Method for high-throughput screening of compounds and combinations of compounds for discovery and quantification of actions, particularly unanticipated therapeutic or toxic actions, in biological systems

Abstract

The invention enables high-throughput screening of compounds in living systems to detect unanticipated or unintended biological actions. The invention also allows for screening, detection, and confirmation of new indications for approved drugs. Screening and detection of toxic effects of compounds also can be achieved by using the methods of the invention. The methods comprise administering isotope-labeled substrates to a living system so that the label is incorporated into molecules in a manner that reveals flux rates through metabolic pathways thought to be involved in a disease. Comparisons between living systems exposed to compounds and living systems not so exposed reveals the effects of the compounds on the flux rates through the metabolic pathways. Combinations or mixtures of compounds can be systematically screened to detect unanticipated or unintended biological actions, including synergistic actions, in the same manner.

1 . A method for high-throughput screening (HTS) of one or more compounds for actions on molecular flux rates in one or more metabolic pathways, said method comprising: 
 a) exposing said one or more compounds to a living system;    b) administering an isotope-labeled substrate to said living system for a period of time sufficient for said isotope-labeled substrate to enter into and pass through a metabolic pathway of interest and thereby enter into and label a targeted molecule or molecules of interest within said one or more metabolic pathways in said living system;    c) obtaining one or more samples from said living system, wherein said one or more samples comprise one or more isotope-labeled targeted molecules of interest;    d) measuring the content, rate of incorporation and/or pattern or rate of change in content and/or pattern of isotope labeling of said targeted molecule or molecules of interest;    e) calculating molecular flux rates in said one or more metabolic pathways of interest;    f) measuring the molecular flux rates in said one or more metabolic pathways of interest according to steps b) through e) in a living system not administered said one or more compounds; and    g) comparing said molecular flux rates in said one or more metabolic pathways of interest in said living system administered said one or more compounds to said molecular flux rates in said one or more metabolic pathways in said living system not administered said one or more compounds to screen said compounds for one or more actions on said molecular flux rates.    
     
     
         2 . The method of  claim 1 , wherein the molecular flux rates in said one or more metabolic pathways of interest being measured are relevant to an underlying molecular pathogenesis, or causation of, one or more diseases.  
     
     
         3 . The method of  claim 2 , wherein the molecular flux rates in said one or more metabolic pathways of interest contribute to the initiation, progression, severity, pathology, aggressiveness, grade, activity, disability, mortality, morbidity, disease sub-classification or other underlying pathogenic or pathologic feature of the disease of interest.  
     
     
         4 . The method of  claim 2 , wherein the molecular flux rates in said metabolic pathways of interest contribute to the prognosis, survival, morbidity, mortality, stage, therapeutic response, symptomology, disability or other clinical factor of the disease of interest.  
     
     
         5 . The method of  claim 2 , wherein the molecular flux rates of said one or more metabolic pathways of two or more diseases are measured concurrently.  
     
     
         6 . The method of  claim 5 , wherein the concurrent measurement of the molecular flux rates from said metabolic pathways of interest is achieved by use of stable isotopic labeling techniques.  
     
     
         7 . The method of  claim 6 , wherein the isotope label used is a stable, non-radioactive isotope.  
     
     
         8 . The method of  claim 7 , wherein the stable isotope used is stable isotope-labeled water.  
     
     
         9 . The method of  claim 8 , wherein the stable isotope-labeled water is  2 H 2 O.  
     
     
         10 . The method of  claim 5 , wherein the concurrent measurement of the molecular flux rates from said metabolic pathways of interest is achieved by use of radioisotope labeling techniques.  
     
     
         11 . The method of  claim 1 , wherein said one or more compounds comprise one or more known drug agents.  
     
     
         12 . The method of  claim 11 , wherein the one or more known drug agents are Federal Food and Drug Administration-approved drug agents.  
     
     
         13 . The method of  claim 11 , wherein said one or more drug agents are selected randomly.  
     
     
         14 . The method of  claim 11 , wherein said one or more drug agents are selected on the basis of a specific biochemical rationale or hypothesis concerning a hypothesized role in the molecular pathogenesis of one or more diseases.  
     
     
         15 . The method of  claim 1 , wherein said one or more compounds comprise one or more new chemical entities.  
     
     
         16 . The method of  claim 1 , wherein said one or more compounds comprise one or more biological factors.  
     
     
         17 . The method of  claim 1 , wherein said action on molecular flux rates in one or more metabolic pathways of interest comprises unintended, unexpected, or unanticipated actions.  
     
     
         18 . The method of  claim 17 , wherein said unintended, unexpected, or unanticipated actions comprise one or more secondary therapeutic indications for said one or more compounds.  
     
     
         19 . The method of  claim 18 , wherein one or more animal models of disease are used for evaluating said one or more secondary therapeutic indications.  
     
