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WO2025062016A2 - Biomarqueurs pour le traitement par inhibiteur de tlr - Google Patents

Biomarqueurs pour le traitement par inhibiteur de tlr Download PDF

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Publication number
WO2025062016A2
WO2025062016A2 PCT/EP2024/076541 EP2024076541W WO2025062016A2 WO 2025062016 A2 WO2025062016 A2 WO 2025062016A2 EP 2024076541 W EP2024076541 W EP 2024076541W WO 2025062016 A2 WO2025062016 A2 WO 2025062016A2
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tlr inhibitor
treatment
biomarker
individual
autoantibody
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WO2025062016A3 (fr
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Andrew Bender
Yin Wu
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Merck Patent GmbH
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Merck Patent GmbH
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Priority claimed from PCT/EP2023/081191 external-priority patent/WO2024100139A1/fr
Application filed by Merck Patent GmbH filed Critical Merck Patent GmbH
Publication of WO2025062016A2 publication Critical patent/WO2025062016A2/fr
Publication of WO2025062016A3 publication Critical patent/WO2025062016A3/fr
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4709—Non-condensed quinolines and containing further heterocyclic rings
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the present invention provides biomarkers, in particular autoantibodies and RNAs, for the treatment of patients with toll-like receptor (TLR) inhibitors and related uses and methods.
  • TLR toll-like receptor
  • the TLR family comprises several members with different specificities and is part of the cellular pathogen pattern recognition system, which has evolved as a defense against a variety of infections.
  • the functional expression of selected TLRs in tissues is highly different. Some receptors are located at the cell surface such as TLR4 (stimulated by E. coli lipopolysaccharide LPS), e.g., on epithelial cells, whereas others, such as TLR3, 7, 8 and 9, are located at endosomal membranes of specific immune cells. The latter are all activated by nucleic acids but recognize various types of them.
  • TLR9 is activated by single stranded DNA containing CpG subsequences
  • TLR7 and 8 are activated by single stranded RNA
  • TLR3 is activated by double- stranded RNA.
  • the activation of the TLRs triggers various downstream signaling cascades, including the signaling via nuclear factor-kB (NF-kB), interferon (IFN) response factors (IRFs), and mitogen-activated protein (MAP) kinases, to result in the transciption of various immune response genes, including inflammatory cytokines, stimulatory immune cytokines, chemokines and co-stimulatory molecules (Farrugia and Baron, Int Jinflam. 2017; 2017: 8391230).
  • NF-kB nuclear factor-kB
  • IRFs interferon response factors
  • MAP mitogen-activated protein
  • RNA molecules are proposed as the primary mechanism of TLR7/8 activation in lupus, although microRNA and other single-stranded RNA species may also play a role in lupus pathogenesis (Bender AT, etal., Immunohorizons 2020; 4: 93-107).
  • the present invention relates to autoantibodies and RNAs as biomarkers for identifying patients that are likely to benefit from treatment with TLR inhibitors.
  • the invention provides the use of one or more biomarkers described herein as a predictive biomarker for the treatment of an individual with a disease with a TLR inhibitor.
  • the invention provides a method for predicting therapeutic effectiveness of a TLR inhibitor in an individual with a disease comprising determining the expression level of one or more biomarkers described herein in the individual, wherein the biomarker expression level indicates the therapeutic effectiveness of the TLR inhibitor in the individual.
  • the invention provides a method for predicting the suitability of an individual with a disease to initiate treatment with a TLR inhibitor comprising determining the expression level of one or more biomarkers described herein in the individual, wherein the biomarker expression level indicates the suitability of the individual to initiate the treatment.
  • the invention provides a method for predicting the suitability of an individual with a disease who is receiving treatment with a TLR inhibitor to continue the treatment comprising determining the expression level of one or more biomarkers described herein in the individual, wherein the biomarker expression level indicates the suitability of the individual to continue the treatment.
  • the invention provides a TLR inhibitor for use in a method of treating a disease in an individual comprising administering the TLR inhibitor to an individual that is likely to respond to such treatment as reflected by the expression level of one or more biomarkers described herein.
