THERAPY FOR NEUROLOGICAL DISEASES/DISORDERS
20210330611 · 2021-10-28
Inventors
- Katerina Akassoglou (San Francisco, CA)
- Anke Meyer-Franke (Menlo Park, CA, US)
- Alex Pico (San Francisco, CA, US)
- Kean-Hooi Ang (San Francisco, CA, US)
- Michelle Rose Arkin (San Francisco, CA, US)
Cpc classification
A61K31/7048
HUMAN NECESSITIES
A61K45/06
HUMAN NECESSITIES
A61K31/165
HUMAN NECESSITIES
A61K31/137
HUMAN NECESSITIES
A61K31/5415
HUMAN NECESSITIES
A61K31/165
HUMAN NECESSITIES
A61K31/475
HUMAN NECESSITIES
A61K31/137
HUMAN NECESSITIES
A61K2300/00
HUMAN NECESSITIES
A61K2300/00
HUMAN NECESSITIES
A61K31/475
HUMAN NECESSITIES
A61K31/5415
HUMAN NECESSITIES
A61K31/7048
HUMAN NECESSITIES
International classification
A61K31/137
HUMAN NECESSITIES
A61K31/165
HUMAN NECESSITIES
A61K31/475
HUMAN NECESSITIES
A61K31/5415
HUMAN NECESSITIES
A61K31/7048
HUMAN NECESSITIES
Abstract
Provided herein are compositions and methods to treat or prevent neurodegeneration in a mammal, inhibit microglial activation in the CNS of a mammal, promote survival of CNS neurons in a mammal, prevent or reduce the rate of demyelination and/or neuronal injury in a mammal, promote remyelination in a mammal and/or treat or prevent or decrease oxidative stress in a mammal.
Claims
1. A method to treat or prevent neurodegeneration in a mammal comparing administering to said mammal an effect amount of a composition comprising a modulator of calcium signaling, a modulator of microtubule dynamics, a modulator of chemokine signaling, a modulator of DNA replication, a modulator of dopamine receptor signaling, a modulator of cAMP signaling, a modulator of glucocorticoid-receptor signaling, a modulator of purine nucleotide biosynthesis, a modulator of neurotransmitter transport or a combination thereof or one or more of the small molecules provided in Table 1 and/or Table 2, provided that the small molecule is not acivicin.
2. A method for inhibiting microglial activation in the CNS of a mammal and/or promoting survival of CNS neurons in a mammal, comprising administering to a mammal in need thereof an effective amount of a composition comprising a modulator of calcium signaling, a modulator of microtubule dynamics, a modulator of chemokine signaling, a modulator of DNA replication, a modulator of dopamine receptor signaling, a modulator of cAMP signaling, a modulator of glucocorticoid-receptor signaling, a modulator of purine nucleotide biosynthesis, a modulator of neurotransmitter transport or a combination thereof.
3. A method for preventing or reducing the rate of demyelination and/or neuronal injury in a mammal and/or promoting remyelination in a mammal and/or treating or preventing or decreasing oxidative stress in a mammal in need thereof comprising administering to the mammal an effective amount of a modulator of calcium signaling, a modulator of microtubule dynamics, a modulator of chemokine signaling, a modulator of DNA replication, a modulator of dopamine receptor signaling, a modulator of cAMP signaling, a modulator of glucocorticoid-receptor signaling, a modulator of purine nucleotide biosynthesis, a modulator of neurotransmitter transport or a combination thereof.
4. The method of claim 1, wherein the modulator of calcium signaling is a calcium channel blocker, a vasodilator or an adrenoreceptor agonist.
5. The method of claim 4, wherein the calcium channel blocker comprises fendiline.
6. The method of claim 4, wherein the vasodilator comprises nylidrin.
7. The method of claim 1, wherein the modulator of microtubule dynamics is an inhibitor of microtubule assembly.
8. The method of claim 7, wherein the inhibitor of microtubule assembly comprises vinblastine, colchicine and/or podofilox.
9. The method of claim 1, wherein the modulator of chemokine signaling comprises tannic acid.
10. The method of claim 1, wherein the modulator of DNA replication is an inhibitor of DNA topoisomerase II.
11. The method of claim 10, wherein the inhibitor of DNA topoisomerase II comprises teniposide.
12. The method of claim 1, wherein the modulator of dopamine receptor signaling is blocker of dopamine receptors.
13. The method of claim 12, wherein the blocker of dopamine receptors comprises prochlorperazine or thioridazine.
14. The method of claim 1, wherein the modulator of cAMP signaling comprises nylidrin, prochlorperazine or thioridazine.
15. The method of claim 1, wherein the modulator of glucocorticoid-receptor signaling comprises betamethasone, dexamethasone, dexamethasone acetate, dexamethasone sodium phosphate, fludrocortisone acetate, fluocinolone acetonide, fluocinonide, fluorometholone, flurandrenolide, hydrocortisone, hydrocortisone acetate, hydrocortisone butyrate, hydrocortisone hemisuccinate, hydrocortisone sodium phosphate, methylprednisolone, prednisolone or triamcinolone diacetate.
16. The method of claim 1, wherein the modulator of purine nucleotide biosynthesis is an inhibitor of inosine-5′-monophosphate dehydrogenase (IMPDH).
17. The method of claim 16, wherein the inhibitor of inosine-5′-monophosphate dehydrogenase (IMPDH) comprises mycophenolic acid (MPA).
18. The method of claim 1, wherein the modulator of neurotransmitter transport is an inhibitor of norepinephrine reuptake.
19. The method of claim 18, wherein the inhibitor of norepinephrine reuptake is maprotiline.
20. The method of claim 1, wherein said mammal has been diagnosed with a disease, disorder, or injury involving demyelination, dysinyelination, or neurodegeneration.
Description
BRIEF DESCRIPTION OF THE DRAWINGS AND APPENDICES
[0033] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:
[0034]
[0035]
[0036]
[0037]
[0038]
[0039]
[0040]
[0041]
[0042]
[0043]
[0044]
[0045]
[0046]
[0047]
[0048]
DETAILED DESCRIPTION OF THE INVENTION
[0049] The practice of the methods and compositions described herein may employ, unless otherwise indicated, conventional techniques of pharmaceutical chemistry, drug formulation techniques, dosage regimes, and biochemistry, all of which are within the skill of those who practice in the art. Such conventional techniques include the use of combinations of therapeutic regimes including but not limited to the methods described herein; technologies for formulations of adjunct therapies used in combination with known, conventional therapies and/or new therapies for the treatment of neurodegeneration, delivery methods that are useful for the compositions of the invention, and the like. Specific illustrations of suitable techniques can be had by reference to the examples herein.
[0050] Oxidative stress is a central part of innate-immune induced neurodegeneration. However, the transcriptomic landscape of the central nervous system (CNS) innate immune cells contributing to oxidative stress is unknown, and therapies to target their neurotoxic functions are not widely available.
[0051] Provided herein is the oxidative stress innate immune cell atlas in neuroinflammatory disease and disclosure of the discovery of new druggable pathways. Transcriptional profiling of oxidative stress-producing CNS innate immune cells (Tox-seq) identified a core oxidative stress gene signature coupled to coagulation and glutathione pathway genes shared between a microglia cluster and infiltrating macrophages. Tox-seq followed by a microglia high-throughput screen (HTS) and oxidative stress gene network analysis, identified several candidates including the glutathione regulator acivicin with potent therapeutic effects decreasing oxidative stress and axonal damage in chronic and relapsing models of multiple sclerosis (MS). Thus, oxidative stress transcriptomics identified neurotoxic CNS innate immune populations and can enable the discovery of selective neuroprotective strategies.
[0052] In the following description, numerous specific details are set forth to provide a more thorough understanding of the present invention. However, it will be apparent to one of skill in the art that the present invention may be practiced without one or more of these specific details. In other instances, features and procedures well known/available to those skilled in the art have not been described in order to avoid obscuring the invention.
Definitions
[0053] For the purposes of clarity and a concise description, features can be described herein as part of the same or separate embodiments; however, it will be appreciated that the scope of the invention may include embodiments having combinations of all or some of the features described.
[0054] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The following definitions are intended to aid the reader in understanding the present invention but are not intended to vary or otherwise limit the meaning of such terms unless specifically indicated.
[0055] As used herein, the indefinite articles “a”, “an” and “the” should be understood to include plural reference unless the context clearly indicates otherwise. Thus, for example, reference to “an inhibitor” refers to one or more agents with the ability to inhibit a target molecule, and reference to “the method” includes reference to equivalent steps and methods known to those skilled in the art, and so forth.
[0056] The phrase “and/or,” as used herein, should be understood to mean “either or both” of the elements so conjoined, e.g., elements that are conjunctively present in some cases and disjunctively present in other cases.
[0057] As used herein, “or” should be understood to have the same meaning as “and/or” as defined above. For example, when separating a listing of items, “and/or” or “or” shall be interpreted as being inclusive, e.g., the inclusion of at least one, but also including more than one, of a number of items, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of” or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e., “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.”
[0058] As used herein, the term “about” means plus or minus 10% of the indicated value. For example, about 100 means from 90 to 110.
[0059] Where a range of values is provided, it is understood that each intervening value, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either both of those included limits are also included in the invention.
[0060] A “CNS disorder” can be any disease, disorder or injury associated with the toxicity of a population of cells within the CNS. In one example, the CNS disorder is associated with a pathological process such as neurodegeneration, demyelination, dysmyelination, axonal injury, and/or dysfunction or death of an oligodendrocyte or a neuronal cell, or loss of neuronal synapsis/connectivity. In other examples, the CNS disorder is a disease associated with plaque formation, e.g., amyloid plaque formation. CNS disorders include neurodegenerative disorders that affect the brain or spinal cord of a mammal. In certain embodiments, the CNS disorder has one or more inflammatory components.
[0061] The term “neurodegenerative diseases” includes any disease or condition characterized by problems with movements, such as ataxia, and conditions affecting cognitive abilities (e.g., memory) as well as conditions generally related to all types of dementia. “Neurodegenerative diseases” may be associated with impairment or loss of cognitive abilities, potential loss of cognitive abilities and/or impairment or loss of brain cells. Exemplary “neurodegenerative diseases” include Alzheimer's disease (AD), diffuse Lewy body type of Alzheimer's disease, Parkinson's disease, Down syndrome, progressive multiple sclerosis (MS), dementia, mild cognitive impairment (MCI), amyotrophic lateral sclerosis (ALS), traumatic brain injuries, ischemia, stroke, cerebral ischemic brain damage, ischemic or hemorrhaging stroke, multi-infarct dementia, hereditary cerebral hemorrhage with amyloidosis of the Dutch-type, cerebral amyloid angiopathy (including single and recurrent lobar hemorrhages), neurodegeneration induced by viral infection (e.g. AIDS, encephalopathies) and other degenerative dementias, including dementias of mixed vascular and degenerative origin, dementia associated with Parkinson's disease, dementia associated with progressive supranuclear palsy and dementia associated with cortical basal degeneration, epilepsy, seizures, and Huntington's disease.
[0062] As used herein, a disease, disorder or condition is “treated” if at least one pathophysiological measurement associated with the disease is decreased and/or progression of a pathophysiological process is reversed, halted or reduced. For example, a disease, disorder or condition can be “treated” if the number of plaques present in the CNS of a patient with a neurodegenerative disease is reduced, remains constant, or the creation of new plaques is slowed by the administration of an agent. In another example, a disease, disorder or condition can be “treated” if one or more symptoms of the disease or disorder is reduced, alleviated, terminated, slowed, or prevented. Measurement of one or more exemplary clinical hallmarks and/or symptoms of a disease can be used to aid in determining the disease status in an individual and the treatment of one or more symptoms associated therewith.
[0063] The term “administering” as used herein refers to administering to a subject and/or contacting a neuron or portion thereof with an inhibitor as described herein. This includes administration of the inhibitor to a subject in which the neuron is present, as well as introducing the inhibitor into a medium in which a neuron is cultured. Administration “in combination with” one or more further agents includes concurrent and consecutive administration, in any order.
[0064] The term “neuron” as used herein denotes nervous system cells that include a central cell body or soma, and two types of extensions or projections: dendrites, by which, in general, the majority of neuronal signals are conveyed to the cell body, and axons, by which, in general, the majority of neuronal signals are conveyed from the cell body to effector cells, such as target neurons or muscle. Neurons can convey information from tissues and organs into the central nervous system (afferent or sensory neurons) and transmit signals from the central nervous systems to effector cells (efferent or motor neurons). Other neurons, designated interneurons, connect neurons within the central nervous system (the brain and spinal column). Certain specific examples of neuron types that may be subject to treatment according to the invention include cerebellar granule neurons, dorsal root ganglion neurons, and cortical neurons.
[0065] The terms “mammal” and “mammalian subject” as used herein refers to any animal classified as a mammal, including humans, higher non-human primates, rodents, and domestic and farm animals, such as cows, horses, dogs, and cats. In some embodiments of the invention, the mammal is a human.
[0066] The term “pharmaceutical composition” refers to a formulation containing the disclosed compounds in a form suitable for administration to a subject. In one embodiment, the pharmaceutical composition is in bulk or in unit dosage form. The unit dosage form is any of a variety of forms, including, for example, a tablet, capsule, or a vial. The quantity of active ingredient in a unit dose of composition is an effective amount and is varied according to the particular treatment involved.
[0067] The phrase “therapeutically effective amount” or “effective amount” used in reference to an agent of the invention is an art-recognized term. In certain embodiments, the term refers to an amount of an agent that produces some desired effect at a reasonable benefit/risk ratio applicable to any medical treatment. In certain embodiments, the term refers to that amount necessary or sufficient to eliminate, reduce or maintain a target of a particular therapeutic regimen. The effective amount may vary depending on such factors as the disease or condition being treated, the particular targeted constructs being administered, the size of the subject or the severity of the disease or condition. One of ordinary skill in the art may empirically determine the effective amount of a particular compound without necessitating undue experimentation.
[0068] “Inhibitors,” “activators,” and “modulators” are used to refer to activating, inhibitory, or modulating (increase, inhibit, decrease or activate expression or activity as compared to control (an untreated or healthy subject/mammal) molecules. Inhibitors are compounds that, e.g., bind to, partially or totally block activity, decrease, prevent, delay activation, inactivate, desensitize, or down regulate the activity or expression. “Activators” are compounds that increase, open, activate, facilitate, enhance activation, sensitize, agonize, or up regulate activity, e.g., agonists.
[0069] In certain embodiments, a therapeutically effective amount of an agent for in vivo use will likely depend on a number of factors, including: the rate of release of an agent from a polymer matrix, which will depend in part on the chemical and physical characteristics of the polymer; the identity of the agent; the mode and method of administration; and any other materials incorporated in the polymer matrix in addition to the agent. In certain embodiments, a therapeutically effective amount is the amount effective to induce endogenous oligodendrocyte precursor cell differentiation and/or maturation, thereby promoting myelination in the subject's central nervous system.
[0070] As used herein, the terms “including”, “includes”, “having”, “has”, “with”, or variants thereof, are intended to be inclusive similar to the term “comprising.”
[0071] As used herein, said “contain”, “have” or “including” include “comprising”, “mainly consist of”, “basically consist of” and “formed of”; “primarily consist of”, “generally consist of” and “comprising of” belong to generic concept of “have” “include” or “contain”.
GENERAL DESCRIPTION OF THE INVENTION
[0072] Discovery of small molecule compounds that inhibit innate immune activation: To identify druggable pathways shared by both microglia and macrophages, function-selective transcriptomics was combined with drug network analysis of pathways identified through a small molecule screen in primary microglia. A high-content, high-throughput screen (HTS) was developed to discover new small molecule inhibitors that suppress microglia activation. Primary microglia were stimulated by the innate immune activators fibrin or lipopolysaccharide (LPS). 1,907 clinical drugs and bioactive compounds were screened using increased cell size (≥800 μm.sup.2) as a marker of activation and decreased size (<150 μm.sup.2) as a marker of toxicity. 128 compounds were identified that inhibited fibrin-induced microglia activation by ≥50% without toxicity (≤3% cell death) (Table 1). A total of 31 compounds, of which 27 of them inhibited fibrin- and/or LPS-induced microglia activation, with known molecular targets and potential clinical relevance for neurological diseases (Table 2) were selected to generate a “microglia drug-target network” containing ten subnetworks targeting different pathways: chemokine signaling, GGT, calcium signaling, purine nucleotide biosynthesis, cAMP signaling, neurotransmitter transport, DNA replication, dopamine-receptor signaling, microtubule dynamics, multidrug resistance, and glucocorticoid-receptor signaling.
[0073] Agents/Compounds
[0074] Agents of the invention include, but are not limited to: Modulators of calcium signaling include, but are not limited to, a calcium channel blocker, a vasodilator, β2 adrenoreceptor agonist, fendiline (fendiline hydrochloride) and/or nylidrin (buphenine, nylidrin hydrochloride).
[0075] Modulators of microtubule dynamics include, but are not limited to, inhibitors of microtubule assembly, vinblastine (vinblastine sulfate), colchicine and/or podofilox.
[0076] Modulators of chemokine signaling include, but are not limited to, tannic acid.
[0077] Modulators of DNA replication include, but are not limited to, teniposide (an inhibitor or DNA topoisomerase II).
[0078] Modulators of beta2-adrenoreceptor signaling, alpha 2-adrenergic receptor signaling, and alpha 1 adrenergic receptor signaling include, but are not limited to, Ritodrine Hydrochloride, Levonordefrin, Salmeterol, Xylazine Hydrochloride, Idazoxan Hydrochloride, Naftopidil Dihydrochloride
[0079] Modulators of progesterone include, but are not limited to Medroxyprogesterone Acetate, Melengestrol Acetate.
[0080] Modulators of dopamine receptor signaling include, but are not limited to, prochlorperazine (prochlorperazine dimaleate) (blocker of dopamine receptors) and/or thioridazine (thioridazine hydrochloride)(antagonist of dopamine; blocks dopamine receptors).
[0081] Modulators of cAMP signaling include, but are not limited to, nylidrin (buphenine, nylidrin hydrochloride), prochlorperazine (prochlorperazine dimaleate) and/or thioridazine (thioridazine hydrochloride).
[0082] Modulators of glucocorticoid-receptor signaling (including glucocorticoid receptor (NR3C1)) include, but are not limited to, betamethasone, dexamethasone, dexamethasone acetate, dexamethasone sodium phosphate, fludrocortisone acetate, fluocinolone acetonide, fluocinonide, fluorometholone, flurandrenolide, hydrocortisone, hydrocortisone acetate, hydrocortisone butyrate, hydrocortisone hemisuccinate, hydrocortisone sodium phosphate, methylprednisolone, prednisolone, and/or triamcinolone diacetate.
[0083] Modulators of purine nucleotide biosynthesis include, but are not limited to, inhibitors of inosine-5′-monophosphate dehydrogenase (IMPDH) and/or mycophenolic acid (MPA; mycophenolate).
[0084] Modulators of neurotransmitter transport include, but are not limited to, an inhibitor of norepinephrine reuptake and/or maprotiline (maprotiline hydrochloride).
[0085] Administration
[0086] Pharmaceutical formulations of the agents described herein are prepared by combining the agent having the desired degree of purity with optional physiologically acceptable carriers, excipients, or stabilizers (see, e.g., Remington's Pharmaceutical Sciences (18th edition), ed. A. Gennaro, 1990, Mack Publishing Co., Easton, Pa.). Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and can include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid, BHA, and BHT; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt forming counter-ions such as sodium; and/or nonionic surfactants such as Tween, Pluronics, or PEG.
[0087] Agents to be used for in vivo administration can be sterile, which can be achieved by filtration through sterile filtration membranes, prior to or following lyophilization and reconstitution. Therapeutic compositions may be placed into a container having a sterile access port, for example, an intravenous solution bag or vial.
[0088] Agents described herein can be optionally combined with or administered in concert with each other or other agents known to be useful in the treatment of the relevant disease or condition.
[0089] Thus, in the treatment of demyelinating diseases, the agents can be administered in combination with injectable compositions including interferon beta 1a inhibitors or interferon beta 1b inhibitors, glatiramer acetate, and daclizumab; oral medications such as teriflunomide, fingolimod, and dimethyl fumarate; or infused medications such as alemtuzumab, mitoxantrone, or natalizumab.
[0090] In the treatment of Alzheimer's disease, agents can be administered with acetylcholinesterase inhibitors (e.g., donepezil, galantamine, and rivastigmine) and/or NMDA receptor antagonists (e.g., memantine).
[0091] In the treatment of ALS, for example, agents can be administered in combination with Riluzole (Rilutek), minocycline, insulin-like growth factor 1 (IGF-1), and/or methylcobalamin.
[0092] In another example, in the treatment of Parkinson's disease, agents can be administered with L-dopa, dopamine agonists (e.g., bromocriptine, pergolide, pramipexole, ropinirole, cabergoline, apomorphine, and lisuride), dopa decarboxylase inhibitors (e.g., levodopa, benserazide, and carbidopa), and/or MAO-B inhibitors (e.g., selegiline and rasagiline).
[0093] The combination therapies can involve concurrent or sequential administration, by the same or different routes, as determined to be appropriate by those of skill in the art. The invention also includes pharmaceutical compositions and kits.
[0094] The route of administration of the agents is selected in accordance with known methods, e.g., injection or infusion by intravenous, intraperitoneal, intracerebral, intramuscular, intraocular, intraarterial or intralesional routes, topical administration, or by sustained release systems as described below.
[0095] For intracerebral use, the agents can be administered continuously by infusion into the fluid reservoirs of the CNS, although bolus injection may be acceptable. The agents can be administered into the ventricles of the brain or otherwise introduced into the CNS or spinal fluid. Administration can be performed by use of an indwelling catheter and a continuous administration means such as a pump, or it can be administered by implantation, e.g., intracerebral implantation of a sustained-release vehicle. More specifically, the agents can be injected through chronically implanted cannulas or chronically infused with the help of osmotic minipumps. Subcutaneous pumps are available that deliver proteins through a small tubing to the cerebral ventricles. Highly sophisticated pumps can be refilled through the skin and their delivery rate can be set without surgical intervention. Examples of suitable administration protocols and delivery systems involving a subcutaneous pump device or continuous intracerebroventricular infusion through a totally implanted drug delivery system are those used for the administration of dopamine, dopamine agonists, and cholinergic agonists to Alzheimer's disease patients and animal models for Parkinson's disease, as described by Harbaugh, J. Neural Transm. Suppl. 24:271, 1987; and DeYebenes et al., Mov. Disord. 2:143, 1987.
[0096] Suitable examples of sustained release preparations include semipermeable polymer matrices in the form of shaped articles, e.g., films or microcapsules. Sustained release matrices include polyesters, hydrogels, polylactides (U.S. Pat. No. 3,773,919; EP 58,481), copolymers of L-glutamic acid and gamma ethyl-L-glutamate (Sidman et al., Biopolymers 22:547, 1983), poly (2-hydroxyethyl-methacrylate) (Langer et al., J. Biomed. Mater. Res. 15:167, 1981; Langer, Chem. Tech. 12:98, 1982), ethylene vinyl acetate (Langer et al., Id), or poly-D-(−)-3-hydroxybutyric acid (EP 133,988A). Sustained release compositions also include liposomally entrapped compounds, which can be prepared by methods known per se (Epstein et al., Proc. Natl. Acad. Sci. U.S.A. 82:3688, 1985; Hwang et al., Proc. Natl. Acad. Sci. U.S.A. 77:4030, 1980; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324A). Ordinarily, the liposomes are of the small (about 200-800 Angstroms) unilamelar type in which the lipid content is greater than about 30 mol % cholesterol, the selected proportion being adjusted for the optimal therapy.
[0097] A therapeutically effective amount of an agent will depend, for example, upon the therapeutic objectives, the route of administration, and the condition of the patient. Accordingly, it will be necessary for the therapist to titer the dosage and modify the route of administration as required to obtain the optimal therapeutic effect. A typical daily dosage might range from, for example, about 1 μg/kg to up to 100 mg/kg or more (e.g., about 1 μg/kg to 1 mg/kg, about 1 μg/kg to about 5 mg/kg, about 1 mg/kg to 10 mg/kg, about 5 mg/kg to about 200 mg/kg, about 50 mg/kg to about 150 mg/mg, about 100 mg/kg to about 500 mg/kg, about 100 mg/kg to about 400 mg/kg, and about 200 mg/kg to about 400 mg/kg), depending on the factors mentioned above. Typically, the clinician will administer an active inhibitor until a dosage is reached that results in improvement in or, optimally, elimination of, one or more symptoms of the treated disease or condition. The progress of this therapy is easily monitored by conventional assays. One or more agent provided herein may be administered together or at different times (e.g., one agent is administered prior to the administration of a second agent). One or more agent may be administered to a subject using different techniques (e.g., one agent may be administered orally, while a second agent is administered via intramuscular injection or intranasally). One or more agent may be administered such that the one or more agent has a pharmacologic effect in a subject at the same time. Alternatively, one or more agent may be administered, such that the pharmacological activity of the first administered agent is expired prior the administration of one or more secondarily administered agents.
[0098] One skilled in the art, upon reading the present specification, will appreciate that it is sometimes necessary to make routine variations to the dosage depending on the age and condition of the patient. The dosage will also depend on the route of administration. A variety of routes are contemplated, including oral, pulmonary, rectal, parenteral, transdermal, subcutaneous, intravenous, intramuscular, intraperitoneal, intranasal, inhalational, and the like. Dosage forms for the topical or transdermal administration of a compound described herein includes powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, nebulized compounds, and inhalants. In a preferred embodiment, the active compound is mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants that are required.
[0099] The present invention also provides a therapeutic kit containing materials useful for the treatment or prevention of the disorders and conditions described above is provided. The therapeutic kit may include a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. The containers may be formed from a variety of materials such as glass or plastic. The container holds a pharmaceutical composition that is by itself or when combined with another agent effective for treating or preventing the condition and may have a sterile access port (e.g., an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the pharmaceutical composition is one of the agents described herein above. The label or package insert indicates that the composition is used for treating the condition of choice. Moreover, the kit may include (a) a first container with a pharmaceutical composition contained therein, wherein the composition includes an agent described herein; and (b) a second container with a pharmaceutical composition contained therein, wherein the composition includes a different agent. The therapeutic kit in this embodiment of the invention may further include a package insert indicating that the compositions can be used to treat a particular condition. Alternatively, or additionally, the therapeutic kit may further include a second (or third) container including a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.
[0100] Assessment of Treatment
[0101] In some embodiments, the successful treatment of a subject with an agent described herein is determined by at least about a 10%-100% decrease in one or more symptoms of a CNS disorder. Examples of such symptoms include, but are not limited to, slowness of movement, loss of balance, depression, decreased cognitive function, short-term memory loss, long-term memory loss, confusion, changes in personality, language difficulties, loss of sensory perception, sensitivity to touch, numbness in extremities, tremors, ataxia, muscle weakness, muscle paralysis, muscle cramps, muscle spasms, significant changes in eating habits, excessive fear or worry, insomnia, delusions, hallucinations, fatigue, back pain, chest pain, digestive problems, headache, rapid heart rate, dizziness, and visual changes.
[0102] For example, clinical signs of MS are routinely classified and standardized, e.g., using an EDSS rating system based on neurological examination and long-distance ambulation. As used herein, the “Expanded Disability Status Scale” or “EDSS” is intended to have its customary meaning in the medical practice. EDSS is a rating system that is frequently used for classifying and standardizing MS. The accepted scores range from 0 (normal) to 10 (death due to MS). Typically, patients having an EDSS score of about 4-6 will have moderate disability (e.g., limited ability to walk), whereas patients having an EDSS score of about 7 or 8 will have severe disability (e.g., will require a wheelchair). More specifically, EDSS scores in the range of 1-3 refer to an MS patient who is fully ambulatory, but has some signs in one or more functional systems; EDSS scores in the range higher than 3 to 4.5 show moderate to relatively severe disability; an EDSS score of 5 to 5.5 refers to a disability impairing or precluding full daily activities; EDSS scores of 6 to 6.5 refer to an MS patient requiring intermittent to constant, or unilateral to bilateral constant assistance (cane, crutch or brace) to walk; EDSS scores of 7 to 7.5 means that the MS patient is unable to walk beyond five meters even with aid, and is essentially restricted to a wheelchair; EDSS scores of 8 to 8.5 refer to patients that are restricted to bed; and EDSS scores of 9 to 10 mean that the MS patient is confined to bed, and progressively is unable to communicate effectively or eat and swallow, until death due to MS.
[0103] In certain embodiments, the evaluation of disease progression includes a measure of upper extremity function (e.g., a 9HP assessment). Alternatively, or in combination, disease progression includes a measure of lower extremity function. Alternatively, or in combination, disease progression includes a measure of ambulatory function, e.g., short distance ambulatory function (e.g., T25FW). Alternatively, or in combination, disease progression includes a measure of ambulatory function, e.g., longer distance ambulatory function (e.g., a 6-minute walk test). In one embodiment, the disease progression includes a measure of ambulatory function other than EDSS ambulatory function. In one embodiment, disease progression includes a measure of upper extremity function e.g., a 9HP assessment, and a measure of ambulatory function, e.g., short distance ambulatory function (e.g., T25FW). In one embodiment, disease progression includes a measure of upper extremity function (e.g., a 9HP assessment) and a measure of lower extremity function. In one embodiment, disease progression includes a measure of upper extremity function (e.g., a 9HP assessment), a measure of lower extremity function, and a measure of ambulatory function, e.g., short distance ambulatory function (e.g., T25FW) and/or longer distance ambulatory function (e.g., a 6-minute timed walk test (e.g., 6MWT)). In one embodiment, one, two or the combination of the T25FW, 6MWT and 9HP assessments can be used to acquire a disease progression value. The measure of ambulatory function (e.g., short distance ambulatory function (e.g., T25FW) or longer distance ambulatory function (e.g., a timed (e.g., 6-minute) walk test (e.g., 6MWT)) and/or measure of upper extremity function (e.g., a 9HP assessment) can further be used in combination with the EDSS to evaluate MS, e.g., progressive forms of MS.
[0104] Alzheimer's disease (AD) is a neurodegenerative disorder that results in the loss of cortical neurons, especially in the associative neocortex and hippocampus which in aim leads to slow and progressive loss of cognitive functions, ultimately leading to dementia and death. Major hallmarks of the disease are aggregation and deposition of misfolded proteins such as aggregated beta-amyloid peptide as extracellular senile or neuritic ‘plaques’, and hyperphosphorylated tau protein as intracellular neurofibrillary tangles.
[0105] Genetic predispositions for AD are divided into two forms: early-onset familial AD (<60 years), and late-onset sporadic AD (>60 years). Rare, disease causing mutations in Amyloid precursor protein (APP), Presenilin 1 (PSEN1), and Presenilin 2 (PSEN2) genes are known to result in early-onset familial AD while, APOE (allele 4) is the single most important risk factor for late-onset AD. In specific embodiments, the methods of the invention are used to treat subjects with a genetic predisposition for wither early onset familial AD or late-onset sporadic AD.
[0106] Although Alzheimer's disease develops differently for every individual, there are many common symptoms. In the early stages, the most common symptom is difficulty in remembering recent events. As the disease advances, symptoms can include confusion, irritability, aggression, mood swings, trouble with language, and long-term memory loss.
