracellular matrix, increased susceptibility to infection, increased risk of heat stroke or hypothermia, thinning and wrinkling of skin, fractures occurring more easily, joint changes ranging from mild stiffness to severe arthritis, slowing and limiting movement, overall decreased energy, constipation, urinary incontinence, cognitive impairment (e.g., slowing of thinking, memory, and thought), decreased reflexes and coordination, difficulty with balance, decreased vision, decreased peripheral vision, hearing loss, whitening or graying of hair, loss of smell, and weight loss, partly due to loss of muscle tissue.

[0018] Serine protease activity in organs outside the GI tract has been found to serve as a mechanism for the chronic and gradual loss of cellular and organ function (e.g., "autodigestion") during aging. Digestive enzymes are synthesized in the pancreas and then released into the small intestine, where they degrade a large number of biomolecules. In the small intestine, digestive enzymes are concentrated (e.g., at submillimolar levels), fully activated, and relatively nonspecific, aiding in the degradation of diverse polymeric food sources into lower molecular weight monomeric nutrients. Furthermore, intestinal autodigestion itself is largely prevented by compartmentalization of digestive enzymes in the intestinal lumen by a mucin / epithelial barrier that is always permeable to small molecule nutrients (e.g., ions, amino acids, or simple sugars), but which generally has low permeability to larger molecules such as pancreatic serine proteases. However, sometimes the mucin / epithelial barrier is compromised due to disease or condition, and sometimes the mucin / epithelial barrier becomes compromised during aging, as older individuals tend to have weaker mucin / epithelial barriers than younger individuals.

[0019] The present disclosure provides a mechanism for autodigestive aging involving serine proteases. The methods of the present disclosure block serine proteases outside the gastrointestinal (GI) tract while minimizing effects on serine protease activity inside the GI tract to ameliorate autodigestive aging symptoms and diseases.

[0020] Serine proteases / serine protease inhibitors Serine proteases, sometimes referred to as serine endopeptidases, are enzymes that can cleave peptide bonds in proteins. There are two main categories of serine proteases based on their structure: chymotrypsin-like (trypsin-like) and subtilisin-like. Subtilisin-like serine proteases can be found in prokaryotes and share the same catalytic mechanism as trypsin-like serine proteases. Chymotrypsin-like / trypsin-like serine proteases contain two beta-barrel domains that converge at the catalytic site. Serine proteases undergo folding in such a way that they utilize a catalytic triad located at the active site of the enzyme, which consists of three amino acids: histidine 57, serine 195, and aspartic acid 102. Furthermore, elastase is a serine protease produced by the pancreas that catalyzes the cleavage of carboxyl groups present on small hydrophobic amino acids, e.g., glycine, alanine, and valine. The primary role of elastase is to break down elastin, the protein that gives connective tissue its elasticity.

[0021] Serine proteases can be inhibited by serine protease inhibitors, which can include proteinaceous inhibitors as well as chemical inhibitors. In a non-limiting embodiment, low molecular weight inhibitors can leave the small intestine and enter blood, plasma, or other tissues. Serine protease inhibitors are sometimes referred to as SERPINs. Serine protease inhibitors can include competitive inhibitors, noncompetitive inhibitors, permeable inhibitors, reversible inhibitors, and irreversible inhibitors. Sometimes, serine protease inhibitors block serine proteases by changing the conformational shape of the serine protease to destroy the active site of the serine protease. Sometimes, serine protease inhibitors bind to the active site of the serine protease to block it.

[0022] Non-limiting examples of serine protease inhibitors include lepirudin, bivalirudin, argatroban, chymostatin, benzamidine, ximelagatran, rivaroxaban, idraparinux, apixaban, otamixaban, aprotinin, dabigatran etexilate, edoxaban, retaxaban, urinastatin, daraxaban, nafamostat, gabexate, sivelestat, melagatran, cholesterol sulfate, dabigatran, fondaparinux, desirudin, betrixaban, CGS-27023, GW-813893, berotralstat, evolocumab, conestat alfa, rosmarinic acid, alpha-1 antitrypsin, alpha-2 antiplasmin, BIA 10-2472, C1-inhibitor, camostat, cospin, CU-2010, CU-2020, kallistatin, Kazal domain, maspin, methoxyarachidonyl fluorophosphonate, microviridin, plasminogen activator inhibitor-1, plasminogen activator inhibitor-2, PMSF, protein C inhibitor, protein Z-related protease inhibitor, SERPINA9, SERPINB1, SERPINB3, SERPINB4, SERPINB6, SERPINB7, SERPINB8, SERPINB9, SERPINB13, SERPINE2, SPINT1, spaostat, and uterine serpin.

[0023] In some embodiments, the serine protease inhibitor of the disclosed method comprises nafamostat mesylate (Futhan), camostat mesylate (FOY 305), gabexate mesylate (FOY) or derivatives, serine protease inhibitor Kazal-1 type (SPINK1), tranexamic acid, granzyme A, granzyme B, UAMC-00050, 4-(2 aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF), soybean trypsin inhibitor, meprin inhibitor, setomelanotide, or alpha-1-antitrypsin. In some embodiments, the serine protease inhibitor can comprise a derivative of any one of the serine protease inhibitors described herein.

[0024] Pharmaceutical Compositions for the Treatment of Age-Related Conditions The methods described herein involve the use of pharmaceutical compositions that contain one or more serine protease inhibitors as active ingredients.

[0025] As used herein, the term "pharmaceutical composition" refers to a composition in which an active agent is formulated together with one or more pharma- ceutically acceptable carriers.In some embodiments, the composition is suitable for administration to human or animal subjects.In some embodiments, the active agent is present in a unit dose suitable for administration in a treatment regimen that shows a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population.

[0026] A pharmaceutical composition is typically formulated to be compatible with its intended route of administration, which includes parenteral, e.g., intravenous, subcutaneous, oral (e.g., capsule or inhalation), transmucosal, and rectal administration.

[0027] Methods for formulating suitable pharmaceutical compositions are known in the art, see, for example, Remington: The Science and Practice of Pharmacy, 21st ed., 2005, and Drugs and the Pharmaceutical Sciences: a Series of Textbooks and Monographs (Dekker, NY). For example, solutions or suspensions used for parenteral or subcutaneous application may contain the following components: sterile diluents, such as water for injection, saline, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents, antibacterial agents, such as benzyl alcohol or methylparabens, antioxidants, such as ascorbic acid or sodium bisulfite, chelating agents, such as ethylenediaminetetraacetic acid, buffers, such as acetates, citrates or phosphates, and agents for adjusting tonicity, such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0028] Pharmaceutical compositions suitable for injectable use can include sterile aqueous solutions (where water soluble) or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, sterile purified water, Cremophor EL™ (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it is preferable to include an isotonic agent in the composition, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride. Prolonged absorption of the injectable composition can be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin.

[0029] Sterile injectable solution can be prepared by incorporating the required amount of active compound into suitable solvent with one or combination of the above-listed components as required, followed by filtration sterilization.Generally, dispersion is prepared by incorporating active compound into sterile vehicle that contains basic dispersion medium and other components that are required among the above-listed components.For the preparation of sterile powder for sterile injectable solution, the preferred preparation method is vacuum drying and freeze-drying, which allows the powder of active ingredient and any additional desired ingredients to be obtained from the solution that has been previously sterilized by filtration.

[0030] Oral compositions generally contain an inert diluent or edible carrier. For the purpose of oral therapeutic administration, the active compound can be incorporated with an excipient and can be used in the form of tablets, troches, or capsules, such as gelatin capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash. Pharmaceutically compatible binders and / or adjuvant materials can be included as part of the composition. Tablets, pills, capsules, troches, etc. can contain any of the following ingredients, or compounds of similar nature: binders, such as microcrystalline cellulose, tragacanth or gelatin, excipients, such as starch or lactose, disintegrants, such as alginic acid, Primogel, or corn starch, lubricants, such as magnesium stearate or Sterotes, glidants, such as colloidal silicon dioxide, sweeteners, such as sucrose or saccharin, or flavorings, such as peppermint, methyl salicylate, or orange flavoring.

[0031] For administration by inhalation, the compounds can be delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer. Such methods include those described in U.S. Patent No. 6,468,798.

[0032] Systemic administration of pharmaceutical compositions as described herein can also be by transmucosal means.For transmucosal administration, a penetrant suitable for the barrier to be permeated is used in the formulation.Such penetrants are generally known in the art, and include, for example, for transmucosal administration, surfactants, bile salts, and fusidic acid derivatives.Transmucosal administration can be achieved by using nasal sprays or suppositories.