  • the TLR inhibitor is a TLR7 and/or TLR8 inhibitor.
  • the TLR7 and/or TLR8 inhibitor could be selected from the group consisting of 5- [(3R, 5 S)-3 -amino-5-(trifluoromethyl)piperidin- 1 -yl] quinoline-8-carbonitrile; (3R, 5 S)- 1 -(8- methoxy-1 ,7-naphthyridin-5-yl)-5-methylpiperidin-3-amine; 2- ⁇ 4-[2-(7,8- dimethyl[l,2,4]triazolo[l,5-a]pyridin-6-yl)-3-(propan-2-yl)-lH-indol-5-yl]piperidin-l- yl ⁇ acetamide; rel-(2R,6R)-4-(8-cyanoquinolin-5yl)-N-((3R,4S)-4-fluoropyrroli
  • Figure 1 shows the cumulative distribution function of a) time to recovery and b) time to clinical deterioration in unstratified patient populations receiving either placebo or 50 mg or 100 mg enpatoran BID (safety analysis set).
  • Figure 2 shows the cumulative distribution function of time to recovery for patients with a) high and b) low IFN-I signature scores at baseline and receiving either placebo or 50 mg or 100 mg enpatoran BID.
  • FIG. 3 shows TLR7/8 activation in human PBMCs by patient-derived immune complexes.
  • IgG was isolated from plasma samples from HC and patients with SLE, LN, IBM, PM and DM and combined with necrotic cell lysate to form immune complexes that were used to stimulate healthy donor PBMCs.
  • the PBMCs were pre-treated with enpatoran for 30 minutes before the immune complex was added to the cells and IFN-a was measured following 24 hours of treatment. All IgG samples were tested with 2-4 healthy donor PBMCs. DM dermatomyositis, HC healthy control, IBM inclusion body myositis, IC immune complex, IFN-a interferon-alpha, IgG immunoglobulin G, PBMCs peripheral blood mononuclear cells, PM polymyositis, SLE systemic lupus erythematosus, TLR7/8 toll-like receptor 7/8.
  • Figure 4 shows gene expression changes induced by patient-derived immune complexes.
  • IgG was isolated from plasma samples from HC and patients with SLE, LN, IBM, PM and DM and then combined with necrotic cell lysate to form immune complexes that were used to stimulate healthy donor PBMCs. After 24 hours of treatment, the cells were collected and analyzed by NanoString to measure changes in gene expression.
  • A) Heat map shows the Log2 FC compared to HC IgG samples. Each column represents a separate IgG sample, and the patient group is indicated by shading. The samples that stimulated IFN-a protein production are indicated by black the black bar above the columns.
  • IFN-GS Score An IFN-I signature score (IFN-GS Score) was calculated using the ISGs indicated in the heat map and scores were plotted for each individual sample. Data are averaged from independent experiments run with 2 PBMC donors for each IgG sample. DM dermatomyositis, FC fold change, HC healthy control, IBM inclusion body myositis, IFN-a interferon-alpha, IgG immunoglobulin G, ISGs interferon-stimulated genes, PBMCs peripheral blood mononuclear cells, PM polymyositis, SLE systemic lupus erythematosus.
  • FIG. 5 shows an autoantibody profiling.
  • Autoreactivity was profiled on a 1581- feature array using IgG purified from plasma samples from HC or patients with SLE, LN, IBM, PM and DM.
  • the Log2 MFI values for the 300 reactivities with the largest CV% are plotted and notable reactivities are identified by boxes. Data for each patient is shown per row and the shaded bar indicates the disease group.
  • FIG. 6 shows autoantibodies associated with TLR7 and/or TLR8 stimulation by immune complexes.
  • A) Autoreactivity of IgG from plasma samples from HC and patients with SLE, LN, IBM, PM and DM was profiled on a 1581 -feature array and correlation analysis was performed to identify the reactivities that correlated most highly with positivity in the ex vivo assay. The 10 reactivities that correlated most highly with SLE/LN (A) or DM/PM (B) patients’ positivity are shown.