[0107] Clinical Decision Support Systems (CDSS) comprising computer hardware, software, and/or systems can be used to determine a diagnosis for a patient who has certain symptoms associated with Alzheimer's disease. CDSS often include at least three component parts: a knowledge basis, an inference engine, and a communication mechanism. The knowledge base may comprise compiled information about symptoms, pharmaceuticals, and other medical information. The inference engine may comprise formulas, algorithms, etc. for combining information in the knowledge base with actual patient data. The communication mechanism may be ways to input patient data and to output helpful information based on the knowledge base and inference engine. For example, information may be inputted by a physician using a computer keyboard or tablet and displayed to the physician on a computer monitor or portable device.
[0108] In certain aspects, the assessment of treatment includes radiological assessment, e.g., single photon emission computed tomography (SPECT), Positron Emission Tomography (PET), Magnetic Resonance Imaging (MRI) and scintigraphy. For example, multiple sclerosis can be assessed using radiologic assessment of CNS plaques, e.g. by MRI. In another example, AD plaque load can be assessed, e.g., using Aβ-PET.
[0109] The assessment of treatment according to the present invention may also be performed using scanning database systems and methods such as those described in US Appln. No. 20150039346.
EXAMPLE
[0110] The following example is put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and is not intended to limit the scope of what the inventors regard as their invention, nor is the example intended to represent or imply that the experiments below are all of or the only experiments performed. It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific aspects without departing from the spirit or scope of the invention as broadly described. The present aspects are, therefore, to be considered in all respects as illustrative and not restrictive.
[0111] Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees centigrade, and pressure is at or near atmospheric.
Example 1
[0112] Transcriptional Profiling and Therapeutic Targeting of Oxidative Stress in Neuroinflammation
INTRODUCTION
[0113] Oxidative injury is a pathologic feature linked to neurodegeneration, myelin damage and disease progression in MS and other neurodegenerative diseases (1-6). Oxidative stress mediated by reactive oxygen species (ROS) release from CNS innate immune cells promotes neurodegeneration and demyelination (1,3,7-10). Innate immune-mediated oxidative injury has been proposed as a critical process underlying the progression of MS from the relapsing phenotype to relentless neurodegeneration (11,12). In progressive MS, neurodegeneration is associated with robust microglia activation and oxidative stress (12,13). However, the mechanisms in innate immune cells that trigger oxidative injury in neuroinflammation remain poorly understood. Single-cell technology has led to an appreciation of the heterogeneity of CNS innate immune responses with distinct gene profiles between microglia and CNS infiltrating macrophages in MS, Alzheimer's disease (AD), and related animal models (14-21). However, the functional transcriptomic landscape of oxidative stress inducing innate immune cells is unknown. Furthermore, the discovery of drugs capable of selectively suppressing innate immune-driven neurodegeneration has been hindered by the lack of molecular understanding of the neurotoxic functions of CNS innate immune cells.
[0114] Here, the innate immune cell atlas of oxidative stress in neuroinflammatory disease and the discovery of new therapeutic targets is reported. To functionally dissect the oxidative stress signature of CNS innate immunity at the single-cell level, a Toxic-RNA-seq (Toxseq) was developed to transcriptionally profile ROS+innate immune cells. A core oxidative stress signature shared among a microglia cluster and subsets of infiltrating myeloid cells in mice were identified, as well as microglia from MS lesions. Tox-seq followed by microglia HTS of a library of 1,907 clinical drugs and bioactive compounds and oxidative stress gene network analysis identified glutathione transferase activity and the compound acivicin, which inhibits the degradation of the antioxidant glutathione by targeting γ-glutamyl transferase (GGT). Therapeutic administration of acivicin reversed clinical signs, decreased oxidative stress, and protected from neurodegeneration in chronic EAE, even when administered eighty days after disease onset. Thus, these studies determine the transcriptomic landscape of oxidative stress in CNS innate immunity and provide druggable pathways for therapeutic targeting of neurotoxic innate immune populations.
[0115] Materials and Methods
[0116] Animals SJL/J, NOD, C57BL/6, and Ggt1.sup.dwg/dwg 38 mice were purchased from The Jackson Laboratory, and Sprague-Dawley rat Po litters were purchased from Charles River Laboratories. Ccr2.sup.RFP/RFP mice (56) on C57BL/6 background (provided by I. F. Charo, Gladstone Institutes) were crossed with Cx3cr1.sup.GFP/GFP mice (57) to generate Cx3cr1.sup.GFP/+ Ccr2.sup.RFP/+ mice. Mice were housed under IACUC guidelines in a temperature and humidity-controlled facility with 12 h light-12 h dark cycle and ad libitum feeding. All animal protocols were approved by the Committee of Animal Research at the University of California, San Francisco, and were in accordance with the National Institutes of Health guidelines.
[0117] EAE. Active EAE was induced in 8- to 10-week-old female SJL/J mice, C57BL/6 mice, and Cx3cr1.sup.GFP/+ Ccr2.sup.RFP/+ mice by subcutaneous immunization with 50 μg MOG.sub.35-55 peptide (MEVGWYRSPFSRVVHLYRNGK; Auspep) or 100 μg PLP139-151 peptide (HSLGKWLGHPDKF; Auspep) in complete Freund's Adjuvant (Sigma-Aldrich) supplemented with 400 μg of heat-inactivated Mycobacterium tuberculosis H37Ra (Difco Laboratories). At day 0 and 2 after immunization, mice were given intraperitoneal injection of 200 ng (C57BL/6) or 75 ng (SJL/J) pertussis toxin (Sigma-Aldrich). For chronic NOD EAE model, 10- to 12-week-old NOD mice were immunized with 150 μg MOG.sub.35-55 peptide, followed by administration of 200 ng pertussis toxin on days 0 and 2 as described 43. For adoptive transfer of EAE in SJL/J mice, donor SJL/J mice were immunized as described above, and on day 10 post immunization, cells were isolated from the draining lymph nodes and spleen. Lymphocytes were re-stimulated with 20 μg/ml PLP.sub.139-151 and 10 ng/ml IL-12 (eBioscience) for 4 d, 3×10.sup.7 restimulated cells were transferred to healthy SJL/J recipient mice as described (9). For prophylactic treatment, mice were each administered with 5 mg/kg acivicin (Santa Cruz Biotechnology, dissolved at 1 mg/ml in saline), 5 mg/kg GGsTop (Tocris) or saline daily from day 0. For therapeutic treatment, acivicin or saline was injected daily starting at the peak of the initial paralytic episode or at day 80 during the chronic phase of EAE in NOD mice. Mice were randomly assigned to treatment groups, scored and drug treated in a blinded manner Mice that did not develop symptoms of EAE were not excluded from the analysis. Experimental groups were unblinded to treatment assignment at the end of the experiments. Mice were observed daily, and clinical scores were assigned as follows by observers blinded to treatment: 0, no symptoms; 1, loss of tail tone; 2, ataxia; 3, hindlimb paralysis; 4 hindlimb and forelimb paralysis; 5, moribund. EAE onset was defined by weight loss (>1 gram) and first day of symptoms (score >0).
[0118] Cell isolation from spinal cord. Mice were perfused with ice-cold phosphate buffered saline (PBS) and spinal cord tissue was flushed out from the spinal column with PBS. Spinal cords were incubated in HBSS with 0.2% collagenase, type 3 (Worthington) gently shaking for 30 min at 37° C. under trypsin-free, mild dissociation conditions. Tissue were mechanically dissociated and passed through a 40 μm cell strainer (Falcon). Single-cell suspension was collected in 5 ml RPMI-1640 medium without phenol red supplemented with 25 mM HEPES, 1% Penicillin/Streptavidin and 10% heat-inactivated fetal bovine serum (FBS; Thermo Fisher Scientific) and myelin was depleted following the myelin removal beads II manufactures guidelines (Miltenyi Biotec). Myelin-depleted cell suspensions were processed for flow cytometry, bulk RNA-seq or scRNA-seq as described below. For bulk RNA-seq, myelin depleted (myelin removal beads II, Miltenyi Biotec) cell suspensions from spinal cords of five mice per experiment were pooled. For scRNAseq, single cell suspensions from individual animals were processed independently.
[0119] ROS labeling for Tox-seq. Following surface antigen flow cytometry staining, live cells isolated from spinal cord were stained for intracellular ROS in vitro using membrane permeable, fluorescent reagent 2′,7′-dichlorofluorescein diacetate (DCFDA; Abcam) as described (22). DCFDA recognizes ROS and is also a redox indicator probe that responds to cellular oxidant stress including reactive nitrogen species and elevated iron. For Bulk RNA-seq, cells were incubated with 10 μM DCFDA in PBS supplemented with 2% FBS, 2 mM EDTA (Gibco) for 30 min at 37° C., and directly analyzed by flow cytometry. For scRNA-seq, to minimize cell activation and cell death cells were incubated with 10 μM DCFDA in PBS supplemented with 2% FBS without EDTA at 4° C. for 30 min prior to cell sorting.
[0120] Fluorescence-activated cell sorting analysis for Tox-seq. For the scRNA-seq experiment, myelin-depleted cell suspensions were treated for 5 min at 4° C. with Fc-block in BSA staining buffer (BD) and then incubated for 30 min at 4° C. with CD11 b APC-Cy7 (M1/70) antibodies. Cells were washed with stain buffer once and then in vitro ROS labeling (DCFDA) was performed as described above. Cells were incubated for 5 min at 4° C. with 1 μM sytox blue live/dead stain and then cells were sorted using FACSAria II. After centrifugation at 300 g and 4° C. for 10 min, cell pellets were resuspended in cold PBS supplemented with 2% FBS at 333 cells/μl and immediately processed for scRNA-seq as described below.
[0121] Droplet-based scRNA-seq. For Tox-seq, 30 μL of each live sytox blue .sup.−CD11b.sup.+ROS.sup.− and live sytox blue .sup.−CD11b.sup.+ROS.sup.+ sorted cell population (233 cells/μl) from healthy and EAE spinal cords (disease score 1.0) were run on the 10× Genomics Chromium platform, and libraries were prepared following manufactures instructions for the Chromium Single Cell 3′ v2 Reagent Kit (
[0122] Unbiased graph-based clustering analysis of scRNA-seq data. The R toolkit Seurat (23) was used for quality control processing, graph-based clustering, visualizations, and differential gene expression analyses of scRNA-seq data and performed in R version 3.4.2. Cellranger Aggr aggregated dataset of 9,079 cells were filtered through quality control parameters that included parameters to keep cells with 200-5,000 nFeature_RNA per cell, and eliminate unhealthy cells with >5% and >25% mitochondrial and ribosomal genes, respectively. The percent of mitochondrial (percent.mito) and ribosomal (percent.ribo) genes were regressed out. All remaining variable genes were used for downstream analyses, including immediate early response genes induced by cell isolation procedure 24. Following QC, 17,814 genes across 8,701 single cells were subjected to downstream analyses following Seurat version 3 default parameters unless otherwise stated (Supp
[0123] Bulk RNA-seq and data analysis. For bulk RNA-seq experiment, surface stained cells were resuspended in 100 μl PBS with 1% FBS for cell sorting using FACSAria II (BD Bioscience). CompBeads Compensation Particles (BD Biosciences) individually stained with each of the fluorescently labeled antibodies were used for color compensation. In each experiment, the maximal cell number of sorted microglia and macrophages was assessed based on CD11b (M1/70) and CD45 (30-F11) signal intensities. The sum of sorted cells from three independent experiments (n=15 mice total) was 240,606 microglia and 183,530 macrophages. ROS.sup.+ (DCFDA) cells were gated based on FITC signals (cut-off 2×10.sup.3), and MHC II.sup.+ cells gated based on PE-Cy7 signal (cut-off 3×10.sup.3) compared to the unstained cells. Microglia and macrophage subpopulations were classified into MHC II.sup.+ ROS.sup.+, MHC II.sup.− ROS.sup.−(baseline), MHC II.sup.− ROS.sup.+ and MHC II.sup.+ROS.sup.−. In each sorting experiment, eight distinct cell populations were collected separately on ice. After centrifugation at 300 g and 4° C. for 6 min, cell pellets were stored at −80° C. in 150 μl RLT buffer (Qiagen) adding 1% 2-mercaptoethanol (Gibco) prior to RNA isolation for bulk RNA-seq.
[0124] Total RNA was isolated using the RNeasy Plus Micro Kit (Qiagen) according to the manufacturer's instructions. cDNA was generated from full-length RNA using the NuGEN Ovation RNA-seq V2 kit, and then sheared by the Covaris S2 Sonicator to yield uniform size fragments. The NuGen Ovation Ultralow V2 kit was used to add adapters, barcoding and amplification. Libraries were purified using Agencourt XP magnetic beads and quantified by KAPA qPCR (Illumina) Four libraries per lane were pooled for a single end (SE 50 bp) run on the Illumnia HiSeq 4000 platform. Input sequences were provided in FASTQ format. Trimming of known adapters and low-quality regions of reads was performed using Fastq-mcf59. Sequence quality control was assessed using the program FastQC and RSeQC. Reads were aligned to the mm9 mouse genome assembly using Tophat 2.0.13, and the number of reads mapping to each gene were counted using “featureCounts”, part of the Subread suite. Differential analysis was performed using edgeR60 Bioconductor package. The data set was filtered by including all genes which had at least in two replicates a CPM (counts per million) between 0.5 and 5000. The remaining genes were normalized using calcNormFactors (TMM) (“weighted trimmed mean of M-values”) in edgeR 61. Calculation of P-values was performed in edgeR for the differential expression between samples. The built-in R function “p.adjust” was used to calculate the FDR (false discovery rate) for each P value using the Benjamini-Hochberg method. To identify differentially expressed genes that were overrepresented in existing annotated genes, the data was analyzed with GO Elite (62). Clustering was performed first by doing k-means clustering with 100 clusters on log transform expression levels then by Hierarchical Ordered Partitioning and Collapsing Hybrid (HOPACH) (63) on the k-means clusters to prune highly similar clusters.
[0125] Co-expression clusters from enriched GO terms for bulk RNA dataset. Functional enrichment analysis of a data set with 2,145 differentially expressed genes in ROS.sup.+ versus MHC II.sup.+ microglia was performed in Cytoscape (32) using BiNGO plugin and GO annotations downloaded on 8 Dec. 2016. Filtering for GO term results with <2000 genes, 592 Biological Process terms with a corrected p-value <0.05 were found. GO term enrichment was also performed on the exclusive subsets of ROS-expressed genes (1,613) and MHC II-expressed genes (924), resulting in 58 and 783 filtered Biological Process terms, respectively. From these GO term results, 6 were selected that represented processes of interest, maximum specificity (i.e., low total gene membership) and exclusive significance (i.e., non-overlapping terms). Interaction networks were constructed for each set of differentially expressed genes associated with these 6 terms using GeneMANIA (64). Default co-expression and physical interaction sources were selected from the network construction; the option to add a number of related genes was set to zero; all other settings were default values. The interaction data from GeneMANIA was imported into Cytoscape for visualization and data overlay. Co-expression clusters were defined as the largest connected set of either up- or down-regulated genes and extracted as subnetworks.
[0126] Preparation of fibrin plates for HTS. Fibrin-coated 384-well plates were prepared as follows: Using a EL406 liquid handler (BioTek), columns 1-22 received 30 μL of 2 U/mL Thrombin (Sigma-Aldrich) in a buffer of 20 mM HEPES, pH 7.4, and 14 mM CaCl2, followed by 30 μL of 12 μg/mL human plasminogen-free fibrinogen (EMD Milipore) in 20 mM HEPES, pH 7.4 for a final concentration of 6 μg/mL of fibrinogen. After incubation for 90 min in 37° C. to allow fibrin formation, plates were dried overnight in a 37° C. incubator equipped with fans to circulate the air.
[0127] Image-based HTS of small molecule inhibitors of microglia activation. Primary rat microglia were isolated and cultured in the presence of heat-inactivated Performance Plus FBS (Thermo Fisher Scientific) as described (18,65). FBS in microglia culture was used to obtain a sufficiently high yield of microglia that was required for the HTS of 2,000 compounds. Since microglia activation can be influenced by the culture conditions (18,65), FBS was batch-tested with three quality control criteria: high cell yield, no effect on morphologic activation at baseline, and response to LPS induced morphologic activation by at least 50%. Microglia HTS was performed in 384-well PDL coated plates (Greiner). To screen for compounds that inhibited fibrin-activated microglia, 50 μL of DMEM containing 10% FBS was added to the fibrin-coated plate using an EL406 liquid dispenser-aspirator (Biotek). A library of 1,907 clinical drugs and bioactive compounds, compiled by the Small Molecule Discovery Center at the University of California, San Francisco, was screened at 10 μM. 100 nL of each compound (10 mM stock solution in DMSO) was added using a Biomek FXP automated laboratory workstation (Beckman Coulter) outfitted with a 50 nL pintool (V&P Scientific). Columns 1-2 contained DMSO-treated control cells (defining maximal activation/0% inhibition); columns 3-22 contained test compounds, and columns 23-24 contained unstimulated control cells (defining minimal activation/100% inhibition). 3,000 microglia cells were added to each well in 50 μL of DMEM containing 10% FBS, giving a final compound concentration of 10 μM in 0.1% DMSO. Assay plates were incubated at 37° C., 5% CO.sub.2 for 48 h. Using the EL406 liquid dispenser-aspirator, cells were then fixed with 4% paraformaldehyde solution, permeabilized with 0.1% Triton-X100, and stained with 0.5 μg/mL CellMask Red (Thermo Fisher Scientific) and 2 μg/mL Hoechst nuclear dye (Thermo Fisher Scientific) with PBS washes between steps. The plates were stored in PBS for readout by imaging.
[0128] To screen for inhibitors of LPS (Sigma-Aldrich)-activated microglia, PDL-precoated 384-well plates (Greiner) were used. 50 μL of microglia cell suspension (3,000 cells per well) were added to each well with a WellMate multi-channel liquid dispenser (Thermo Fisher Scientific). 100 nL of each test compound (10 mM stock solutions in DMSO) was then added to columns 3-22 using the Biomek FXP and 50 nL pintool. One hour after the addition of compounds, 50 μL of a 1 ng/mL LPS solution was added to columns 1-22 using the EL406 liquid dispenser-aspirator, yielding an assay concentration of 0.5 ng/mL LPS in 100 μL assay volume. Columns 1-2 contained stimulated cells treated with DMSO (defining maximal activation/0% inhibition) and columns 22-24 contained unstimulated cells (defining minimal activation/100% inhibition). Assay plates were incubated at 37° C., 5% CO.sub.2 for 48 h. Cells were then fixed, permeabilized, stained and washed as described for the fibrin assay (9).
[0129] To measure microglial activation, assay plates were imaged using an INCell Analyzer 2000 automated fluorescent microscope (GE Healthcare) equipped with a 10× objective and excitation/emission filter pairs of 350 nm/455 nm (Hoechst stain) and 579 nm/624 nm (CellMask Red). Images were analyzed with the INCell Developer Toolbox feature extraction software (GE Healthcare). Cell nuclei stained with Hoechst dye were segmented using a “nuclear” segmentation method, with a minimum target area of 30 μm2 and sensitivity of 75%. Exclusion criteria for cell segmentation was set to intensity <120 units, or area >1000 μm2 The CellMask Red-stained cell bodies were segmented using an “intensity” segmentation method with a set threshold between 200-4095 intensity units. The borders of adjacent contacting cells were resolved using the “clump breaking” post-processing segmentation method that utilized discrete nuclei as seeds. Only cells containing a nucleus within the cell body area were analyzed. Activated microglia were defined as cells with a size ≥800 μm.sup.2, whereas cell size <150 μm.sup.2 was classified as dead cells. The number of activated microglia was divided by the total number of cells in the well to yield a fraction of activation. This activated fraction was normalized to the stimulated/untreated wells (columns 1-2) and unstimulated wells (columns 23-24) to determine the percentage of inhibition of microglial activation. Similarly, the percentage of dead cells was calculated by comparing the fraction of dead cells in a well to the stimulated and unstimulated controls. For each assay plate, Z-prime values were calculated as described (9). The average Z′ value was 0.5 for the six fibrin screening plates and 0.63 for the six LPS screening plates.
[0130] Compounds that were toxic to fewer than 3% of microglial cells and inhibited activation by >50% in either LPS or fibrin-treated plates were considered active. The 50% inhibition cutoff value was set as 3 standard deviation from the mean value of untreated control. Active compounds were reconfirmed in dose-response assays conducted in triplicates of 10 concentrations, with 3-fold serial dilutions ranging from 0.001 μM-20 μM. IC.sub.50 values were estimated from normalized % inhibition values, using the four-parameter non-linear regression analysis (Graphpad Prism).
[0131] Oxidative stress pathway modeling. A novel oxidative stress pathway was constructed based on compiling information from the literature and existing pathway diagrams, that included glutathione metabolism, redox, biosynthesis, uptake, breakdown, glutathionylation and acivicin inhibition. The gamma-glutamyl cycle was simplified for clarity. PathVisio (33) was used to construct the pathway model, which was deposited at WikiPathways (WP4466) (66) and then imported into Cytoscape for RNA-seq data overlay.
[0132] Immunohistochemistry. For immunohistochemical analysis, spinal cords were processed as Described (9,29,30,67,68). Antibodies used were as follows: mouse anti-gp91 (CYBB, 1:200; 53, BD Biosciences), mouse anti-iNOS (1:500, 610329, BD Biosciences), rabbit anti-Iba-1 (1:1000; 019-19741, Wako), rat anti-MHC II (1:300; M5/114.15.2, Thermo Fisher Scientific), rabbit coagulation factor X (F10; NBP1-33320, NOVUS), mouse anti-CLEC4E (1:700; AT16E3, abcam), mouse anti-neurofilament H non-phosphorylated (1:100; SMI-32, BioLegend), mouse anti-myelin basic protein (1:100; SMI-99, BioLegend), rabbit polyclonal anti-GGT1 (1:100; SAB2701966, Sigma), or goat polyclonal anti-4HNE (1:200; ab46544, Abcam) and Alexa 647, 488, 405 (1:500; Jackson ImmunoResearch) the Vector-Red and Vector-Blue alkaline phosphatase substrate kit (Vector Labs) for detection. Sections were stained with DAPI (1:1000, Thermo Fisher Scientific) for 3 min at room temperature. For mouse primary antibodies, the Mouse on Mouse (M.O.M.) kit (Vector Labs) was used according to the manufacturer's protocol. Analysis was based on an established method of neuropathology for sampling multiple spinal cord sections and comparing similar anatomical regions as described (68-70). Images were acquired with an Axioplan II epifluorescence microscope (Zeiss) equipped with Plan-Neofluar objectives (10×0.3 NA, 20×0.5 NA, or 40×0.75 NA) or all-in-one BZ-X700 fluorescence microscope (Keyence), Fluoview FV 1000 (Olympus) confocal microscope and Fluoview software v3.1b with Olymus 40× and 0.8 NA water-immersion lens as described (9), or Aperio Versa scanner (Leica) with Aperio Imagescope 12.4 and 1.25×, 10×, and 20× lenses. Images of similar anatomical locations were quantified using NIH ImageJ (version 1.50) by observers blinded to experimental conditions.
[0133] Flow cytometry. Cells were incubated with anti-mouse CD16/CD32 antibody (2.4G2) diluted in PBS with 2% FBS, and 2 mM EDTA at 4° C. for 15 min to block Fc receptor binding. Cells were stained with fluorescent-conjugated antibodies: CD3 (17A2), CD4 (GK1.5), CD8 (53-6.7), CD62L (MEL-14), CD44 (1M7), CD25 (3C7), CD11b (M1/70), CD45 (30-F11), and MHC II (M5/114.15.2). For surface labeling of GGT1, unconjugated mouse monoclonal anti-GGT1 (1:100; ab55138, Abcam) was incubated with surface markers for 1 hr at 4° C. Then FITC conjugated species-specific secondary antibody (1:100) was added for 1 hr at 4° C. For intracellular staining of IFN-γ and IL-17A, T cells were stimulated for 4 hr with Cell Activation Cocktail (BioLegend). Cells were then fixed with Cytofix/Cytoperm solution (BD Bioscience) and stained with antibodies to IL-17A (TC11-18H10.1), or IFN-γ (XMG1.2). Foxp3 (FJK-16S) staining was performed according to the manufacturer's protocol (eBioscience). Cells were analyzed by flow cytometry on an LSR II (BD Biosciences) with FlowJo software (Tree Star Inc.). Antibodies were purchased from BioLegend, BD Biosciences, or eBioscience.
[0134] Bone marrow—derived macrophage cultures. Bone marrow—derived macrophages (BMDMs) were prepared as described (67). In brief, bone-marrow cells were isolated from tibia and femur of C57BL/6 mice, Ggt1.sup.+/+ mice or Ggt1.sup.dwg/dwg mice and cultured in RPMI-1640 medium supplemented with 10% FBS, 1% penicillin-streptomycin (Corning), and 10 ng/ml murine M-CSF (14-8983-80, Thermo Fisher Scientific). On days 6-7, adherent BMDMs were harvested by adding PBS with 5 mM EDTA to the plates and used for assays.
[0135] Human macrophage cultures. Human peripheral blood mononuclear cells (PBMCs) were purchased from AllCells, LLC (Alameda, Calif.). To differentiate PBMC into monocyte-derived macrophages, 2×10.sup.6 PBMC/mL were plated in RPMI-1640 media supplemented with 10% FBS, 1% penicillin-streptomycin (Corning), and 50 ng/ml human M-CSF (300-25, Peprotech) in tissue-culture treated dishes (Corning). After 24 h, non-adherent cells were removed, and adherent cells were cultivated for 7-8 additional days at 37° C. in 5% CO.sub.2 to promote their full differentiation into macrophages. Oxidant detection with DHE. Intracellular concentrations of ROS were measured using dihydroethidium (DHE) as described (9). Cultured BMDMs or human macrophages were incubated in medium containing 5 μM DHE (Invitrogen) for 30 min Cells were plated on 96-well, black μ-clear-bottom microtiter plates (Greiner Bio-One) pre-coated with 25 μg/ml fibrin. For GGT inhibition, BMDMs or human macrophages were pre-incubated with 5 μM acivicin or GGsTop for 1 h, and the cells were plated on fibrin-coated 96-well plates as described above. PBS was used as vehicle control. Cells were then incubated for 24-48 h and fixed with 4% PFA for 10 min. The DHE fluorescence was detected at an excitation/emission wavelength of 518 nm/605 nm using a SpectraMax M5 microplate reader (Molecular Devices) with SoftMax Pro 5.2 software (Phoenix Technologies Ltd.).
[0136] Real-time imaging of glutathione. The real-time GSH dynamics in living BMDMs was determined with the fluorescent GSH probe, RealThiol RT-AM as described (37). Cells were plated on 2-well chamber slide (Nunc) pre-coated with 25 μg/ml fibrin. Cells were stimulated with fibrin with or without 5 μM acivicin or GGsTop. The GSH synthesis inhibitor, buthionine sulfoximine (BSO, Sigma-Aldrich) was used as positive control for reducing intracellular GSH levels at 100 μM. After 24 h incubation, cells were loaded with 1 μM RT-AM probe and were incubated for 5 min before imaging. The cells were kept at 37° C. during the entire experiment. Following incubation, fluorescence emissions after sequential excitation at 405 nm and 488 nm was acquired using sequential confocal laser scanning microscopy (Olympus FV1000; Olympus, Tokyo, Japan) with a 10× objective and 2× optical magnification. For each independent experiment, laser power and detector settings were set using the control/untreated cells incubated with RT-AM alone as reference. All settings were kept constant throughout the experiment. The ratio bound intracellular RT-AM:unbound intracellular RT-AM for each treatment condition was calculated by subtracting the 488-nm fluorescence signal from the 405 nm fluorescence signal from 30-50 cells/treatment for each independent experiment. The ratio calculated for each treatment was expressed as percentage from that of the untreated cells (control).
[0137] Blood sampling and hematological analysis. Hematological analysis was carried out for mice treated with saline or acivicin for 10 days. Blood samples were collected from the heart of each anesthetized mouse via cardiac puncture. The complete blood cell counts were measured with an automated blood count analyzer (Hemavet, Drew Scientific Inc).
[0138] Protein carbonyl content assay. Blood was isolated from EAE mice via terminal cardiac puncture, and serum levels of protein carbonylation were measured with OxiSelect™ Protein Carbonyl ELISA (Cell Biolabs) according to the manufacturer's protocol. The absorbance at 450 nm was measured using a SpectraMax M5 microplate reader (Molecular Devices) with SoftMax Pro 5.2 software (Phoenix Technologies Ltd.).
[0139] Real time qPCR. Total RNA was isolated from fibrin- or LPS-stimulated BMDMs (Ggt1.sup.dwg/dwg or Ggt1.sup.+/+ mice) with the RNAeasy Mini kit (Qiagen) according to the manufacturer's instructions. cDNA was prepared with High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Real-time qPCR analysis was performed on spinal cord tissues prepared from MOG.sub.35-55 EAE mice. SYBR green-based qPCR was performed using murine primers to Cxcl10, Nos2, Cxcl3, Ccl5, Il1b, or Il12b. Results were analyzed with the Opticon 2 Software and the comparative CT method. Gene expression was normalized to Gapdh and presented as fold change relative to control.
[0140] GGT activity assay. GGT activity was measured using the MaxDiscovery gamma-Glutamyl Transferase (GGT) Enzymatic Assay Kit (Bioo Scientific) according to the manufacturer's protocol. BMDMs were pre-incubated for 1 h with 10 μM GGT inhibitor acivicin (Santa Cruz Biotechnology) before plating cells on 25 μg/ml fibrin-coated 6-well culture plates that were prepared as described (67). For in vivo GGT activity assay, spinal cord tissues from the onset of MOG35-55 EAE or LPS-injected substantia nigra area at 12 h were prepared. Tissues were homogenized in 0.1 M Tris-HCl and centrifuged at 13,000 g for 30 min at 4° C. The supernatant was collected and subsequently assessed for GGT activity. The absorbance at 405 nm was detected with a SpectraMax M5 microplate reader (Molecular Devices) with SoftMax Pro 5.2 software (Phoenix Technologies Ltd.). The GGT activity in IU/1 was calculated following the manufacturer's protocol by multiplying the average increase in absorbance at 405 nm over 10 min GGT activity was also measured using a fluorescent probe, ProteoGREEN-gGlu (Goryo Chemical, Hokkaido, Japan), according to the manufacturer's protocol. Cultured BMDMs were plated on 96-well, black μ-clear-bottom microtiter plates (Greiner Bio-One) pre-coated with 25 μg/ml fibrin. BMDMs were incubated with ProteoGREEN-gGlu with acivicin or GGsTop (diluted at threefold concentrations from 0.01 μM to 8.3 μM). The fluorescence intensity (excitation/emission filter pairs of 488 nm/520 nm) was measured using a SpectraMax M5 microplate reader (Molecular Devices) with SoftMax Pro 5.2 software (Phoenix Technologies).
[0141] Stereotactic LPS injection and drug treatment. Mice were anaesthetized with isoflurane and placed in a stereotactic apparatus (David Kopf Instruments). LPS (Sigma-Aldrich) was dissolved in endotoxin-free distilled water (HyClone) and was diluted to 1 μg/ml with PBS. LPS (2 μl of 1 μg/ml) or PBS was slowly injected (0.3 μl/min) using a 10-μl Hamilton syringe attached to a 33-G needle into the substantia nigra at coordinates (anteroposterior, 3.0 mm; mediolateral, 1.3 mm; dorsoventral, 4.7 mm from the bregma, according to Paxinos and Watson) Animals received acivicin (5 mg/kg) or saline intraperitoneally beginning 5 days before LPS injection and for 7 days after LPS injection. After 12 h of the LPS injection, the substantia nigra area was immediately collected on ice and kept at −80° C. for later analysis of GGT activity. After 7 d of the LPS injection, mice were perfused with 4% PFA, and the brains were post-fixed in 4% PFA overnight at 4° C. Immunohistochemistry in coronal brain sections (30 μm) were performed using antibodies to tyrosine hydroxylase (TH, 1:2000; P40101, Pel Freez) and Iba-1 (1:1000; 019-19741, Wako). Images were acquired with all-in-one BZ-X700 fluorescence microscope. Images were quantified using NIH ImageJ (version 1.50) by blinded observers.