[0033] Pharmaceutical compositions can also be prepared in the form of suppositories (eg, with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[0034] In one embodiment, the pharmaceutical composition is prepared with a carrier that protects the pharmaceutical composition against rapid elimination from the body, for example, a controlled release formulation, including implants and microencapsulated delivery systems. In some embodiments, the pharmaceutical composition includes a serine protease inhibitor linked, conjugated, or fused to another molecule. In some embodiments, the other molecule changes the properties of the pharmaceutical composition. In some embodiments, the pharmaceutical composition can be delivered by using nanoparticle encapsulation. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Such formulations can be prepared using standard techniques or purchased, for example, from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to selected cells with monoclonal antibodies against cellular antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.

[0035] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

[0036] Methods for reducing serine protease activity outside the gastrointestinal (GI) tract - Patent Application 20070233633 Aging and / or age-related diseases or conditions may cause the intestinal barrier to increase in permeability to digestive serine proteases, so that serine protease activity may be detected in the circulating blood of subjects.Digestive enzymes may leak across the mucin-epithelial barrier into tissues and organs outside the pancreas and intestine, where they may damage extracellular matrix and cell membranes.In some embodiments, damage may include cleavage of ectodomain receptors.

[0037] Digestive enzymes can cause multiple forms of tissue damage, including cleavage of membrane receptors (e.g., insulin receptor, growth hormone receptor) and degradation of collagen in target organs. Pancreatic trypsin can also activate prohormones, and may disrupt physiological signaling due to its ability to cleave a wide range of humoral mediators and their receptors. Treatment by administering digestive enzyme inhibitors (e.g., serine protease inhibitors, e.g., trypsin inhibitors) can attenuate the disruption of mucin barriers, reducing the accumulation of digestive enzymes in peripheral organs, as well as the cleavage of collagen. For example, intervention in pancreatic trypsin outside the small intestine not only blocks the activation of secondary proteases, but also maintains a range of cellular functions, including but not limited to immune response, mitochondrial function, stem cell proliferation and differentiation, DNA repair mechanisms, epigenetics, protein folding, intracellular and intercellular signaling, and nutrient utilization.

[0038] The compositions described herein can be administered to a subject to treat or prevent the diseases, disorders or conditions described herein.In some embodiments, the present disclosure describes a method of selecting a subject at risk of damage to an organ and administering a therapeutically effective amount of a serine protease inhibitor to reverse the accumulation of serine proteases in the organ of the subject, reverse the cell damage in the organ of the subject, and / or preserve the extracellular matrix in the organ of the subject.In some embodiments, the present disclosure describes a method of treating a disease or condition of the brain, spinal cord, heart, muscle, kidney, liver, or lung.

[0039] In some embodiments, the present disclosure describes a method for reducing serine protease activity outside the gastrointestinal (GI) tract of a subject.In some embodiments, the method can inhibit or reduce the activity of serine proteases outside the gastrointestinal (GI) tract of a subject, or can alleviate symptoms of biological aging in a subject.In some embodiments, the method comprises administering to a subject in need thereof a therapeutically effective amount of a serine protease inhibitor that results in a reduction in the activity of serine proteases outside the GI tract.

[0040] In some embodiments, the method includes a step of prophylactically treating age-related disease or condition.As used herein, the term "prophylactically treating" can refer to taking preventive measures to maintain health or prevent the progression or occurrence of disease or condition (e.g., reverse the accumulation of serine proteases in a subject's organ, reverse cell damage in a subject's organ, and / or preserve extracellular matrix in a subject's organ).For example, a subject can be prophylactically treated when the subject is at risk of experiencing a disease or condition (e.g., has a biomarker that increases susceptibility to a particular condition, e.g., dementia).

[0041] subject As used herein, the term "subject" refers to an organism, typically a mammal (e.g., a human). In some embodiments, the subject is afflicted with the relevant disease, disorder, or condition. In some embodiments, the subject is susceptible to the disease, disorder, or condition. In some embodiments, the subject exhibits one or more symptoms or characteristics of the disease, disorder, or condition. In some embodiments, the subject does not exhibit any symptoms or characteristics of the disease, disorder, or condition. In some embodiments, the subject is one who has one or more characteristics characteristic of susceptibility to or risk for a disease, disorder, or condition. In some embodiments, the subject is a patient. In some embodiments, the subject is an individual to whom and / or to whom diagnosis and / or therapy is administered.

[0042] In some embodiments, the subject may be an animal, human or non-human. Non-limiting examples of non-human subjects may include mice, rats, hamsters, rabbits, cats, dogs, horses, pigs, donkeys, monkeys, and / or other non-human primates, such as apes and lemurs.

[0043] In some embodiments, the subject is a human. In some embodiments, the human patient can be an adult human or a young human (e.g., a human under 18 years of age). In some embodiments, the subject is a patient suffering from an age-related disease, disorder, or condition. In some embodiments, the subject is a patient with a susceptibility to an age-related disease, disorder, or condition. In some embodiments, the subject is a patient exhibiting one or more signs or symptoms or characteristics of an age-related disease, disorder, or condition. In some embodiments, the subject is exhibiting symptoms of an age-related disease, disorder, or condition when the subject is considered biologically old, e.g., over 50 years old, over 55 years old, over 60 years old, over 65 years old, over 70 years old, over 75 years old, over 80 years old, over 85 years old, over 90 years old, or over 95 years old. In some embodiments, the subject is exhibiting symptoms of an age-related disease, disorder, or condition when the subject is not considered biologically old, e.g., under 45 years old, under 40 years old, under 35 years old, under 30 years old, or under 25 years old. In some embodiments, the subject is an adult over 18 years of age. In some embodiments, the subject is older than 20 years of age. In some embodiments, the subject is older than 30 years of age. In some embodiments, the subject is older than 40 years of age. In some embodiments, the subject is older than 50 years of age. In some embodiments, the subject is older than 60 years of age. In some embodiments, the subject is older than 70 years of age. In some embodiments, the subject is older than 80 years of age. In some embodiments, the subject is older than 90 years of age. In some embodiments, the subject is older than 100 years of age.

[0044] Treating / Treatment As used herein, the term "treat" means a reduction in the number, frequency, severity, or duration of one or more (e.g., two, three, four, five, or six) symptoms of a disease or disorder in a subject (e.g., any of the subjects described herein) and / or which results in a reduction in the onset and / or worsening of one or more symptoms of a disease or disorder in a subject.

[0045] As used herein, the term "therapeutically effective amount" refers to an amount sufficient to treat a disease, disorder, and / or condition when administered according to a therapeutic dosing regimen to a population suffering from or susceptible to the disease, disorder, and / or condition. In some embodiments, a therapeutically effective amount is an amount that reduces the incidence and / or severity of one or more symptoms, stabilizes one or more characteristics, and / or delays the onset of a disease, disorder, and / or condition. Those skilled in the art will appreciate that the term "therapeutically effective amount" does not actually require that successful treatment be achieved in a particular individual. Instead, a therapeutically effective amount may be an amount that, when administered to a patient in need of such treatment, results in a particular desired pharmacological response in a significant number of subjects. For example, in some embodiments, the term "therapeutically effective amount" in the context of the therapy of the present invention refers to an amount that, when administered to an individual in need thereof, blocks, stabilizes, attenuates, or reverses the process underlying aging occurring in said individual, or enhances or increases the process of anti-aging in said individual. A "therapeutically effective amount" of the compositions described herein can reverse the occurrence of serine protease accumulation in a subject's organ (in a therapeutic treatment), reverse cellular damage in a subject's organ, or preserve the extracellular matrix structure in a subject's organ. A therapeutically effective amount can include preserving the molecular structure of organ tissue, as detected by hybridizing a peptide that can bind to collagen structure at the cleavage site. A therapeutically effective amount administered to an individual to treat a disease or condition in the individual can be the same or different from a therapeutically effective amount administered for prophylactic purposes.The therapeutic methods described herein are not intended to be interpreted as, limited to, or limited to "curing" aging, but rather are directed to the use of the compositions described to "treat" aging-related pathologies, i.e., to reverse the accumulation of serine proteases in the subject's organ, ongoing extracellular matrix protein (e.g., collagen) cleavage in the subject's organ, cell damage (e.g., membrane receptor cleavage) and cell dysfunction (e.g., integrin binding to extracellular matrix and reduced intracellular integrin signaling) in the subject's organ, or to preserve the extracellular matrix in the subject's organ, and other desirable or beneficial changes to the health of individuals with aging-related pathologies, such as, but not limited to.As understood in the art, the effective amount of serine protease inhibitors may vary depending on, among other things, the patient's medical history and other factors, such as the type (and / or dosage) of serine protease inhibitor used.