  • C-I Reactivities that correlated highly with TLR7 and/or TLR8 stimulation of PBMCs are plotted by disease subgroup.
  • DM dermatomyositis CENPJ centromere protein J
  • HC healthy control HIST1H4A histone cluster 1 H4 family member A
  • IBM inclusion body myositis IFN-a interferon-alpha, IgG immunoglobulin G, ISGs interferon-stimulated genes
  • MFI mean fluorescence intensity
  • PBMCs peripheral blood mononuclear cells PM polymyositis, SLE systemic lupus erythematosus, TLR7/8 toll-like receptor 7/8, TRIM21 or SSA/Ro52 tripartite motif containing 21, TROVE2 or Ra/SSA TROVE domain family member 2.
  • FIG. 7 shows that immune complex-associated RNAs stimulate TLR7 and/or TLR8.
  • RNA molecules associated with various autoantibodies were synthesized in vitro. The RNAs were complexed with LyoVec and PBMCs were treated with 1 pg/mL of the RNA/LyoVec complexes. PBMCs were also pre-treated with 1 pM enpatoran for 30 minutes prior to treatment with the complexes. After 24 hours, supernatants were collected and the production of A) IFN-a B) IL-6, C) IL-1 ⁇ and D) IFN-y was measured using an MSD U-PLEX biomarker group assay. Means and standard error of the means plotted.
  • Data are from 2 independent experiments using 4 healthy donor PBMCs. His-tRNA histidyl-transfer RNA, IFN-a2 interferon-alpha 2, IL- 1ft interleukin- 1 beta, IL-6 interleukin-6, IFN-y interferon gamma, PBMCs peripheral blood mononuclear cells, TLR7/8 toll-like receptor 7/8.
  • the term “about” when used to modify a numerically defined parameter refers to any minimal alteration in such parameter that does not change the overall effect, e.g., the efficacy of the agent in treatment of a disease or disorder. In some embodiments, the term “about” means that the parameter may vary by as much as 10% below or above the stated numerical value for that parameter.
  • the term “around” when used in relationship to a value refers to any value that is reasonably close to the value referred to, for instance, any value that is less than 10% below or above the value referred to. In some embodiments, the term “around” means the exact value.
  • administering or “administration of’ a drug to a patient (and grammatical equivalents of this phrase) refers to direct administration, which may be administration to a patient by a medical professional or may be self-administration, and/or indirect administration, which may be the act of prescribing a drug, e.g., a physician who instructs a patient to self- administer a drug or provides a patient with a prescription for a drug is administering the drug to the patient. It is understood that the therapeutic agents mentioned herein, such as the TLR inhibitor, are administered in a therapeutically effective amount.
  • An “autoantibody” is an antibody directed to a self-antigen.
  • Biomarker generally refers to biological molecules, and quantitative and qualitative measurements of the same, that are indicative of a disease state. “Prognostic biomarkers” correlate with disease outcome, independent of therapy. For example, tumor hypoxia is a negative prognostic marker - the higher the tumor hypoxia, the higher the likelihood that the outcome of the disease will be negative. “Predictive biomarkers” indicate whether a patient is likely to respond positively to a particular therapy, e.g., HER2 profiling is commonly used in breast cancer patients to determine if those patients are likely to respond to Herceptin (trastuzumab, Genentech). “Response biomarkers” provide a measure of the response to a therapy and so provide an indication of whether a therapy is working.
  • decreasing levels of prostate-specific antigen generally indicate that anti-cancer therapy for a prostate cancer patient is working.
  • the marker can be measured before and/or during treatment, and the values obtained are used by a clinician in assessing any of the following: (a) probable or likely suitability of an individual to initially receive treatment(s); (b) probable or likely unsuitability of an individual to initially receive treatment(s); (c) responsiveness to treatment; (d) probable or likely suitability of an individual to continue to receive treatment(s); (e) probable or likely unsuitability of an individual to continue to receive treatment(s); (f) adjusting dosage; (g) predicting likelihood of clinical benefits; or (h) toxicity.