[0142] T-cell proliferation assay. For antigen-specific T cell proliferation, näive CD4+ T cells were magnetically sorted from the TCR-transgenic 2D2 mice using naïve CD4+ T cell isolation kits (Miltenyi Biotec). BMDMs were incubated with 5 μM acivicin, primed with MOG.sub.35-55 peptide (10 μg/ml) and then stimulated with LPS (10 ng/ml) for 24 h. CD4 T cells were cultured with MOG.sub.35-55 peptide primed BMDMs for 3 days and BrdU incorporation was assessed as described previously (67). For non-antigen-specific T cell proliferation, naïve CD4.sup.+ T cells were treated with acivicin and stimulated with mouse T-activator CD3/CD28 Dynabeads (Thermo Fisher Scientific) for 3 d. Cells were then fixed, permeabilized, and stained with FITC-conjugated anti-BrdU (BrdU Flow Kits, BD Biosciences).
[0143] Statistical analyses. Statistical analyses were performed with GraphPad Prism (Version 7). Data are presented as mean±s.e.m. No statistical methods were used to predetermine sample size, but sample sizes are similar to those reported previously. Statistical significance was determined with two-sided unpaired student's t-test, or non-parametric, two-sided Mann-Whitney test, or a oneway or two-way ANOVA analysis of variance followed by Bonferroni or Tukey's post-test (multiple comparisons). Mice were age and sex-matched and were randomly assigned to experimental groups. The assignment of EAE scores, histopathological analysis and quantification were done in a blinded manner. The statistical significance of the changes in the mean clinical score for each day of the EAE experiment was estimated using permutation tests (9). The corresponding P values were estimated using 1000 permutations. In each permutation, mice were randomly permuted.
[0144] Results
[0145] Single cell oxidative stress transcriptome of CNS innate immunity: To functionally profile the oxidative stress transcriptome of CNS innate immunity and identify neuroprotective drugs, a strategy for single-cell RNA-seq (scRNA-seq) transcriptional profiling of ROS+ CNS innate immune cells was developed and termed Tox-seq and performed microglia HTS of a small molecule library, followed by network analysis (
[0146] CNS innate immune clusters with oxidative stress and antigen presenting signatures: Single cells were overlaid with gene markers from the core oxidative stress signature and combined with unbiased GO analysis of DEGs from microglia and monocyte/macrophage subclusters (
[0147] Co-expression of oxidative stress, coagulation, and glutathione pathway genes: Tox-seq identified the Mg5 microglia cluster as a ROS.sup.+ CNS innate immune population enriched with oxidative stress genes. The transcriptomic signature of Mg5 showed the highest expression for oxidative stress, coagulation, inflammatory, antigen presenting, and pattern recognition receptor markers and the lowest expression of homeostatic markers (
[0148] Selection of acivicin by microglia HTS and oxidative stress gene network analysis: Tox-seq identified the coagulation pathway as mechanistically coupled to oxidative stress (
TABLE-US-00001 TABLE 1 IC.sub.50 of hits selected from fibrin screen. Dose response inhibition activity of 128 hits picked from the high-throughput screen that revealed >50% inhibition of fibrin-induced microglia activation. IC.sub.50 values were calculated for these 128 hits tested in both fibrin and LPS assay. The list in Table 1 are compounds originated from Q2 and Q3 in FIG. 5b (Q1 is LPS, Q3 is fibrin, Q2 is both LPS and fibrin, and Q4 is inactive). Fibrin LPS IC.sub.50 Quadrant No. Name IC.sub.50 (μM) (μM) (FIG. 5b) 1 2-methoxyestradiol 3 5 Q2 2 Acivicin 2 1 Q2 3 Acrisorcin 7 5 Q2 4 Albendazole 4 6 Q3 5 Alclometazone dipropionate <0.001 2 Q2 6 Algestone acetophenide 1 9 Q2 7 Almotriptan >20 >20 Q2 8 Amisulpride 20 >20 Q3 9 Amoxapine 20 11 Q3 10 Artesunate 7 >20 Q3 11 Atorvastatin calcium 0.6 3 Q2 12 Bacitracin >20 >20 Q3 13 Benzoyl peroxide 20 >20 Q2 14 Benzydamine hydrochloride 6 3 Q2 15 Benzyl isothiocyanate 10 >20 Q2 16 Betamethasone <0.001 0.01 Q2 17 Betamethasone sodium 0.05 0.2 Q2 phosphate 18 Betamethasone valerate 20 >20 Q2 19 Betazole hydrochloride 12 >20 Q3 20 Chlormadinone acetate 17 >20 Q3 21 Chlorthalidone >20 >20 Q3 22 Clobetasol propionate <0.001 <0.001 Q2 23 Cloperastine hydrochloride 7 9 Q3 24 Colistimethate sodium >20 >20 Q2 25 Cyclosporin A 20 19 Q3 26 Cyproterone 17 >20 Q3 27 Deflazacort <0.001 0.04 Q2 28 Demeclocycline hydrochloride 6 15 Q3 29 Desoxycorticosterone acetate 0.5 5 Q3 30 Dexamethasone 15 >20 Q3 31 Dexamethasone sodium 0.004 2 Q2 phosphate 32 Dichlorisone acetate 0.03 4 Q2 33 Diflorasone diacetate 0.01 >20 Q3 34 Docetaxel 0.002 >20 Q3 35 Docetaxel 20 >20 Q3 36 Eplerenore 3 >20 Q2 37 Ethacrynic acid >20 >20 Q3 38 Ethinyl estradiol >20 >20 Q3 39 Fenbendazole 3 4 Q3 40 Fludrocortisone acetate <0.001 0.4 Q2 41 Flumethasone <0.001 <0.001 Q2 42 Flumethazone pivalate <0.001 0.004 Q2 43 Flunisolide <0.001 <0.001 Q2 44 Fluocinolone acetonide <0.001 <0.001 Q2 45 Fluorouracil 7 >20 Q3 46 Ftaxilide >20 >20 Q3 47 Fusidic acid 19 >20 Q2 48 Gentamicin sulfate 4 >20 Q2 49 Hycanthone 1 1 Q2 50 Hydrocortisone 0.008 0.1 Q2 51 Hydrocortisone 0.03 18 Q2 52 Hydrocortisone butyrate 0.02 2 Q2 53 Hydrocortisone hemisuccinate 0.02 7 Q2 54 Hydrocortisone valerate 0.05 1 Q2 55 Hydroxytoluic acid 20 >20 Q3 56 Ibudilast >20 >20 Q2 57 Idazoxan hydrochloride 0.4 2 Q3 58 Imipenem >20 >20 Q3 59 Iopanic acid 5 >20 Q3 60 Irbesartan >20 >20 Q3 61 Isoxicam 7 >20 Q3 62 Levonordefrin 0.9 >20 Q3 63 Mebendazole 0.2 6 Q2 64 Mechlorethamine 7 0.1 Q2 65 Medroxyprogesterone >20 8 Q3 66 Medroxyprogesterone acetate 0.3 >20 Q2 67 Medroxyprogesterone acetate 0.5 >20 Q2 68 Megestrol acetate 5 >20 Q3 69 Melengestrol acetate 0.07 13 Q2 70 Mephentermine sulfate >20 >20 Q3 71 Mequinol >20 >20 Q2 72 Metampicillin sodium 20 >20 Q3 73 Methylphenidate >20 >20 Q2 hydrochloride 74 Methylprednisolone <0.001 0.02 Q2 75 Miconazole nitrate 20 >20 Q3 76 Midodrine hydrochloride >20 >20 Q2 77 Milnacipran hydrochloride 8 20 Q3 78 Mirtazapine >20 >20 Q3 79 Molsidomine >20 >20 Q2 80 Monensin 0.004 0.2 Q2 81 Mycophenolate mofetil 2 1 Q2 82 Mycophenolic acid 1 1 Q2 83 Naftopidil dihydrochloride 7 >20 Q3 84 Nortriptyline 13 3 Q2 hydrochloride 85 Oxaliplatin 5 15 Q3 86 Oxiglutatione disodium salt >20 >20 Q3 87 Oxybendazole 7 12 Q3 88 Perhexiline maleate 8 3 Q2 89 Phenytoin sodium >20 >20 Q2 90 Pimozide >20 18 Q3 91 Pitavastatin calcium 1 5 Q2 92 Prednicarbate 0.002 >20 Q2 93 Prednisolone sodium 0.1 4 Q2 phosphate 94 Prednisone 3 >20 Q3 95 Prednisone 12 >20 Q3 96 R(−)-Apomorphine >20 14 Q3 hydrochloride 97 Ractopamine hydrochloride 20 >20 Q3 98 Rapamycin <0.001 <0.001 Q2 99 Rasagiline 20 >20 Q3 100 Rebamipide 2 11 Q2 101 Ribavirin 1 2 Q2 102 Risedronate sodium 19 >20 Q3 103 Ritodrine hydrochloride 0.6 >20 Q3 104 Rosuvastatin 0.3 3 Q2 105 Rubitecan 2 9 Q3 106 Salicin >20 >20 Q2 107 Salinomycin, sodium 2 2 Q2 108 Salmeterol 1 14 Q3 109 Saquinavir >20 >20 Q2 110 Securinine 2 6 Q2 111 Selamectin 6 >20 Q3 112 Sulbactam >20 >20 Q3 113 Sulfaquinoxaline sodium >20 >20 Q2 114 Tegaserod maleate 1 19 Q2 115 Teniposide 2 1 Q2 116 Tepoxalin 3 18 Q2 117 Thiabendazole >20 >20 Q2 118 Tolazamide 12 >20 Q3 119 Tolnaftate 2 17 Q2 120 Toltrazuril >20 >20 Q2 121 Triamcinolone 0.04 0.4 Q2 122 Triamcinolone diacetate 0.04 0.8 Q2 123 Trifluoperazine 12 6 Q3 dihydrochloride 124 Trimipramine maleate 7 5 Q2 125 Tyrothricin 3 0.5 Q2 126 Vinblastine sulfate 0.02 0.2 Q2 127 Xylazine hydrochloride 2 8 Q2 128 Zoledronic acid 2 8 Q3
TABLE-US-00002 TABLE 2 Selection of 31 small molecules from the HTS, of which 27 compounds inhibited fibrin- and/or LPS-induced microglia activation (i.e. compounds originated from Q1, Q2, Q3 in FIG. 5b; Q1 is LPS, Q3 is fibrin, Q2 is both LPS and fibrin, and Q4 is inactive). Molecular targets and biological functions for each compound were retrieved from the KEGG database. Quadrant # Name of small molecule Primary target (gene name) Biological Function Reference (FIG. 5b) 1 Acivicin Gamma-glutamyl transferase Gamma-glutamyl cycle, glutathione KEGG: D02755; Q2 (GGT) metabolism PMID: 7914892 2 Nylidrin hydrochloride Adrenergic receptor beta Calcium signaling pathway, cAMP KEGG: D01543 Q4 (C19H26ClNO2) (ADRB) signaling pathway 3 Vinblastine sulfate ATP-binding cassette transporter Multidrug resistance, Microtuble KEGG: D01068 Q2 (ABCB1), Tubulin beta chain dynamics (TUBB) 4 Colchicine Tubulin beta chain (TUBB) Cell motility, Microtuble dynamics, KEGG: D00570 Q2 Mitosis 5 Podofilox Tubulin beta chain (TUBB) Cell motility, Microtuble dynamics, KEGG: D05529 Q2 Mitosis 6 Fendiline hydrochloride Calcium channel L type Calcium signaling KEGG: D07943 Q4 (CACNA1) 7 Tannic acid Chemokine ligand 12 (CXCL12) Chemokine signaling PMID: 12912963 Q4 8 Teniposide DNA Topoisomerase II (Top2) DNA replication, DNA damage KEGG: D02698 Q2 checkpoint 9 Prochlorperazine dimaleate Dopamine receptor (DRD2) Dopamine Receptor Signaling, cAMP- KEGG: D00493 Q4 mediated signaling, GPCR signaling 10 Thioridazine hydrochloride Dopamine receptor (DRD2) Dopamine Receptor Signaling, cAMP- KEGG: D00798 Q2 mediated signaling, GPCR signaling 11 Betamethasone Glucocorticoid receptor (NR3C1) Glucocorticoid Receptor Signaling KEGG: D00244 Q2 12 Dexamethasone Glucocorticoid receptor (NR3C1) Glucocorticoid Receptor Signaling KEGG: D00292 Q3 13 Dexamethasone acetate Glucocorticoid receptor (NR3C1) Glucocorticoid Receptor Signaling KEGG: D02174 Q2 14 Dexamethasone sodium phosphate Glucocorticoid receptor (NR3C1) Glucocorticoid Receptor Signaling KEGG: D00975 Q2 15 Fludrocortisone acetate Glucocorticoid receptor (NR3C1) Glucocorticoid Receptor Signaling KEGG: D00986 Q2 16 Fluocinolone acetonide Glucocorticoid receptor (NR3C1) Glucocorticoid Receptor Signaling KEGG: D01825 Q2 17 Fluocinonide Glucocorticoid receptor (NR3C1) Glucocorticoid Receptor Signaling KEGG: D00325 Q2 18 FluoromethoIone Glucocorticoid receptor (NR3C1) Glucocorticoid Receptor Signaling KEGG: D01367 Q2 19 Flurandrenolide Glucocorticoid receptor (NR3C1) Glucocorticoid Receptor Signaling KEGG: D00328 Q2 20 Hydrocortisone Glucocorticoid receptor (NR3C1) Glucocorticoid Receptor Signaling KEGG: D00088 Q2 21 Hydrocortisone acetate Glucocorticoid receptor (NR3C1) Glucocorticoid Receptor Signaling KEGG: D00165 Q2 22 Hydrocortisone butyrate Glucocorticoid receptor (NR3C1) Glucocorticoid Receptor Signaling KEGG: D01619 Q2 23 Hydrocortisone hemisuccinate Glucocorticoid receptor (NR3C1) Glucocorticoid Receptor Signaling KEGG: D04467 Q2 24 Hydrocortisone sodium phosphate Glucocorticoid receptor (NR3C1) Glucocorticoid Receptor Signaling KEGG: D00977 Q2 25 Methylprednisolone Glucocorticoid receptor (NR3C1) Glucocorticoid Receptor Signaling KEGG: D00407 Q2 26 Prednisolone Glucocorticoid receptor (NR3C1) Glucocorticoid Receptor Signaling KEGG: D00472 Q2 27 Prednisolone acetate Glucocorticoid receptor (NR3C1) Glucocorticoid Receptor Signaling KEGG: D00980 Q2 28 Triamcinolone diacetate Glucocorticoid receptor (NR3C1) Glucocorticoid Receptor Signaling KEGG: D00984 Q2 29 Mycophenolic acid Inosine-5′-monophosphate Purine Nucleotide Biosynthesis KEGG: D05096 Q2 dehydrogenase (IMPDH) 30 Maprotiline hydrochloride Solute carrier family 6, member 2 Neurotransmitter transport KEGG: D00818 Q1 (SLC6A2) 31 Desipramine hydrochloride Solute carrier family 6 member 2, Serotonin Receptor Signaling; KEGG: D00812 Q1 4 (SLC6A2; SLC6A4) neurotransmitter transport
TABLE-US-00003 TABLE 3 Top 64 compounds sorted for selectivity to fibrin vs LPS. Fibrin LPS Cell Cell Cherry- IC50 IC50 KEGG Fibrin Toxicity Toxicity picked Hit (uM) (uM) Analysis Fibrin Selec- IC50 (uM) IC50 (uM) Quadrant (Suppl. (Suppl. (Suppl. (Suppl. Selec- tivity [cFib [LPS No. Name (FIG. 5b) Table 7) Table 7) Table 7) Table 8) tivity (LOG10) assay] assay] Therapy 1 Docetaxel Q3 HIT 0.002 >20 9814 3.99 >20 >20 antineoplastic 2 Prednicarbate Q2 HIT 0.002 >20 8510 3.93 >20 >20 antiinflammatory, glucocorticoid 3 Diflorasone Q3 HIT 0.01 >20 2188 3.34 >20 >20 antiinflammatory, Diacetate glucocorticoid 4 Alclometazone Q2 HIT <0.001 2 2153 3.33 >20 >20 antiinflammatory, Dipropion glucocorticoid 5 Hydrocortisone Q2 HIT 0.03 18 KEGG 568 2.75 >20 >20 glucocorticoid 6 Dexamethasone Q2 HIT 0.004 2 KEGG 383 2.58 >20 >20 glucocorticoid, Sodium antiinflammatory 7 Fludrocortisone Q2 HIT <0.001 0.4 KEGG 362 2.56 >20 >20 mineralocorticoid Acetate 8 Hydrocortisone Q2 HIT 0.02 7 KEGG 350 2.54 >20 >20 glucocorticoid Hemisuc 9 Melengestrol Q2 HIT 0.07 13 196 2.29 >20 >20 antineoplastic, Acetate progestin 10 Dichlorisone Q2 HIT 0.03 4 169 2.23 >20 >20 antipruretic Acetate 11 Hydrocortisone Q2 HIT 0.02 2 KEGG 89 1.95 >20 >20 glucocorticoid, Butyrate antiinflammatory 12 Medroxypro- Q2 HIT 0.3 >20 70 1.84 >20 >20 progestogen gesterone Ac 13 Monensin Q2 HIT 0.004 0.2 49 1.69 >20 2 antibiotic, Sodium antibacterial; antibacterial 14 Deflazacort Q2 HIT <0.001 0.04 42 1.63 20 >20 antiinflammatory 15 Medroxy- Q2 HIT 0.5 >20 41 1.61 >20 >20 contraceptive progesterone Ac 16 Ritodrine Q3 HIT 0.6 >20 33 1.51 >20 >20 muscle relaxant Hydrochloride (smooth) 17 Prednisolone Q2 HIT 0.1 4 31 1.49 >20 >20 antiinflammatory, Sodium Ph glucocorticoid 18 Mebendazole Q2 HIT 0.2 6 31 1.49 >20 >20 anthelmintic 19 Hydrocortisone Q2 HIT 0.05 1 28 1.45 >20 >20 antiinflammatory, Valerate glucocorticoid 20 Levonordefrin Q3 HIT 0.9 >20 21 1.33 >20 >20 vasoconstrictor 21 Tegaserod Q2 HIT 1 19 20 1.29 >20 >20 5HT4 receptor Maleate agonist, peristaltic stimula 22 Triamcinolone Q2 HIT 0.04 0.8 KEGG 17 1.23 >20 >20 antiinflammatory Diacetate 23 Hydrocortisone Q2 HIT 0.008 0.1 KEGG 16 1.21 >20 >20 glucocorticoid, antiinflammatory 24 Methylprednisolone Q2 HIT <0.001 0.02 KEGG 15 1.19 >20 >20 glucocorticoid 25 Rosuvastatin Q2 HIT 0.3 3 12 1.09 20 >20 antihyperlipidemic 26 Vinblastine Q2 HIT 0.02 0.2 KEGG 11 1.05 >20 >20 antineoplastic, Sulfate spindle poison 27 Salmeterol Q3 HIT 1 14 10 1.02 >20 >20 Bronchodilator 28 Triamcinolone Q2 HIT 0.04 0.4 10 1.00 >20 >20 glucocorticoid 29 Betamethasone Q2 HIT <0.001 0.01 KEGG 10 0.99 >20 >20 glucocorticoid, antiinflammatory 30 Desoxycortico- Q3 HIT 0.5 5 10 0.98 >20 >20 mineralocorticoid sterone Ac 31 Algestone Q2 HIT 1 9 9 0.95 >20 >20 antiacne, progestin Acetophenide 32 Eplerenore Q2 HIT 3 >20 8 0.88 >20 >20 antihypertensive 33 Tolnaftate Q2 HIT 2 17 7 0.87 >20 >20 antifungal 34 Rebamipide Q2 HIT 2 11 6 0.80 >20 >20 antiulcer, antioxidant 35 Prednisone Q3 HIT 3 >20 6 0.78 >20 >20 glucocorticoid 36 Tepoxalin Q2 HIT 3 18 6 0.75 >20 >20 antipsoratic 37 Xylazine Q2 HIT 2 8 5 0.72 >20 >20 ″alpha2 Hydrochloride Adrenoceptor agonist; anesthetic 38 Gentamicin Q2 HIT 4 >20 5 0.71 >20 >20 antibacterial Sulfate 39 Idazoxan Q3 HIT 0.4 2 5 0.69 >20 >20 alpha2- Hydrochloride adrenergic blocker 40 Atorvastatin Q2 HIT 0.6 3 5 0.67 >20 >20 antihyperlipidemic, Calcium HMGCoA reductase i 41 Rubitecan Q3 HIT 2 9 4 0.63 >20 >20 antineoplastic 42 Megestrol Q3 HIT 5 >20 4 0.63 >20 >20 progestogen, Acetate antineoplastic 43 Betamethasone Q2 HIT 0.05 0.2 4 0.62 >20 >20 antiinflammatory, Sodium glucocorticoid 44 Pitavastatin Q2 HIT 1 5 4 0.61 >20 >20 HMGA reductase Calcium inhibitor 45 Flumethazone Q2 HIT <0.001 0.004 4 0.58 >20 >20 glucocorticoid, Pivalate antiinflammatory 46 Iopanic Acid Q3 HIT 5 >20 4 0.58 >20 >20 radioopaque agent 47 Zoledronic Q3 HIT 2 8 4 0.55 >20 >20 Antiosteoporotic Acid 48 Selamectin Q3 HIT 6 >20 3 0.51 >20 >20 anthelmintic, antiparasitic, antimite 49 Oxaliplatin Q3 HIT 5 15 3 0.48 >20 >20 antineoplastic 50 Artesunate Q3 HIT 7 >20 3 0.45 >20 >20 Antimalarial 51 Naftopidil Q3 HIT 7 >20 3 0.45 >20 >20 ″alpha1 Dihydrochlorid Adrenoceptor antagonist; antihy 52 Fluorouracil Q3 HIT 7 >20 3 0.44 >20 >20 antineoplastic, pyrimidine antimetabolite 53 Isoxicam Q3 HIT 7 >20 3 0.43 >20 >20 antiinflammatory 54 Milnacipran Q3 HIT 8 20 3 0.40 >20 >20 inhibitor of Hydrochlorid norepinephrine and seritonin 55 Securinine Q2 HIT 2 6 3 0.40 >20 12 GABAA receptor blocker, CNS stimulant 56 Demeclocycline Q3 HIT 6 15 2 0.38 >20 >20 antibacterial Hydroch 57 Benzyl Q2 HIT 10 >20 2 0.29 >20 >20 antineoplastic, Isothiocyanate antibacterial, antifungal 58 Ribavirin Q2 HIT 1 2 2 0.29 >20 >20 antiviral 59 Tolazamide Q3 HIT 12 >20 2 0.23 >20 >20 ″Oral hypoglycemic agent; stimulates pa 60 Oxybendazole Q3 HIT 7 12 2 0.22 >20 >20 anthelmintic 61 Albendazole Q3 HIT 4 6 2 0.22 >20 >20 anthelmintic 62 Betazole Q3 HIT 12 >20 2 0.22 >20 >20 gastric secretion Hydrochloride stimulant 63 Prednisone Q3 HIT 12 >20 2 0.21 >20 >20 antiinflammatory, glucocorticoid 64 2- Q2 HIT 3 5 2 0.21 >20 >20 angiogenesis Methoxyestradiol inhibitor
TABLE-US-00004 Fibrin % LPS % Fibrin LPS Fibrin % cell LPS % cell Quadrant IC50 IC50 KEGG Name PAINS inhibition toxicity inhibition toxicity (FIG. 5b) Hit (uM) (uM) Analysis Acivicin 0 90 2 93 4 Q2 HIT 2 1 KEGG Betamethasone 0 103 2 94 5 Q2 HIT <0.001 0.01 KEGG Dexamethasone Sodium Phosphate 0 112 2 100 3 Q2 HIT 0.004 2 KEGG Fludrocortisone Acetate 0 104 2 101 4 Q2 HIT <0.001 0.4 KEGG Fluocinolone Acetonide 0 105 2 104 4 Q2 HIT <0.001 <0.001 KEGG Hydrocortisone 0 105 2 101 5 Q2 HIT 0.008 0.1 KEGG Hydrocortisone 0 104 2 96 2 Q2 HIT 0.03 18 KEGG Hydrocortisone Butyrate 0 108 2 78 2 Q2 HIT 0.02 2 KEGG Hydrocortisone Hemisuccinate 0 100 1 64 1 Q2 HIT 0.02 7 KEGG Methylprednisolone 0 110 3 86 2 Q2 HIT <0.001 0.02 KEGG Mycophenolic Acid 0 94 1 91 2 Q2 HIT 1 1 KEGG Teniposide 0 91 2 100 18 Q2 HIT 2 1 KEGG Triamcinolone Diacetate 0 110 2 100 5 Q2 HIT 0.04 0.8 KEGG Vinblastine Sulfate 0 106 2 108 8 Q2 HIT 0.02 0.2 KEGG Dexamethasone 0 57 2 17 0 Q3 HIT 15 >20 KEGG 2-Methoxyestradiol 0 63 2 65 2 Q2 HIT 3 5 Acrisorcin 2 87 2 113 52 Q2 HIT 7 5 Alclometazone Dipropionate 0 105 2 82 2 Q2 HIT <0.001 2 Algestone Acetophenide 0 62 1 61 2 Q2 HIT 1 9 Almotriptan 0 101 2 85 1 Q2 HIT >20 >20 Atorvastatin Calcium 0 107 3 88 2 Q2 HIT 0.6 3 Benzoyl Peroxide 0 75 1 82 2 Q2 HIT 20 >20 Benzydamine Hydrochloride 0 75 1 102 6 Q2 HIT 6 3 Benzyl Isothiocyanate 0 77 2 68 2 Q2 HIT 10 >20 Betamethasone Sodium Phosphate 0 107 2 82 3 Q2 HIT 0.05 0.2 Betamethasone Valerate 0 69 1 55 1 Q2 HIT 20 >20 Clobetasol Propionate 0 108 3 105 3 Q2 HIT <0.001 <0.001 Colistimethate Sodium 0 72 1 112 4 Q2 HIT >20 >20 Deflazacort 0 111 2 105 4 Q2 HIT <0.001 0.04 Dichlorisone Acetate 0 108 3 74 1 Q2 HIT 0.03 4 Eplerenore 0 63 2 69 2 Q2 HIT 3 >20 Flumethasone 0 109 2 101 6 Q2 HIT <0.001 <0.001 Flumethazone Pivalate 0 101 1 94 3 Q2 HIT <0.001 0.004 Flunisolide 0 105 2 102 5 Q2 HIT <0.001 <0.001 Fusidic Acid 0 77 1 74 2 Q2 HIT 19 >20 Gentamicin Sulfate 0 105 1 101 4 Q2 HIT 4 >20 Hycanthone 2 103 2 71 2 Q2 HIT 1 1 Hydrocortisone Valerate 0 95 1 73 1 Q2 HIT 0.05 1 Ibudilast 0 75 1 57 1 Q2 HIT >20 >20 Mebendazole 0 57 1 72 2 Q2 HIT 0.2 6 Mechlorethamine 0 77 1 125 68 Q2 HIT 7 0.1 Medroxyprogesterone Acetate 0 72 1 56 1 Q2 HIT 0.5 >20 Medroxyprogesterone Acetate 0 57 1 54 1 Q2 HIT 0.3 >20 Melengestrol Acetate 0 86 2 67 2 Q2 HIT 0.07 13 Mequinol 0 98 2 76 1 Q2 HIT >20 >20 Methylphenidate Hydrochloride 0 81 2 75 1 Q2 HIT >20 >20 Midodrine Hydrochloride 0 95 3 52 1 Q2 HIT >20 >20 Molsidomine 0 103 2 124 5 Q2 HIT >20 >20 Monensin Sodium 0 101 3 117 6 Q2 HIT 0.004 0.2 Mycophenolate Mofetil 0 94 2 79 2 Q2 HIT 2 1 Nortriptyline Hydrochloride 1 98 2 114 81 Q2 HIT 13 3 Perhexiline Maleate 0 99 3 123 69 Q2 HIT 8 3 Phenytoin Sodium 0 82 1 63 1 Q2 HIT >20 >20 Pitavastatin Calcium 0 100 1 66 1 Q2 HIT 1 5 Prednicarbate 0 61 1 73 1 Q2 HIT 0.002 >20 Prednisolone Sodium Phosphate 0 108 3 96 3 Q2 HIT 0.1 4 Rapamycin 0 86 3 53 2 Q2 HIT <0.001 <0.001 Rebamipide 0 65 1 72 2 Q2 HIT 2 11 Ribavirin 0 102 2 107 3 Q2 HIT 1 2 Rosuvastatin 0 93 2 95 4 Q2 HIT 0.3 3 Salicin 0 76 1 61 1 Q2 HIT >20 >20 Salinomycin, Sodium 0 100 3 101 1 Q2 HIT 2 2 Saquinavir 0 51 1 56 1 Q2 HIT >20 >20 Securinine 0 104 2 106 1 Q2 HIT 2 6 Sulfaquinoxaline Sodium 0 106 3 104 3 Q2 HIT >20 >20 Tegaserod Maleate 0 100 2 86 2 Q2 HIT 1 19 Tepoxalin 0 76 1 66 2 Q2 