[0046] The phrases "decreased," "reduced," "reduced level," or "reduced level" and similar phrases generally refer to a decrease or reduction of at least 1% (e.g., at least 2%, at least 4%, at least 6%, at least 8%, at least 10%, at least 12%, at least 14%, at least 16%, at least 18%, at least 20%, at least 22%, at least 24%, at least 26%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) as compared to a reference level or value. The phrases "increased," "greater," "increased level," or "greater level" and similar phrases generally refer to an increase of at least 1% (e.g., at least 2%, at least 4%, at least 6%, at least 8%, at least 10%, at least 12%, at least 14%, at least 16%, at least 18%, at least 20%, at least 22%, at least 24%, at least 26%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, 100%, 150%, 200%, or more) compared to a reference level or value.

[0047] In some embodiments, the therapeutically effective amount can be formulated and / or administered in multiple doses, for example, as part of a dosing regimen. In some embodiments, the effective amount and schedule for administering the serine protease inhibitors described herein can be empirically determined, and making such a determination is within the skill of the art. Those skilled in the art will understand that the dosage that must be administered will vary depending, for example, on the subject receiving the serine protease inhibitors disclosed herein, the route of administration, the specific type of serine protease inhibitor, and other drugs administered to the subject. In some embodiments, administering a therapeutically effective amount includes chronic administration, while in other embodiments, administering a therapeutically effective amount includes scheduled administration.

[0048] In some embodiments, the scheduled administration comprises a predetermined schedule. Non-limiting examples of scheduled bases include every other day, every 2 days, every 3 days, every 4 days, every 5 days, every 6 days, or once a week. Other non-limiting examples of scheduled bases include 1 day on: 6 days off, 2 days on: 5 days off, 3 days on: 4 days off, 4 days on: 3 days off, 5 days on: 2 days off, 6 days on: 1 day off. Further non-limiting examples of scheduled bases include 2 days on: 1 day off, 2 days on: 2 days off, 2 days on: 3 days off, 2 days on-4 days off, 2 days on: 5 days off, 3 days on: 1 day off, 3 days on: 2 days off, 3 days on: 3 days off, 3 days on: 4 days off, 4 days on: 1 day off, 4 days on-2 days off, 4 days on: 3 days off, 5 days on: 1 day off, 5 days on: 2 days off, 6 days on: 1 day off. Further alternative non-limiting examples of a scheduled basis include 1 day every 7 days, 2 days every 7 days, 3 days every 7 days, 4 days every 7 days, 5 days every 7 days, or 6 days every 7 days. The method may include administering the composition according to a weekly protocol consisting of daily administration of the maintenance dose composition for 3-5 consecutive days, followed by 1-3 consecutive days of no administration.

[0049] In some embodiments, the subject may be administered the serine protease inhibitor for an extended period of time (e.g., for a period of at least 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1 year, 2 years, 3 years, 4 years, or 5 years). A skilled medical professional may determine the length of the treatment period using any of the methods described herein for diagnosing or tracking the effectiveness of the treatment (e.g., observation of at least one symptom of aging). As described herein, a skilled medical professional may also change (e.g., increase or decrease) the identity and number of serine protease inhibitors administered to the subject, and may adjust (e.g., increase or decrease) the dose or frequency of administration of the serine protease inhibitor to the subject based on the evaluation of the effectiveness of the treatment. In some embodiments, the serine protease inhibitor is administered for more than 1 week. In some embodiments, the serine protease inhibitor is administered for more than 2 weeks. In some embodiments, the serine protease inhibitor is administered for more than 4 weeks, hi some embodiments, the serine protease inhibitor is administered for more than 1 month, more than 2 months, more than 3 months, more than 4 months, more than 5 months, more than 6 months, more than 7 months, more than 8 months, more than 9 months, more than 10 months, more than 11 months, more than 12 months, or more.

[0050] In some embodiments, the serine protease inhibitor can be administered at a concentration lower than the serine protease concentration in the GI tract. In some embodiments, the serine protease inhibitor can be administered at a concentration less than 10% of the serine protease concentration in the GI tract. In some embodiments, the serine protease inhibitor can be administered at a concentration less than 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the serine protease concentration in the GI tract. For example, a low molecular weight serine protease competitive inhibitor (e.g., TXA or FOY) is administered at a concentration (e.g., 10 μM) lower than the serine protease concentration inside the small intestine (e.g., 100 μM) but matching and / or exceeding the protease concentration in plasma (e.g., 5 μM). In some embodiments, the administered serine protease inhibitor blocks the activity of serine proteases in plasma. In some embodiments, most of the digestive activity of the small intestine is preserved. In some embodiments, the concentration of the serine protease inhibitor does not interfere with or reduce the digestive or functional activity of the stomach and / or small intestine. The concentration of serine proteases in the GI tract can be determined empirically or with reference to standardized and accepted sources of such information.

[0051] In some embodiments, the concentration of the serine protease inhibitor to be administered to the subject can be determined by measuring serine protease activity in the subject. In some embodiments, the concentration of the serine protease inhibitor to be administered to the subject can be determined by measuring serine protease activity in the subject at a particular time point. In some embodiments, the concentration of the serine protease inhibitor to be administered to the subject can be determined by measuring serine protease activity outside the subject's GI tract (e.g., in plasma, in peripheral tissues). In some embodiments, the serine protease concentration in the GI tract is determined by mass spectrometry determination of peptide occurrence in plasma. For example, a sample of the patient's plasma can be passed through a mass spectrometer and proteolysis of plasma proteins can be determined. In some embodiments, the serine protease concentration can be determined by receptor cleavage with an antibody against the extracellular domain using cells taken from the subject or from the subject's plasma or other bodily fluids (e.g., lymph).

[0052] In some embodiments, the serine protease inhibitor can be administered at a concentration that is lower than the serine protease concentration in the GI tract, but higher than the serine protease concentration outside the GI tract (e.g., in plasma or peripheral tissues). In some embodiments, the serine protease inhibitor can be administered at about the same concentration as the serine protease concentration outside the GI tract. In some embodiments, the serine protease inhibitor can be administered at a concentration that is higher than the serine protease inhibitor concentration outside the GI tract. In some embodiments, the serine protease inhibitor can be administered at a concentration that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the concentration of serine protease in the plasma and / or tissues of the subject.

[0053] In some embodiments, the serine protease inhibitor may be administered at a concentration of less than 10 mM, less than 9 mM, less than 8 mM, less than 7 mM, less than 6 mM, less than 5 mM, less than 4 mM, less than 3 mM, less than 2 mM, less than 1 mM, less than 0.5 mM, or less than 0.1 mM.

[0054] In some embodiments, the serine protease inhibitor can be administered at a concentration of less than 50 μM (e.g., less than 48 μM, less than 46 μM, less than 44 μM, less than 42 μM, less than 40 μM, less than 38 μM, less than 36 μM, less than 34 μM, less than 32 μM, less than 30 μM, less than 28 μM, less than 26 μM, less than 24 μM, less than 22 μM, less than 20 μM, less than 18 μM, less than 16 μM, less than 14 μM, less than 12 μM, less than 10 μM, less than 8 μM, less than 6 μM, less than 5 μM, less than 4 μM, less than 3 μM, less than 2 μM, less than 1 μM, less than 0.5 μM, less than 0.25 μM, or less than 0.1 μM). In some embodiments, the serine protease inhibitor is administered at a concentration of less than 5 μM. In some embodiments, the serine protease inhibitor is less than 1 μM (e.g., less than 0.8 μM, less than 0.6 μM, less than 0.4 μM, less than 0.2 μM, less than 0.1 μM, less than 90 nM, less than 80 nM, less than 70 nM, less than 60 nM, less than 50 nM, less than 40 nM, less than 30 nM, less than 20 nM, less than 10 nM, less than 5 nM, less than 3 nM, less than 1 nM, less than 0.8 nM, less than 0 and / or less than 0.6 nM, less than 0.4 nM, less than 0.2 nM, less than 0.1 nM, less than 90 pM, less than 80 pM, less than 70 pM, less than 60 pM, less than 50 pM, less than 40 pM, less than 30 pM, less than 20 pM, less than 10 pM, less than 5 pM, less than 3 pM, less than 1 pM, less than 0.8 pM, less than 0.6 pM, less than 0.4 pM, less than 0.2 pM, or less than 0.1 pM).