  • Combination treatment or “in combination with” as used herein denotes any form of concurrent, parallel, simultaneous, sequential or intermittent treatment with at least two distinct treatment modalities (i.e., compounds, components, targeted agents, therapeutic agents or therapies). As such, the terms refer to administration of one treatment modality before, during, or after administration of the other treatment modality to the subject.
  • the modalities in combination can be administered in any order.
  • the therapeutically active modalities are administered together (e.g., simultaneously in the same or separate compositions, formulations or dosage forms) or separately (e.g., on the same day or on different days and in any order as according to an appropriate dosing protocol for the separate compositions, formulations or dosage forms) in a manner and dosing regimen prescribed by a medical caretaker or according to a regulatory agency.
  • each treatment modality will be administered at a dose and/or on a time schedule determined for that treatment modality.
  • four or more modalities may be used in a combination treatment.
  • the combination treatments provided herein may be used in conjunction with other types of treatment.
  • other anti-cancer treatment may be selected from the group consisting of chemotherapy, surgery, radiotherapy (radiation) and/or hormone therapy, amongst other treatments associated with the current standard of care for the subject.
  • compositions and methods include the recited elements, but not excluding others.
  • Consisting essentially of when used to define compositions and methods, shall mean excluding other elements of any essential significance to the composition or method.
  • Consisting of shall mean excluding more than trace elements of other ingredients for claimed compositions and substantial method steps. Embodiments defined by each of these transition terms are within the scope of this invention. Accordingly, it is intended that the methods and compositions can include additional steps and components (comprising) or alternatively including steps and compositions of no significance (consisting essentially of) or alternatively, intending only the stated method steps or compositions (consisting of).
  • Dose and “dosage” refer to a specific amount of active or therapeutic agents for administration. Such amounts are included in a “dosage form,” which refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active agent calculated to produce the desired onset, tolerability, and therapeutic effects, in association with one or more suitable pharmaceutical excipients such as carriers.
  • an “expression level” of a biomarker refers to the mRNA or protein expression level in case of a protein biomarker and the RNA expression level in the case of an RNA biomarker.
  • the “expression level” of a protein biomarker refers to the protein expression level.
  • Methods for measuring mRNA and protein levels are well known in the art. For instance, mRNA expression levels may be determined using Northern blotting, quantitative polymerase chain reaction (qPCR) or microarray and protein expression levels may be determined using ELISA, Western blotting and mass spectrometry.
  • IFN-I signature refers to one or more genes whose expression is modulated by IFN-I and whose expression pattern is reflective of IFN-I activity.
  • IFN-I signature score refers to the arithmetic mean of the normalized expression levels of the genes in an IFN-I signature.
  • a patient refers to a mammal in need of treatment for a disease or disorder.
  • the “patient”, “subject” or “individual” is a human diagnosed or at risk for suffering from one or more symptoms of a disease or disorder.
  • a “patient”, “subject” or “individual” may refer to a non-human mammal, such as a non-human primate, a dog, cat, rabbit, pig, mouse, or rat, or animals used, e.g., in screening, characterizing, and evaluating drugs and therapies.
  • “Pharmaceutically acceptable” indicates that the substance or composition must be compatible chemically and/or toxicologically, with the other ingredients comprising a formulation, and/or the mammal being treated therewith.
  • “Pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Examples of pharmaceutically acceptable carriers include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof.
  • a “prediction” as referred to herein of, e.g., the therapeutic effectiveness of a TLR inhibitor, the suitability of an individual with a disease to initiate treatment with a TLR inhibitor or the suitability of an individual with a disease who is receiving treatment with a TLR inhibitor to continue the treatment only provides an indication about the likelihood of a treatment outcome with a TLR inhibitor but does not predict the treatment outcome with certainty. For instance, an individual for which the TLR inhibitor is predicted to be therapeutically effective just has a higher likelihood of therapeutic effectiveness, whereas an individual for which the TLR inhibitor is predicted not to be therapeutically effective just has a lower likelihood of therapeutic effectiveness.