HIT 3 18 Thiabendazole 0 58 1 62 1 Q2 HIT >20 >20 Tolnaftate 0 76 1 60 1 Q2 HIT 2 17 Toltrazuril 0 104 3 90 2 Q2 HIT >20 >20 Triamcinolone 0 107 3 99 4 Q2 HIT 0.04 0.4 Trimipramine Maleate 0 52 1 92 5 Q2 HIT 7 5 Tyrothricin 0 89 2 102 2 Q2 HIT 3 0.5 Xylazine Hydrochloride 0 102 1 72 2 Q2 HIT 2 8 Albendazole 0 64 1 16 1 Q3 HIT 4 6 Amisulpride 0 54 2 31 1 Q3 HIT 20 >20 Amoxapine 0 58 0 43 1 Q3 HIT 20 11 Artesunate 0 63 1 20 0 Q3 HIT 7 >20 Bacitracin 0 82 1 21 0 Q3 HIT >20 >20 Betazole Hydrochloride 0 59 1 38 1 Q3 HIT 12 >20 Chlormadinone Acetate 0 72 1 43 0 Q3 HIT 17 >20 Chlorthalidone 0 77 2 35 1 Q3 HIT >20 >20 Cloperastine Hydrochloride 0 64 2 44 0 Q3 HIT 7 9 Cyclosporin A 0 62 1 8 0 Q3 HIT 20 19 Cyproterone 0 51 1 5 0 Q3 HIT 17 >20 Demeclocycline Hydrochloride 0 75 1 31 1 Q3 HIT 6 15 Desoxycorticosterone Acetate 0 83 1 47 1 Q3 HIT 0.5 5 Diflorasone Diacetate 0 76 1 39 1 Q3 HIT 0.01 >20 Docetaxel 0 64 2 32 1 Q3 HIT 20 >20 Docetaxel 0 78 1 32 1 Q3 HIT 0.002 >20 Ethacrynic Acid 0 57 2 17 1 Q3 HIT >20 >20 Ethinyl Estradiol 0 58 1 37 2 Q3 HIT >20 >20 Fenbendazole 0 75 1 10 1 Q3 HIT 3 4 Fluorouracil 0 56 1 40 1 Q3 HIT 7 >20 Ftaxilide 0 78 2 42 1 Q3 HIT >20 >20 Hydroxytoluic Acid 0 69 1 33 0 Q3 HIT 20 >20 Idazoxan Hydrochloride 0 70 2 14 0 Q3 HIT 0.4 2 Imipenem 0 53 1 20 0 Q3 HIT >20 >20 Iopanic Acid 0 51 1 35 0 Q3 HIT 5 >20 Irbesartan 0 66 2 31 1 Q3 HIT >20 >20 Isoxicam 0 67 1 30 1 Q3 HIT 7 >20 Levonordefrin 2 70 1 36 0 Q3 HIT 0.9 >20 Medroxyprogesterone 0 61 2 6 0 Q3 HIT >20 8 Megestrol Acetate 0 66 1 24 0 Q3 HIT 5 >20 Mephentermine Sulfate 0 60 2 31 1 Q3 HIT >20 >20 Metampicillin Sodium 0 68 1 35 0 Q3 HIT 20 >20 Miconazole Nitrate 0 53 1 23 0 Q3 HIT 20 >20 Milnacipran Hydrochloride 0 72 1 31 1 Q3 HIT 8 20 Mirtazapine 0 57 2 36 1 Q3 HIT >20 >20 Naftopidil Dihydrochloride 0 59 1 32 0 Q3 HIT 7 >20 Oxaliplatin 0 96 7 41 1 Q3 HIT 5 15 Oxiglutatione Disodium Salt 0 84 1 38 1 Q3 HIT >20 >20 Oxybendazole 0 78 2 15 1 Q3 HIT 7 12 Pimozide 0 70 2 45 0 Q3 HIT >20 18 Prednisone 0 66 1 45 1 Q3 HIT 3 >20 Prednisone 0 74 2 38 0 Q3 HIT 12 >20 R(−)-Apomorphine Hydrochloride 2 63 1 18 0 Q3 HIT >20 14 Ractopamine Hydrochloride 0 50 1 39 0 Q3 HIT 20 >20 Rasagiline 0 76 1 28 1 Q3 HIT 20 >20 Risedronate Sodium 0 81 2 16 0 Q3 HIT 19 >20 Ritodrine Hydrochloride 0 51 1 36 1 Q3 HIT 0.6 >20 Rubitecan 0 73 2 30 0 Q3 HIT 2 9 Salmeterol 0 56 1 29 1 Q3 HIT 1 14 Selamectin 0 51 1 42 1 Q3 HIT 6 >20 Sulbactam 0 51 1 33 0 Q3 HIT >20 >20 Tolazamide 0 88 1 42 0 Q3 HIT 12 >20 Trifluoperazine Dihydrochloride 1 69 2 26 0 Q3 HIT 12 6 Zoledronic Acid 0 83 2 49 1 Q3 HIT 2 8 Desipramine hydrochloride 0 −2 1 83 4 Q1 KEGG Maprotiline hydrochloride 0 31 1 67 1 Q1 KEGG Colchicine 0 114 12 113 10 Q2 KEGG Dexamethasone Acetate 0 112 4 105 7 Q2 KEGG Fluocinonide 0 112 3 106 6 Q2 KEGG Fluorometholone 0 104 6 84 1 Q2 KEGG Flurandrenolide 0 110 6 98 5 Q2 KEGG Hydrocortisone Acetate 0 112 4 89 4 Q2 KEGG Podofilox/Podophyllotoxin 0 110 8 107 9 Q2 KEGG Prednisolone 0 114 4 100 4 Q2 KEGG Prednisolone Acetate 0 109 4 111 6 Q2 KEGG Thioridazine hydrochloride 1 116 72 118 73 Q2 KEGG Fendiline hydrochloride 0 −33 1 10 0 Q4 KEGG Nylidrin hydrochloride 0 6 0 24 1 Q4 KEGG Prochlorperazine 1 6 0 17 1 Q4 KEGG Tannic Acid 2 −60 0 32 1 Q4 KEGG Amlodipine 0 −29 0 59 2 Q1 Amodiaquine Dihydrochloride 3 4 1 114 85 Q1 Astermizole 0 10 1 69 2 Q1 Bronopol 0 43 2 119 61 Q1 Chlorhexidine, Chlorhexidine 0 15 1 69 8 Q1 Dihydrochloride Chlormidazole 0 38 1 70 2 Q1 Colistin Sulfate 0 19 1 58 1 Q1 Deslanoside 0 −3 1 67 1 Q1 Fluoxetine Hydrochloride, Fluoxetine 0 46 1 65 2 Q1 Imipramine 0 −4 0 60 1 Q1 Iodamide 0 41 0 67 1 Q1 Loperamide 0 12 1 63 2 Q1 Maprotiline 0 −4 1 78 2 Q1 Mesoridazine 1 −5 0 66 2 Q1 Methylene Blue 3 25 1 73 1 Q1 Oxyphenbutazone 1 2 1 114 97 Q1 Phenacemide 0 −9 1 66 1 Q1 Polymyxin B Sulfate 0 −25 1 76 2 Q1 Polymyxin B1 0 −21 1 60 1 Q1 Prazosin 0 43 3 118 77 Q1 Promazine 1 46 1 117 64 Q1 Quinacrine 2 24 1 118 65 Q1 Raloxifene 0 7 0 60 1 Q1 Reserpine 3 −4 3 64 7 Q1 Sanguinarine Chloride, Sanguinarine 0 24 0 101 55 Q1 Sulfate Tamsulosin 0 20 1 59 2 Q1 Tilorone 0 −21 0 60 1 Q1 Trichlormethine, Trichlormethine 0 −10 1 96 31 Q1 Hydrochloride Acarbose 0 81 11 62 5 Q2 Acriflavinium Hydrochloride 2 110 57 129 63 Q2 Alexidine Hydrochloride 0 108 5 120 9 Q2 Alprostadil 0 100 9 87 3 Q2 Amcinonide 0 112 7 119 6 Q2 Aminacrine 2 117 72 118 67 Q2 Amsacrine 0 70 3 120 54 Q2 Auranofin 0 106 94 106 98 Q2 Benzalkonium Chloride 0 112 71 122 31 Q2 Benzethonium Chloride 0 111 85 114 86 Q2 Benzoylpas 0 107 4 87 2 Q2 Betamethasone Acetate 0 107 3 100 4 Q2 Bismuth Subsalicylate 0 93 8 98 27 Q2 Bopindolol 0 105 6 81 2 Q2 Bortezomib 0 106 86 106 56 Q2 Budesonide 0 110 3 108 5 Q2 Budesonide 0 105 6 79 1 Q2 Cepharanthine 0 114 85 130 58 Q2 Cerivastatin, Cerivastatin Na 0 103 13 101 5 Q2 Cetrimonium 0 114 28 132 12 Q2 Cetylpyridinium Bromide 0 112 25 114 39 Q2 Cinoctramide 0 90 4 53 1 Q2 Cinoxacin 0 117 50 115 12 Q2 Compactin, Mevastatin 0 105 3 89 2 Q2 Cycloheximide 0 108 45 123 45 Q2 Dactinomycin 3 106 88 106 50 Q2 Dactinomycin 3 112 75 114 66 Q2 Danazol 0 105 28 99 14 Q2 Dasatinib 0 109 14 70 10 Q2 Dasatinib 0 105 65 64 8 Q2 Daunorubicin 3 116 77 114 57 Q2 Desonide 0 108 17 112 5 Q2 Desoximetasone 0 101 5 91 3 Q2 Desoxymetasone 0 108 3 105 3 Q2 Dexamethasone 0 112 4 111 6 Q2 Dexamethasone Phosphate 0 103 5 93 2 Q2 Dinoprostone 0 102 9 85 2 Q2 Dipyrone 0 106 26 101 36 Q2 Disulfiram 0 114 90 131 75 Q2 Doxorubicin 3 106 75 108 −1 Q2 Doxorubicin 3 113 82 118 32 Q2 Emetine, Emetine Dihydrochloride 0 117 82 115 46 Q2 Enoxacin 0 113 84 119 12 Q2 Epirubicin, Epirubicin Hydrochloride 3 113 79 123 58 Q2 Estrone 0 106 93 106 25 Q2 Ethylmercurithiosalicylic acid 0 109 92 127 95 Q2 Flunisolide 0 104 5 101 4 Q2 Fluocinonide 0 103 4 88 2 Q2 Fluorometholone 0 112 3 106 7 Q2 Flurandrenolide 0 102 5 103 5 Q2 Fluticasone Propionate 0 108 7 117 3 Q2 Fluticasone Propionate 0 99 5 96 4 Q2 Fluvastatin 0 110 10 101 3 Q2 Fluvastatin 0 102 12 102 7 Q2 Gentian Violet 3 117 38 118 73 Q2 Gramicidin 0 114 43 118 64 Q2 Halcinonide 0 103 8 95 2 Q2 Haloprogin 0 78 42 106 70 Q2 Hexamethonium 0 108 9 100 2 Q2 Homidium Bromide 2 84 17 103 9 Q2 Homoharringtonie 0 106 90 106 67 Q2 Hydrocortisone 0 104 4 57 1 Q2 Idarubicin, Idarubicin Hydrochloride 3 106 90 106 40 Q2 Inosine 0 111 7 115 2 Q2 Isoflupredone Acetate 0 106 3 90 2 Q2 Isotretinoin 0 112 13 55 1 Q2 Lovastatin 0 106 3 87 2 Q2 Medrysone 0 111 4 87 4 Q2 Menadione 3 113 87 123 62 Q2 Mestranol 0 106 90 90 27 Q2 Methylbenzethonium Chloride 0 105 17 108 10 Q2 Mitomycin C, Mitomycin 3 112 74 110 49 Q2 Mitoxantrone 3 113 88 122 40 Q2 Mometasone Furoate 0 100 3 106 4 Q2 Nadolol 0 107 4 108 3 Q2 Naltrexone Hydrochloride 0 107 4 79 2 Q2 Nifenazone 0 114 85 132 78 Q2 Nifuroxazide 0 112 6 120 14 Q2 Ondansetron 0 106 20 92 3 Q2 Oxyquinoline 0 117 80 116 57 Q2 Phenylmercuric Acetate 0 109 82 127 80 Q2 Pimethixene Maleate 1 112 6 92 2 Q2 Piperacillin 0 103 6 80 2 Q2 Prednisolone 0 97 3 87 3 Q2 Prednisolone Hemisuccinate 0 106 3 96 3 Q2 Proflavine 0 113 82 132 59 Q2 Puromycin Hydrochloride, 0 107 66 124 52 Q2 Puromycin Dihydrochloride Pyrithione Zinc 0 109 86 127 87 Q2 Pyrvinium 2 113 24 115 45 Q2 Rimantadine 0 106 93 106 56 Q2 Simvastatin 0 108 7 102 4 Q2 Sirolimus 0 108 4 81 1 Q2 Sunitinib 0 106 75 101 27 Q2 Tetramizole Hydrochloride 0 107 8 112 5 Q2 Thiostrepton 0 109 64 65 1 Q2 Thiram 0 109 91 126 64 Q2 Topotecan, Topotecan 3 112 63 112 40 Q2 Hydrochloride Triamcinolone Acetonide 0 111 4 104 4 Q2 Vincristine 0 105 33 104 10 Q2 Vincristine Sulfate 0 108 16 119 7 Q2 Vindesine, Vindesine Sulfate 0 106 30 102 9 Q2 Vinorelbine 0 108 3 106 9 Q2 Vorinostat 0 112 89 112 50 Q2 Digoxin 0 99 4 26 1 Q3 Oxaliplatin 0 66 0 21 0 Q3 Suloctidil 0 112 90 3 0 Q3 (−)-ephedrine, N-methyl, N- 0 −52 0 7 0 Q4 methyl (−)ephedrine [1r,2s] (¬±)-atenolol, Atenolol 0 6 0 −7 0 Q4 (¬±)-baclofen, Baclofen 0 −8 0 9 0 Q4 (¬±)-gamma-vinyl Gaba, Vigabatrin 0 8 1 12 0 Q4 (¬±)-ibuprofen, Ibuprofen 0 −20 0 16 0 Q4 (¬±)-octopamine Hydrochloride, 0 −48 1 16 0 Q4 Octopamine Hydrochloride (¬±)-p-aminoglutethimide, 0 3 0 6 0 Q4 Aminoglutethimide (¬±)-p-chlorophenylalanine, 0 16 1 5 0 Q4 Fenclonine (r)-bicalutamide, Bicalutamide 0 −1 0 20 0 Q4 1-(Isopropylamino)-3-(1- 0 −18 1 −5 0 Q4 Naphthyloxy)-2-Propanol 2-Aminoethanesulfonic Acid 0 −13 0 19 0 Q4 2-Phenyl-Ethanol 0 −6 1 15 0 Q4 2-thiouracil 0 −15 1 7 0 Q4 2,6-di-t-butyl-4-methylphenol, 0 −4 1 5 0 Q4 Butylated Hydroxytoluene 2H-1-BENZOPYRAN-2-ONE 0 −35 0 13 0 Q4 3-acetamidophenol 0 4 1 5 0 Q4 3-Hydroxy-3-Methyl-Glutaric Acid 0 9 0 8 0 Q4 3-methyl-1-phenyl-2-pyrazolin-5-one 0 −15 1 15 0 Q4 (mci-186), Edaravone 4-acetamidobenzoic acid 0 32 1 42 0 Q4 4-Aminobenzoic Acid 0 −31 1 13 1 Q4 5-azacytidine, Azacitidine 0 9 1 −7 0 Q4 5-fluoro-5′-deoxyuridine, 0 −27 1 9 0 Q4 Doxifluridine 7-hydroxy-4-methyl-2H-chromen-2- 0 12 0 4 0 Q4 one Abacavir Sulfate 0 −2 1 3 0 Q4 Acamprosate Calcium 0 12 1 22 0 Q4 Acarbose 0 −22 1 −5 0 Q4 Acebutolol 0 −11 1 19 0 Q4 Aceclidine Hydrochloride, Aceclidine 0 2 1 0 0 Q4 Aceclofenac 0 −2 1 8 0 Q4 Acedapsone 0 0 0 4 0 Q4 Aceglutamide 0 36 1 20 0 Q4 Acemetacin 0 −16 1 17 0 Q4 Acenocoumarol, Acenocoumarin 0 −38 0 14 0 Q4 Acepromazine 1 9 1 31 1 Q4 Acesulfame Potassium 0 −3 0 16 0 Q4 Acetaminophen 0 14 0 22 0 Q4 Acetaminosalol 0 −15 1 20 1 Q4 Acetanilide 0 −31 0 9 0 Q4 Acetarsol 0 −12 0 13 0 Q4 Acetazolamide 0 −12 0 4 1 Q4 Acetohexamide 0 12 1 14 0 Q4 Acetohydroxamic Acid 0 −3 0 12 0 Q4 Acetophenazine Maleate 0 34 1 15 0 Q4 Acetrizoic acid 0 14 1 9 0 Q4 Acetyl-l-leucine 0 −10 5 45 3 Q4 Acetylcholine 0 −39 0 9 0 Q4 Acetylcysteine 0 −19 0 7 0 Q4 Acetylcysteine 0 −30 1 13 0 Q4 Acetylsalicylic acid 0 −21 1 17 0 Q4 Acexamic Acid 0 −25 1 13 0 Q4 Aciclovir 0 −20 1 3 1 Q4 Acipimox 0 −11 1 20 0 Q4 Aconitine 0 −23 0 12 0 Q4 Actarit 0 11 1 9 0 Q4 Adapalene 0 −12 2 45 4 Q4 Adenine 0 −7 1 22 1 Q4 Adenosine 0 −6 1 3 1 Q4 Adenosine monophosphate 0 −30 1 4 0 Q4 Adenosine Phosphate 0 6 1 31 2 Q4 Adiphenine Hydrochloride 0 −30 1 12 0 Q4 Adipic Acid, Hexanedioic Acid 0 −24 0 8 0 Q4 Adrenaline Bitartrate, Epinephrine 2 35 0 19 0 Q4 Bitartrate Adrenolone Hydrochloride 2 28 0 32 0 Q4 Afalanine 0 0 1 10 0 Q4 Ajmaline 0 −6 1 13 0 Q4 Aklomide 0 −18 0 −2 0 Q4 Alaproclate Hydrochloride, 0 −7 1 15 0 Q4 Alaproclate Alendronic acid 0 5 1 18 0 Q4 Alfacalcidol 0 −26 1 4 0 Q4 Alfuzosin Hydrochloride, Alfuzosin 0 1 1 18 0 Q4 Alimemazine 1 32 1 39 0 Q4 Aliskiren 0 2 1 21 0 Q4 Allantoin 0 −21 1 6 0 Q4 Allopurinol 0 −17 1 16 0 Q4 Allylisothiocyanate 0 11 1 18 0 Q4 Allylthiourea 0 −7 1 11 0 Q4 Aloin 0 −35 1 14 0 Q4 Alpha-cypermethrin 0 −15 1 11 0 Q4 Alpha-tochopherol 0 18 0 21 0 Q4 Alpha-tochopheryl Acetate 0 22 1 8 0 Q4 Alprazolam 0 33 1 20 0 Q4 Alprenolol Hydrochloride, Alprenolol 0 1 0 13 1 Q4 Alrestatin 0 11 1 12 0 Q4 Althiazide 0 −11 1 12 1 Q4 Altrenogest 0 6 1 15 0 Q4 Altretamine 0 −29 1 16 0 Q4 Alverine 0 −17 0 6 0 Q4 Amantadine 0 −7 1 3 0 Q4 Amantadine 0 −32 1 −5 0 Q4 Ambroxol Hydrochloride 0 −19 0 9 0 Q4 Amifostine 0 −38 0 15 0 Q4 Amikacin Sulfate 0 3 1 1 0 Q4 Amiloride 0 20 1 7 0 Q4 Aminocaproic Acid 0 5 1 7 0 Q4 Aminoguanidine 0 −25 0 12 0 Q4 Aminohippuric Acid 0 −7 1 16 1 Q4 Aminohydroxybutyric Acid 0 −4 1 17 0 Q4 Aminolevulinic acid 0 −4 1 10 0 Q4 Aminopentamide Sulfate 0 3 1 12 0 Q4 Aminophenazone 0 −41 1 20 1 Q4 Aminophylline, Theophylline 0 35 1 25 0 Q4 Aminopterin 0 −12 1 17 0 Q4 Aminosalicylic Acid 0 9 0 9 0 Q4 Aminothiazole 0 −31 1 17 1 Q4 Amiodarone 0 −4 1 13 0 Q4 Amisulpride 0 −5 1 12 0 Q4 Amitraz 0 −18 0 9 0 Q4 Amitriptyline 1 14 1 30 1 Q4 Amlexanox 0 −17 0 −4 0 Q4 Amlodipine 0 −17 0 36 2 Q4 Amorolfine 0 6 1 22 0 Q4 Amoxicillin 0 −14 0 12 0 Q4 Amphotericin B 0 −25 1 29 1 Q4 Amphotericin B 0 −39 1 −6 0 Q4 Ampicillin 0 1 1 15 0 Q4 Ampiroxicam 0 −9 1 14 0 Q4 Amprenavir 0 −10 1 6 0 Q4 Amprolium 0 −10 1 8 0 Q4 Ampyzine Sulfate 0 −18 0 11 1 Q4 Amrinone, Inamrinone 0 1 1 18 0 Q4 Amylene Hydrate 0 −18 1 18 1 Q4 Anagrelide 0 −2 1 19 1 Q4 Anagrelide 0 −22 1 −2 0 Q4 Anastrozole 0 31 1 16 0 Q4 Ancitabine Hydrochloride 0 −4 1 27 1 Q4 Anethole 0 −37 0 13 0 Q4 Aniracetam 0 6 1 4 0 Q4 Anisindione 2 −15 0 13 0 Q4 Anisodamine Hydrobromide 0 −41 1 4 0 Q4 Antazoline 0 −2 1 17 1 Q4 Anthralin 0 −10 1 13 1 Q4 Antipyrine 0 −8 0 13 1 Q4 Apomorphine Hydrochloride 2 11 1 −4 0 Q4 Apramycin Sulfate 0 11 1 25 1 Q4 Aprepitant 0 20 1 1 0 Q4 Arecoline 0 17 1 14 0 Q4 Argatroban 0 −10 1 8 0 Q4 Arginine Hydrochloride 0 9 0 42 2 Q4 Aripiprazole 0 −21 0 16 0 Q4 Aripiprazole 0 −48 1 3 0 Q4 Armodafinil 0 −10 0 19 1 Q4 Arsanilic Acid 0 −42 0 12 0 Q4 Arsenic Trioxide 0 14 1 14 0 Q4 Artemether 0 −31 1 0 0 Q4 Artemether 0 −1 0 4 0 Q4 Artemisinin 0 −25 0 12 0 Q4 Artenimol 0 −56 0 11 0 Q4 Artesunate 0 47 1 7 0 Q4 Ascorbic Acid 0 −8 0 12 1 Q4 Ascorbyl Palmitate 0 38 1 20 0 Q4 Aspartame 0 −15 1 7 0 Q4 Atazanavir 0 −17 0 13 1 Q4 Atomoxetine Hydrochloride 0 15 1 7 1 Q4 Atovaquone 3 −15 0 13 0 Q4 Atracurium Besylate 0 −3 0 5 0 Q4 Atracurium Besylate 0 2 1 28 0 Q4 Atropine 0 −21 0 4 0 Q4 Atropine Oxide 0 −30 1 6 0 Q4 Avermectin B1, Abamectin 0 20 1 9 0 Q4 (avermectin B1a Shown) Avobenzone 0 22 1 20 0 Q4 Azaperone 0 −4 0 12 0 Q4 Azaserine 3 8 1 2 0 Q4 Azatadine 0 −9 1 7 0 Q4 Azathioprine 0 −14 0 16 0 Q4 Azelaic Acid 0 6 0 19 0 Q4 Azelastine, Azelastine Hydrochloride 0 5 0 11 0 Q4 Azilsartan medoxomil 0 5 0 31 1 Q4 Azithromycin 0 −16 0 11 0 Q4 Azlocillin 0 −14 1 6 1 Q4 Azlocillin Sodium 0 −5 0 17 0 Q4 Aztreonam 0 3 1 1 0 Q4 Aztreonam 0 44 0 34 0 Q4 Bacampicillin Hydrochloride 0 −12 1 8 0 Q4 Bacitracin 0 −7 0 6 1 Q4 Balsalazide 3 −2 0 11 0 Q4 Bambuterol 0 −24 1 9 0 Q4 Bambuterol 0 −10 1 13 1 Q4 Barbital 0 −5 0 11 0 Q4 Becanamycin Sulfate, Bekanamycin 0 −10 1 16 0 Q4 Sulfate Beclamide 0 19 1 2 0 Q4 Beclomethasone 0 −8 1 −3 0 Q4 Beclomethasone Dipropionate 0 7 1 28 1 Q4 Bemotrizinol 0 −23 1 16 0 Q4 Benazepril 0 0 1 26 1 Q4 Bendrofumethiazide, 0 −15 1 12 1 Q4 Bendroflumethiazide Benfluorex 0 −26 1 −3 0 Q4 Benfotiamine 0 −47 0 18 0 Q4 Benserazide 3 −16 0 8 0 Q4 Benurestat 0 6 1 22 1 Q4 Benzbromarone 0 0 0 24 1 Q4 Benzocaine 0 −24 1 7 0 Q4 Benzoclidine 0 −55 1 5 0 Q4 Benzoic Acid 0 −6 0 6 0 Q4 Benzonatate 0 0 1 12 0 Q4 Benzoxiquine 0 −2 1 14 0 Q4 Benzthiazide 0 −8 1 7 0 Q4 Benztropine 0 −7 1 30 0 Q4 Benzydamine 0 −16 1 30 1 Q4 Benzyl Alcohol 0 −54 0 11 0 Q4 Benzyl Benzoate 0 35 1 32 1 Q4 Benzylpenicillin 0 29 1 42 0 Q4 Bephenium Hydroxynapthoate 0 25 1 19 0 Q4 Bepridil 0 1 1 15 0 Q4 Berberine 0 −62 0 4 0 Q4 Bergapten 0 −5 1 22 0 Q4 Beta-carotene 0 −15 0 −3 0 Q4 Beta-escin 0 −11 0 11 0 Q4 Beta-naphthol 0 −17 0 15 0 Q4 Beta-propiolactone, Propiolactone 0 −12 1 8 0 Q4 Betahistine 0 −29 1 13 0 Q4 Betamethasone 0 −4 1 −9 0 Q4 Betamethasone 17,21-dipropionate 0 21 1 29 0 Q4 Betamipron 0 −6 1 14 0 Q4 Betaxolol 0 −42 0 13 0 Q4 Betaxolol 0 2 1 1 0 Q4 Bethanechol 0 14 1 15 0 Q4 Bexarotene 0 −3 1 10 1 Q4 Bezafibrate 0 −4 0 18 0 Q4 Bifonazole 0 −3 1 14 0 Q4 Biotin 0 24 1 12 0 Q4 Biperiden 0 2 1 17 1 Q4 BIPHENYL-4-YL-ACETALDEHYDE 0 −26 0 7 0 Q4 Bisacodyl 0 10 0 8 0 Q4 Bisoctrizole 0 33 0 20 0 Q4 Bisoprolol 0 1 0 13 0 Q4 Bithionol 0 11 1 14 0 Q4 Bitoscanate 0 −9 1 3 1 Q4 Bleomycin (bleomycin B2 Shown) 0 27 0 12 0 Q4 Bornyl Acetate 0 −38 0 2 0 Q4 Bosentan 0 −14 1 10 0 Q4 Bretylium 0 −26 0 −9 0 Q4 Brimonidine 0 −36 1 −9 0 Q4 Brinzolamide 0 −3 0 5 0 Q4 Bromfenac 2 −12 1 0 0 Q4 Bromhexine 0 −16 0 11 0 Q4 Bromindione 2 17 1 27 0 Q4 Bromocriptine Mesylate 0 26 1 24 0 Q4 Bromopride 0 −2 1 4 0 Q4 Bromperidol 0 −6 1 7 0 Q4 Brompheniramine 0 −20 1 −7 −1 Q4 Broxaldine 0 19 1 6 0 Q4 Broxyquinoline 0 −25 0 8 0 Q4 Brucine 0 6 1 13 0 Q4 Bucetin 0 −25 0 12 0 Q4 Bucladesine 0 10 1 24 0 Q4 Bufexamac 0 −2 1 17 0 Q4 Buflomedil, Buflomedil Hydrochloride 0 −37 1 13 1 Q4 Bumetanide 0 −3 1 18 1 Q4 Bupivacaine 0 12 1 23 0 Q4 Bupropion Hydrochloride, 0 −15 1 27 2 Q4 Bupropion, Amfebutamone Hydrochloride Buramate 0 9 1 17 0 Q4 Buspirone 0 −13 0 8 0 Q4 Busulfan 0 1 0 2 0 Q4 Butacaine 0 −18 0 3 0 Q4 Butamben 0 −25 0 14 1 Q4 Butenafine 0 −4 1 −8 0 Q4 Butoconazole 0 −51 0 10 0 Q4 Butylated Hydroxyanisole 0 −17 1 26 0 Q4 Butylparaben 0 −16 1 20 0 Q4 Cabergoline 0 20 1 33 1 Q4 Caffeine 0 2 0 10 0 Q4 Calcidiol 0 14 1 8 0 Q4 Calcipotriol 0 2 1 −8 0 Q4 Calcitriol 0 −46 0 0 1 Q4 Calcium Gluceptate 0 8 1 8 0 Q4 Camylofine Dihydrochloride 0 −27 1 15 1 Q4 Candesartan 0 9 1 11 0 Q4 Candesartan Cilextil 0 −5 1 10 0 Q4 Candicidin 0 −41 1 31 0 Q4 Canrenoic Acid, Potassium Salt 0 −8 0 8 0 Q4 Canrenone 0 −10 1 19 0 Q4 Capecitabine 0 43 1 25 1 Q4 Capobenic Acid 0 9 1 16 0 Q4 Capreomycin Sulfate 0 −32 1 3 0 Q4 Capsaicin 0 −45 1 9 0 Q4 Capsaicin, NGX-4010 0 5 1 10 0 Q4 Captamine 0 9 1 19 0 Q4 Captopril 0 −11 1 12 1 Q4 Carbachol 0 −18 1 6 0 Q4 Carbadox 2 −2 1 11 1 Q4 Carbamazepine 0 −25 0 4 0 Q4 Carbarsone 0 −4 1 17 0 Q4 Carbenicillin 0 −4 1 4 0 Q4 Carbenoxolone Sodium 0 16 0 6 0 Q4 Carbetapentane Citrate 0 −13 1 7 0 Q4 Carbimazole 0 −32 0 8 0 Q4 Carbinoxamine 0 6 0 −3 0 Q4 Carboplatin 0 −23 1 4 0 Q4 Carboplatin 0 −4 1 7 0 Q4 Carglumic Acid 0 1 1 6 0 Q4 Carisoprodol 0 1 0 −1 0 Q4 Carmofur 0 −13 0 17 0 Q4 Carmustine 0 1 1 9 0 Q4 Carnitine Hydrochloride, Carnitine 0 −23 1 1 0 Q4 (dl) Hydrochloride Carprofen 0 −2 0 15 0 Q4 Carsalam 0 −7 0 11 1 Q4 Carteolol 0 12 0 7 0 Q4 Carvedilol, Carvedilol Phosphate 0 10 1 15 1 Q4 Carvedilol, Carvedilol Phosphate 0 −17 0 19 0 Q4 Carzenide 0 −24 1 14 0 Q4 Casanthranol [cascaroside A 0 −49 0 9 0 Q4 Shown] Cefaclor 0 −14 1 19 0 Q4 Cefadroxil 0 1 0 3 0 Q4 Cefalonium 0 9 1 11 0 Q4 Cefalotin 0 1 1 1 0 Q4 Cefamandole Nafate 0 −3 0 12 0 Q4 Cefamandole Sodium 0 −12 1 16 0 Q4 Cefapirin 0 −26 0 7 0 Q4 Cefazolin 0 0 1 11 0 Q4 Cefdinir 0 −9 1 16 0 Q4 Cefditorin Pivoxil 0 2 1 21 1 Q4 Cefepime 0 −26 1 4 0 Q4 Cefepime Hydrochloride 0 7 1 16 0 Q4 Cefixime 0 0 1 −8 0 Q4 Cefmenoxime Hydrochloride 0 −15 0 2 0 Q4 Cefmetazole 0 −50 0 8 0 Q4 Cefonicid Sodium 0 −11 0 15 0 Q4 Ceforanide 0 −2 1 14 1 Q4 Cefotaxime 0 −20 1 −8 0 Q4 Cefotaxime Sodium 0 −1 0 3 0 Q4 Cefotetan 0 −11 0 −5 0 Q4 Cefoxitin 0 6 0 8 0 Q4 Cefpiramide 0 12 1 20 0 Q4 Cefpodoxime Proxetil 0 −39 0 12 0 Q4 Cefprozil 0 3 0 14 0 Q4 Cefsulodin Sodium 0 3 0 14 0 Q4 Ceftazidime 0 14 1 17 1 Q4 Ceftazidime 0 −3 1 30 1 Q4 Ceftibuten 0 −7 1 12 0 Q4 Ceftiofur Hydrochloride 0 4 1 14 0 Q4 Ceftriaxone 0 −16 1 12 0 Q4 Cefuroxime 0 16 1 2 0 Q4 Cefuroxime Axetil 0 17 1 13 0 Q4 Cefuroxime Sodium 0 −12 1 19 1 Q4 Celecoxib 0 −31 0 21 0 Q4 Cephalexin 0 −1 0 17 0 Q4 Cephalosporin C Sodium 0 −34 1 8 0 Q4 Cephradine 0 −26 0 −1 0 Q4 Cetirizine 0 5 1 0 0 Q4 Chenodiol 0 −32 0 4 0 Q4 Chiniofon 0 −52 1 −1 1 Q4 Chlomezanone, Chlormezanone 0 −52 1 12 0 Q4 Chloralose 0 −14 1 9 0 Q4 Chlorambucil 3 8 1 −7 0 Q4 Chloramine-t 0 4 1 11 0 Q4 Chloramphenicol 0 19 1 6 0 Q4 Chloramphenicol Palmitate 0 15 1 9 1 Q4 CHLORAMPHENICOL SUCCINATE 0 24 2 14 1 Q4 Chlorazanil Hydrochloride 0 0 1 3 0 Q4 Chlorcyclizine 0 9 1 6 0 Q4 Chlorindanol 0 23 1 15 0 Q4 Chlorindione 2 −7 1 10 0 Q4 Chlorobutanol 0 −20 1 3 0 Q4 Chlorocresol 0 −25 1 11 0 Q4 Chlorophyllide Cu Complex Na Salt 0 −36 0 9 0 Q4 Chloropyramine 0 25 1 13 0 Q4 Chloroquine 0 2 0 7 0 Q4 Chlorothiazide 0 20 0 3 0 Q4 Chloroxine 0 −17 1 8 0 Q4 Chloroxylenol 0 −15 1 7 0 Q4 Chlorphenamine 0 0 1 13 0 Q4 Chlorphenesin Carbamate 0 −41 1 −4 0 Q4 Chlorpromazine Hydrochloride, 1 9 2 25 1 Q4 Chlorpromazine Chlorpropamide 0 3 1 17 1 Q4 Chlorprothixene 1 29 1 41 1 Q4 Chlorprothixene 1 11 1 −19 0 Q4 Chlorpyrifos 0 −7 1 1 0 Q4 Chlorquinaldol 0 −14 1 15 0 Q4 Chlortetracycline 0 −15 1 −6 0 Q4 Chlortetracycline, Chlortetracycline 0 27 2 23 0 Q4 Hydrochloride Chlorzoxazone 0 30 1 17 1 Q4 Cholecalciferol 0 −10 1 4 0 Q4 Cholecalciferol 0 −17 1 −10 0 Q4 Cholesterol 0 4 0 9 0 Q4 Cholic Acid 0 −31 0 −5 0 Q4 Choline 0 0 0 12 0 Q4 Chromocarb 0 −7 1 5 0 Q4 Cianidanol 2 −19 0 7 0 Q4 Ciclopirox 0 −1 1 40 1 Q4 Cilastatin 0 −6 1 2 0 Q4 Cilostazol 0 5 1 17 1 Q4 Cimetidine 0 −1 0 22 1 Q4 Cinalukast 0 −21 1 −8 0 Q4 Cinchocaine 0 −57 0 7 0 Q4 Cinchonidine 0 13 1 4 0 Q4 Cinchonine 0 −6 1 28 0 Q4 Cinchophen 0 −9 1 36 0 Q4 Cinnarazine 0 −17 1 6 0 Q4 Cinnarizine 0 −32 1 −2 0 Q4 Cinromide 0 −10 1 