[0055] In some embodiments, the serine protease inhibitor can be administered according to the patient's weight. In some embodiments, the serine protease inhibitor can be administered anywhere between 0.01 and 1.0 gm / kg / day. In some embodiments, the therapeutically effective amount of the serine protease inhibitor is 0.01 gm / kg / day, 0.02 gm / kg / day, 0.03 gm / kg / day, 0.04 gm / kg / day, 0.05 gm / kg / day, 0.06 gm / kg / day, or more. 0.07gm / kg / day, 0.08gm / kg / day, 0.09gm / kg / day, 0.1gm / kg / day, 0.11gm / kg / day, 0.12gm / kg / day, 0.13gm / kg / day, 0.14gm / kg / day, 0.15 gm / kg / day, 0.16gm / kg / day, 0.17gm / kg / day, 0.18gm / kg / day, 0.19gm / kg / day, 0.20gm / kg / day, 0.21gm / kg / day, 0.22gm / kg / day, 0.23gm / kg / day, 0.24gm / kg / day, 0.25gm / kg / day, 0.26gm / kg / day, 0.27gm / kg / day, 0.28gm / kg / day, 0.29gm / kg / day, 0.30gm / kg / day, 0.31gm / kg / day day, 0.32gm / kg / day, 0.33gm / kg / day, 0.34gm / kg / day, 0.35gm / kg / day, 0.36gm / kg / day, 0.37gm / kg / day, 0.38gm / kg / day, 0.39gm / kg / day, 0 .40gm / kg / day, 0.41gm / kg / day, 0.42gm / kg / day, 0.43gm / kg / day, 0.44gm / kg / day, 0.45gm / kg / day, 0.46gm / kg / day, 0.47gm / kg / day, 0.48 gm / kg / day, 0.49gm / kg / day, 0.50gm / kg / day, 0.51gm / kg / day, 0.52gm / kg / day, 0.53gm / kg / day, 0.54gm / kg / day, 0.55gm / kg / day, 0.56gm / day kg / day, 0.57gm / kg / day, 0.58gm / kg / day, 0.59gm / kg / day, 0.60gm / kg / day, 0.61gm / kg / day, 0.62gm / kg / day, 0.63gm / kg / day, 0.64gm / kg / day day, 0.65gm / kg / day, 0.66gm / kg / day, 0.67gm / kg / day, 0.68gm / kg / day, 0.69gm / kg / day, 0.70gm / kg / day, 0.71gm / kg / day, 0.72gm / kg / day, 0.73gm / kg / day, 0.74gm / kg / day, 0.75gm / kg / day, 0.76gm / kg / day, 0.77gm / kg / day, 0.78gm / kg / day, 0.79gm / kg / day, 0. 80gm / kg / day, 0.81gm / kg / day, 0.82gm / kg / day, 0.83gm / kg / day, 0.84gm / kg / day, 0.85gm / kg / day, 0.86gm / kg / day, 0.8 7 gm / kg / day, 0.88 gm / kg / day, 0.89 gm / kg / day, 0.90 gm / kg / day, 0.91 gm / kg / day, 0.92 gm / kg / day, 0.93 gm / kg / day, 0.94 gm / kg / day, 0.95 gm / kg / day, 0.96 gm / kg / day, 0.97 gm / kg / day, 0.98 gm / kg / day, 0.99 gm / kg / day, 1.0 gm / kg / day.

[0056] In some embodiments, depending on the identity of the serine protease inhibitor, a therapeutically effective amount of the serine protease inhibitor may include an amount greater than 1.0 gm / kg / day. For example, a therapeutically effective amount of the serine protease inhibitor may include an amount greater than 1.0 gm / kg / day, such as 1 gm / kg / day, 2 gm / kg / day, 3 gm / kg / day, 4 gm / kg / day, 5 gm / kg / day, 6 gm / kg / day, 7 gm / kg / day, 8 gm / kg / day, 9 gm / kg / day, 10 gm / kg / day, 11 gm / kg / day, 12 gm / kg / day, 13 gm / kg / day, 14 gm / kg / day, 15 gm / kg / day, 16 gm / kg / day, 17 gm / kg / day, 18 gm / kg / day, 19 gm / kg / day, 20 gm / kg / day, 21 gm / kg / day, 22 gm / kg / day, 23 gm / kg / day, 24 gm / kg / day, 25 gm / kg / day, 26 gm / kg / day, 27 gm / kg / day, 28 gm / kg / day, 29 gm / kg / day, 30 gm / kg / day, 31 gm / kg / day, 32 gm / kg / day, 33 gm / kg / day, 34 gm / kg / day, 35 gm / kg / day, 36 gm / kg / day, 37 gm / kg / day, 38 gm / kg / day, 39 gm / kg / day, 40 gm / kg / day, 41 gm / kg / day, 42 gm / kg / day, 43 gm / kg / day, 44 gm / kg / g / day, 23gm / kg / day, 24gm / kg / day, 25gm / kg / day, 26gm / kg / day, 27gm / kg / day, 28gm / kg / day, 29gm / kg / day, 30gm / kg / day, 31gm / kg / day, 32gm / kg / day, 33gm / kg / day, 3 4gm / kg / day, 35gm / kg / day, 36gm / kg / day, 37gm / kg / day, 38gm / kg / day, 39gm / kg / day, 40gm / kg / day, 41gm / kg / day, 42gm / kg / day, 43gm / kg / day, 44gm / kg / day, 45gm / kg / day, 46gm / kg / day, 47gm / kg / day, 48gm / kg / day, 49gm / kg / day, 50gm / kg / day, 51gm / kg / day, 52gm / kg / day, 53gm / kg / day, 54gm / kg / day, 55gm / kg / day, 56gm / kg / day, 57 gm / kg / day, 58gm / kg / day, 59gm / kg / day, 60gm / kg / day, 61gm / kg / day, 62gm / kg / day, 63gm / kg / day, 64gm / kg / day, 65gm / kg / day, 66gm / kg / day, 67gm / kg / day, 68gm / kg / day day, 69gm / kg / day, 70gm / kg / day, 71gm / kg / day, 72gm / kg / day, 73gm / kg / day, 74gm / kg / day, 75gm / kg / day, 76gm / kg / day, 77gm / kg / day, 78gm / kg / day, 79gm / kg / day, 80gm / kg / day, 81gm / kg / day, 82gm / kg / day, 83gm / kg / day, 84gm / kg / day, 85gm / kg / day, 86gm / kg / day, 87gm / kg / day, 88gm / kg / day, 89gm / kg / day, 90gm / kg / day, 91gm / kg / day,The dosage may include 92 gm / kg / day, 93 gm / kg / day, 94 gm / kg / day, 95 gm / kg / day, 96 gm / kg / day, 97 gm / kg / day, 98 gm / kg / day, 99 gm / kg / day, or 100 gm / kg / day.

[0057] Administration As used herein, the term "administration" typically refers to administration of a composition to a subject or system to achieve delivery of an agent that is or is contained in the composition. Those skilled in the art will be aware of the various routes that may be utilized for administration to a subject (e.g., a human) in the appropriate circumstances. For example, in some embodiments, administration may be ocular, oral, enteral, parenteral, and the like. In some specific embodiments, administration may be bronchial (e.g., by bronchial instillation), buccal, enteral, intra-arterial, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, intravesical, intracerebral, intravesical, intraspecific organ (e.g., intrahepatic), mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (e.g., by intratracheal instillation), vaginal, vitreous, by patch, and the like. In some embodiments, administration may involve only a single administration. In some embodiments, administration may involve the application of a certain number of doses. In some embodiments, administration may involve dosing that is intermittent (e.g., multiple doses separated by time) and / or periodic (e.g., individual doses separated by a common period of time) dosing. In some embodiments, administration may involve continuous dosing (e.g., perfusion) over at least a selected period of time. In some embodiments, administration may involve delivery methods including, but not limited to, the use of external and / or implanted infusion pumps, liquid formulations, encapsulated formulations, or sustained release encapsulations.