  • an individual that is predicted to be suitable to initiate treatment with a TLR inhibitor just has a higher likelihood that the treatment in such individual with a TLR inhibitor will be therapeutically effective, whereas an individual that is predicted not to be suitable to initiate treatment with a TLR inhibitor just has a lower likelihood that the treatment in such individual with a TLR inhibitor will be therapeutically effective.
  • an individual that is predicted to be suitable to continue treatment with a TLR inhibitor just has a higher likelihood that the treatment in such individual with a TLR inhibitor will be therapeutically effective, whereas an individual that is predicted not to be suitable to continue treatment with a TLR inhibitor just has a lower likelihood that the treatment in such individual with a TLR inhibitor will be therapeutically effective.
  • An individual defined to “likely benefit from TLR inhibitor treatment” or which is “predicted to benefit from TLR inhibitor treatment” refers to an individual predicted to have a higher likelihood of response of a TLR inhibitor treatment or a higher likelihood of therapeutic efficacy of TLR inhibitor treatment.
  • an individual defined to “unlikely benefit from TLR inhibitor treatment” or which is “predicted not to benefit from TLR inhibitor treatment” refers to an individual predicted to have a lower likelihood of response of a TLR inhibitor treatment or a lower likelihood of therapeutic efficacy of TLR inhibitor treatment.
  • a “reference expression level” of a biomarker allows to distinguish patients that are more likely to respond to treatment with a TLR inhibitor from those that are less likely to respond based on a comparison of the biomarker expression level in the respective patients to the reference expression level. Any expression of a biomarker above a corresponding reference expression level is also referred to as “high” expression of such biomarker and any expression of a biomarker below a corresponding reference expression level is also referred to as “low” expression of such biomarker herein.
  • Biomarkers showing a positive correlation between their expression level and the likelihood to respond to TLR inhibitor treatment indicate a likely positive treatment outcome in a patient if, based on a comparison to the reference expression level, the biomarker expression level in such patient is considered to be high and a likely negative treatment outcome if, based on a comparison to the reference expression level, the biomarker expression level in such patient is considered to be low.
  • Biomarkers showing a negative correlation between their expression level and the likelihood to respond to TLR inhibitor treatment indicate a likely positive treatment outcome in a patient if, based on a comparison to the reference expression level, the biomarker expression level in such patient is considered to be low and a likely negative treatment outcome if, based on a comparison to the reference expression level, the biomarker expression level in such patient is considered to be high.
  • the person of skill in the art is well aware of how to select a suitable reference expression level for a biomarker in order to distinguish between the patients that are more and less likely to respond to TLR inhibitor treatment. Which expression level indicates which treatment outcome depends on how the corresponding reference expression level is defined.
  • biomarker expression level of the different patients before treatment is compared to the treatment outcome of the different patients to then divide the population into patients where treatment was more effective and patients where treatment was less effective and, e.g., define a threshold value for the biomarker expression level that divides these patient populations or define a characteristic biomarker expression level for either or both of these patient populations, e.g., the arithmetic mean or median of the expression levels in the respective group.
  • the so-defined biomarker expression level may then serve as as the reference expression level.
  • the biomarker expression level of the different individuals in such healthy population can be determined and the expression level that is, e.g., around the upper limit of the determined range or corresponding to the arithmetic mean or median may then be defined as the reference expression level.
  • the reference expression level may also be defined as the level of expression in a certain subpopulation of a reference group.
  • the range of expression levels of a biomarker can be determined in a reference group and, e.g., the upper 25% percent of this range may be defined as the reference expression level. Patients whose biomarker expression level falls within this upper 25% range may then be determined to be more likely to respond to treatment with a TLR inhibitor than patients falling outside of it.
  • a reference expression level may be derived from an individual with or without a disease.