13 0 Q4 Cintriamide 0 −2 1 15 0 Q4 Ciprofibrate 0 −16 0 16 1 Q4 Ciprofloxacin 0 −30 0 17 0 Q4 Cisapride 0 −38 0 5 0 Q4 Cisplatin 0 −13 0 20 0 Q4 Citalopram 0 −12 0 10 0 Q4 Citalopram 0 −30 0 8 0 Q4 Citicoline 0 5 1 16 0 Q4 Citiolone 0 −32 0 7 0 Q4 Citric Acid 0 −36 1 −6 0 Q4 Cladribine 0 −23 1 6 0 Q4 Clarithromycin 0 −7 1 18 0 Q4 Clavulanate 0 −16 0 5 0 Q4 Clemastine, Clemastine Fumarate 0 9 1 27 0 Q4 Clemizole Hydrochloride 0 −38 0 21 1 Q4 Clenbuterol 0 11 1 25 0 Q4 Clidinium Bromide 0 −20 1 9 0 Q4 Climbazole 0 −10 1 1 0 Q4 Clinafoxacin Hydrochloride 0 −22 0 9 0 Q4 Clindamycin Hydrochloride 0 −44 0 7 0 Q4 Clindamycin Palmitate Hydrochloride 0 −23 0 11 0 Q4 Clioquinol 0 −11 1 22 1 Q4 Clobetasol Propionate 0 −22 1 30 2 Q4 Clodronate 0 −15 1 1 0 Q4 Clofarabine 0 −4 1 −2 0 Q4 Clofarabine 0 26 1 8 −1 Q4 Clofazimine 3 23 1 15 0 Q4 Clofibrate 0 −20 0 18 1 Q4 Clofibric Acid 0 −27 1 15 0 Q4 Clofoctol 0 −26 0 2 0 Q4 Clomiphene 0 11 1 7 0 Q4 Clomipramine 0 −1 1 32 1 Q4 Clonazepam 0 −12 0 −9 0 Q4 Clonidine 0 −22 1 14 0 Q4 Clopidogrel 0 −24 1 7 0 Q4 Clopidol 0 −41 1 9 0 Q4 Clorgyline Hydrochloride, Clorgiline 0 −28 0 6 0 Q4 Hydrochloride Clorsulon 0 0 0 17 0 Q4 Closantel 0 −9 0 5 0 Q4 Clotrimazole 0 −13 1 13 0 Q4 Cloxacillin 0 −13 1 17 1 Q4 Cloxyquin 0 −4 0 18 0 Q4 Clozapine 0 3 0 9 0 Q4 Colesevalam Hydrochloride (high 0 8 1 3 0 Q4 Mol Wt Copolymer @10 mg/ml) Colforsin 0 46 1 26 0 Q4 Cortisone 1 37 1 44 1 Q4 Cortisone Acetate 1 0 1 7 0 Q4 Cotinine 0 −39 1 6 1 Q4 Coumophos 0 −12 1 16 0 Q4 Creatinine 0 −3 1 10 0 Q4 Cresopyrine, Cresopirine 0 13 1 15 1 Q4 Cromoglicic acid 0 −28 0 8 0 Q4 Crotamiton 0 12 0 3 0 Q4 Cryoflurane 0 −4 1 12 0 Q4 Cyanocobalamin 0 −1 1 11 0 Q4 Cyclamic Acid 0 4 0 13 0 Q4 Cyclandelate 0 −57 0 4 0 Q4 Cyclizine 0 −23 0 34 1 Q4 Cyclobenzaprine 0 −29 0 35 1 Q4 Cyclopentolate 0 −8 1 9 0 Q4 Cyclophosphamide Hydrate, 0 −15 0 12 0 Q4 Cyclophosphamide, Cytoxan Cycloserine 0 −4 0 19 0 Q4 Cyproheptadine 0 24 1 43 1 Q4 Cyproterone Acetate 0 −10 1 12 1 Q4 Cyproterone Acetate 0 −24 1 3 0 Q4 Cyromazine 0 −9 1 9 0 Q4 Cysteamine 0 9 0 12 0 Q4 Cytarabine 0 −19 0 14 0 Q4 Cytisine 0 23 1 16 1 Q4 D-(+)-maltose 0 −7 1 14 0 Q4 D-camphor 0 −32 0 6 0 Q4 D-lactitol Monohydrate 0 11 1 16 0 Q4 D-limonene 0 −19 1 11 1 Q4 D-phenylalanine 0 1 1 23 0 Q4 Dabigatran etexilate 0 3 0 9 0 Q4 Dacarbazine 3 −13 0 17 0 Q4 Dalfampridine 0 8 2 2 0 Q4 Danazol 0 −14 1 5 0 Q4 Dantrolene 0 −6 1 14 0 Q4 Dantron, Danthron 3 −9 0 13 0 Q4 Dapiprazole 0 −9 1 14 0 Q4 Dapsone 0 −23 1 11 0 Q4 Daptomycin (5 Millimolar/dmso) 0 −6 1 17 0 Q4 Darifenacin Hydrobromide 0 1 1 0 0 Q4 Debrisoquin 0 8 1 17 0 Q4 Decamethonium 0 1 0 17 0 Q4 Decoquinate 0 7 1 20 1 Q4 Deet, Diethyltoluamide 0 −38 0 6 0 Q4 Deferiprone 0 31 1 18 0 Q4 Deferoxamine 0 2 1 32 0 Q4 Dehydroacetic Acid 0 −36 0 2 0 Q4 Dehydrocholic Acid 0 −30 1 7 0 Q4 Delavirdine 0 −11 1 9 1 Q4 Denatonium Benzoate 0 2 0 12 0 Q4 Dequadin 0 −32 0 2 0 Q4 Deracoxib 0 −34 0 8 0 Q4 Desloratadine 0 19 1 45 0 Q4 Desvenlafaxine 0 13 1 3 1 Q4 Dexbrompheniramine, 0 0 0 10 0 Q4 Dexbrompheniramine Maleate Dexchlorpheniramine 0 −8 1 11 0 Q4 Dexfenfluramine 0 −15 1 5 0 Q4 Dexibuprofen 0 −3 1 13 0 Q4 Dexketoprofen 0 −23 0 −8 0 Q4 Dexlansoprazole 0 −23 0 −12 0 Q4 Dexpanthenol 0 −1 0 11 0 Q4 Dexpropranolol Hydrochloride 0 −21 0 19 0 Q4 Dextromethorphan 0 −18 1 4 0 Q4 Dextromethorphan 0 −20 1 −5 0 Q4 Dextropropoxyphene 0 −25 1 15 0 Q4 Diacerin 3 −30 1 10 0 Q4 Diacetamate 0 −25 1 21 0 Q4 Diatrizoate 0 4 0 5 0 Q4 Diaveridine 0 −8 1 13 1 Q4 Diazepam 0 −19 1 5 0 Q4 Diazinon, Dimpylate 0 −7 1 −8 0 Q4 Diazoxide 0 3 0 24 0 Q4 Dibekacin 0 10 1 20 1 Q4 Dibenzothiophene 0 −21 0 14 0 Q4 Dibutyl Phthalate 0 −30 1 9 0 Q4 Dichlorophene, Dichlorophen 0 −23 1 15 0 Q4 Dichlorvos 0 −1 1 19 1 Q4 Diclazuril 0 48 1 21 0 Q4 Diclofenac 0 −27 0 12 0 Q4 Dicloxacillin 0 −23 0 14 0 Q4 Dicoumarol 0 −24 1 18 0 Q4 Dicyclomine 0 −36 0 16 0 Q4 Didanosine 0 −8 1 −2 0 Q4 Dienestrol 0 17 1 21 0 Q4 Diethylcarbamazine 0 −26 0 11 0 Q4 Diethylstilbestrol 0 −26 1 16 0 Q4 Diflorasone Diacetate 0 42 1 31 0 Q4 Difloxacin Hydrochloride 0 25 1 33 1 Q4 Diflunisal 0 −62 0 15 0 Q4 Digitoxin 0 −13 1 5 1 Q4 Digitoxin 0 −1 1 20 0 Q4 Digoxin 0 −5 1 10 0 Q4 Dihydroergotamine 0 7 1 39 1 Q4 Dihydroergotamine 0 −3 0 22 0 Q4 Dihydrostreptomycin Sulfate 0 −7 1 12 0 Q4 Diiodohydroxyquinoline 0 −6 1 22 0 Q4 Diloxanide Furoate 0 −3 1 17 0 Q4 Diltiazem 0 21 1 9 0 Q4 Diltiazem Hydrochloride 0 −5 0 15 0 Q4 Dimenhydrinate 0 −17 1 11 0 Q4 Dimercaptopropanol, Dimercaprol 0 49 1 16 0 Q4 Dimesna 0 −19 1 13 0 Q4 Dimethadione 0 −13 1 14 0 Q4 Dimethyl Fumarate 0 4 0 16 0 Q4 Dimetridazole 0 −16 1 15 1 Q4 Diminazene 3 −58 0 11 0 Q4 Dinitolmide 0 −45 1 10 0 Q4 Dinoprost Tromethamine 0 36 1 15 0 Q4 Diosmin 0 −12 1 10 0 Q4 Dioxybenzone 0 −4 1 16 0 Q4 Diperodon Hydrochloride 0 42 1 26 1 Q4 Diphenhydramine 0 −47 0 15 0 Q4 Diphenidol 0 −34 1 −5 0 Q4 Diphenylpyraline 0 −69 0 6 0 Q4 Dipyridamole 0 11 1 6 0 Q4 Dipyrocetyl 0 −1 1 7 0 Q4 Dirithromycin 0 7 1 27 1 Q4 Disopyramide 0 −13 1 1 0 Q4 Dixanthogen 0 4 1 17 0 Q4 Dobutamine 2 −29 1 −7 0 Q4 Dobutamine Hydrochloride 2 22 1 27 0 Q4 Doconexent 0 3 1 11 0 Q4 Docosanol 0 −10 0 −2 0 Q4 Docusate Sodium 0 −39 0 −4 0 Q4 Dofetilide 0 −26 1 5 0 Q4 Dolasetron 0 −19 1 −3 0 Q4 Domperidone 0 20 1 15 0 Q4 Donepezil 0 5 1 17 0 Q4 Dopamine 2 −17 1 9 0 Q4 Doramectin 0 −9 1 7 0 Q4 Dorzolamide 0 −40 1 15 1 Q4 Doxapram Hydrochloride 0 −18 0 9 0 Q4 Doxazosin 0 −26 0 6 0 Q4 Doxepin 0 −10 1 16 0 Q4 Doxepin 0 −36 1 13 0 Q4 Doxofylline 0 −42 0 18 0 Q4 Doxycycline 0 −19 0 18 0 Q4 Doxylamine 0 −45 1 17 0 Q4 Drofenine Hydrochloride 0 −44 1 9 0 Q4 Droperidol 0 −3 1 20 1 Q4 Dropropizine 0 −26 1 11 0 Q4 Drospirenone 0 11 1 6 1 Q4 Duloxetine 0 22 0 24 0 Q4 Dutasteride 0 7 1 22 0 Q4 Dyclonine 0 7 1 12 0 Q4 Dyclonine 0 −8 1 5 0 Q4 Dydrogesterone 0 −11 0 4 0 Q4 Dyphylline 0 −7 0 −6 0 Q4 Ebselen 0 −49 0 −1 0 Q4 Ecamsule 0 20 1 29 0 Q4 Econazole 0 0 0 18 0 Q4 Edetic Acid 0 −54 0 2 0 Q4 Edetic Acid 0 0 0 16 1 Q4 Editol 0 24 1 3 0 Q4 Edoxudine 0 −10 0 1 0 Q4 Edrophonium 0 −9 1 −2 0 Q4 Efaroxan, Efaroxan Hydrochloride 0 1 1 5 0 Q4 Efavirenz 0 −7 1 −5 0 Q4 Efloxate 0 −8 1 −1 0 Q4 Eletriptan Hydrobromide 0 −14 1 12 0 Q4 Ellagic Acid 2 −5 1 24 0 Q4 Eltanolone 0 −6 1 4 0 Q4 Emtricitabine 0 −73 1 11 1 Q4 Enalapril 0 24 1 6 1 Q4 Enalaprilat 0 25 1 18 0 Q4 Enilconazole 0 24 1 15 1 Q4 Enoxolone 0 −40 1 7 0 Q4 Enrofloxacin 0 5 1 23 1 Q4 Entacapone 2 −3 1 11 0 Q4 Entacapone 2 −19 1 17 1 Q4 Ephedrine (1r,2s) Hydrochloride 0 −26 1 10 0 Q4 Epiestriol 0 −25 0 6 0 Q4 Epinastine 0 −38 1 −15 0 Q4 Eprinomectin 0 −3 1 8 1 Q4 Eprobemide 0 −24 0 21 0 Q4 Eprodisate Disodium 0 −3 1 12 0 Q4 Eprosartan 0 −29 1 −8 0 Q4 Equilin 0 1 0 15 0 Q4 Erdosteine 0 2 1 13 0 Q4 Ergocalciferol 0 −6 1 7 0 Q4 Ergonovine 0 −23 1 −10 0 Q4 Ergonovine Maleate 0 9 1 7 0 Q4 Ergotamine 0 6 1 −4 0 Q4 Ergotamine Tartrate 0 31 0 22 1 Q4 Erlotinib 0 −52 1 −8 0 Q4 Erythritol 0 −25 1 10 0 Q4 Erythromycin 0 9 1 20 0 Q4 Erythromycin 0 −20 1 13 1 Q4 Erythromycin Ethylsuccinate 0 −14 0 14 0 Q4 Erythromycin Propionate 0 23 1 27 0 Q4 Erythrosine Sodium 2 1 1 0 0 Q4 Esmolol, Esmolol Hydrochloride 0 −15 1 2 0 Q4 Esomeprazole; Omeprazole 0 −17 0 24 0 Q4 Esomeprazole; Omeprazole 0 45 1 13 1 Q4 Estradiol 0 3 0 16 0 Q4 Estradiol 0 −10 1 −5 0 Q4 Estradiol Benzoate 0 −3 1 8 0 Q4 Estradiol Cypionate 0 −25 0 8 0 Q4 Estradiol Dipropionate 0 0 1 17 1 Q4 Estradiol Valerate 0 −8 1 16 1 Q4 Estramustine 0 8 1 16 1 Q4 Estriol 0 −17 1 8 1 Q4 Estriol 0 −17 1 16 1 Q4 Estrone 0 35 2 16 0 Q4 Estropipate 0 10 1 10 0 Q4 Eszopiclone 0 1 0 17 0 Q4 Etamivan 0 −16 1 16 0 Q4 Ethacridine Lactate 2 −64 1 −8 0 Q4 Ethambutol 0 12 1 14 1 Q4 Ethaverine Hydrochloride 0 41 1 24 1 Q4 Ethenzamide 0 −15 1 5 0 Q4 Ethinyl Estradiol 0 9 1 5 0 Q4 Ethionamide 0 17 1 14 0 Q4 Ethisterone 0 −5 0 13 1 Q4 Ethopabate 0 −43 0 6 0 Q4 Ethopropazine 1 31 1 30 0 Q4 Ethosuximide 0 −9 0 19 0 Q4 Ethotoin 0 −36 1 12 0 Q4 Ethoxzolamide 0 29 1 16 1 Q4 Ethyl carbamate 0 44 1 27 0 Q4 Ethyl Vanillin 0 −15 0 18 0 Q4 Ethylparaben 0 −5 1 8 1 Q4 Ethynodiol Diacetate 0 −29 1 −6 0 Q4 Ethynodiol Diacetate 0 −6 1 16 1 Q4 Etidronic acid 0 −16 1 18 0 Q4 Etodolac 0 −87 0 14 1 Q4 Etomidate 0 −19 0 12 0 Q4 Etomidate 0 −24 0 −7 0 Q4 Etoposide 0 30 1 15 0 Q4 Etoricoxib 0 15 1 −4 0 Q4 Etretinate 0 −19 1 9 0 Q4 Eucalyptol 0 −15 0 9 0 Q4 Eucatropine Hydrochloride 0 1 1 24 0 Q4 Eugenol 0 37 0 20 0 Q4 Evans Blue 3 −37 0 7 0 Q4 Exalamide 0 −15 1 18 0 Q4 Exemestane 0 22 1 21 1 Q4 Exemestane 0 31 1 34 0 Q4 Ezetimibe 0 41 1 27 0 Q4 Famciclovir 0 −22 0 14 1 Q4 Famotidine 0 −82 0 9 0 Q4 Famprofazone 0 −3 1 15 0 Q4 Fasudil Hydrochloride 0 −72 1 −21 0 Q4 Febuxostat 0 3 1 10 0 Q4 Felbamate 0 −32 1 0 0 Q4 Felodipine 0 2 1 14 0 Q4 Fenaclon 0 8 1 16 0 Q4 Fenamisal, Phenyl Aminosalicylate 0 −8 1 16 0 Q4 Fenbufen 0 3 0 25 0 Q4 Fenipentol 0 −1 1 25 0 Q4 Fenofibrate 0 −37 1 0 0 Q4 Fenofibric Acid 0 6 1 9 0 Q4 Fenoldopam 2 6 1 20 0 Q4 Fenoldopam 2 −23 0 5 0 Q4 Fenoprofen 0 −7 0 −4 0 Q4 Fenoterol 0 40 0 26 0 Q4 Fenretinide 0 −42 0 35 0 Q4 Fenspiride 0 −43 1 −1 0 Q4 Fexofenadine 0 13 1 14 0 Q4 Finasteride 0 −46 1 42 1 Q4 Finasteride 0 14 0 −16 0 Q4 Fipexide Hydrochloride 0 −15 0 11 0 Q4 Fipronil 0 −5 1 12 0 Q4 Firocoxib 0 −10 1 11 1 Q4 Flecainide 0 −35 0 5 0 Q4 Flopropione 0 −35 1 10 0 Q4 Florfenicol 0 −12 1 16 1 Q4 Floxuridine 0 −16 1 5 0 Q4 Floxuridine 0 7 1 −16 0 Q4 Fluconazole 0 3 0 19 0 Q4 Flucytosine, 5-fluorocytosine 0 13 1 17 1 Q4 Fludarabine Phosphate 0 1 1 11 0 Q4 Flufenamic Acid 0 −20 0 −1 0 Q4 Flumazenil 0 5 1 14 1 Q4 Flumequine 0 −1 0 16 1 Q4 Flunarizine 0 −10 1 12 1 Q4 Flunixin Meglumine 0 −6 1 8 0 Q4 Fluocinolone Acetonide 0 −35 1 10 0 Q4 Fluorescein 0 −3 0 4 0 Q4 Fluphenazine 1 7 1 20 0 Q4 Flurbiprofen 0 17 0 33 0 Q4 Flurofamide 0 3 0 −12 0 Q4 Flurothyl 0 0 1 28 0 Q4 Fluroxene 0 −19 0 11 0 Q4 Flutamide 0 15 1 23 0 Q4 Fluvoxamine 0 −8 0 −3 0 Q4 Folic Acid 0 30 1 22 0 Q4 Folic Acid 0 11 1 17 1 Q4 Fomepizole Hydrochloride, 0 8 1 11 0 Q4 Fomepizole Fomepizole Hydrochloride, 0 −7 1 15 0 Q4 Fomepizole Formestane 0 −6 1 10 0 Q4 Formoterol 0 −6 1 32 1 Q4 Foscarnet 0 5 1 10 0 Q4 Fosfosal 0 −29 0 16 1 Q4 Fosinopril 0 −52 1 −1 0 Q4 Fosphomycin 0 4 1 12 0 Q4 Fulvestrant 0 −27 0 15 0 Q4 Fulvestrant 0 −11 1 11 1 Q4 Fumaric Acid 0 19 1 17 0 Q4 Furaltadone 0 −60 1 0 0 Q4 Furazolidone 0 3 0 8 −1 Q4 Furosemide 0 −5 1 25 1 Q4 Gaba, Gamma-aminobutyric Acid 0 −14 1 18 0 Q4 Gabapentin 0 −17 1 19 1 Q4 Gabapentin 0 −3 1 16 0 Q4 Gaboxadol 0 −43 0 3 0 Q4 Gadoteridol 0 −18 1 15 0 Q4 Galanthamine Hydrobromide, 0 10 0 9 0 Q4 Galantamine Gallamine Triethiodide 0 −11 1 24 1 Q4 Gallic Acid 2 −32 0 10 0 Q4 Gamma-Phenyl-Butyric Acid 0 −36 0 9 0 Q4 Ganaxolone 0 −38 1 12 0 Q4 Ganciclovir 0 −2 0 13 0 Q4 Gatifloxacin 0 −19 1 19 0 Q4 Gefitinib 0 39 1 17 0 Q4 Gefitinib 0 −52 0 32 0 Q4 Gefitinib 0 −10 1 23 0 Q4 Gemcitabine 0 −31 1 0 0 Q4 Gemfibrozil 0 −47 0 10 0 Q4 Gemifloxacin 0 −34 0 6 0 Q4 Glafenine 0 8 1 18 0 Q4 Glibenclamide, Glyburide 0 −23 1 8 0 Q4 Gliclazide 0 1 1 20 0 Q4 Glimepiride 0 0 1 21 0 Q4 Glipizide 0 12 1 18 0 Q4 Gluconolactone 0 −1 0 17 0 Q4 Glucosamine Hydrochloride 0 −2 1 19 0 Q4 Glutathione 0 −5 1 13 0 Q4 Glycopyrrolate 0 −33 0 3 0 Q4 Granisetron 0 −38 1 4 0 Q4 Granisetron Hydrochloride 0 −1 0 14 0 Q4 Griseofulvin 0 9 1 13 0 Q4 Guaiacol 0 −51 0 11 0 Q4 Guaifenesin 0 −15 0 11 0 Q4 Guanabenz 0 −46 1 −18 0 Q4 Guanabenz Acetate 0 −46 0 −2 0 Q4 Guanadrel 0 −1 1 5 0 Q4 Guanethidine 0 −44 0 12 0 Q4 Guanfacine 0 −5 1 15 0 Q4 Guanfacine 0 −34 1 −11 0 Q4 Guanidine 0 −32 0 −1 0 Q4 Halazone 0 −13 0 19 0 Q4 Haloperidol 0 −22 1 12 1 Q4 Halothane 0 17 1 17 0 Q4 Heptaminol Hydrochloride 0 −19 1 13 0 Q4 Hetacillin Potassium 0 −15 0 15 1 Q4 Hexachlorophene 0 −19 0 15 0 Q4 Hexamethylenetetramine 0 −9 1 20 0 Q4 Hexestrol 0 −29 1 7 0 Q4 Hexetidine 0 −85 1 19 0 Q4 Hexylene Glycol 0 −8 1 12 0 Q4 Hexylresorcinol 0 −6 1 18 0 Q4 Histamine 0 21 1 27 0 Q4 Homatropine Bromide, Homatropine 0 26 1 30 0 Q4 Hydrobromide Homatropine Methylbromide 0 −18 1 12 0 Q4 Homosalate 0 −3 1 18 1 Q4 Hydralazine 0 −13 1 22 0 Q4 Hydrastine (1r,9s) 0 −27 0 10 0 Q4 Hydrastinine Hydrochloride 0 0 1 22 0 Q4 Hydrochlorothiazide 0 −29 0 21 0 Q4 Hydroflumethiazide 0 −9 0 11 0 Q4 Hydroquinidine 0 −8 1 18 0 Q4 Hydroquinone 0 14 1 12 0 Q4 Hydroxyamphetamine 0 −12 1 −2 0 Q4 Hydroxychloroquine 0 35 1 17 0 Q4 Hydroxyprogesterone 0 9 1 33 1 Q4 Hydroxyprogesterone Caproate 0 22 1 25 0 Q4 Hydroxyurea 0 1 1 25 1 Q4 Hydroxyzine 0 −28 0 10 0 Q4 Hydroxyzine 0 22 1 35 0 Q4 Hyoscyamine 0 −20 0 13 0 Q4 Hyoscyamine 0 −42 1 10 0 Q4 Ibandronate 0 40 0 −12 0 Q4 Ibandronate 0 −7 1 2 0 Q4 Icosapent ethyl 0 −13 0 1 0 Q4 Idebenone 3 −24 1 2 0 Q4 Idoxuridine 0 −8 1 −20 0 Q4 Idoxuridine 0 −12 1 15 1 Q4 Idramantone 0 −7 1 8 0 Q4 Ifosfamide 0 −11 1 10 0 Q4 Iloprost 0 23 1 −1 0 Q4 Imatinib, Imatinib Mesylate 0 −4 1 11 0 Q4 Imexon 0 −3 1 12 1 Q4 Imipenem 0 −39 1 2 0 Q4 Imiquimod 0 −23 0 15 0 Q4 Imiquimod 0 −25 0 −5 0 Q4 Indapamide 0 −14 0 11 0 Q4 Indinavir 0 −22 1 5 0 Q4 Indomethacin 0 −56 0 13 0 Q4 Indoprofen 0 −34 1 18 0 Q4 Inosine 0 −31 1 −11 0 Q4 Inositol 0 −21 0 11 0 Q4 Iodixanol 0 −15 0 10 0 Q4 Iopamidol 0 −14 1 −4 0 Q4 Iothalamic Acid 0 5 1 7 0 Q4 Ioversol 0 −1 1 18 1 Q4 Ioxilan 0 7 0 8 0 Q4 Ipratropium Bromide 0 6 1 8 0 Q4 Ipriflavone 0 −29 1 7 0 Q4 Iproheptine 0 −34 1 11 0 Q4 Iproniazid 0 −44 1 10 0 Q4 Irinotecan, Irinotecan Hydrochloride 0 −6 0 10 1 Q4 (trihydrate) Irsogladine Maleate 0 −27 1 14 0 Q4 Isaxonine 0 −46 0 −1 0 Q4 Isobutamben 0 −5 1 −1 0 Q4 Isoetarine 2 21 1 23 0 Q4 Isoniazid 0 −29 1 19 0 Q4 Isoprenaline 2 29 0 37 0 Q4 Isopropamide 0 19 0 24 0 Q4 Isosorbide Dinitrate 0 −14 0 10 0 Q4 Isosorbide Mononitrate 0 −17 0 15 1 Q4 Isovaleramide 0 −10 1 1 0 Q4 Isoxsuprine Hydrochloride 0 −13 0 23 0 Q4 Isradipine 0 9 1 2 0 Q4 Itopride, Itopride Hydrochloride 0 −49 0 25 0 Q4 Itraconazole 3 −27 1 1 0 Q4 Itraconazole 3 −26 1 3 1 Q4 Ivermectin 0 −15 1 10 0 Q4 Ivermectin 0 6 0 19 0 Q4 Kainic Acid 0 −77 0 14 0 Q4 Kanamycin Sulfate, Kanamycin A 0 −13 0 20 1 Q4 Sulfate Ketamine 0 −86 1 −2 0 Q4 Ketanserin Tartrate 0 17 1 15 0 Q4 Ketoconazole 3 −57 0 −1 0 Q4 Ketoprofen 0 34 0 25 0 Q4 Ketorolac 0 15 0 26 0 Q4 Ketotifen 0 1 1 15 0 Q4 Khellin 0 −16 0 5 0 Q4 L-alpha-methyl Dopa, Methyldopa 2 4 0 15 0 Q4 L-aspartic Acid, Aspartic Acid (I) 0 5 1 13 1 Q4 L-glutamine, Glutamine (I) 0 4 0 14 0 Q4 L-Isoleucine 0 −35 0 17 0 Q4 L(−)-norephedrine, 0 −15 1 7 0 Q4 Phenylpropanolamine Hydrochloride Labetalol 0 32 1 30 1 Q4 Lacitol 0 −24 1 18 1 Q4 Lactic Acid 0 0 1 14 1 Q4 Lactose Monohydrate 0 −22 1 9 0 Q4 Lactulose 0 −14 0 4 0 Q4 Lamivudine 0 −14 1 −20 0 Q4 Lamivudine 0 38 1 28 0 Q4 Lamotrigine 0 −12 1 12 0 Q4 Lanatoside C 0 17 1 35 1 Q4 Lansoprazole 0 3 0 15 0 Q4 Lapatinib 0 −54 1 10 0 Q4 Lapatinib 0 −20 1 −1 0 Q4 Latamoxef 0 −18 1 22 0 Q4 Latanoprost 0 10 1 4 0 Q4 Lefunamide, Leflunomide 0 −22 0 10 0 Q4 Letrozole 0 15 1 16 0 Q4 Leucovorin 0 −29 1 8 0 Q4 Leuprolide 0 5 1 −3 −1 Q4 Levamisole 0 −15 0 12 0 Q4 Levcycloserine 0 2 1 12 0 Q4 Levetiracetam 0 −24 1 7 0 Q4 Levobunolol 0 −28 1 30 0 Q4 Levobunolol Hydrochloride 0 −19 0 12 0 Q4 Levobupivacaine 0 34 1 −10 0 Q4 Levocabastine 0 7 1 4 0 Q4 Levocarnitine 0 8 1 15 0 Q4 Levocarnitine Propionate 0 −16 1 12 1 Q4 Hydrochloride Levocetirizine Dihydrochloride 0 −44 0 −2 0 Q4 Levodopa 2 17 1 −5 0 Q4 Levofloxacin 0 12 1 31 0 Q4 Levonorgestrel 0 16 1 −13 0 Q4 Levosalbutamol 0 −29 0 11 0 Q4 Levosimendan 2 46 1 42 0 Q4 Levosulpiride 0 −36 1 18 0 Q4 Lidocaine 0 −23 0 8 0 Q4 Linagliptin 0 2 1 14 1 Q4 Lincomycin 0 −29 1 −5 0 Q4 Lincomycin Hydrochloride 0 −4 1 14 0 Q4 Lindane 0 42 0 11 0 Q4 Linezolid 3 −18 1 19 0 Q4 Liothyronine 0 0 1 21 1 Q4 Liothyronine 0 29 1 20 1 Q4 Lipoamide 0 −18 0 8 0 Q4 Lipoic Acid 0 −38 1 39 0 Q4 Lisinopril 0 1 1 3 0 Q4 Lisuride 0 −27 1 −9 0 Q4 Lithium hydride 0 −6 1 20 0 Q4 Lobeline 0 −38 1 −5 0 Q4 Lobendazole 0 −4 0 20 0 Q4 Lofexidine 0 −30 0 0 0 Q4 Lofexidine 0 −22 1 10 0 Q4 Lomefloxacin 0 7 1 15 0 Q4 Lomerizine, Lomerizine 0 −5 1 8 0 Q4 Dihydrochloride Lomustine 0 0 1 19 1 Q4 Lonidamine 0 7 1 19 0 Q4 Loratadine 0 7 0 8 0 Q4 Lorazepam 0 −13 1 −1 0 Q4 Lorglumide Sodium 0 −29 1 13 1 Q4 Lornoxicam 0 22 0 10 1 Q4 Losartan, Losartan Potassium 0 −3 1 19 1 Q4 Loxapine 0 −41 0 9 0 Q4 Loxoprofen 0 −19 1 10 0 Q4 Lufenuron 0 15 1 13 0 Q4 Lumiracoxib 0 14 1 18 0 Q4 Mafenide 0 10 1 −3 0 Q4 Malathion 0 −8 0 8 0 Q4 Mangafodipir Trisodium 0 1 1 11 0 Q4 Manidipine Hydrochloride 0 −43 1 12 1 Q4 Mannitol 0 5 1 0 0 Q4 Mebeverine 0 −11 0 7 0 Q4 Mebhydrolin Naphthalenesulfonate 3 4 1 17 0 Q4 Meclizine 0 −33 0 12 0 Q4 Meclocycline Sulfosalicylate 0 20 1 18 0 Q4 Meclofenamic Acid 0 12 1 14 0 Q4 Meclofenoxate 0 −23 1 24 0 Q4 Mecysteine Hydrochloride 0 20 1 22 0 Q4 Mefenamic Acid 0 −6 1 3 1 Q4 Mefexamide, Mefexamide 0 −13 0 −2 0 Q4 Hydrochloride Mefloquine 0 −18 1 13 0 Q4 Megestrol Acetate 0 11 1 24 1 Q4 Meglumine 0 25 0 5 0 Q4 Meglumine 0 −19 1 11 1 Q4 Melatonin 0 4 1 −3 0 Q4 Meloxicam 0 −1 1 20 0 Q4 Melphalan 3 28 0 3 0 Q4 Memantine 0 2 0 5 0 Q4 Menthol 0 −20 1 0 0 Q4 Meparfylon 0 −49 0 3 0 Q4 Mepartricin 0 6 1 −1 0 Q4 Mepenzolate 0 45 1 34 1 Q4 Mephenesin 0 14 0 13 0 Q4 Mepiroxol 0 −6 1 11 0 Q4 Mepivacaine 0 7 0 10 0 Q4 Meprylcaine Hydrochloride 0 −10 0 7 0 Q4 Mepyramine 0 −11 0 13 0 Q4 Merbromin 2 −6 0 36 0 Q4 Mercaptopurine 0 2 0 9 0 Q4 Meropenem 0 47 1 28 0 Q4 Meropenem 0 −1 1 3 0 Q4 Mesalazine 0 9 1 19 0 Q4 Mesna 0 18 1 5 0 Q4 Mesoridazine 1 −5 1 40 1 Q4 Mestranol 0 −6 0 10 1 Q4 Meta-cresyl Acetate 0 −7 1 −3 0 Q4 Metaraminol 0 −30 0 6 1 Q4 Metaraminol 0 39 1 25 1 Q4 Metaxalone 0 13 1 6 0 Q4 Metergoline 2 −12 1 16 0 Q4 Metformin 0 −15 0 0 0 Q4 Methacholine 0 2 1 9 0 Q4 Methacycline 0 −10 0 11 0 Q4 Methapyrilene 0 17 1 13 0 Q4 Methazolamide 0 2 1 10 0 Q4 Methazolamide 0 −32 1 11 0 Q4 Methimazole 0 −29 0 12 0 Q4 Methiothepin Mesylate, Metitepine 1 11 1 −5 0 Q4 Maleate Methocarbamol 0 4 0 10 0 Q4 Methoprene (s) 0 −9 1 12 0 Q4 Methotrexate 0 −32 1 −4 0 Q4 Methotrexate(+/−) 0 0 0 0 0 Q4 Methoxamine 0 3 1 1 0 Q4 Methoxsalen 0 −31 0 14 0 Q4 Methscopolamine Bromide 0 1 1 12 0 Q4 Methscopolamine Bromide 0 14 1 −15 0 Q4 Methsuximide 0 −39 0 19 0 Q4 Methyclothiazide 0 25 1 17 0 Q4 Methylatropine Nitrate 0 −2 1 14 0 Q4 Methylergometrine 0 −43 0 7 0 Q4 Methyltestosterone 0 −40 1 −2 0 Q4 Methylthiouracil 0 31 0 16 0 Q4 Methysergide Maleate 2 1 0 16 0 Q4 Methysergide Maleate 2 26 1 7 0 Q4 Meticillin 0 −19 0 18 0 Q4 Meticrane 0 −33 0 9 0 Q4 Metoclopramide 0 −2 1 12 0 Q4 Metolazone 0 −2 1 19 0 Q4 Metoprolol 0 4 1 22 0 Q4 Metrifonate 0 −34 0 8 0 Q4 Metronidazole 0 −12 0 15 1 Q4 Metyrapone 0 −60 1 39 0 Q4 Mexeneone 0 −8 1 9 0 Q4 Mexiletine 0 2 1 13 0 Q4 Mianserin 0 −18 0 17 0 Q4 Mifepristone 3 12 1 −3 0 Q4 Mifepristone 3 8 1 7 0 Q4 Miglitol 0 −9 0 13 0 Q4 Miglustat 0 −6 1 0 0 Q4 Milrinone 0 4 0 23 0 Q4 Miltefosine 0 −31 1 11 0 Q4 Minaprine 0 −22 0 10 0 Q4 Minocycline 0 −6 1 16 0 Q4 Minoxidil 0 −5 1 9 1 Q4 Misoprostol 0 13 1 32 0 Q4 Mitotane 0 −19 0 −1 0 Q4 Moclobemide 0 −6 1 14 0 Q4 Modafinil 0 −5 1 25 0 Q4 Modaline Sulfate 0 −9 1 25 0 Q4 Moexipril 0 −29 1 9 1 Q4 Moguisteine 0 −2 1 15 0 Q4 Monobenzone 0 7 1 16 0 Q4 Montelukast 0 −5 1 12 0 Q4 Montelukast Sodium 0 21 1 16 0 Q4 Morantel Citrate 0 −11 1 11 0 Q4 Moroxydine Hydrochloride 0 −22 1 4 0 Q4 Moxidectin 0 1 0 14 1 Q4 Moxifloxacin Hydrochloride 0 7 1 14 0 Q4 Moxisylyte 0 −22 0 3 0 Q4 Mupirocin 0 11 1 20 0 Q4 N-acetylneuramic Acid, 0 −9 1 19 1 Q4 Aceneuramic Acid N-acetylprocainamide 0 −11 0 9 1 Q4 Hydrochloride, Acecainide Hydrochloride Nabumetone 0 −13 1 15 0 Q4 Nadide 0 −14 1 5 0 Q4 Nadifloxacin 0 −14 1 10 0 Q4 Nafcillin Sodium 0 −7 0 15 0 Q4 Nafronyl Oxalate 0 15 1 15 0 Q4 Naftifine 0 −12 0 3 0 Q4 Nalbuphine 0 −8 1 16 0 Q4 Nalbuphine Hydrochloride 0 −11 1 15 0 Q4 Nalidixic Acid 0 −1 1 13 1 Q4 Naloxone 0 −41 1 21 1 Q4 Naloxone Hydrochloride 0 −16 0 11 0 Q4 Nanofin 0 17 1 12 0 Q4 Naphazoline 0 −1 1 13 1 Q4 Naproxen 0 −25 0 15 0 Q4 Naproxol 0 −10 0 16 0 Q4 Natamycin 0 −7 1 35 0 Q4 Nateglinide 0 −20 1 −2 1 Q4 Nateglinide 0 −18 1 13 0 Q4 Nebivolol 0 −10 1 6 0 Q4 Nefazodone 0 −37 0 3 0 Q4 Nefiracetam 0 −21 0 13 0 Q4 Nefopam 