[0058] In some embodiments, the administration of the serine protease inhibitor can be ophthalmic, oral, parenteral, bronchial (e.g., by bronchial instillation), buccal, enteral, intra-arterial, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, intravesical, intracapsular, in a particular organ (e.g., in the liver), mucosal, nasal, oral, rectal, subcutaneous, sublingual, tracheal (e.g., by intratracheal instillation), vaginal, or vitreous. In some embodiments, the serine protease inhibitor is administered enterally, intraperitoneally, intravenously, intramuscularly, subcutaneously, intradermally, orally, intranasally, intrapulmonary, intrarectally, or by an external or implanted infusion pump controlled by telemetry. In some embodiments, the serine protease inhibitor is administered by oral administration. In some embodiments, the serine protease inhibitor is administered by an infusion pump controlled by telemetry. In some embodiments, the telemetry-controlled infusion pump is directed to a target tissue. In some embodiments, the target tissue may include adipose tissue, pancreatic tissue, liver tissue, kidney tissue, lung tissue, vasculature, bone tissue, central nervous system (CNS) tissue, eye tissue, muscle tissue, and secondary lymphoid organ tissue. In some embodiments, the target tissue may include a vein, an artery, a lymphatic vessel, cerebrospinal fluid, subcutaneous tissue, or a joint. In some embodiments, the telemetry-controlled infusion pump is directed to a target organ. In some embodiments, the target organ may include the brain, spinal cord, heart, kidney, muscle, liver, lung, pancreas, or any other organ. In some embodiments, the serine protease inhibitor is administered in the form of eye drops or liposome composition.

[0059] In some embodiments, the subject can be administered two or more serine protease inhibitors.In some embodiments, the administration of one or more serine protease inhibitors is sequential (e.g., administer one serine protease inhibitor, stop, and administer a second different serine protease inhibitor).In some embodiments, the subject can be administered a combination of serine protease inhibitors simultaneously.

[0060] In some embodiments, the method of the present disclosure can be administered before, in conjunction with, or after other methods or therapeutic treatments, either for the condition treated by administration of a serine protease inhibitor, or for another condition.In one non-limiting example, a subject can be treated by surgery for a cancerous mass in the intestine, and after surgery, the subject can be administered a serine protease inhibitor to limit protease leakage from the intestine.Preventive / prophylactic administration of a serine protease inhibitor can be used to delay and / or prevent the autodigestion of the subject's organs due to intestinal permeability.In another non-limiting example, a subject can be administered a serine protease inhibitor for the treatment of suspected dementia, while simultaneously receiving another medication for either the subject's dementia or another condition (e.g., diabetes).

[0061] In some embodiments, serine protease activity is increased during the postprandial period. To block this activity, a serine protease inhibitor can be administered before food intake (e.g., eating). In diabetes or prediabetes, this postprandial period during which serine protease activity is elevated can last for several hours longer than in nondiabetics. In some embodiments, the amount or concentration of serine protease inhibitor administered will depend on the measurement of protease activity in plasma or peripheral tissues, such as peritoneal fluid, heart, brain, intestine, kidney, liver, lung, eye, or other tissues disclosed herein. In some embodiments, a serine protease inhibitor is administered during the diurnal cycle, and the administration of the serine protease inhibitor depends on the serine protease activity measured in the subject.

[0062] Organ / Condition / Disease In some embodiments, the methods provided herein can alleviate symptoms of biological aging in a subject. In some embodiments, the subject can exhibit one or more signs or symptoms or characteristics of an age-related disease, disorder, or condition.

[0063] In some embodiments, the method involved selecting a subject at risk of damage to an organ. The subject may be at risk of damage to an organ because they exhibit symptoms consistent with a known disease or condition affecting that organ. The subject may be at risk of damage to an organ because they have a biomarker known to predispose to a known disease or condition affecting that organ. The subject may be at risk of damage to an organ because they have a family history believed to predispose to a known disease or condition affecting that organ. In some embodiments, the subject exhibits symptoms and / or biomarkers of elevated serine protease activity outside the GI tract (e.g., in plasma or in peripheral tissues).

[0064] With respect to the methods disclosed herein, the organ may be selected from the group consisting of the brain, spinal cord, heart, kidney, muscle, intestine, liver, eye, skeletal muscle, abdominal and subcutaneous adipose tissue, small and large intestine walls, connective tissue (including mesentery), breast tissue, tissue from the male and female reproductive tract, bone, cartilage, ear, nasal passages, and lung. In some embodiments, the organ may be selected from the group consisting of the brain, heart, intestine, and muscle. In some embodiments, the organ is the heart. In some embodiments, the organ is the brain. In some embodiments, the organ is the kidney. In some embodiments, the organ is the liver. In some embodiments, the organ is the intestine (e.g., small intestine and / or large intestine). In some embodiments, the organ is the eye. In some embodiments, the organ is the lung. For clarity, the methods of the present disclosure are not intended to treat shock (e.g., septic shock) or HIV. Additionally, the methods of the disclosure may further include screening the subject for shock (e.g., septic shock) and / or HIV, and not administering the serine protease inhibitor to the subject if the subject is currently experiencing shock or biomarkers for HIV.

[0065] In some embodiments, the age-related disease is Alzheimer's disease, age-related loss of neurological function including but not limited to memory loss, loss of balance, and loss of sensory function, pain including but not limited to back pain, aneurysms, chronic venous disease, cystic fibrosis, fibrosis in pancreatitis, glaucoma, hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease, degenerative disc disease, osteoarthritis, type 2 diabetes, lipodystrophy, lipodystrophy, atherosclerosis, cataracts, COPD, kidney transplant failure, liver cirrhosis, and the like. Fibrosis, bone loss, myocardial infarction, sarcopenia, wound healing, hair loss, myocardial hypertrophy, osteoarthritis, Parkinson's disease, age-related loss of lung tissue elasticity, age-related macular degeneration, cachexia, glomerulosclerosis, cirrhosis, nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), hepatitis A, hepatitis B, hepatitis C, autoimmune hepatitis, primary biliary cholangitis, primary sclerosing cholangitis, hemochromatosis, Wilson's disease, alpha-1 antitrypsin deficiency, liver cancer, bile duct Cancer, hepatic adenoma, osteoporosis, Huntington's disease, spinocerebellar ataxia, psychiatric disorders, neurodegeneration, epilepsy or other seizure disorders, stroke, cancer, dementia including frontotemporal dementia, meningitis, encephalitis, vascular disease, coronary heart disease, angina, unstable angina, heart failure, valvular disease, hypertension, cardiac arrhythmias, endocarditis, pericardial disease, cardiomyopathy, susceptibility to infection, chronic inflammation, renal dysfunction, rheumatoid arthritis, inflammatory bowel disease, lupus erythematosus, lupus nephritis, diabetic nephropathy, CNS damage, amyotrophic lateral sclerosis, fibromyalgia ... The conditions may include fibromyalgia, myositis including polymyositis and dermatomyositis, muscular dystrophy, myasthenia gravis, rhabdomyolysis, sarcopenia, Charcot-Marie-Tooth disease, myopathy, peripheral neuropathy, and spinal muscular atrophy, acute kidney injury, kidney stones, kidney infections including pyelonephritis, renal cysts, kidney cancer, a condition in which the subject requires kidney dialysis, pancreatic cancer, Crohn's disease, multiple sclerosis, Guillain-Barré syndrome, psoriasis, Graves' disease, ulcerative colitis, mood disorders, and / or cognitive disorders.

[0066] kit Also provided herein is a kit for use in the methods described herein.For example, the kit can include a composition comprising a serine protease inhibitor for oral administration.Instructions for use can also be included in the kit. EXAMPLES

[0067] [Example 1] Sample collection and serine protease inhibition in various organs of rats Animals and tissue collection Adult (4 months, 300-350 gm) and aged (24 months, 375-450 gm) male Wistar rats (Harlan Sprague Dawley Inc., Indianapolis, IN) were included in the study. Animals were maintained on a standard laboratory chow diet (8604 Teklad rodent diet; Harlan Laboratories, Indianapolis, IN) with ad libitum access to water and were maintained in separate rooms without sterile conditions. They were confirmed to exhibit normal motility, water and food consumption, and excretion of fecal material. Animals showing signs of morbidity were excluded. A subgroup of aged animals received a serine protease inhibitor (tranexamic acid, 14 days) in the drinking water (137 mM, changed daily), which resulted in a minimum dose of 0.39 gm / kg / day for a 350 gm body weight (BW) with a minimum water consumption of 40 ml / day.