  • sample as referred to herein is any biological sample from an individual that allows the determination of the biomarker expression level.
  • the sample may, for instance, refer to fluids, cells and tissues of an individual. It may refer to a blood sample and extracted RNA of an individual.
  • a “small molecule” is a chemical, usually organic, compound with low molecular weight, such as ⁇ 1000 daltons or ⁇ 900 daltons.
  • “Therapeutically effective amount” of a therapeutic agent refers to an amount effective, at dosages and for periods of time necessary, that, when administered to a patient, will have the intended therapeutic effect, e.g., alleviation, amelioration, palliation, or elimination of one or more manifestations of the disease or disorder in the patient, or any other clinical result in the course of treating the patient.
  • a therapeutic effect does not necessarily occur by administration of one dose, and may occur only after administration of a series of doses.
  • a therapeutically effective amount may be administered in one or more administrations.
  • Such therapeutically effective amount may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of a therapeutic agent to elicit a desired response in the individual.
  • a therapeutically effective amount is also one in which any toxic or detrimental effects of a therapeutic agent are outweighed by the therapeutically beneficial effects.
  • “Therapeutic effectiveness” refers to a favorable treatment response, e.g., alleviation, amelioration of one or more symptoms of a disease; diminishment of extent of disease; delay or slowing of disease progression; amelioration, palliation, or stabilization of the disease state; or other beneficial results.
  • therapeutic effectiveness may refer to the time to recovery from a certain disease, e.g., COVID-19.
  • TLR inhibitor refers to a compound that inhibits the activity of one or more members of the human TLR family of proteins through direct interaction between the TLR inhibitor and the TLR.
  • the TLR inhibitor may function, for instance, by stabilizing the TLR in its resting, inactive state.
  • the TLR inhibitor inhibits the activity of human TLR7 (also referred to as a “TLR7 inhibitor”).
  • TLR7 inhibitor also referred to as a “TLR7 inhibitor”.
  • TLR8 inhibitor also referred to as a “TLR8 inhibitor”.
  • the TLR inhibitor inhibits the activity of human TLR7 and/or TLR8 (also referred to as a “TLR7 and/or TLR8 inhibitor”).
  • the TLR inhibitor inhibits the activity of human TLR7 and TLR8 (also referred to as a “TLR7 and TLR8 inhibitor”). In some embodiments, the TLR inhibitor selectively inhibits human TLR7 and/or TLR8.
  • the TLR inhibitor may, for example, be a small molecule, a nucleic acid, such as an oligonucleotide, or a polypeptide, such as an antibody. In some embodiments, the TLR inhibitor is a small molecule. Possible effects of the inhibition of the TLR pathway include the suppression of inflammatory processes. Inhibition in this context need not be complete or 100%. Instead, inhibition means reducing, decreasing or abrogating the activity of the TLR pathway or inflammatory processes, respectively.
  • the IC50 value of the TLR inhibitor for the inhibition of a TLR is below 10 pM, below 2 pM, below 1 pM, below 250 nM, below 100 nM, below 50 nM or below 25 nM.
  • the IC50 value is determined in HEK293 cells.
  • HEK293 cells are stably transfected with either TLR7 or TLR8 and an NF-kappaB-luciferase reporter gene.
  • TLR inhibitor For testing the TLR inhibitor, cells are seeded in 384- well black, clear bottom plates and after an overnight incubation at 37°C and 5% CO 2 , TLR inhibtor dilutions are added in duplicate. The cells are then stimulated with 10 pM R848 or 30 pM R848 for testing in HEK TLR7 or HEK TLR8 cells respectively. Following incubation for 5 hours at 37°C and 5% CO 2 , luciferase substrate, such as SteadyGlo substrate reagent (Promega, Madison, WI), is added to each well and luminescence is measured, e.g., using the Perkin Elmer Envision Multilabel Reader.
  • SteadyGlo substrate reagent Promega, Madison, WI
  • Treating” or “treatment of’ a condition or patient refers to taking steps to obtain beneficial or desired results, including clinical results.