0 −15 0 3 0 Q4 Nelarabin 0 −10 0 0 0 Q4 Nelfinavir 0 16 1 13 0 Q4 Neomycin Sulfate 0 15 0 18 0 Q4 Neostigmine 0 −15 0 15 0 Q4 Netilmicin Sulfate 0 −6 1 12 0 Q4 Nevirapine 0 3 0 15 0 Q4 Niacin 0 8 1 13 0 Q4 Nialamide 0 −6 1 11 0 Q4 Nicardipine 0 11 0 12 0 Q4 Nicergoline 2 6 1 7 0 Q4 Nicolsamide, Niclosamide 0 −2 1 21 1 Q4 Nicopholine 0 26 0 21 0 Q4 Nicotinamide 0 −8 1 16 0 Q4 Nicotine 0 2 1 21 1 Q4 Nicotinyl Alcohol Tartrate 0 −5 0 4 0 Q4 Nifedipine 0 −36 0 2 0 Q4 Niflumic Acid 0 −14 1 9 0 Q4 Nifursol 0 47 1 20 0 Q4 Nikethamide 0 12 0 −4 0 Q4 Nilotinib 0 −35 1 17 1 Q4 Nilutamide 0 33 1 15 0 Q4 Nilutamide 0 −30 1 4 0 Q4 Nimesulide 0 −19 1 13 0 Q4 Nimodipine 0 −6 1 2 0 Q4 Nimustine 0 −1 1 10 0 Q4 Nisoldipine 0 6 1 25 1 Q4 Nitarsone 0 −3 1 19 0 Q4 Nitazoxanide 0 −16 1 22 0 Q4 Nithiamide 0 3 1 21 1 Q4 Nitrazepam 0 −1 1 −4 0 Q4 Nitrendipine 0 −12 1 18 0 Q4 Nitrofural 0 −14 0 1 0 Q4 Nitrofurantoin 0 −3 1 11 0 Q4 Nitroglycerin 0 2 1 17 1 Q4 Nitromide 0 −7 0 11 0 Q4 Nitroxoline 0 −5 1 −6 0 Q4 Nizatidine 0 −18 0 14 0 Q4 Nomifensine 0 −12 0 13 0 Q4 Nonoxynol-9 0 −7 0 23 0 Q4 Norepinephrine 2 44 1 32 0 Q4 Norepinephrine 2 6 1 −1 0 Q4 Norethindrone 0 9 0 15 1 Q4 Norethindrone Acetate 0 −5 1 18 0 Q4 Norethisterone 0 18 1 1 0 Q4 Norethynodrel 0 1 0 10 0 Q4 Norfloxacin 0 0 1 9 0 Q4 Norgestimate 0 −5 0 12 0 Q4 Norgestrel 0 4 1 22 1 Q4 Norgestrel, Levonorgestrel 0 13 0 14 1 Q4 Noscapine Hydrochloride 0 −15 1 23 0 Q4 Novobiocin 0 −17 0 8 0 Q4 Nystatin 0 −34 0 39 1 Q4 Nystatin 0 −76 1 −15 1 Q4 Octisalate 0 −17 0 5 0 Q4 Octodrine 0 2 0 −2 0 Q4 Octreotide 0 3 1 −5 1 Q4 Ofloxacin 0 −13 1 16 0 Q4 Olanzapine 0 6 1 9 0 Q4 Oleandomycin Phosphate 0 −9 1 16 0 Q4 Oleic Acid 0 23 1 25 1 Q4 Olmesartan 0 −13 0 18 0 Q4 Olmesartan Medoxomil 0 −5 1 12 0 Q4 Olopatadine 0 −42 1 −9 0 Q4 Olsalazine 3 −36 1 22 1 Q4 Oltipraz 0 −2 1 −1 0 Q4 Ondansetron 0 −6 1 14 0 Q4 Orbifloxacin 0 −18 0 15 0 Q4 Orciprenaline 0 15 0 25 0 Q4 Orlistat 0 17 1 5 1 Q4 Orlistat 0 15 1 −1 0 Q4 Ornidazole 0 −22 0 12 0 Q4 Ornithine Aketoglutarate, Ornithine 0 −36 1 14 0 Q4 Hydrochloride Orotic Acid 0 −5 0 16 0 Q4 Orphenadrine 0 −17 0 10 0 Q4 Oseltamivir 0 −28 1 26 1 Q4 Oseltamivir Phosphate 0 −7 1 13 0 Q4 Ouabain 0 −5 0 11 0 Q4 Ouabain 0 −45 0 −13 0 Q4 Oxacillin Sodium 0 −20 0 11 0 Q4 Oxaprozin 0 0 1 15 1 Q4 Oxcarbazepine, Trileptal 0 3 1 35 0 Q4 Oxeladin 0 −27 1 6 0 Q4 Oxethazaine 0 25 1 42 0 Q4 Oxfendazole 0 10 0 20 1 Q4 Oxiconazole 0 12 1 15 1 Q4 Oxidopamine Hydrochloride 2 1 0 19 0 Q4 Oxiniacic Acid 0 −6 1 12 0 Q4 Oxolamine Citrate 0 −1 1 10 0 Q4 Oxolinic Acid 0 7 0 13 1 Q4 Oxybenzone 0 −15 0 12 0 Q4 Oxybuprocaine 0 7 1 11 0 Q4 Oxybutynin 0 −9 0 0 1 Q4 Oxymetazoline 0 −13 1 20 0 Q4 Oxymetholone 0 −18 1 −7 0 Q4 Oxyphencyclimine 0 15 1 11 0 Q4 Oxyphenonium 0 −14 1 30 1 Q4 Oxytetracycline 0 0 1 10 0 Q4 P-Cresol 0 −15 0 15 0 Q4 Paliperidone 0 −26 1 9 0 Q4 Palmidrol 0 −40 0 7 0 Q4 Pamidronate 0 0 1 −5 0 Q4 Pancuronium 0 −4 1 1 0 Q4 Pancuronium Bromide 0 9 1 11 0 Q4 Pantethine 0 −53 1 7 0 Q4 Panthenol 0 −32 1 6 0 Q4 Pantoprazole, Pantoprazole Sodium 0 −8 0 −1 0 Q4 Salt Pantothenic Acid 0 7 0 5 1 Q4 Papaverine 0 12 0 27 0 Q4 Parachlorophenol 0 21 0 15 0 Q4 Paramethadione 0 −15 0 3 0 Q4 Pargyline 0 −25 1 11 0 Q4 Paromomycin Sulfate 0 9 1 14 0 Q4 Paroxetine 0 38 1 15 0 Q4 Paroxypropione 0 −11 1 17 0 Q4 Pasiniazid 0 −61 1 17 0 Q4 Pazufloxacin Mesylate, Pazufloxacin 0 −10 1 11 0 Q4 Pefloxacin 0 −22 0 21 0 Q4 Pefloxacin 0 −16 0 13 1 Q4 Pemetrexed 0 −8 0 9 0 Q4 Pempidine Tartrate 0 −19 1 13 0 Q4 Penbutolol 0 −8 1 17 0 Q4 Penciclovir 0 −44 1 4 0 Q4 Penfluridol 0 3 1 8 0 Q4 Penicillamine 0 −15 0 16 0 Q4 Pentagastrin 0 −6 1 14 1 Q4 Pentamidine 0 6 1 33 1 Q4 Pentetic Acid, 0 11 0 −2 0 Q4 Diethylenetriaminepentaacetic Acid Pentetrazole, Pentylenetetrazol 0 −12 1 18 1 Q4 Pentoxifylline 0 4 0 6 0 Q4 Perflubron emulsion 0 16 1 −10 0 Q4 Pergolide 0 −18 0 16 0 Q4 Perindopril 0 −27 0 18 0 Q4 Permethrin 0 −21 0 11 0 Q4 Perphenazine 1 23 1 23 1 Q4 Phenacetin 0 −5 1 16 0 Q4 Phenazopyridine 3 9 0 6 0 Q4 Phenelzine 0 2 1 −4 0 Q4 Phenethicillin Potassium 0 −2 0 3 0 Q4 Phenformin 0 −30 1 13 0 Q4 Phenindione 2 −10 1 0 0 Q4 Pheniramine 0 −9 1 1 0 Q4 Phenobarbital 0 −22 0 −7 0 Q4 Phenolphthalein 0 10 1 13 0 Q4 Phenothiazine 1 −84 0 24 1 Q4 Phenothrin 0 −50 0 11 0 Q4 Phenoxybenzamine 0 8 1 −5 0 Q4 Phenoxymethylpenicillin 0 0 1 14 0 Q4 Phensuximide 0 −7 1 4 0 Q4 Phentolamine 0 −21 0 17 0 Q4 Phenylacetic acid 0 −1 1 13 0 Q4 Phenylacetic acid 0 −54 1 3 0 Q4 Phenylbutazone 1 −23 0 5 0 Q4 Phenylephrine 0 38 1 31 0 Q4 Phenylpropanolamine 0 −2 1 −6 1 Q4 Phosphonoserine 0 −6 1 10 0 Q4 Phthalylsulfacetamide 0 1 1 −1 0 Q4 Phthalylsulfathiazole 0 3 1 13 1 Q4 Phylloquinone 3 4 0 31 2 Q4 Physostigmine 0 −73 0 18 0 Q4 Picolamine 0 −1 1 4 0 Q4 Piconol 0 −1 1 26 1 Q4 Pidolic Acid 0 −57 1 15 0 Q4 Pidotimod 0 −10 1 14 0 Q4 Pilocarpine 0 −2 1 14 0 Q4 Pinacidil 0 15 1 26 1 Q4 Pindolol 0 −13 1 24 1 Q4 Pioglitazone 0 2 1 33 1 Q4 Pipamperone 0 −4 1 8 0 Q4 Pipemidic Acid 0 −27 1 12 0 Q4 Piperacetazine 1 −32 0 48 2 Q4 Piperacillin Sodium 0 −25 0 17 0 Q4 Piperazine 0 −3 0 16 0 Q4 Piperidolate Hydrochloride 0 −21 0 8 0 Q4 Piperine 0 −13 1 10 0 Q4 Piperonyl Butoxide 0 0 0 12 0 Q4 Pipobroman 0 −28 0 0 0 Q4 Piracetam 0 −21 0 9 0 Q4 Pirenperone 0 2 1 20 0 Q4 Pirenzepine 0 16 0 19 0 Q4 Piroctone Olamine 0 44 1 39 1 Q4 Piromidic Acid 0 −30 1 7 0 Q4 Piroxicam 0 4 1 19 0 Q4 Pizotyline Malate 0 8 1 37 1 Q4 Praesterone Acetate, Prasterone 0 −3 1 5 0 Q4 Acetate Pralidoxime 0 −12 1 17 0 Q4 Pramipexole 0 −19 1 1 0 Q4 Pramocaine 0 14 0 13 0 Q4 Pranoprofen 0 −14 1 8 0 Q4 Prasterone 0 1 1 15 0 Q4 Prasugrel 0 15 1 22 1 Q4 Pravastatin 0 10 1 10 0 Q4 Pravastatin Sodium 0 21 1 11 0 Q4 Praziquantel 0 −3 1 21 0 Q4 Pregabalin 0 −5 1 1 0 Q4 Pregnenolone 0 −11 1 12 0 Q4 Pregnenolone Succinate 0 4 1 21 1 Q4 Pridinol 0 −18 1 4 0 Q4 Prilocaine 0 −3 0 12 0 Q4 Primaquine 0 −58 0 7 0 Q4 Primidone 0 −15 1 12 0 Q4 Probenecid 0 14 1 13 0 Q4 Probucol 0 12 0 −1 0 Q4 Procainamide, Pronestyl 0 −4 0 10 0 Q4 Procaine, Novocain 0 −10 0 25 0 Q4 Procarbazine 0 −21 1 9 0 Q4 Procaterol 0 16 1 21 0 Q4 Procodazole 0 −15 1 15 0 Q4 Procyclidine 0 0 1 26 1 Q4 Progesterone 0 36 1 36 0 Q4 Proglumide 0 −4 1 −1 0 Q4 Proguanil; Chlorguanide 0 −7 0 20 1 Q4 Hydrochloride Promethazine 1 −30 0 5 0 Q4 Pronetalol Hydrochloride 0 −12 1 10 0 Q4 Propafenone 0 16 1 21 1 Q4 Propantheline 0 −7 1 20 0 Q4 Proparacaine 0 −6 1 13 0 Q4 Propofol 0 −18 0 11 0 Q4 Propoxur 0 −51 1 8 0 Q4 Propoxycaine 0 13 0 14 0 Q4 Propranolol 0 −30 0 2 0 Q4 Propylthiouracil 0 −4 0 8 0 Q4 Proscillaridin 0 8 1 16 0 Q4 Protionamide, Ektebin 0 −18 1 12 0 Q4 Protirelin 0 2 1 15 1 Q4 Protoporphyrin Ix 0 −70 1 2 0 Q4 Protriptyline 0 21 1 15 0 Q4 Proxyphylline 0 −21 1 11 0 Q4 Prulifloxacin 0 7 1 16 0 Q4 Pseudoephedrine 0 −13 1 5 0 Q4 Pyrantel Pamoate, Oxantel Pamoate 0 −4 1 14 0 Q4 Pyrantel Pamoate, Oxantel Pamoate 0 5 1 17 1 Q4 Pyrazinamide 0 −20 0 13 0 Q4 Pyrethrins 0 4 0 11 0 Q4 Pyridostigmine 0 4 1 9 0 Q4 Pyridoxine 0 1 1 21 0 Q4 Pyrimethamine 0 −11 1 12 1 Q4 Pyrithyldione 0 −24 1 9 0 Q4 Pyritinol 0 −15 1 4 0 Q4 Pyronaridine Tetraphosphate 3 16 1 9 0 Q4 Quetiapine 0 −2 1 13 0 Q4 Quinapril 0 −24 1 13 0 Q4 Quinaprilat 0 −5 1 5 0 Q4 Quinestrol 0 15 0 24 0 Q4 Quinethazone 0 15 1 17 1 Q4 Quinidine 0 −6 1 20 0 Q4 Quinine 0 −32 1 −7 0 Q4 Quinine Ethyl Carbonate 0 −21 0 −11 0 Q4 Quinine Sulfate 0 −30 1 14 1 Q4 Quipazine Dimaleate, Quipazine 0 −18 1 9 0 Q4 Maleate Rabeprazole 0 −46 0 3 0 Q4 Racecadotril 0 −51 1 18 0 Q4 Racephedrine Hydrochloride 0 −26 1 17 0 Q4 Racepinephrine 2 27 1 29 0 Q4 Ramelteon 0 25 0 20 0 Q4 Ramifenazone 0 −11 0 9 0 Q4 Ramipril 0 8 0 16 0 Q4 Ramoplanin [a2 Shown; 2 mm] 0 −3 1 12 0 Q4 Ranitidine 0 35 1 15 0 Q4 Ranitidine 0 23 1 16 0 Q4 Ranolazine, Ranolazine 0 −53 0 12 0 Q4 Dihydrochloride Ranolazine, Ranolazine 0 −18 1 2 0 Q4 Dihydrochloride Reboxetine Mesylate 0 −33 1 8 0 Q4 Remacemide Hydrochloride 0 −21 1 13 0 Q4 Remoxipride 0 −14 1 3 0 Q4 Repaglinide 0 −14 1 18 0 Q4 Resorcinol 0 −4 0 8 0 Q4 Resorcinol Monoacetate 0 12 1 26 1 Q4 Retinyl Acetate 0 3 1 15 0 Q4 Retinyl Palmitate 0 −15 0 11 0 Q4 Riboflavin 0 −23 1 −8 0 Q4 Riboflavin 0 5 0 13 0 Q4 Riboflavin 5-phosphate Sodium 0 11 0 24 0 Q4 Ribostamycin Sulfate 0 −16 0 13 0 Q4 Rifabutin 1 −51 0 2 0 Q4 Rifampicin 3 −21 1 7 0 Q4 Rifampin 3 8 1 20 0 Q4 Rifapentine 3 −23 1 −7 0 Q4 Rifaximin 0 6 0 22 2 Q4 Riluzole 0 0 1 7 0 Q4 Rimantadine 0 −19 0 2 0 Q4 Risedronate 0 47 1 15 0 Q4 Risperidone 0 −20 0 18 0 Q4 Risperidone 0 −40 1 −13 0 Q4 Ritonavir 0 −8 0 11 0 Q4 Rivastigmine 0 −35 1 5 0 Q4 Rivastigmine 0 −37 1 10 0 Q4 Rizatriptan 0 −9 1 10 0 Q4 Rocuronium 0 −22 1 5 0 Q4 Rofecoxib 0 15 0 14 0 Q4 Rolipram 0 11 0 11 0 Q4 Rolitetracycline 0 4 1 9 0 Q4 Ronidazole 0 31 1 20 0 Q4 Ropinirole 0 −26 1 15 0 Q4 Ropivacaine, Ropivacaine 0 18 1 5 0 Q4 Hydrochloride Rosiglitazone, Rosiglitazone 0 5 1 20 1 Q4 Maleate Roxarsone 0 −18 1 17 1 Q4 Roxatidine acetate 0 22 1 6 0 Q4 Roxithromycin 0 −4 0 17 1 Q4 Rufloxacin Hydrochloride 0 −3 1 21 0 Q4 Rutin 2 1 1 18 0 Q4 S-(−)-carbidopa, Carbidopa 2 −91 0 16 0 Q4 Saccharin 0 −8 0 11 0 Q4 Salbutamol 0 25 1 25 0 Q4 Salicyl Alcohol 0 −5 1 19 0 Q4 Salicylamide 0 −20 1 16 1 Q4 Salicylanilide 0 1 1 13 0 Q4 Salicylic Acid 0 −32 0 14 0 Q4 Salsalate 0 −22 1 14 1 Q4 Sarafloxacin 0 2 0 4 0 Q4 Saxagliptin 0 34 1 38 0 Q4 Scopolamine 0 −26 1 −6 1 Q4 Scopolamine Hydrobromide 0 5 1 3 1 Q4 Secnidazole 0 −24 1 22 2 Q4 Selegiline 0 −36 0 −5 0 Q4 Selegiline 0 9 1 19 0 Q4 Selenomethionine 0 −5 1 10 0 Q4 Semustine 0 −7 1 18 0 Q4 Sennoside A 0 −10 1 14 0 Q4 Seratrodast 3 20 1 14 0 Q4 Sertaconazole 0 −25 1 11 0 Q4 Sertraline 0 16 1 22 0 Q4 Sevoflurane 0 −1 1 17 1 Q4 Sibutramine 0 −12 1 8 0 Q4 Sildenafil, Sildenafil Citrate 0 22 0 20 0 Q4 Sisomicin Sulfate 0 −41 0 10 0 Q4 Sitagliptin 0 −16 0 4 0 Q4 Skf-525a Hydrochloride, Proadifen 0 −10 0 5 0 Q4 Hydrochloride Sodium Dehydrocholate, 0 21 1 28 1 Q4 Dehydrocholate Sodium Sodium Gluconate 0 1 1 11 0 Q4 Sodium Monofluorophosphate 0 −10 0 0 0 Q4 Sodium Nitroprusside 0 27 3 12 1 Q4 Sodium Oxybate 0 −5 1 34 0 Q4 Sodium Tetradecyl Sulfate 0 −5 1 13 0 Q4 Solifenacin Succinate 0 −2 0 −4 0 Q4 Sorafenib 0 −67 1 35 1 Q4 Sorbitol 0 −9 1 14 0 Q4 Sorbitol 0 −65 0 5 0 Q4 Sotalol 0 6 1 6 0 Q4 Spaglumic Acid 0 −15 1 15 0 Q4 Sparfloxacin 0 −12 0 11 0 Q4 Sparfloxacin 0 −14 1 14 1 Q4 Sparteine Sulfate 0 7 1 20 0 Q4 Spectinomycin 0 14 0 19 0 Q4 Spiperone Hydrochloride, Spiperone 0 2 2 12 0 Q4 Spiramycin 0 17 1 14 0 Q4 Spironolactone 0 −9 0 20 0 Q4 Spironolactone 0 −15 1 12 1 Q4 Stanozolol 0 −5 1 33 1 Q4 Stavudine 0 −17 1 19 0 Q4 Streptomycin Sulfate 0 −46 0 13 0 Q4 Streptozocin 0 −30 0 19 1 Q4 Strychnine 0 1 1 18 0 Q4 Succimer 0 −37 0 −5 0 Q4 Succinylcholine 0 −54 1 −5 0 Q4 Succinylsulfathiazole 0 15 1 16 1 Q4 Sucralfate 0 −35 1 22 0 Q4 Sucralose 0 0 1 16 0 Q4 Sulbentine 0 48 1 13 0 Q4 Sulconazole 0 10 1 13 1 Q4 Sulfabenzamide 0 −15 1 19 1 Q4 Sulfacarbamide 0 45 1 46 0 Q4 Sulfacetamide 0 −2 1 10 0 Q4 Sulfachlorpyridazine 0 18 1 14 0 Q4 Sulfadiazine 0 −19 0 13 0 Q4 Sulfadimethoxine 0 42 1 13 0 Q4 Sulfadoxine 0 −12 1 3 0 Q4 Sulfaguanidine 0 −23 1 12 0 Q4 Sulfamerazine 0 −2 1 12 0 Q4 Sulfameter 0 4 1 11 0 Q4 Sulfamethazine 0 −9 0 14 0 Q4 Sulfamethizole 0 −3 1 16 0 Q4 Sulfamethoxazole 0 −17 0 17 0 Q4 Sulfamethoxypyridazine 0 21 1 15 0 Q4 Sulfamonomethoxine 0 38 1 19 1 Q4 Sulfanilamide 0 −14 0 11 0 Q4 Sulfanilate Zinc 0 −6 0 15 0 Q4 Sulfanitran 0 10 1 18 0 Q4 Sulfaphenazole 0 −4 1 14 0 Q4 Sulfapyridine 0 −37 1 20 0 Q4 Sulfasalazine 3 −29 0 13 0 Q4 Sulfathiazole 0 −17 0 9 0 Q4 Sulfinpyrazone 1 −6 1 13 1 Q4 Sulfisoxazole 0 −11 0 6 0 Q4 Sulfisoxazole Acetyl 0 −3 1 17 1 Q4 Sulindac 0 21 1 6 0 Q4 Sulindac Sulfone 0 −27 1 3 0 Q4 Sulisobenzone 0 −19 0 −2 0 Q4 Sulpiride 0 −7 1 11 0 Q4 Sulpiride 0 −52 1 7 0 Q4 Sumatriptan 0 −7 1 8 0 Q4 Suramin 0 −5 0 18 0 Q4 Suxibuzone 1 25 1 23 0 Q4 Symclosene 0 −13 0 11 0 Q4 Synephrine 0 −16 0 12 0 Q4 Tacrine 0 −2 1 5 0 Q4 Tacrolimus 0 27 1 14 0 Q4 Tadalafil 3 −1 1 16 0 Q4 Tamoxifen 0 14 1 5 1 Q4 Tamsulosin Hydrchloride 0 −31 0 12 0 Q4 Tapentadol Hydrochloride 0 −12 1 12 0 Q4 Taxol, Paclitaxel 0 45 1 19 1 Q4 Tazobactam 0 2 1 −9 0 Q4 Tazobactam 0 −11 0 11 0 Q4 Teicoplanin [a(2-1) Shown] 0 43 1 20 0 Q4 Telenzepine Dihydrochloride, 0 3 1 14 0 Q4 Telenzepine Hydrochloride Telithromycin 0 0 1 17 0 Q4 Telmisartan 0 −24 0 −2 0 Q4 Temozolomide 0 −31 0 7 0 Q4 Temozolomide 0 −26 1 −12 0 Q4 Tenatoprazole 0 −17 1 9 0 Q4 Tenofovir 0 −10 0 17 0 Q4 Tenoxicam 0 11 1 15 0 Q4 Tenylidone 2 −9 1 15 0 Q4 Terazosin 0 −1 1 9 0 Q4 Terbinafine, Terbinafine 0 −23 0 6 0 Q4 Hydrochloride Terbutaline 0 39 1 28 0 Q4 Terconazole 3 −1 1 −10 0 Q4 Terconazole 3 27 1 35 0 Q4 Terfenadine 0 19 11 7 0 Q4 Terpene Hydrate 0 −6 1 8 0 Q4 Testosterone 0 −1 0 5 0 Q4 Testosterone Propionate 0 21 1 8 0 Q4 Tetracaine 0 4 1 9 1 Q4 Tetracycline 0 7 0 19 0 Q4 Tetraquinone 3 −10 0 15 0 Q4 Tetryzoline 0 −29 0 16 0 Q4 Thalidomide 0 8 0 23 1 Q4 Theobromine 0 11 0 18 1 Q4 Thiamine 0 27 2 −5 0 Q4 Thiamine 0 −10 0 30 0 Q4 Thiamphenicol 0 −19 0 12 0 Q4 Thiamylal 0 −1 0 26 0 Q4 Thiodiglycol 0 −20 0 9 0 Q4 Thiopental 0 3 0 13 0 Q4 Thiotepa 0 −15 1 15 1 Q4 Thiothixene 1 −4 0 37 0 Q4 Thioxolone, Tioxolone 0 −18 0 12 0 Q4 Thonzonium Bromide 0 −1 0 19 1 Q4 Thonzylamine Hydrochloride 0 −21 0 6 0 Q4 Thymol 0 15 1 20 0 Q4 Thymopentin 0 −20 1 20 0 Q4 Thyroxine, Levothyroxine 0 5 1 25 1 Q4 Tiapride 0 −18 0 6 0 Q4 Tibolone 0 −8 0 4 0 Q4 Ticarcillin Disodium 0 −22 0 7 0 Q4 Ticlopidine 0 −13 1 22 0 Q4 Tigecycline 0 7 0 13 0 Q4 Tiletamine Hydrochloride 0 8 0 22 0 Q4 Timolol 0 11 1 −7 0 Q4 Timolol Maleate 0 −4 0 19 0 Q4 Timonacic 0 −38 0 19 0 Q4 Tinidazole 0 2 0 14 1 Q4 Tioconazole 0 4 1 12 0 Q4 Tioconazole 0 −16 1 8 1 Q4 Tioguanine 0 12 1 18 0 Q4 Tiopronin 0 −8 1 6 0 Q4 Tiotropium 0 −32 0 −1 0 Q4 Tiratricol 0 −51 1 13 0 Q4 Tizanidine 0 2 1 2 1 Q4 Tobramycin 0 1 1 24 0 Q4 Tocainide 0 −25 1 0 0 Q4 Todralazine Hydrochloride 0 0 1 19 0 Q4 Tolazoline, Priscoline 0 −90 0 17 0 Q4 Tolbutamide 0 31 1 24 0 Q4 Tolcapone 2 −43 1 9 0 Q4 Tolfenamic Acid 0 3 1 36 1 Q4 Tolmetin 0 48 0 28 0 Q4 Tolonium Chloride 0 −4 1 43 1 Q4 Tolperisone 0 −7 1 11 0 Q4 Tolterodine 0 −36 1 15 0 Q4 Topiramate 0 4 1 5 0 Q4 Torasemide 0 −29 1 7 0 Q4 Toremifene 0 14 1 19 0 Q4 Tramadol 0 26 1 12 0 Q4 Tramadol Hydrochloride 0 35 0 21 0 Q4 Trandolapril 0 −5 1 15 0 Q4 Tranexamic Acid 0 −4 1 13 0 Q4 Tranilast 0 35 0 22 0 Q4 Tranylcypromine 0 5 1 −5 0 Q4 Tranylcypromine Sulfate 0 3 0 17 0 Q4 Travoprost 0 4 1 15 0 Q4 Trazodone 0 −10 0 −2 0 Q4 Tretinon, Tretinoin 0 27 1 34 1 Q4 Triacetin 0 −32 1 15 0 Q4 Triamcinolone 0 −2 1 −14 0 Q4 Triamterene 0 40 1 24 0 Q4 Trichlormethiazide 0 17 1 13 0 Q4 Triclosan 0 −6 1 16 1 Q4 Triethylenetetramine 0 3 1 10 0 Q4 Triflupromazine 1 49 1 37 1 Q4 Trifluridine 0 10 1 16 1 Q4 Trihexyphenidyl 0 5 1 14 1 Q4 Trilostane 0 −24 0 11 0 Q4 Trimebutine 0 −11 1 13 0 Q4 Trimetazidine 0 −18 1 1 0 Q4 Trimethadione 0 −5 1 14 0 Q4 Trimethobenzamide 0 16 1 17 0 Q4 Trimethoprim 0 −26 0 17 0 Q4 Trimethyl Glycine 0 −14 0 22 0 Q4 Trimetozine 0 2 1 12 0 Q4 Trioxsalen 0 −18 1 18 0 Q4 Tripelennamine 0 14 1 25 0 Q4 Triprolidine 0 −24 1 23 0 Q4 Triptorelin 0 −15 1 4 0 Q4 Tris 0 −18 1 41 1 Q4 Troclosene Potassium 0 −8 1 1 0 Q4 Troglitazone 0 −22 1 4 0 Q4 Tropicamide 0 −4 1 17 0 Q4 Tropisetron 0 −28 0 14 0 Q4 Trospium Chloride 0 −39 0 9 1 Q4 Troxerutin 0 −10 0 8 0 Q4 Tryptophan 0 −6 1 16 0 Q4 Tuaminoheptane Sulfate 0 −7 1 19 0 Q4 Tubocurarine 0 −1 0 19 0 Q4 Tulobuterol, Tulobuterol 0 21 0 36 0 Q4 Hydrochloride Tylosin Tartrate 0 9 1 11 0 Q4 Tyloxapol 0 −1 1 17 0 Q4 Tyrosine 0 −7 1 21 1 Q4 Undecylenic Acid, Zinc 0 −13 1 12 0 Q4 Undecylenate Undecylenic Acid, Zinc 0 −6 1 14 0 Q4 Undecylenate Uracil 0 3 0 11 0 Q4 Urapidil Hydrochloride 0 −6 1 20 0 Q4 Urea 0 −14 1 20 0 Q4 Ursodeoxycholic Acid 0 −18 1 1 0 Q4 Ursodiol 0 −21 0 18 0 Q4 Valaciclovir 0 12 1 15 0 Q4 Valdecoxib 0 −39 1 −4 0 Q4 Valganciclovir 0 −12 0 14 1 Q4 Valproic Acid 0 −28 0 27 0 Q4 Valsartan 0 −17 0 12 0 Q4 Vancomycin 0 −1 1 −2 0 Q4 Vancomycin Hydrochloride 0 −20 1 9 0 Q4 Vardenafil 0 −21 0 12 0 Q4 Vardenafil 0 10 1 13 0 Q4 Vatalanib 0 −26 1 −3 0 Q4 Vecuronium 0 −24 1 0 1 Q4 Vecuronium Bromide 0 7 1 23 0 Q4 Venlafaxine, Venlafaxine 0 3 1 20 0 Q4 Hydrochloride Verapamil 0 5 0 19 0 Q4 Vidarabine 0 −3 1 14 0 Q4 Vilazodone 0 1 1 27 1 Q4 Vincamine 0 1 0 20 0 Q4 Vinorelbine 0 −12 1 −11 0 Q4 Vinpocetine 0 −19 0 0 0 Q4 Viomycin Sulfate 0 −7 0 19 0 Q4 Vitamin A 0 15 1 17 0 Q4 Vitamin C 0 −28 1 18 0 Q4 Voriconazole 0 −42 0 5 0 Q4 Warfarin, Warfarin Sodium 0 −4 1 16 0 Q4 Xylometazoline 0 −5 0 19 1 Q4 Yohimbine 3 4 1 −17 0 Q4 Yohimbine Hydrochloride 3 −8 1 19 1 Q4 Zafirlukast 0 −21 0 3 1 Q4 Zalcitabine 0 −10 0 11 0 Q4 Zaleplon 0 16 1 13 0 Q4 Zaprinast 0 −34 1 13 0 Q4 Zidovudine 3 −20 1 9 0 Q4 Zidovudine 3 −7 0 13 0 Q4 Zileuton 0 −12 0 21 0 Q4 Ziprasidone Mesylate, Ziprasidone 0 14 1 35 1 Q4 Zolmitriptan 0 9 1 18 0 Q4 Zolpidem, Zolpidem Tartrate 0 −12 0 4 0 Q4 Zomepirac 0 −4 1 10 0 Q4 Zonisamide 0 −37 0 −12 0 Q4 Zopiclone 0 11 1 5 0 Q4 Zoxazolamine 0 −14 0 11 0 Q4 0 14 1 6 0 Q4 0 −3 2 −4 1 Q4 0 20 1 7 0 Q4 0 0 1 −2 0 Q4
Table 4 provides a list of all 1907 compounds with labeling Q1, Q2, Q3 and Q4quadrants in