[0068] A femoral vein catheter was placed after general anesthesia (local anesthesia with 2% lidocaine HCl [Hospira, Inc, Lake Forrest, IL] followed by sodium pentobarbital, 50 mg / kg [Abbott Laboratories, North Chicago, IL] intramuscularly). Tissues (intestine, liver, lung, heart, kidney, brain, mesentery (n=1)) were collected immediately after euthanasia (Beuthanasia, intravenous, 120 mg / kg, Schering-Plough Animal Health Corp, Union, NJ), fixed (formalin, 10%, neutral buffered, 1 h), postfixed (in fresh formalin solution, 24 h), and stored in formalin (10%). The period between initial anesthesia and fixation of the mesentery was kept less than 60 min to minimize activation or de novo synthesis of MMPs during tissue collection.

[0069] tissue section Formalin-fixed tissues were cut into 40 μm sections using a vibratome (Pelco Lancer Vibratome Series 1000).

[0070] Additionally, to generate thinner sections of the intestine, segments of the upper jejunum were embedded in Araldite resin (Polysciences, Washington, PA) and cut again into 1 μm sections (Ultramicrotome, LKB Ultratome Nova, diamond knife). The resin was removed (Maxwell Solution (one dip)), rinsed with tap water, incubated in hydrogen peroxide (4%, 1 min), rinsed (in phosphate buffer), and immunolabeled for trypsin.

[0071] Serine protease immunohistochemistry To determine immunolabeling density and distribution of serine proteases on tissue sections, pancreatic trypsin MoAb (D-1):sc-137077 (Santa Cruz) primary antibody was used, followed by secondary antibodies (MP-7601 for anti-rabbit IgG; MP-7602 for anti-mouse IgG; ImmPRESS Excel staining kit peroxidase). Two substrate colors were used, red (ImmPACT™ AEC substrate kit peroxidase, sk-4205; Vector® Laboratories) and brown (ImmPACT™ DAB substrate kit peroxidase, sk4105; and Vectorstain Elite ABC-HRP kit, Vector® Laboratories). Sections without primary antibody served as controls. No counterstain was applied to facilitate quantitative labeling intensity measurements, as cellular and vascular structures were easily identified. The concentration and exposure of the primary and secondary antibodies applied to the sections were adjusted (24 hours, respectively, according to the protocol by Vector® Laboratories) to achieve complete penetration of the antibodies into the tissue sections. All procedures were performed under standardized conditions to allow quantitative comparison of labeling density.

[0072] Whole-mount tissue labeling Small intestine: Full-thickness tissue blocks of the wall of the proximal jejunum (3 × 5 mm) were fixed on all sides in 10% formaldehyde. Serine proteases were detected with primary and secondary antibodies labeled with DAB (Peroxidase Substrate Kit, ab64238, ABCAM).

[0073] The mucin-containing mucus layer on the epithelial cells of the small intestine was stained using Alcian blue (pH 2.5, kt 003; Diagnostic BioSystems, Pleasanton, CA) followed by rinsing with distilled water and mounting on microscope slides (Vector Mount AQ Aqueous Mounting Medium, Vector Laboratories, Burlington, CA).

[0074] Mesentery: Trypsin distribution in intact mesenteric sectors was depicted by biotin / avidin immunolabeling with MoAb (D-1), secondary antibody (anti-mouse IgG, MP-7602, ImmPRESS Excel Staining Kit Peroxidase, Vector® Laboratories) and brown substrate (ImmPACT DAB sk-4105).

[0075] Collagen fragment labeling After removal of the resin, 1 μm sections were labeled with biotin-conjugated collagen hybridization peptide (B-CHP), which binds to unfolded collagen by triple helix formation and localizes molecular defects in collagen with high specificity.

[0076] Tissue sections were stained with B-CHP (stock solution, 150 mM; final applied solution 7.5 mM). Trimeric CHP was thermally dissociated into monomers prior to use (10 min at 80° C.), and this hot CHP solution was rapidly cooled to room temperature (by immersion in 4° C. water for 15 s), diluted, and immediately applied to sections (waiting time less than 1 min). In this way, most CHP peptides were expected to remain as active monomers during the staining process based on kinetic studies of CHP triple helix folding. Sections were incubated overnight at room temperature, and unbound B-CHP was removed by washing (3 times for 30 min in 1 ml 1×PBS at room temperature). To visualize B-CHP, tissue sections were incubated with streptavidin peroxidase (sk-5704, Vector® Laboratories, according to the manufacturer's instructions) followed by incubation with substrate (ImmPact AEC Substrate Kit Peroxidase; sk-4205, Vector Laboratories) at room temperature (1–10 min depending on the tissue). B-CHP labeling intensity in the sections was recorded by digital microscopy.

[0077] To label thin (1 μm) sections of small intestine for both mucin and trypsin, fixed intestines were immersed in a primary antibody against trypsin and stained with DAB substrate. The tissue was then embedded in resin and sectioned. A mucin label (Alcian Blue) was applied to the thin sections, coverslipped and imaged.

[0078] Glucose analysis At the time of tissue collection, fresh femoral arterial blood was used to measure blood glucose levels (Contour, Bayer Diabetes Care, Tarrytown, NY) and percent glycated hemoglobin levels (A1C Home Test; Bristol-Myers-Squibb Co; NY, NY).

[0079] Insulin receptor density (in the mesentery) Measurement of insulin receptor cleavage was performed by labeling its ectodomain with an antibody. Fixed tissue sections (10% formalin, neutral buffered) were identified with a primary antibody against the extracellular domain of the insulin receptor (Rα, N-20, sc-710 polyclonal antibody mapping to the N-terminus, Santa Cruz Biotech). The primary antibody was visualized using a biotin / avidin technique with a peroxidase enzyme substrate (ImmPACT AEC Substrate Kit sk-4205; Vector® Laboratories.). Sections without primary antibody were used as negative controls.

[0080] Digital image analysis Images of immunolabeling density were recorded at different magnifications, ranging from relatively low magnification overviews of tissues (10x objective, numerical aperture 0.25) to higher magnifications of single cells (60x oil immersion objective, numerical aperture 1.4). Images were recorded under standard light conditions with a digital camera (Spot Insight GIGABIT camera, Sterling Height) under a fixed setting of the substage condenser, so that the camera acts as a quantitative light intensity meter. Images were analyzed on a laboratory computer to minimize operator errors (NIH Image, 1.61, public domain software, spatial resolution 640x480 pixels).

[0081] The density of immunosubstrate labeling was measured in terms of light absorption (A) such that A=ln(I / Io), where I is the light intensity on the tissue and Io is the incident light intensity without tissue. For each organ, the mean labeling density per group (3 animals per group) was determined from the mean labeling density per animal (determined from 5 organ tissue sections / animal, 30 images / section).

[0082] statistics Measurements were summarized as the mean ± standard deviation. For comparison between two groups, unpaired two-tailed Student's t-test was used. Analysis of variance (ANOVA) was used to test for differences in outcomes of interest between groups. Results were determined to be significant at p<0.05. Bonferroni's post-hoc multiple comparison test was used to determine significance between individual groups. The number of animals used was estimated to minimize the number of experimental animals required to obtain statistically conclusive results, assuming equal variances between groups, α=0.05 and β=1-0.9. Animals were randomly assigned to test or control groups. No animals were excluded from the analysis and the study was blinded.

[0083] Results – Heart Immunohistochemistry was performed under standard conditions using mAB against pancreatic trypsin in rat myocardium for the young, old, and old-treated groups. Two weeks of treatment with oral trypsin inhibitors reduces pancreatic trypsin accumulation in old rats (100 weeks old; "old-treated") to levels similar to those seen in young rats (16 weeks old) (Figure 1A). Furthermore, two weeks of treatment with oral trypsin inhibitors reduces collagen fragmentation in the myocardium of old-treated rats to levels similar to those seen in young rats (16 weeks old) below those seen in old rats (Figure 1B). The results show that intervention with serine protease inhibitors prevents collagen fragmentation and improves organ function in old rats.

[0084] Results – Brain Immunohistochemistry was performed under standard conditions using mAB against pancreatic trypsin in rat brain for the young, old, and old-treated groups. Two weeks of treatment with oral trypsin inhibitors reduces pancreatic trypsin accumulation in old rats (100 weeks old; "old-treated") to a level similar to that seen in young rats (16 weeks old) (Figure 2A). Furthermore, two weeks of treatment with oral trypsin inhibitors reduces collagen fragmentation in rat brains of old-treated rats to a level similar to that seen in young rats (16 weeks old) below the level seen in old rats (Figure 2B). The results show that intervention with serine protease inhibitors prevents collagen fragmentation and improves organ function in old rats.