     
         20 . The method of  claim 19 , wherein said animal model of disease is chosen from Alzheimer's disease, heart failure, renal disease, diabetic nephropathy, osteoporosis, hepatic fibrosis, cirrhosis, hepatocellular necrosis, pulmonary fibrosis, scleroderma, renal fibrosis, multiple sclerosis, arteriosclerosis, osteoarthritis, rheumatoid arthritis, psoriasis, skin photoaging, skin rashes, breast cancer, prostate cancer, colon cancer, pancreatic cancer, lung cancer, acquired immunodeficiency syndrome, immune defects, multiple myeloma, chronic lymphocytic leukemia, chronic myelocytic leukemia, diabetes, diabetic complications, insulin resistance, obesity, lipodystrophy, metabolic syndrome, muscle wasting, frailty, deconditioning, angiogenesis, hyperlipidemia, infertility, viral or bacterial infections, auto-immune disorders, and immune flares.  
     
     
         21 . The method of  claim 18 , wherein the molecular flux rates in said metabolic pathways of interest related to said one or more secondary therapeutic indications are measured in response to a specific dose or a range of doses of said one or more compounds.  
     
     
         22 . The method of  claim 1 , wherein said one or more metabolic pathways are chosen from hepatocyte proliferation and destruction, renal tubular cell turnover, lymphocyte turnover, spermatocyte turnover, protein synthesis and breakdown in muscle and heart, liver collagen synthesis and breakdown, myelin synthesis and breakdown in brain or peripheral nerves, breast epithelial cell proliferation, colon epithelial cell proliferation, prostate epithelial cell proliferation, ovarian epithelial cell proliferation, endometrial cell proliferation, bronchial epithelial cell proliferation, pancreatic epithelial cell proliferation, keratin synthesis in skin, keratinocyte proliferation, carbohydrate metabolism, immunoglobulin synthesis, synthesis and breakdown of mitochondrial DNA, synthesis and breakdown of mitochondrial phospholipids, synthesis and breakdown of mitochondrial proteins, synthesis and breakdown of adipose lipids, and synthesis and breakdown of adipose cells.  
     
     
         23 . The method of  claim 12 , wherein said approved drug agents are screened for actions on multiple biochemical processes concurrently.  
     
     
         24 . The method of  claim 23 , wherein said approved drug agents are chosen from statins, glitazones, COX-2 inhibitors, NSAIDS, β-blockers, calcium channel blockers, ACE inhibitors, antibiotics, antiviral agents, hypolipidemic agents, antihypertensives, anti-inflammatory agents, antidepressants, anxiolytics, anti-psychotics, sedatives, analgesics, antihistamines, oral hypoglycemic agents, antispasmodics, antineoplastics, cancer chemotherapeutic agents, sex steroids, pituitary hormones, cytokines, chemokines, appetite suppressant agents, thyromimetics, anti-seizure agents, sympathomimetics, sulfa drugs, biguanides, and other classes of agents.  
     
     
         25 . The method of  claim 17 , wherein said unanticipated or unintended action comprises at least one toxic effect of said one or more compounds.  
     
     
         26 . The method of  claim 25 , wherein said toxic effect comprises at least one end-organ toxicity.  
     
     
         27 . The method of  claim 26 , wherein said end-organ toxicity is chosen from hepatocyte proliferation and destruction, renal tubular cell turnover, lymphocyte turnover, spermatocyte turnover, protein synthesis and breakdown in muscle and heart, liver collagen synthesis and breakdown, myelin synthesis and breakdown in brain or peripheral nerves, breast epithelial cell proliferation, colon epithelial cell proliferation, prostate epithelial cell proliferation, ovarian epithelial cell proliferation, endometrial cell proliferation, bronchial epithelial cell proliferation, pancreatic epithelial cell proliferation, keratin synthesis in skin, keratinocyte proliferation, immunoglobulin synthesis, synthesis and breakdown of mitochondrial DNA, synthesis and breakdown of mitochondrial phospholipids, synthesis and breakdown of mitochondrial proteins, synthesis and breakdown of adipose lipids, and synthesis and breakdown of adipose cells.  
     
     
         28 . The method of  claim 26 , wherein the molecular flux rates of said metabolic pathways of interest related to said end-organ toxicity are measured in response to a specific dose or a range of doses of said one or more compounds.  
     
     
         29 . The method of  claim 1 , wherein said living system is chosen from prokaryotic cells, eukaryotic cells, cell lines, cell cultures, isolated tissue preparations, rabbits, dogs, mice, rats, guinea pigs, pigs, non-human primates, and humans.  
     
     
         30 . The method of  claim 1 , wherein said living system is a human.  
     
     
         31 . The method of  claim 1 , wherein said isotope-labeled substrate is chosen from  2 H 2 O, H 2   18 O,  2 H-glucose,  2 H-labeled amino acids,  2 H-labeled organic molecules,  13 C-labeled organic molecules,  13 CO 2 ,  15 N-labeled organic molecules,  3 H 2 O,  3 H-labeled glucose,  3 H-labeled amino acids,  3 H-labeled organic molecules,  14 C-labeled organic molecules, and  14 CO 2 .  
     