  • beneficial or desired clinical results include, but are not limited to, alleviation, amelioration of one or more symptoms of a disease; diminishment of extent of disease; delay or slowing of disease progression; amelioration, palliation, or stabilization of the disease state; or other beneficial results.
  • references to “treating” or “treatment” include prophylaxis as well as the alleviation of established symptoms of a condition.
  • Treating” or “treatment” of a state, disorder or condition therefore includes: (1) preventing or delaying the appearance of clinical symptoms of the state, disorder or condition developing in a subject that may be afflicted with or predisposed to the state, disorder or condition but does not yet experience or display clinical or subclinical symptoms of the state, disorder or condition, (2) inhibiting the state, disorder or condition, i.e., arresting, reducing or delaying the development of the disease or a relapse thereof (in case of maintenance treatment) or at least one clinical or subclinical symptom thereof, or (3) relieving or attenuating the disease, i.e., causing regression of the state, disorder or condition or at least one of its clinical or subclinical symptoms.
  • Chemical compound definitions [0046] For purposes of this invention, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75 th Ed. Additionally, general principles of organic chemistry are described in “Organic Chemistry”, Thomas Sorrell, University Science Books, Sausalito: 1999, and “March’s Advanced Organic Chemistry”, 5 th Ed., Ed.: Smith, M.B. and March, J., John Wiley & Sons, New York: 2001.
  • aliphatic or “aliphatic group”, as used herein, means a straight-chain (i.e., unbranched) or branched, substituted or unsubstituted hydrocarbon chain that is completely saturated or that contains one or more units of unsaturation, or a monocyclic hydrocarbon or bicyclic hydrocarbon that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic (also referred to herein as “carbocycle” “cycloaliphatic” or “cycloalkyl”), that has a single point of attachment to the rest of the molecule.
  • aliphatic groups contain 1-6 aliphatic carbon atoms.
  • aliphatic groups contain 1-5 aliphatic carbon atoms. In other embodiments, aliphatic groups contain 1-4 aliphatic carbon atoms. In still other embodiments, aliphatic groups contain 1-3 aliphatic carbon atoms, and in yet other embodiments, aliphatic groups contain 1-2 aliphatic carbon atoms.
  • “cycloaliphatic” (or “carbocycle” or “cycloalkyl”) refers to a monocyclic C3-C6 hydrocarbon that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic, that has a single point of attachment to the rest of the molecule.
  • Exemplary aliphatic groups are linear or branched, substituted or unsubstituted Ci-Cs alkyl, C2-C8 alkenyl, C2-C8 alkynyl groups and hybrids thereof such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl or (cycloalkyl)alkenyl.
  • heteroatom means one or more of oxygen, sulfur, nitrogen, or phosphorus (including, any oxidized form of nitrogen, sulfur, or phosphorus; the quaternized form of any basic nitrogen or; a substitutable nitrogen of a heterocyclic ring, for example N (as in 3,4-dihydro-2/7- pyrrolyl), NH (as in pyrrolidinyl) or NR + (as in N-substituted pyrrolidinyl)).
  • alkylene refers to a bivalent alkyl group.
  • An “alkylene chain” is a polymethylene group, i.e., -(CH 2 ) n - wherein n is a positive integer, preferably from 1 to 6, from 1 to 4, from 1 to 3, from 1 to 2, or from 2 to 3.
  • a substituted alkylene chain is a polymethylene group in which one or more methylene hydrogen atoms are replaced with a substituent. Suitable substituents include those described below for a substituted aliphatic group.
  • alkenylene refers to a bivalent alkenyl group.
  • a substituted alkenylene chain is a polymethylene group containing at least one double bond in which one or more hydrogen atoms are replaced with a substituent. Suitable substituents include those described below for a substituted aliphatic group.
  • halogen means F, Cl, Br, or I.
  • aryl used alone or as part of a larger moiety as in “aralkyl”, “aralkoxy”, or
  • aryloxyalkyl refers to monocyclic and bicyclic ring systems having a total of five to fourteen ring members, wherein at least one ring in the system is aromatic and wherein each ring in the system contains three to seven ring members.