TABLE-US-00005 TABLE 5 provides which compounds scored for their selectivity for fibrin. This criterion further differentiates the compounds and demonstrates those that are selective. Fibrin LPS Cell Cell Cherry- IC50 IC50 Toxicity Toxicity picked (uM) (uM) KEGG IC50 IC50 Hit (Suppl. (Suppl. Analysis Fibrin (uM) (uM) Quadrant (Suppl. Table Table (Suppl. Fibrin Selectivity [cFib [LPS No. Name (FIG. 5b) Table 7) 7) 7) Table 8) Selectivity (LOG10) assay] assay] Therapy 1 Docetaxel Q3 HIT 0.002 >20 9814 3.99 >20 >20 antineoplastic 2 Prednicarbate Q2 HIT 0.002 >20 8510 3.93 >20 >20 antiinflammatory, glucocorticoid 3 Diflorasone Q3 HIT 0.01 >20 2188 3.34 >20 >20 antiinflammatory, Diacetate glucocorticoid 4 Alclometazone Q2 HIT <0.001 2 2153 3.33 >20 >20 antiinflammatory, Dipropionate glucocorticoid 5 Hydrocortisone Q2 HIT 0.03 18 KEGG 568 2.75 >20 >20 glucocorticoid 6 Dexamethasone Q2 HIT 0.004 2 KEGG 383 2.58 >20 >20 glucocorticoid, Sodium Phosphate antiinflammatory 7 Fludrocortisone Q2 HIT <0.001 0.4 KEGG 362 2.56 >20 >20 mineralocorticoid Acetate 8 Hydrocortisone Q2 HIT 0.02 7 KEGG 350 2.54 >20 >20 glucocorticoid Hemisuccinate 9 Melengestrol Q2 HIT 0.07 13 196 2.29 >20 >20 antineoplastic, Acetate progestin 10 Dichlorisone Q2 HIT 0.03 4 169 2.23 >20 >20 antipruretic Acetate 11 Hydrocortisone Q2 HIT 0.02 2 KEGG 89 1.95 >20 >20 glucocorticoid, Butyrate antiinflammatory 12 Medroxyprogesterone Q2 HIT 0.3 >20 70 1.84 >20 >20 progestogen Acetate 13 Monensin Sodium Q2 HIT 0.004 0.2 49 1.69 >20 2 antibiotic, antibacterial; antibacterial 14 Deflazacort Q2 HIT <0.001 0.04 42 1.63 20 >20 antiinflammatory 15 Medroxyprogesterone Q2 HIT 0.5 >20 41 1.61 >20 >20 contraceptive Acetate 16 Ritodrine Q3 HIT 0.6 >20 33 1.51 >20 >20 muscle relaxant Hydrochloride (smooth) 17 Prednisolone Q2 HIT 0.1 4 31 1.49 >20 >20 antiinflammatory, Sodium Phosphate glucocorticoid 18 Mebendazole Q2 HIT 0.2 6 31 1.49 >20 >20 anthelmintic 19 Hydrocortisone Q2 HIT 0.05 1 28 1.45 >20 >20 antiinflammatory, Valerate glucocorticoid 20 Levonordefrin Q3 HIT 0.9 >20 21 1.33 >20 >20 vasoconstrictor 21 Tegaserod Maleate Q2 HIT 1 19 20 1.29 >20 >20 5HT4 receptor agonist, peristaltic stimulant 22 Triamcinolone Q2 HIT 0.04 0.8 KEGG 17 1.23 >20 >20 antiinflammatory Diacetate 23 Hydrocortisone Q2 HIT 0.008 0.1 KEGG 16 1.21 >20 >20 glucocorticoid, antiinflammatory 24 Methylprednisolone Q2 HIT <0.001 0.02 KEGG 15 1.19 >20 >20 glucocorticoid 25 Rosuvastatin Q2 HIT 0.3 3 12 1.09 20 >20 antihyperlipidemic 26 Vinblastine Sulfate Q2 HIT 0.02 0.2 KEGG 11 1.05 >20 >20 antineoplastic, spindle poison 27 Salmeterol Q3 HIT 1 14 10 1.02 >20 >20 Bronchodilator 28 Triamcinolone Q2 HIT 0.04 0.4 10 1.00 >20 >20 glucocorticoid 29 Betamethasone Q2 HIT <0.001 0.01 KEGG 10 0.99 >20 >20 glucocorticoid, antiinflammatory 30 Desoxycorticosterone Q3 HIT 0.5 5 10 0.98 >20 >20 mineralocorticoid Acetate 31 Algestone Q2 HIT 1 9 9 0.95 >20 >20 antiacne, progestin Acetophenide 32 Eplerenore Q2 HIT 3 >20 8 0.88 >20 >20 antihypertensive 33 Tolnaftate Q2 HIT 2 17 7 0.87 >20 >20 antifungal 34 Rebamipide Q2 HIT 2 11 6 0.80 >20 >20 antiulcer, antioxidant 35 Prednisone Q3 HIT 3 >20 6 0.78 >20 >20 glucocorticoid 36 Tepoxalin Q2 HIT 3 18 6 0.75 >20 >20 antipsoratic 37 Xylazine Q2 HIT 2 8 5 0.72 >20 >20 “alpha2 Hydrochloride Adrenoceptor agonist; anesthetic, analgesic”; analgesic 38 Gentamicin Sulfate Q2 HIT 4 >20 5 0.71 >20 >20 antibacterial 39 Idazoxan Q3 HIT 0.4 2 5 0.69 >20 >20 alpha2-adrenergic Hydrochloride blocker 40 Atorvastatin Q2 HIT 0.6 3 5 0.67 >20 >20 antihyperlipidemic, Calcium HMGCoA reductase inhibitor 41 Rubitecan Q3 HIT 2 9 4 0.63 >20 >20 antineoplastic 42 Megestrol Acetate Q3 HIT 5 >20 4 0.63 >20 >20 progestogen, antineoplastic 43 Betamethasone Q2 HIT 0.05 0.2 4 0.62 >20 >20 antiinflammatory, Sodium Phosphate glucocorticoid 44 Pitavastatin Q2 HIT 1 5 4 0.61 >20 >20 HMGA reductase Calcium inhibitor 45 Flumethazone Q2 HIT <0.001 0.004 4 0.58 >20 >20 glucocorticoid, Pivalate antiinflammatory 46 lopanic Acid Q3 HIT 5 >20 4 0.58 >20 >20 radioopaque agent 47 Zoledronic Acid Q3 HIT 2 8 4 0.55 >20 >20 Antiosteoporotic 48 Selamectin Q3 HIT 6 >20 3 0.51 >20 >20 anthelmintic, antiparasitic, antimite 49 Oxaliplatin Q3 HIT 5 15 3 0.48 >20 >20 antineoplastic 50 Artesunate Q3 HIT 7 >20 3 0.45 >20 >20 Antimalarial 51 Naftopidil Q3 HIT 7 >20 3 0.45 >20 >20 “alpha1 Dihydrochloride Adrenoceptor antagonist; antihypertensive, alpha-blocker, 5HT1a agonist”; antihypertensive, alpha- blocker, 5HT1a agonist 52 Fluorouracil Q3 HIT 7 >20 3 0.44 >20 >20 antineoplastic, pyrimidine antimetabolite 53 Isoxicam Q3 HIT 7 >20 3 0.43 >20 >20 antiinflammatory 54 Milnacipran Q3 HIT 8 20 3 0.40 >20 >20 inhibitor of Hydrochloride norepinephrine and seritonin uptake, treatment of fibromyalgia 55 Securinine Q2 HIT 2 6 3 0.40 >20 12 GABAA receptor blocker, CNS stimulant 56 Demeclocycline Q3 HIT 6 15 2 0.38 >20 >20 antibacterial Hydrochloride 57 Benzyl Q2 HIT 10 >20 2 0.29 >20 >20 antineoplastic, Isothiocyanate antibacterial, antifungal 58 Ribavirin Q2 HIT 1 2 2 0.29 >20 >20 antiviral 59 Tolazamide Q3 HIT 12 >20 2 0.23 >20 >20 “Oral hypoglycemic agent; stimulates pancreatic islet cells to secrete insulin, antidiabetic”; antidiabetic 60 Oxybendazole Q3 HIT 7 12 2 0.22 >20 >20 anthelmintic 61 Albendazole Q3 HIT 4 6 2 0.22 >20 >20 anthelmintic 62 Betazole Q3 HIT 12 >20 2 0.22 >20 >20 gastric secretion Hydrochloride stimulant 63 Prednisone Q3 HIT 12 >20 2 0.21 >20 >20 antiinflammatory, glucocorticoid 64 2- Q2 HIT 3 5 2 0.21 >20 >20 angiogenesis Methoxyestradiol inhibitor 65 Dexamethasone Q3 HIT 15 >20 KEGG 1 0.13 >20 >20 Nuclear receptor ligands: corticosteroid 66 Fenbendazole Q3 HIT 3 4 1 0.12 >20 >20 anthelmintic 67 Cloperastine Q3 HIT 7 9 1 0.09 >20 >20 antitussive Hydrochloride 68 Cyproterone Q3 HIT 17 >20 1 0.08 >20 >20 antiandrogen 69 Chlormadinone Q3 HIT 17 >20 1 0.07 >20 >20 progestin, Acetate antiandrogen 70 Fusidic Acid Q2 HIT 19 >20 1 0.03 >20 >20 antibacterial 71 Risedronate Q3 HIT 19 >20 1 0.02 >20 >20 calcium regulator, Sodium Antibone resorptive 72 Salinomycin, Q2 HIT 2 2 1 0.02 >20 17 antibiotic, Sodium antibacterial; antibacterial 73 Mycophenolic Acid Q2 HIT 1 1 KEGG 1 0.00 >20 >20 immune suppressant, antineoplastic, antiviral 74 Almotriptan Q2 HIT >20 >20 1 0.00 >20 >20 5HT 1B/2D receptor agonist 75 Amisulpride Q3 HIT 20 >20 1 0.00 >20 >20 antipsychotic 76 Bacitracin Q3 HIT >20 >20 1 0.00 >20 >20 antibacterial 77 Benzoyl Peroxide Q2 HIT 20 >20 1 0.00 >20 >20 keratolytic 78 Betamethasone Q2 HIT 20 >20 1 0.00 >20 >20 glucocorticoid Valerate 79 Chlorthalidone Q3 HIT >20 >20 1 0.00 >20 >20 diuretic, antihypertensive 80 Clobetasol Q2 HIT <0.001 <0.001 1 0.00 >20 >20 glucocorticoid, Propionate antiinflammatory 81 Colistimethate Q2 HIT >20 >20 1 0.00 >20 >20 antibacterial Sodium 82 Docetaxel Q3 HIT 20 >20 1 0.00 >20 >20 antineoplastic 83 Ethacrynic Acid Q3 HIT >20 >20 1 0.00 >20 >20 diuretic 84 Ethinyl Estradiol Q3 HIT >20 >20 1 0.00 >20 >20 estrogen, plus progestogen as oral contraceptive 85 Flumethasone Q2 HIT <0.001 <0.001 1 0.00 >20 >20 antiinflammatory 86 Flunisolide Q2 HIT <0.001 <0.001 1 0.00 >20 >20 antiinflammatory 87 Fluocinolone Q2 HIT <0.001 <0.001 KEGG 1 0.00 >20 >20 glucocorticoid, Acetonide antiinflammatory 88 Ftaxilide Q3 HIT >20 >20 1 0.00 >20 >20 antiulcer 89 Hydroxytoluic Acid Q3 HIT 20 >20 1 0.00 >20 >20 analgesic, antiseptic 90 Ibudilast Q2 HIT >20 >20 1 0.00 >20 >20 antiinflammatory 91 Imipenem Q3 HIT >20 >20 1 0.00 >20 >20 antibacterial, Antibiotic; antibacterial 92 Irbesartan Q3 HIT >20 >20 1 0.00 >20 >20 angiotensin 2 receptor antagonist 93 Mephentermine Q3 HIT >20 >20 1 0.00 >20 >20 vasoconstrictor Sulfate 94 Mequinol Q2 HIT >20 >20 1 0.00 >20 >20 skin depigmentor 95 Metampicillin Q3 HIT 20 >20 1 0.00 >20 >20 antimicrobial; Sodium antibacterial 96 Methylphenidate Q2 HIT >20 >20 1 0.00 >20 >20 CNS stimulant Hydrochloride 97 Miconazole Nitrate Q3 HIT 20 >20 1 0.00 >20 >20 antifungal (topical) 98 Midodrine Q2 HIT >20 >20 1 0.00 >20 >20 antihypertensive, Hydrochloride vasoconstrictor 99 Mirtazapine Q3 HIT >20 >20 1 0.00 >20 >20 antidepressant 100 Molsidomine Q2 HIT >20 >20 1 0.00 >20 >20 antianginal 101 Oxiglutatione Q3 HIT >20 >20 1 0.00 >20 >20 antioxidant Disodium Salt 102 Phenytoin Sodium Q2 HIT >20 >20 1 0.00 >20 >20 “Anticonvulsant; anti- epileptic”; anticonvulsant, antieleptic 103 Ractopamine Q3 HIT 20 >20 1 0.00 >20 >20 beta-adrenergic Hydrochloride agonist, growth stimulant 104 Rapamycin Q2 HIT <0.001 <0.001 1 0.00 >20 >20 Inhibitors: FRAP inhibitor 105 Rasagiline Q3 HIT 20 >20 1 0.00 >20 >20 antiparkinsonian 106 Salicin Q2 HIT >20 >20 1 0.00 >20 >20 analgesic, antipyretic 107 Saquinavir Q2 HIT >20 >20 1 0.00 >20 >20 antiviral 108 Sulbactam Q3 HIT >20 >20 1 0.00 >20 >20 b-lactamase inhibitor 109 Sulfaquinoxaline Q2 HIT >20 >20 1 0.00 >20 >20 antibacterial, Sodium coccidiostat 110 Thiabendazole Q2 HIT >20 >20 1 0.00 >20 >20 anthelmintic 111 Toltrazuril Q2 HIT >20 >20 1 0.00 >20 >20 coccidiostat 112 Cyclosporin A Q3 HIT 20 19 1 −0.01 >20 >20 “Calcineurin phosphatase inhibitor; immunosuppressant”; immunosuppressant 113 Hycanthone Q2 HIT 1 1 1 −0.04 >20 11 anthelmintic, hepatotoxic 114 Pimozide Q3 HIT >20 18 1 −0.06 >20 >20 “Ca2+ channel antagonist; antipsychotic; D2 dopamine receptor antagonist”; antipsychotic 115 Acrisorcin Q2 HIT 7 5 1 −0.13 20 6 antifungal 116 Trimipramine Q2 HIT 7 5 1 −0.15 >20 12 antidepressant Maleate 117 R(−)-Apomorphine Q3 HIT >20 14 1 −0.16 >20 >20 Dopamine receptor Hydrochloride agonist 118 Mycophenolate Q2 HIT 2 1 1 −0.23 >20 >20 immunesuppressant, Mofetil antineoplastic, antiviral 119 Amoxapine Q3 HIT 20 11 1 −0.24 >20 >20 “Tricyclic antidepressant; inhibits neuronal uptake of norepinephrine”; antidepressant, inhibits norepinephrine uptake 120 Acivicin Q2 HIT 2 1 KEGG 1 −0.27 >20 >20 antineoplastic 121 Benzydamine Q2 HIT 6 3 1 −0.29 20 8 analgesic, Hydrochloride antipyretic, antiinflammatory 122 Trifluoperazine Q3 HIT 12 6 0 −0.31 >20 12 “Calmodulin Dihydrochloride antagonist; dopamine receptor antagonist; antipsychotic; sedative”; antipsychotic 123 Teniposide Q2 HIT 2 1 KEGG 0 −0.32 20 4 antineoplastic 124 Perhexiline Q2 HIT 8 3 0 −0.35 20 11 coronary Maleate vasodilator 125 Medroxyprogesterone Q3 HIT >20 8 0 −0.42 >20 17 progestogen 126 Nortriptyline Q2 HIT 13 3 0 −0.61 20 10 Tricyclic Hydrochloride antidepressant; antidepressant 127 Tyrothricin Q2 HIT 3 0.5 0 −0.80 >20 20 topical antibacterial (topical) 128 Mechlorethamine Q2 HIT 7 0.1 0 −1.68 20 0.5 antineoplastic, alkylating agent
TABLE-US-00006 TABLE 6 provides compounds listing those compounds in Table 5 with a cut-off of 1.5 log selectivity for fibrin (the top 16 compounds in Table 5). Fibrin LPS Cell Cell Cherry- IC50 IC50 Toxicity Toxicity picked (uM) (uM) KEGG IC50 IC50 Hit (Suppl. (Suppl. Analysis Fibrin (uM) (uM) Quadrant (Suppl. Table Table (Suppl. Fibrin Selectivity [cFib [LPS No. Name (FIG. 5b) Table 7) 7) 7) Table 8) Selectivity (LOG10) assay] assay] Therapy 1 Docetaxel Q3 HIT 0.002 >20 9814 3.99 >20 >20 antineoplastic 2 Prednicarbate Q2 HIT 0.002 >20 8510 3.93 >20 >20 antiinflammatory, glucocorticoid 3 Diflorasone Q3 HIT 0.01 >20 2188 3.34 >20 >20 antiinflammatory, Diacetate glucocorticoid 4 Alclometazone Q2 HIT <0.001 2 2153 3.33 >20 >20 antiinflammatory, Dipropionate glucocorticoid 5 Hydrocortisone Q2 HIT 0.03 18 KEGG 568 2.75 >20 >20 glucocorticoid 6 Dexamethasone Q2 HIT 0.004 2 KEGG 383 2.58 >20 >20 glucocorticoid, Sodium Phosphate antiinflammatory 7 Fludrocortisone Q2 HIT <0.001 0.4 KEGG 362 2.56 >20 >20 mineralocorticoid Acetate 8 Hydrocortisone Q2 HIT 0.02 7 KEGG 350 2.54 >20 >20 glucocorticoid Hemisuccinate 9 Melengestrol Q2 HIT 0.07 13 196 2.29 >20 >20 antineoplastic, Acetate progestin 10 Dichlorisone Q2 HIT 0.03 4 169 2.23 >20 >20 antipruretic Acetate 11 Hydrocortisone Q2 HIT 0.02 2 KEGG 89 1.95 >20 >20 glucocorticoid, Butyrate antiinflammatory 12 Medroxyprogesterone Q2 HIT 0.3 >20 70 1.84 >20 >20 progestogen Acetate 13 Monensin Sodium Q2 HIT 0.004 0.2 49 1.69 >20 2 antibiotic, antibacterial; antibacterial 14 Deflazacort Q2 HIT <0.001 0.04 42 1.63 20 >20 antiinflammatory 15 Medroxyprogesterone Q2 HIT 0.5 >20 41 1.61 >20 >20 contraceptive Acetate 16 Ritodrine Q3 HIT 0.6 >20 33 1.51 >20 >20 muscle relaxant Hydrochloride (smooth)
TABLE-US-00007 TABLE 7 provides compounds and noting those that have been indicated for MS or EAE. log ratio Reported effects dead_IC50 fib_IC50 LPS_IC50 fib LPS brain/ brain/ brain/ in MS or EAE or Name (uM) (uM) fib_HS fib_R-sq (uM) LPS_HS LPS_R-sq Activity specificity specificity blood blood blood_scale Therapy neuroinflammation flurandrenolide 0.002 0.00 0.002 0.00 fib/LPS 1.0 1.0 −1.37 0.04 3 antiinflammatory none flumethazone 0.002 −0.23 0.12 0.002 0.00 fib/LPS 1.0 1.0 4 glucocorticoid, none pivalate antiinflammatory fluocinolone 0.002 0.94 0.71 0.002 0.53 0.33 fib/LPS 1.0 1.0 −1.34 0.05 3 glucocorticoid, Protection in acetonide antiinflammatory retinal neuroinflammation (microglia!) triamcinolone 0.002 ~−12 0.96 0.002 −0.42 0.94 fib/LPS 1.0 1.0 4 antiinflammatory none diacetate dexamethasone 0.002 0.00 0.002 0.14 0.07 fib/LPS 1.0 1.0 −1.16 0.07 3 glucocorticoid Protection in several rodent EAE models, optical neuritis, EAN, EAU, BBB disruption, reactive oxygen species generation in microglia dexamethasone 0.002 0.00 0.002 0.00 fib/LPS 1.0 1.0 −1.13 0.07 3 glucocorticoid, see acetate antiinflammatory dexamethasone dexamethasone 0.002 −0.49 0.68 0.005 −0.60 0.97 fib/LPS 2.3 0.4 4 glucocorticoid, see sodium antiinflammatory dexamethasone phosphate Nylidrin 0.002 −0.24 0.48 0.007 −0.51 0.86 fib/LPS 3.3 0.3 0.14 1.37 1 beta none hydrochloride Adrenoceptor agonist; peripheral vasodilator fludrocortisone 0.002 −1.10 0.76 0.002 −0.29 0.83 fib/LPS 1.0 1.0 −1.22 0.06 3 mineralocorticoid none acetate fluocinonide 0.002 −0.30 0.77 0.002 −0.41 0.37 fib/LPS 1.0 1.0 −1.30 0.05 3 antiinflammatory, none glucocorticoid prednisolone 0.002 −0.78 0.94 0.002 −0.80 0.94 fib/LPS 1.0 1.0 −1.30 0.05 3 glucocorticoid see methylprednisolone 6alpha- 0.002 −0.26 0.36 0.002 −0.40 0.84 fib/LPS 1.0 1.0 −1.19 0.06 3 glucocorticoid see methylprednisolone methylprednisolone acetate fluoromethoIone 0.002 −0.27 0.65 0.002 0.00 fib/LPS 1.0 1.0 −0.65 0.23 3 glucocorticoid, none antiinflammatory prednisolone 0.003 −0.66 0.89 0.003 −0.53 0.95 fib/LPS 1.2 0.9 −1.27 0.05 3 glucocorticoid Protection in EAE acetate and collagen- induced arthritis + see methylprednisolone clopamide 0.003 −0.88 0.88 0.002 −1.30 0.91 fib/LPS 0.7 1.5 −0.98 0.11 3 diuretic none methylprednisolone 0.003 −0.68 0.87 0.002 −0.45 0.82 fib/LPS 0.6 1.6 −1.22 0.06 3 glucocorticoid Treatment for MS; Protection in EAE hydrocortisone 0.004 −0.39 0.84 0.036 −1.10 0.83 fib/LPS 8.4 0.1 4 glucocorticoid Beneficial in hemisuccinate combination therapy in MS (1970's) hydrocortisone 0.010 −0.67 0.97 0.017 −0.95 0.98 fib/LPS 1.6 0.6 −1.26 0.05 3 glucocorticoid, Protection in EAE acetate antiinflammatory (1970's) podofilox 0.014 −1.20 0.98 0.002 −0.50 0.96 fib/LPS 0.2 5.8 −0.95 0.11 3 antineoplastic, none inhibits microtubule assembly, and human DNA topoisomerasell; antimitotic agent colchicine 0.015 −3.20 0.94 0.009 −1.10 0.97 fib/LPS 0.6 1.8 −0.84 0.15 3 antimitotic, Beneficial with antigout agent polyunsaturated FA in MS (1970's) hydrocortisone 0.018 −0.65 0.90 0.002 −0.58 0.97 fib/LPS 0.1 9.1 −1.29 0.05 3 glucocorticoid, see hydrocortisone antiinflammatory hemisuccinate/ acetate podophyllotoxin 0.022 −1.10 0.99 0.012 −0.87 0.93 fib/LPS 0.5 1.8 −0.92 0.12 3 none acetate dihydromundulone 0.022 −0.67 0.96 0.006 −0.56 0.99 fib/LPS 0.3 3.6 −0.03 0.94 2 none cefamandole 0.052 −0.66 0.91 0.002 −0.35 0.83 fib/LPS 0.0 33.2 4 antimicrobial none sodium hydrocortisone 0.071 −0.60 0.95 0.005 −0.45 0.93 fib/LPS 0.1 15.4 −0.91 0.12 3 glucocorticoid, see hydrocortisone butyrate antiinflammatory hemisuccinate/ acetate hydrocortisone 0.128 −0.52 0.73 0.161 −0.69 0.95 fib/LPS 1.3 0.8 4 glucocorticoid see hydrocortisone sodium hemisuccinate/ phosphate acetate 5,7- 0.172 −0.56 0.90 0.021 −0.59 0.75 fib/LPS 0.1 8.0 −0.49 0.32 2 none dihydroxyisoflavone nigericin 0.206 −2.30 0.91 0.065 −0.35 0.54 fib/LPS 0.3 3.2 4 antibiotic none sodium mycophenolic 0.289 −1.70 0.98 0.193 −1.50 0.96 fib/LPS 0.7 1.5 −0.67 0.22 3 antineoplastic Promising in co- acid therapy with IFNbeta1a in MS; Improvement in neuromyelitis optica beta-peltatin 0.505 −2.10 0.97 0.252 −1.50 0.92 fib/LPS 0.5 2.0 −0.69 0.21 3 antineoplastic, none cytotoxic acivicin 0.570 −0.83 0.93 1.662 ~−19 0.76 fib/LPS 2.9 0.3 −1.68 0.02 3 antineoplastic none mycophenolic 0.606 −0.83 0.92 0.771 −1.60 0.79 fib/LPS 1.3 0.8 −0.67 0.22 3 antineoplastic Promising in co- acid therapy with IFNbeta1a in MS; Improvement in neuromyelitis optica hycanthone 0.619 −0.84 0.94 0.354 −0.67 0.88 fib/LPS 0.6 1.7 0.09 1.22 1 anthelmintic, none hepatotoxic colchiceine 0.625 −2.00 0.96 0.820 −3.00 0.99 fib/LPS 1.3 0.8 −1.10 0.08 3 antimitotic Patent on use of colchic(e)ine in MS + see clochicine teniposide 0.821 −1.10 0.91 0.240 −0.66 0.94 fib/LPS 0.3 3.4 4 antineoplastic none 2,4- 0.906 −1.00 0.84 1.006 −0.63 0.84 fib/LPS 1.1 0.9 1.12 13.21 0 herbicide none dichlorophenoxyacetic acid, isooctyl ester Thioridazine 1.012 −1.30 0.83 0.677 −0.38 0.53 fib/LPS 0.7 1.5 1.46 28.77 0 Dopamine none hydrochloride receptor antagonist; Ca2+ channel antagonist; antipsychotic monensin 1.143 −3.80 0.97 0.212 −0.48 0.76 fib/LPS 0.2 5.4 4 antibiotic none sodium (monensin a is shown) dehydrodeoxysappanone 1.237 −0.96 0.91 0.657 −0.89 0.97 fib/LPS 0.5 1.9 −0.22 0.61 2 none b dimethyl ether vinblastine 1.394 −0.80 0.85 0.628 −0.57 0.84 fib/LPS 0.5 2.2 4 antineoplastic, none sulfate spindle poison maprotiline 1.399 −0.63 0.50 0.289 −0.39 0.68 fib/LPS 0.2 4.8 0.92 8.22 0 antidepressant none hydrochloride brazilein 1.466 −0.89 0.95 2.174 −1.70 0.92 fib/LPS 1.5 0.7 −1.22 0.06 3 none Desipramine 1.616 −0.36 0.65 0.002 0.00 fib/LPS 0.0 1037.2 0.78 6.04 0 Antidepressant none hydrochloride fendiline 1.747 −0.72 0.80 0.900 −0.51 0.25 fib/LPS 0.5 1.9 1.31 20.37 0 coronary none hydrochloride vasodilator colforsin 2.083 −0.94 0.97 1.346 −0.65 0.90 fib/LPS 0.6 1.5 4 adenylate none cyclase activator, antiglaucoma neomycin 2.297 −0.75 0.93 5.000 fib/LPS 2.2 0.5 4 antibacterial Reduction EAE via sulfate alteration gut microflora 6,7-dichloro-3- 2.648 −1.30 0.85 5.000 fib/LPS 1.9 0.5 −0.52 0.30 3 NMDA none hydroxy-2- and quinoxalinecarboxylic kainate acid receptor antagonist salinomycin, 2.970 −4.80 0.77 4.575 ~−11 0.76 fib/LPS 1.5 0.6 4 antibiotic none sodium Prochlorperazine 4.381 ~−11 0.80 2.014 −2.30 0.83 fib/LPS 0.5 2.2 1.14 13.77 0 Antipsychotic none dimaleate agent; used in the treatment of spastic gastrointestinal disorders tannic acid 4.955 ~−21 0.54 4.379 −3.70 0.91 fib/LPS 0.9 1.1 4 nonspecific none enzyme/ receptor blocker salinomycin, 3.862 0.061 −0.53 0.89 0.056 −0.31 0.62 tox/fib/ 0.9 1.1 4 antibiotic none sodium LPS pomiferin 3.445 0.251 −0.43 0.50 0.123 −0.29 0.51 tox/fib/ 0.5 2.0 4 antioxidant none LPS betamethasone 2.085 0.002 0.00 0.002 0.00 tox/fib/ 1.0 1.0 −1.16 0.07 3 glucocorticoid, none LPS antiinflammatory colchicine 0.520 0.022 ~−20 0.94 0.054 ~−21 0.31 tox/fib/ 2.5 0.4 −0.84 0.15 3 antimitotic, Beneficial with LPS antigout agent polyunsaturated FA in MS (1970's)
[0149] Acivicin suppresses oxidative stress in innate immune cells: Although acivicin has been studied primarily in cancer cells (34), its functions in inflammation and neurological diseases are poorly understood. The effects of acivicin were tested in innate immune cell activation using a series of secondary assays, such as ROS generation, glutathione regulation, gene expression of prooxidant and inflammatory genes, GGT activity, and antigen presentation. Acivicin inhibited fibrin- and LPS-induced microglial activation in a dose-dependent manner, and decreased ROS generation in mouse and human macrophages (
[0150] Therapeutic effects of acivicin in neuroinflammation: In patients with MS and neuromyelitis optica, serum GGT levels correlate with clinical disability, BBB disruption, and inflammatory markers (39). GGTLC1, which encodes the GGT light chain 1, is detected in the cortex of MS patients and is associated with oxidative damage and neuronal injury (40). Serum GGT activity positively correlates with dementia risk and glutathione S-transferase alpha in the plasma of AD patients correlates with late-onset AD progression (41,42). The effects of acivicin treatment were tested in autoimmune acute and chronic progressive models of neuroinflammation, as well as in models of microglia-mediated neurodegeneration. Using immunohistochemistry and FACS, the expression of GGT in EAE spinal cord was first tested.
[0151] GGT was not detected in healthy spinal cord but increased in microglia and infiltrating monocyte/macrophages in EAE lesions (
[0152] Immunization of non-obese diabetic (NOD) mice with MOG.sub.35-55 results in an acute neurological impairment followed by a chronic phase of progressive accumulation of disability (43). In chronic NOD MOG.sub.35-55 EAE, therapeutic administration of acivicin during the chronic phase even eighty days after EAE induction suppressed progression as indicated by decreased clinical signs (
DISCUSSION
[0153] The study revealed the oxidative stress transcriptome of CNS innate immune cells in neuroinflammation and identified druggable pathways to suppress neurotoxic innate immunity. By developing a functional transcriptomic and drug discovery pipeline consisting of deep sequencing, small molecule screening, and pathway analysis, novel innate immune cell populations involved in oxidative stress were identified and discovered upstream targeting of glutathione metabolism and redox homeostasis as a therapeutic strategy in neuroinflammation. Using Tox-seq, it was discovered that innate immune cells share a core oxidative stress gene signature mechanistically coupled to coagulation, antigen presentation, and glutathione pathways. The findings introduce the concept of distinct molecular circuits governing oxidative stress and immune-mediated neurodegeneration and reveal their molecular signatures. Given that oxidative stress producing resident and infiltrating innate immune cells are players in MS progression (3,12,45), the oxidative stress signature could enable the identification of specific cell subpopulations contributing to neurotoxicity that could be further characterized with cell-fate mapping studies. Molecular convergence of innate immune cells to an oxidative stress core signature is a springboard for the development of therapies to selectively target CNS innate immune populations that promote oxidative cell injury. Given the broad range of diseases with oxidative stress, the findings have implications for a wide range of diseases including MS, AD, and traumatic brain injury.