     
         32 . The method of  claim 1 , wherein said isotope-labeled substrate is  2 H 2 O.  
     
     
         33 . The method of  claim 1 , wherein said one or more compounds are administered according to established or hypothesized dose ranges that have the potential for biological activity in said living system.  
     
     
         34 . The method of  claim 1 , wherein said one or more samples are collected at known times or intervals after administration or contacting said living system to said isotope-labeled substrate and after exposing said living system to said one or more compounds.  
     
     
         35 . The method of  claim 1 , wherein combinations of two or more compounds are exposed to said living system.  
     
     
         36 . The method of  claim 35 , wherein synergistic, complementary, or antagonistic actions of combinations of compounds on molecular flux rates through the metabolic pathway of interest are determined by comparing said molecular flux rates in said living systems exposed to the combination of compounds to said molecular flux rates in said living systems exposed to a single compound alone or not exposed to any of said compounds being tested.  
     
     
         37 . The method of  claim 35 , wherein said combinations of compounds are selected randomly.  
     
     
         38 . The method of  claim 35 , wherein said combinations of compounds are selected on the basis of a specific biochemical rationale or hypothesis concerning a hypothesized role of one or more of said compounds in the molecular pathogenesis of one or more of said diseases.  
     
     
         39 . The method of  claim 17 , wherein unanticipated or unintended actions identified for said one or more compounds is developed for approval as a potential new medical indication for said one or more compounds.  
     
     
         40 . Compounds so identified by use of the methods disclosed herein.  
     
     
         41 . An information storage device comprising data obtained from the method according to  claim 1 .  
     
     
         42 . The device of  claim 41 , wherein said device is a printed report.  
     
     
         43 . The printed report of  claim 42 , wherein the medium in which said report is printed on is chosen from paper, plastic, and microfiche.  
     
     
         44 . The device of  claim 41 , wherein said device is a computer disc.  
     
     
         45 . The disc of  claim 44 , wherein said disc is chosen from a compact disc, a digital video disc, and a magnetic disc.  
     
     
         46 . The device of  claim 41 , wherein said device is a computer.  
     
     
         47 . An isolated isotopically-perturbed molecule generated by the method according to  claim 1 .  
     
     
         48 . The isolated isotopically-perturbed molecule of  claim 47 , wherein said molecule is chosen from protein, lipid, nucleic acid, glycosaminoglycan, proteoglycan, porphyrin, and carbohydrate molecules.  
     
     
         49 . The isolated isotopically-perturbed molecule of  claim 47 , wherein said molecule is myelin.  
     
     
         50 . The isolated isotopically-perturbed molecule of  claim 47 , wherein said molecule is amyloid-β.  
     
     
         51 . The isolated isotopically-perturbed molecule of  claim 47 , wherein said molecule is deoxyribonucleic acid.  
     
     
         52 . The isolated isotopically-perturbed molecule of  claim 47 , wherein said molecule is ribonucleic acid.  
     
     
         53 . The isolated isotopically-perturbed molecule of  claim 47 , wherein said molecule is collagen.  
     
     
         54 . The isolated isotopically-perturbed molecule of  claim 47 , wherein said molecule is triglyceride.  
     
     
         55 . A kit for determining high-throughput screening (HTS) of one or more compounds for actions on molecular flux rates in one or more metabolic pathways potentially related to disease in a subject, comprising: 
 a) one or more isotope-labeled precursors, and    b) instructions for use of the kit.    
     
     
         56 . The kit of  claim 55  further comprising a tool for administration of precursor molecules.  
     
     
         57 . The kit of  claim 55  further comprising an instrument for collecting a sample from the subject.  
     
     
         58 . The method of  claim 1 , further comprising the manufacture of one or more drugs at least partially identified by said method of  claim 1 .  
     
     
         59 . A method of determining whether a compound alters the metabolic flux rate of one or more metabolic pathways, comprising: 
 a) treating a living system with a compound;    b) administering an isotope-labeled substrate to said living system for a period of time sufficient for said isotope-labeled substrate to enter into and pass through a metabolic pathway of interest and thereby enter into and label a targeted molecule or molecules of interest within said one or more metabolic pathways in said living system;    c) obtaining one or more samples from said living system, wherein said one or more samples comprise one or more isotope-labeled targeted molecules of interest;    d) measuring the content, rate of incorporation and/or pattern or rate of change in content and/or pattern of isotope labeling of said targeted molecule or molecules of interest;    e) calculating molecular flux rates in said one or more metabolic pathways of interest;    f) measuring the molecular flux rates in said one or more metabolic pathways of interest according to steps b) through e) in a living system not administered said compound; and    g) comparing said molecular flux rates in said one or more metabolic pathways of interest in said living system administered said compound to said molecular flux rates in said one or more metabolic pathways in said living system not administered said compound to determine whether said compound alters said metabolic flux rate.    
     
     
         60 . The method of  claim 59  wherein the isotope label is a stable, non-radioactive isotope.  
     
     
         61 . The method of  claim 60  wherein said stable isotope is stable isotope-labled water.