[0085] Results – Intestines Immunohistochemistry was performed under standard conditions using mAbs against pancreatic trypsin in rat intestine for young, old, and old-treated animals. Two weeks of treatment with oral trypsin inhibitors reduced pancreatic trypsin accumulation in old rats (100 weeks old) to levels similar to those seen in young rats (16 weeks old) (Figure 3A).

[0086] Villi in rat small intestine consist of elongated, pleated villi. The pleats are aligned parallel to the long axis of the intestine. Mucin in the villi of the small intestine is a barrier to digestive enzymes. Mucin labeling density is significantly decreased in aged rats, especially at the tips of the villi. Mucin labeling density was partially restored by orally administered trypsin inhibitor for 2 weeks in aged treated rats (Figure 3B).

[0087] [Example 2] Exemplary Treatment of Possible Conditions in Patients - Cardiac A patient visits a physician with shortness of breath and fatigue. The physician evaluates the patient and determines that the patient is likely to have symptoms of heart failure. The physician prescribes chronic administration of the pharmaceutical composition disclosed herein for the treatment of the heart failure symptoms. The patient's heart failure symptoms stabilize and begin to improve.

[0088] [Example 3] Exemplary Treatment of Possible Pathologies in Patients - Brain A patient visits a doctor with amnesia and memory problems.The doctor evaluates the patient and determines that the patient is likely to have mild cognitive impairment.The doctor prescribes chronic administration of the pharmaceutical composition disclosed herein for the treatment of mild cognitive impairment.The patient's mild cognitive impairment symptoms are stabilized.

[0089] [Example 4] Exemplary Treatment of Possible Pathologies in Patients - Kidneys A patient visits a physician with reduced urinary output and fatigue. The physician evaluates the patient and determines that the patient is likely to have symptoms of renal failure. The physician prescribes chronic administration of a pharmaceutical composition disclosed herein for treatment of the symptoms of renal failure. The patient's symptoms of renal failure improve.

[0090] [Example 5] Exemplary Treatment of Possible Pathologies in Patients - Liver A patient visits a physician with jaundice and nausea. The physician evaluates the patient and determines that the patient is likely to have symptoms of liver failure. The physician prescribes chronic administration of the pharmaceutical composition disclosed herein for treatment of the liver failure symptoms. The patient's liver failure symptoms stabilize.

[0091] [Example 6] Exemplary Treatment of Possible Conditions in Patients - Pulmonary A patient visits a physician with shortness of breath and cough. The physician evaluates the patient and determines that the patient is likely to have a pulmonary disease symptom. The physician prescribes chronic administration of the pharmaceutical composition disclosed herein for treatment of the pulmonary disease symptom. The patient's pulmonary disease symptom stabilizes.

[0092] [Example 7] Exemplary Treatment of Possible Pathologies in Patients - Intestine A patient visits a physician with indigestion, bloating, and nausea. The physician evaluates the patient and determines that the patient is likely to have intestinal perforation / leaky gut symptoms. The physician prescribes chronic administration of the pharmaceutical composition disclosed herein for treating the leaky gut symptoms. The patient's leaky gut symptoms stabilize.

[0093] [Example 8] Exemplary Treatment of Possible Conditions in Patients - Tremor A patient visits a doctor with tremors in the hands and difficulty in writing or drawing.The doctor evaluates the patient and determines that the patient is likely to have tremor symptoms.The doctor prescribes chronic administration of the pharmaceutical composition disclosed herein for treating the patient's tremor symptoms.One or more of the patient's tremor symptoms generally stabilize.

[0094] [Example 9] Exemplary Treatment of Possible Conditions in Patients - Unstable Blood Pressure A patient visits a physician with unstable blood pressure. The physician evaluates the patient and determines that the patient has unstable blood pressure requiring treatment. The physician prescribes chronic administration of a pharmaceutical composition disclosed herein for treatment of the patient's unstable blood pressure. The patient's blood pressure stabilizes.

[0095] [Example 10] Exemplary Treatment of General Biological Aging in Elderly Patients A relatively old patient visits a doctor with mild loss of muscle strength, mild loss of vision and hearing, and mild loss of energy compared to several years ago.The doctor evaluates the patient and determines that the patient does not currently meet the criteria for any particular disease or condition, and that these symptoms are likely due to biological aging.The doctor prescribes chronic administration of the pharmaceutical composition disclosed herein to treat the patient's symptoms.One or more of the patient's symptoms generally stabilize.

[0096] Other embodiments Although the invention has been described in conjunction with its detailed description, it is to be understood that the foregoing description is intended to illustrate, but not to limit, the scope of the invention as defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. 1. A method of reversing accumulation of a serine protease in an organ of a subject, comprising: (a) selecting a subject having or at risk of accumulation of serine proteases in said organ; and (b) administering a therapeutically effective amount of a serine protease inhibitor. thereby reversing accumulation of said serine protease in said organ of said subject.

2. 1. A method of reversing cellular damage in an organ of a subject, comprising: (a) selecting a subject having or at risk of cellular damage to said organ; and (b) administering a therapeutically effective amount of a serine protease inhibitor. thereby reversing cellular damage in said organ of said subject.

3. 1. A method for preserving extracellular matrix in an organ of a subject, comprising: (a) selecting a subject having or at risk of losing extracellular matrix in said organ; and (b) administering a therapeutically effective amount of a serine protease inhibitor. thereby preserving extracellular matrix in said organ of said subject.

4. 2. The method of any one of the preceding claims, wherein the subject is at least 40 years old.

5. 2. The method of any one of the preceding claims, wherein the subject is at least 50 years old.

6. 2. The method of any one of the preceding claims, wherein the subject is at least 60 years of age.

7. 10. The method according to any one of the preceding claims, wherein the subject is not at risk of developing shock and / or septic shock.

8. 2. The method of any one of the preceding claims, wherein the subject does not have HIV.

9. 10. The method of any one of the preceding claims, wherein the organ is selected from the group consisting of the brain, spinal cord, heart, kidney, muscle, liver, and lung.

10. 10. The method of any one of the preceding claims, wherein the organ is selected from the group consisting of the brain, the heart, and the muscle.

11. 10. The method of any one of the preceding claims, wherein the organ is the brain.

12. 2. The method of any one of the preceding claims, wherein the selecting step comprises selecting a subject having a brain disease or brain pathology.

13. 13. The method of claim 12, wherein the brain disease or pathology is selected from the group consisting of Alzheimer's disease, dementia including frontotemporal dementia, epilepsy or other seizure disorders, psychiatric disorders, multiple sclerosis, Huntington's disease, Parkinson's disease, amyotrophic lateral sclerosis, meningitis, encephalitis, brain tumors, Creutzfeldt-Jakob disease, chronic traumatic encephalopathy, long-term COVID-related dementia, and stroke.

14. 10. The method according to any one of the preceding claims, wherein the organ is the heart.

15. 10. The method of any one of the preceding claims, wherein selecting a subject comprises selecting a subject having a cardiac disease or condition.

16. 17. The method of claim 16, wherein the cardiac disease or condition is selected from the group consisting of coronary heart disease, angina, unstable angina, heart failure, cardiac arrhythmia, valvular disease, hypertension, cardiac arrhythmia, endocarditis, pericardial disease, and cardiomyopathy.

17. 10. The method according to any one of the preceding claims, wherein the organ is a muscle.

18. 10. The method of any one of the preceding claims, wherein selecting a subject comprises selecting a subject having a muscle disease or muscle pathology.

19. 19. The method of claim 18, wherein the muscle disease or condition is selected from the group consisting of fibromyalgia, myositis including polymyositis and dermatomyositis, muscular dystrophy, myasthenia gravis, amyotrophic lateral sclerosis, rhabdomyolysis, cardiomyopathy, sarcopenia, Charcot-Marie-Tooth disease, multiple sclerosis, myopathy, peripheral neuropathy, and spinal muscular atrophy.

20. 10. The method according to any one of the preceding claims, wherein the organ is a kidney.

21. 2. The method of any one of the preceding claims, wherein selecting a subject at risk comprises selecting a subject with a renal disease or condition.

22. 22. The method of claim 21, wherein the kidney disease or condition is selected from the group consisting of chronic kidney disease, diabetic kidney disease, acute kidney injury, kidney stones, kidney infections including pyelonephritis, kidney cysts, and kidney cancer.

23. 10. The method according to any one of the preceding claims, wherein the organ is the liver.

24. 10. The method of any one of the preceding claims, wherein the step of selecting a subject comprises the step of selecting a subject having a liver disease or liver pathology.