[0154] The study revealed previously unknown molecular links between coagulation and oxidative stress. In neuroinflammatory lesions, in situ expression of coagulation genes promoting fibrin formation were identified, such as genes encoding coagulation factors IX and X, Vitamin K Dependent Plasma Glycoprotein, and the von Willebrand factor receptors glycoproteins IX and Ib. Intriguingly, coagulation gene expression was differentially increased in ROS.sup.+ CNS innate immune cells that co-expressed genes regulating oxidative stress, such as the NADPH oxidase subunit gp91-phox and GGT, and iron metabolism. Dysregulation of the coagulation pathway and fibrin deposition correlates with cortical damage, microglia activation, and neuronal loss in MS and EAE (29,30,46-48). Administration of anti-coagulants or inhibition of the interaction of fibrin with the CD11b-CD18 integrin receptor (also known as Mac-1, complement receptor 3, αMβ2) reduces clinical signs, oxidative stress, and neurodegeneration in MS animal models (9,10,31). Fibrin signaling via the CD11b-CD18 receptor may potentiate the crosstalk of NADPH oxidase with the GGT pathway (49) leading to redox regulation. Indeed, fibrin activates NADPH oxidase (9) and GGT (herein) to promote degradation of glutathione and oxidative stress in innate immune cells. Furthermore, inhibition of fibrin interaction with CD11b-CD18 or inhibition of NADPH oxidase (9,10), or GGT (herein) suppresses fibrin-induced ROS generation. NADPH oxidase activation and degradation of glutathione by fibrin can be a prooxidant mechanisms in diseases with blood-brain barrier disruption and vascular pathology. Thus, it is possible that there is a positive-feedback loop between coagulation, oxidative stress, and the pro-inflammatory response, whereby subpopulations of innate immune cells promote the local synthesis of coagulation factors to increase fibrin deposition and promote oxidative injury. Local increases in coagulation activity by innate immune cell subpopulations as an oxidative stress mechanism could be relevant for other diseases in the brain and periphery with vascular damage associated with fibrin deposition and oxidative injury (5,7,10,31,50,51).
[0155] Acivicin was selected as an upstream regulator of glutathione and redox homeostasis. GGT mediated cleavage of glutathione causes iron redox cycling, which stimulates the release of hydroxyl radicals (34). Thus, redox restoration by acivicin may protect against EAE progression by homeostatic regulation of glutathione in oxidative stress-producing innate immune cells. In accordance, pharmacologic inhibition of GGT by GGsTop reduced oxidative stress markers and protected from renal reperfusion injury (52). In EAE, acivicin suppressed inflammatory and prooxidant pathways and decreased axonal damage, demyelination, and peripheral cell recruitment into the CNS. Suppression of oxidative stress and reduction of chemokines that facilitate cell recruitment by acivicin might reduce myeloid cell numbers and decrease lesion size. Acivicin also regulates glutamate metabolism and leukotriene responses with potential effects on immune cell recruitment, neuronal and T cell functions (34,53,54). Cell sorting and scRNA-seq studies may be used to determine the drug selectivity of acivicin at the single-cell level and its effects on oxidative stress resistance and additional prooxidant and inflammatory markers. These studies could decipher mechanisms linking oxidative stress and peripheral cell recruitment into the CNS in EAE and other models of neurologic disease. Glutamine analogues like acivicin exhibit dose limiting toxicity in anti-cancer trials potentially due to interference with recycling of glutamine (34). Given the toxicity of high doses of acivicin in the clinic or the consequences of global depletion of GGT1 in mice (34), identification of safe drugs modulating glutathione metabolism might facilitate the restoration of redox hemostasis in neuroinflammatory disease.
[0156] In summary, by generating the first oxidative stress cell atlas of innate immunity, cell populations and molecular mechanisms involved in oxidative injury and neurotoxicity in neuroinflammatory disease were identified. Transcriptional signatures of oxidative stress genes that can be used as a resource and follow-up studies to validate additional gene targets, cell populations, and drugs from the HTS screen were defined. Furthermore, a method, Tox-seq, was advanced, which can be used to determine the functional role of oxidative stress producing cells in a wide range of disease states. Given the multiple roles of ROS in oxidative damage and redox regulation (55), Tox-seq could reveal molecular pathways governing ROS-mediated functions in physiology and pathology. Integration of functional transcriptomics and HTS may prioritize druggable pathways that could enhance cherry-picking or in silico screens to identify compounds of interest for preclinical testing. Thus, oxidative stress transcriptomics and drug discovery approaches could identify and target neurotoxic CNS innate immune populations and lead to the development of selective neuroprotective strategies.
BIBLIOGRAPHY
[0157] 1. Nikic, I. et al. A reversible form of axon damage in experimental autoimmune encephalomyelitis and multiple sclerosis. Nat Med 17, 495-499 (2011). [0158] 2. Locatelli, G. et al. Mononuclear phagocytes locally specify and adapt their phenotype in a multiple sclerosis model. Nat Neurosci 21, 1196-1208 (2018). [0159] 3. Fischer, M. T. et al. NADPH oxidase expression in active multiple sclerosis lesions in relation to oxidative tissue damage and mitochondrial injury. Brain 135, 886-899 (2012). [0160] 4. Heppner, F. L., Ransohoff, R. M. & Becher, B. Immune attack: the role of inflammation in Alzheimer disease. Nat Rev Neurosci 16, 358-372 (2015). [0161] 5. Nortley, R. et al. Amyloid beta oligomers constrict human capillaries in Alzheimer's disease via signaling to pericytes. Science 365 (2019). [0162] 6. Park, L. et al. NADPH-oxidase-derived reactive oxygen species mediate the cerebrovascular dysfunction induced by the amyloid beta peptide. J Neurosci 25, 1769-1777 (2005). [0163] 7. Park, L. et al. NADPH-oxidase-derived reactive oxygen species mediate the cerebrovascular dysfunction induced by the amyloid beta peptide. J Neurosci 25, 1769-1777 (2005). [0164] 8. Back, S. A., Gan, X., Li, Y., Rosenberg, P. A. & Volpe, J. J. Maturation-dependent vulnerability of oligodendrocytes to oxidative stress-induced death caused by glutathione depletion. J Neurosci 18, 6241-6253 (1998). [0165] 9. Ryu, J. K. et al. Fibrin-targeting immunotherapy protects against neuroinflammation and neurodegeneration. Nat Immunol 19, 1212-1223 (2018). [0166] 10. Merlini, M. et al. Fibrinogen induces microglia-mediated spine elimination and cognitive impairment in Alzheimer's Disease. Neuron 101, 1099-1108 (2019). [0167] 11. Weiner, H. L. A shift from adaptive to innate immunity: a potential mechanism of disease progression in multiple sclerosis. J Neurol 255 Suppl 1, 3-11 (2008). [0168] 12. Lassmann, H., van Horssen, J. & Mahad, D. Progressive multiple sclerosis: pathology and pathogenesis. Nat. Rev. Neurol. 8, 647-656 (2012). [0169] 13. Mahad, D. H., Trapp, B. D. & Lassmann, H. Pathological mechanisms in progressive multiple sclerosis. Lancet Neurol 14, 183-193 (2015). [0170] 14. Keren-Shaul, H. et al. A Unique Microglia Type Associated with Restricting Development of Alzheimer's Disease. Cell 169, 1276-1290 e1217 (2017). [0171] 15. Hammond, T. R. et al. Single-Cell RNA Sequencing of Microglia throughout the Mouse Lifespan and in the Injured Brain Reveals Complex Cell-State Changes. Immunity 50, 253-271 e256 (2019). [0172] 16. Schirmer, L. et al. Neuronal vulnerability and multilineage diversity in multiple sclerosis. Nature (2019). [0173] 17. Mathys, H. et al. Single-cell transcriptomic analysis of Alzheimer's disease. Nature 570, 332-337 (2019). [0174] 18. Gosselin, D. et al. An environment-dependent transcriptional network specifies human microglia identity. Science 356 (2017). [0175] 19. Krasemann, S. et al. The TREM2-APOE Pathway Drives the Transcriptional Phenotype of Dysfunctional Microglia in Neurodegenerative Diseases. Immunity 47, 566-581.e569 (2017). [0176] 20. Jordão, M. J. C. et al. Single-cell profiling identifies myeloid cell subsets with distinct fates during neuroinflammation. Science 363, eaat7554 (2019). [0177] 21. Van Hove, H. et al. A single-cell atlas of mouse brain macrophages reveals unique transcriptional identities shaped by ontogeny and tissue environment. Nat Neurosci 22, 1021-1035 (2019). [0178] 22. Diehn, M. et al. Association of reactive oxygen species levels and radioresistance in cancer stem cells. Nature 458, 780-783 (2009). [0179] 23. Butler, A., Hoffman, P., Smibert, P., Papalexi, E. & Satija, R. Integrating single-cell transcriptomic data across different conditions, technologies, and species. Nat Biotechnol 36, 411-420 (2018). [0180] 24. Li, Q. et al. Developmental Heterogeneity of Microglia and Brain Myeloid Cells Revealed by Deep Single-Cell RNA Sequencing. Neuron 101, 207-223 e210 (2019). [0181] 25. Ajami, B. et al. Single-cell mass cytometry reveals distinct populations of brain myeloid cells in mouse neuroinflammation and neurodegeneration models. Nat Neurosci 21, 541-551 (2018). [0182] 26. Mrdjen, D. et al. High-Dimensional Single-Cell Mapping of Central Nervous System Immune Cells Reveals Distinct Myeloid Subsets in Health, Aging, and Disease. Immunity 48, 380-395.e386 (2018). [0183] 27. Geirsdottir, L. et al. Cross-Species Single-Cell Analysis Reveals Divergence of the Primate Microglia Program. Cell 179, 1609-1622 e1616 (2019). [0184] 28. Choi, B. Y. et al. Inhibition of NADPH oxidase activation reduces EAE-induced white matter damage in mice. J Neuroinflammation 12, 104 (2015). [0185] 29. Davalos, D. et al. Early detection of thrombin activity in neuroinflammatory disease. Ann Neurol 75, 303-308 (2014). [0186] 30. Davalos, D. et al. Fibrinogen-induced perivascular microglial clustering is required for the development of axonal damage in neuroinflammation. Nat. Commun. 3, 1227 (2012). [0187] 31. Petersen, M. A., Ryu, J. K. & Akassoglou, K. Fibrinogen in neurological diseases: mechanisms, imaging and therapeutics. Nat Rev Neurosci 19, 283-301 (2018). [0188] 32. Shannon, P. et al. Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res 13, 2498-2504 (2003). [0189] 33. Kutmon, M. et al. PathVisio 3: an extendable pathway analysis toolbox. PLoS Comput. Biol. 11, e1004085 (2015). [0190] 34. Corti, A., Franzini, M., Paolicchi, A. & Pompella, A. Gamma-glutamyltransferase of cancer cells at the crossroads of tumor progression, drug resistance and drug targeting. Anticancer Res 30, 1169-1181 (2010). [0191] 35. Koga, M. et al. Glutathione is a physiologic reservoir of neuronal glutamate. Biochem Biophys Res Commun 409, 596-602 (2011). [0192] 36. Drozdz, R. et al. gamma-Glutamyltransferase dependent generation of reactive oxygen species from a glutathione/transferrin system. Free Radic Biol Med 25, 786-792 (1998). [0193] 37. Jiang, X. et al. Quantitative real-time imaging of glutathione. Nat. Commun. 8, 16087 (2017). [0194] 38. Tsuji, T., Yamada, K. & Kunieda, T. Characterization of the dwg mutations: dwg and dwg(Bayer) are new mutant alleles of the Ggt1 gene. Mamm Genome 20, 711-719 (2009). [0195] 39. Shu, Y. et al. Association of serum gamma-glutamyltransferase and C-reactive proteins with neuromyelitis optica and multiple sclerosis. Mult. Scler. Relat. Disord. 18, 65-70 (2017). [0196] 40. Fischer, M. T. et al. Disease-specific molecular events in cortical multiple sclerosis lesions. Brain 136, 1799-1815 (2013). [0197] 41. Iturria-Medina, Y. et al. Early role of vascular dysregulation on late-onset Alzheimer's disease based on multifactorial data-driven analysis. Nat. Commun. 7, 11934 (2016). [0198] 42. Kunutsor, S. K. & Laukkanen, J. A. Gamma glutamyltransferase and risk of future dementia in middle-aged to older Finnish men: A new prospective cohort study. Alzheimers Dement 12, 931-941 (2016). [0199] 43. Mayo, L. et al. Regulation of astrocyte activation by glycolipids drives chronic CNS inflammation. Nat Med 20, 1147-1156 (2014). [0200] 44. Herrera, A. J., Castano, A., Venero, J. L., Cano, J. & Machado, A. The single intranigral injection of LPS as a new model for studying the selective effects of inflammatory reactions on dopaminergic system. Neurobiol. Dis. 7, 429-447 (2000). [0201] 45. International Multiple Sclerosis Genetics Consortium. Multiple sclerosis genomic map implicates peripheral immune cells and microglia in susceptibility. Science 365, eaav7188 (2019). [0202] 46. Magliozzi, R. et al. Iron homeostasis, complement, and coagulation cascade as CSF signature of corical lesions in early multiple sclerosis. Ann. Clin. Transl. Neurol. 6, 2150-2163 (2019). [0203] 47. Yates, R. L. et al. Fibrin(ogen) and neurodegeneration in the progressive multiple sclerosis cortex. Ann Neurol 82, 259-270 (2017). [0204] 48. Han, M. H. et al. Proteomic analysis of active multiple sclerosis lesions reveals therapeutic targets. Nature 451, 1076-1081 (2008). [0205] 49. Ravuri, C., Svineng, G., Pankiv, S. & Huseby, N. E. Endogenous production of reactive oxygen species by the NADPH oxidase complexes is a determinant of gammaglutamyltransferase expression. Free Radic. Res. 45, 600-610 (2011). [0206] 50. Rodriguez-Rodriguez, A., Egea-Guerrero, J. J., Murillo-Cabezas, F. & Carrillo-Vico, A. Oxidative stress in traumatic brain injury. Curr Med Chem 21, 1201-1211 (2014). [0207] 51. Davalos, D. & Akassoglou, K. Fibrinogen as a key regulator of inflammation in disease. Semin Immunopathol 34, 43-62 (2012). [0208] 52. Yamamoto, S. et al. Preventive effect of GGsTop, a novel and selective gamma-glutamyl transpeptidase inhibitor, on ischemia/reperfusion-induced renal injury in rats. J Pharmacol Exp Ther 339, 945-951 (2011). [0209] 53. Birkner, K. et al. beta1-Integrin- and KV1.3 channel-dependent signaling stimulates glutamate release from Th17 cells. J Clin Invest (2020). [0210] 54. Sedlak, T. W. et al. The glutathione cycle shapes synaptic glutamate activity. Proc Natl Acad Sci USA 116, 2701-2706 (2019). [0211] 55. Schieber, M. & Chandel, N. S. ROS function in redox signaling and oxidative stress. Curr Biol 24, R453-462 (2014). [0212] 56. Saederup, N. et al. Selective chemokine receptor usage by central nervous system myeloid cells in CCR2-red fluorescent protein knock-in mice. PLoS One 5, e13693 (2010). [0213] 57. Jung, S. et al. Analysis of fractalkine receptor CX(3)CR1 function by targeted deletion and green fluorescent protein reporter gene insertion. Mol Cell Biol 20, 4106-4114 (2000). [0214] 58. Huang da, W., Sherman, B. T. & Lempicki, R. A. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat. Protoc. 4, 44-57 (2009). [0215] 59. Aronesty, E. ea-utils: “Command-line tools for processing biological sequencing data”. (2011). [0216] 60. Robinson, M. D., McCarthy, D. J. & Smyth, G. K. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26, 139-140 (2010). [0217] 61. Robinson, M. D. & Oshlack, A. A scaling normalization method for differential expression analysis of RNA-seq data. Genome Biol. 11, R25 (2010). [0218] 62. Zambon, A. C. et al. GO-Elite: a flexible solution for pathway and ontology overrepresentation. Bioinformatics 28, 2209-2210 (2012). [0219] 63. Van der Laan, M. J. & Pollard, K. S. A new algorithm for hybrid clustering of gene expression data with visualization and the bootstrap. J. Stat. Plan. Interference 117, 275-303 (2003). [0220] 64. Zuberi, K. et al. GeneMANIA prediction server 2013 update. Nucleic Acids Res 41, W115-122 (2013). [0221] 65. Butovsky, O. et al. Identification of a unique TGF-beta-dependent molecular and functional signature in microglia. Nat Neurosci 17, 131-143 (2014). [0222] 66. Kutmon, M. et al. WikiPathways: capturing the full diversity of pathway knowledge. Nucleic Acids Res 44, D488-494 (2016). [0223] 67. Ryu, J. K. et al. Blood coagulation protein fibrinogen promotes autoimmunity and demyelination via chemokine release and antigen presentation. Nat. Commun. 6, 8164 (2015). [0224] 68. Adams, R. A. et al. The fibrin-derived gamma377-395 peptide inhibits microglia activation and suppresses relapsing paralysis in central nervous system autoimmune disease. J Exp Med 204, 571-582 (2007). [0225] 69. Akassoglou, K. et al. Oligodendrocyte apoptosis and primary demyelination induced by local TNF/p55TNF receptor signaling in the central nervous system of transgenic mice: models for multiple sclerosis with primary oligodendrogliopathy. Am J Pathol 153, 801-813 (1998). [0226] 70. Akassoglou, K. et al. Fibrin depletion decreases inflammation and delays the onset of demyelination in a tumor necrosis factor transgenic mouse model for multiple sclerosis. Proc Natl Acad Sci USA 101, 6698-6703 (2004).
[0227] PCT/US2018/052694
[0228] All publications, nucleotide and amino acid sequence identified by their accession nos., patents and patent applications are incorporated herein by reference. While in the foregoing specification this invention has been described in relation to certain embodiments thereof, and many details have been set forth for purposes of illustration, it will be apparent to those skilled in the art that the invention is susceptible to additional embodiments and that certain of the details described herein may be varied considerably without departing from the basic principles of the invention.
[0229] The specific methods and compositions described herein are representative of embodiments and are exemplary and not intended as limitations on the scope of the invention. Other objects, aspects, and embodiments will occur to those skilled in the art upon consideration of this specification and are encompassed within the spirit of the invention as defined by the scope of the claims. It will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. The invention illustratively described herein suitably may be practiced in the absence of any element or elements, or limitation or limitations, which is not specifically disclosed herein as essential. The methods and processes illustratively described herein suitably may be practiced in differing orders of steps, and the methods and processes are not necessarily restricted to the orders of steps indicated herein or in the claims. As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to “a nucleic acid” or “a polypeptide” includes a plurality of such nucleic acids or polypeptides (for example, a solution of nucleic acids or polypeptides or a series of nucleic acid or polypeptide preparations), and so forth. In this document, the term “or” is used to refer to a nonexclusive or, such that “A or B” includes “A but not B,” “B but not A,” and “A and B,” unless otherwise indicated.
[0230] Under no circumstances may the patent be interpreted to be limited to the specific examples or embodiments or methods specifically disclosed herein. Under no circumstances may the patent be interpreted to be limited by any statement made by any Examiner or any other official or employee of the Patent and Trademark Office unless such statement is specifically and without qualification or reservation expressly adopted in a responsive writing by Applicants.
[0231] The terms and expressions that have been employed are used as terms of description and not of limitation, and there is no intent in the use of such terms and expressions to exclude any equivalent of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention as claimed. Thus, it will be understood that although the present invention has been specifically disclosed by embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims and statements of the invention.