25. 25. The method of claim 24, wherein the liver disease or condition is selected from the group consisting of hepatitis A, hepatitis B, hepatitis C, autoimmune hepatitis, primary biliary cholangitis, primary sclerosing cholangitis, hemochromatosis, Wilson's disease, alpha-1 antitrypsin deficiency, hepatocarcinoma, cholangiocarcinoma, hepatic adenoma, nonalcoholic fatty liver disease, and nonalcoholic steatohepatitis.

26. 2. The method of any one of the preceding claims, wherein the serine protease comprises at least one of trypsin, subtilisin, or a combination thereof.

27. 2. The method of any one of the preceding claims, wherein the serine protease comprises at least one of trypsin, elastase, chymotrypsin, or a combination thereof.

28. 10. The method of any one of the preceding claims, wherein the serine protease comprises trypsin.

29. 10. The method of any one of the preceding claims, wherein the serine protease inhibitor is a competitive inhibitor.

30. 13. The method of any one of the preceding claims, wherein the serine protease inhibitor is selected from the group consisting of nafamostat mesylate (Futhan), camostat mesylate (FOY 305), gabexate mesylate (FOY) or derivatives, serine protease inhibitor Kazal type 1 (SPINK1), aprotinin, tranexamic acid, urinastatin, granzyme A, granzyme B, UAMC-00050, 4-(2 aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF), soybean trypsin inhibitor, meprin inhibitor, setomelanotide, alpha-1-antitrypsin, and serpin.

31. 2. The method of any one of the preceding claims, wherein the serine protease inhibitor comprises tranexamic acid.

32. 10. The method of any one of the preceding claims, wherein the therapeutically effective amount of the serine protease inhibitor is less than 10% of the subject's digestive enzyme activity.

33. 2. The method of any one of the preceding claims, wherein the therapeutically effective amount of said serine protease inhibitor is less than 10 μM.

34. 2. The method of any one of the preceding claims, wherein the therapeutically effective amount of said serine protease inhibitor is less than 5 μM.

35. 20. The method of any one of the preceding claims, wherein the serine protease inhibitor is administered enterally, intraperitoneally, intravenously, intramuscularly, subcutaneously, intradermally, orally, intranasally, pulmonary, rectally, or by a telemetry-controlled external or implanted infusion pump.

36. 13. The method of any one of the preceding claims, wherein the serine protease inhibitor is administered orally.

37. 10. The method of any one of the preceding claims, wherein the serine protease inhibitor is administered by a telemetry-controlled infusion pump.

38. 10. The method of any one of the preceding claims, wherein the serine protease inhibitor is administered as a liposomal composition or as a nanoparticle encapsulation.

39. 13. The method of any one of the preceding claims, wherein the serine protease inhibitor is administered as an eye drop.

40. 10. The method of any one of the preceding claims, wherein the telemetry-controlled infusion pump is directed to the organ.

41. 10. The method of any one of the preceding claims, wherein the serine protease inhibitor is administered for more than one week.

42. 10. The method of any one of the preceding claims, wherein the serine protease inhibitor is administered for more than two weeks.

43. 10. The method of any one of the preceding claims, wherein the serine protease inhibitor is administered for more than four weeks.

44. A pharmaceutical composition for the treatment of aging or age-related conditions comprising a serine protease inhibitor.

45. 20. The pharmaceutical composition of any one of the preceding claims, wherein the age-related pathology affects an organ selected from the group consisting of the brain, spinal cord, heart, kidneys, muscles, liver, and lungs.

46. 20. The pharmaceutical composition of any one of the preceding claims, wherein the age-related pathology affects an organ selected from the group consisting of the brain, the heart, and muscles.

47. 2. The pharmaceutical composition according to any one of the preceding claims, wherein the organ is the brain.

48. 10. The pharmaceutical composition of any one of the preceding claims, wherein the age-related condition is selected from the group consisting of Alzheimer's disease, dementia including frontotemporal dementia, age-related loss of neuronal function including but not limited to memory, balance, sensation, pain, epilepsy or other seizure disorders, psychiatric disorders, multiple sclerosis, Huntington's disease, Parkinson's disease, amyotrophic lateral sclerosis, meningitis, encephalitis, brain tumors, and transient ischemic attacks.

49. 2. The pharmaceutical composition according to any one of the preceding claims, wherein the organ is the heart.

50. 20. The pharmaceutical composition of any one of the preceding claims, wherein the age-related pathology is selected from the group consisting of coronary heart disease, angina, unstable angina, heart failure, valvular disease, hypertension, cardiac arrhythmias, endocarditis, pericardial disease, and cardiomyopathy.

51. 2. The pharmaceutical composition according to any one of the preceding claims, wherein the organ is a muscle.

52. 10. The pharmaceutical composition of any one of the preceding claims, wherein the age-related pathology is selected from the group consisting of fibromyalgia, myositis including polymyositis and dermatomyositis, muscular dystrophy, myasthenia gravis, amyotrophic lateral sclerosis, rhabdomyolysis, cardiomyopathy, sarcopenia, Charcot-Marie-Tooth disease, multiple sclerosis, myopathy, peripheral neuropathy, and spinal muscular atrophy.

53. 2. The pharmaceutical composition according to any one of the preceding claims, wherein the organ is the kidney.

54. 20. The pharmaceutical composition of any one of the preceding claims, wherein the age-related condition is selected from the group consisting of acute kidney injury, kidney stones, kidney infections including pyelonephritis, kidney cysts, subjects requiring renal dialysis, and kidney cancer.

55. 2. The pharmaceutical composition according to any one of the preceding claims, wherein the organ is the liver.

56. 10. The pharmaceutical composition of any one of the preceding claims, wherein the age-related pathology is selected from the group consisting of hepatitis A, hepatitis B, hepatitis C, autoimmune hepatitis, primary biliary cholangitis, primary sclerosing cholangitis, hemochromatosis, Wilson's disease, alpha-1 antitrypsin deficiency, hepatocarcinoma, cholangiocarcinoma, hepatic adenoma, nonalcoholic fatty liver disease, and nonalcoholic steatohepatitis.

57. 2. The pharmaceutical composition according to any one of the preceding claims, wherein the serine protease inhibitor is a competitive inhibitor.

58. 10. The pharmaceutical composition of any one of the preceding claims, wherein the serine protease inhibitor is selected from the group consisting of nafamostat mesylate (Futhan), camostat mesylate (FOY 305), gabexate mesylate (FOY) or derivatives, serine protease inhibitor Kazal type 1 (SPINK1), aprotinin, tranexamic acid, urinastatin, granzyme A, granzyme B, UAMC-00050, 4-(2 aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF), soybean trypsin inhibitor, meprin inhibitor, setomelanotide, alpha-1-antitrypsin, and serpin.

59. 2. The pharmaceutical composition of any one of the preceding claims, wherein the serine protease inhibitor comprises tranexamic acid.

60. 2. The pharmaceutical composition of any one of the preceding claims, wherein the serine protease inhibitor is administered at less than 10% of the subject's digestive enzyme activity.

61. 2. The pharmaceutical composition of any one of the preceding claims, wherein the serine protease inhibitor is less than 10 μM.

62. 2. The pharmaceutical composition of any one of the preceding claims, wherein the serine protease inhibitor is less than 5 μM.

63. 10. The pharmaceutical composition of any one of the preceding claims, wherein the serine protease inhibitor is administered enterally, intraperitoneally, intravenously, intramuscularly, subcutaneously, intradermally, orally, intranasally, pulmonary, rectally, or by a telemetry-controlled external or implanted infusion pump.

64. 20. The pharmaceutical composition of any one of the preceding claims, wherein the serine protease inhibitor is administered orally.

65. 10. The pharmaceutical composition according to any one of the preceding claims, wherein the serine protease inhibitor is administered by a telemetry-controlled infusion pump.

66. 10. The pharmaceutical composition according to any one of the preceding claims, wherein the serine protease inhibitor is administered as a liposomal composition or nanoparticles.

67. 10. The pharmaceutical composition of any one of the preceding claims, wherein the serine protease inhibitor is administered as an eye drop.

68. 2. The pharmaceutical composition of any one of the preceding claims, wherein the telemetry-controlled infusion pump is directed to the organ.

69. 10. The pharmaceutical composition according to any one of the preceding claims, wherein the serine protease inhibitor is administered for more than one week.

70. 10. The pharmaceutical composition according to any one of the preceding claims, wherein the serine protease inhibitor is administered for more than two weeks.

71. 10. The pharmaceutical composition according to any one of the preceding claims, wherein the serine protease inhibitor is administered for more than four weeks.

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