  • aryl is used interchangeably with the term “aryl ring”.
  • aryl refers to an aromatic ring system.
  • Exemplary aryl groups are phenyl, biphenyl, naphthyl, anthracyl and the like, which optionally includes one or more substituents.
  • aryl is a group in which an aromatic ring is fused to one or more non-aromatic rings, such as indanyl, phthalimidyl, naphthimidyl, phenanthridinyl, or tetrahydronaphthyl, and the like.
  • heteroatom refers to nitrogen, oxygen, or sulfur, and includes any oxidized form of nitrogen or sulfur, and any quaternized form of a basic nitrogen.
  • heteroaryl and “heteroar-”, as used herein, also include groups in which a heteroaromatic ring is fused to one or more aryl, cycloaliphatic, or heterocyclyl rings, where the radical or point of attachment is on the heteroaromatic ring.
  • Nonlimiting examples include indolyl, isoindolyl, benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzthiazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4/7 quinolizinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and pyrido[2,3-b]-l,4-oxazin-3(4H)-one.
  • heteroaryl group is optionally mono- or bicyclic.
  • heteroaryl is used interchangeably with the terms “heteroaryl ring”, “heteroaryl group”, or “heteroaromatic”, any of which terms include rings that are optionally substituted.
  • heteroarylkyl refers to an alkyl group substituted by a heteroaryl, wherein the alkyl and heteroaryl portions independently are optionally substituted.
  • heterocycle As used herein, the terms “heterocycle”, “heterocyclyl”, “heterocyclic radical”, and “heterocyclic ring” are used interchangeably and refer to a stable 5- to 7-membered monocyclic or 7-10-membered bicyclic heterocyclic moiety that is either saturated or partially unsaturated, and having, in addition to carbon atoms, one or more, preferably one to four, heteroatoms, as defined above.
  • nitrogen includes a substituted nitrogen.
  • the nitrogen is N (as in 3,4-dihydro- 2H- pyrrolyl), NH (as in pyrrolidinyl), or + NR (as in N- substituted pyrrolidinyl).
  • a heterocyclic ring can be attached to its pendant group at any heteroatom or carbon atom that results in a stable structure and any of the ring atoms can be optionally substituted.
  • saturated or partially unsaturated heterocyclic radicals include, without limitation, tetrahydrofuranyl, tetrahydrothiophenyl pyrrolidinyl, piperidinyl, pyrrolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, oxazolidinyl, piperazinyl, dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl, morpholinyl, and quinuclidinyl.
  • heterocycle refers to an alkyl group substituted by a heterocyclyl, wherein the alkyl and heterocyclyl portions independently are optionally substituted.
  • partially unsaturated refers to a ring moiety that includes at least one double or triple bond.
  • partially unsaturated is intended to encompass rings having multiple sites of unsaturation, but is not intended to include aryl or heteroaryl moieties, as herein defined.
  • substituents contain “optionally substituted” moieties.
  • substituted whether preceded by the term “optionally” or not, means that one or more hydrogens of the designated moiety are replaced with a suitable substituent. “Substituted” applies to one or more hydrogens that are either explicit or implicit from the structure (e.g., Unless otherwise indicated, an “optionally substituted” group has a suitable substituent at each substitutable position of the group, and when more than one position in any given structure is substituted with more than one substituent selected from a specified group, the substituent is either the same or different at every position. Combinations of substituents envisioned by this invention are preferably those that result in the formation of stable or chemically feasible compounds.
  • stable refers to compounds that are not substantially altered when subjected to conditions to allow for their production, detection, and, in certain embodiments, their recovery, purification, and use for one or more of the purposes disclosed herein.
  • Suitable monovalent substituents on R° are independently deuterium, halogen, - (CH 2 ) 0-2 R ⁇ , -(haloR ⁇ ), -(CH 2 ) 0-2 OH, -(CH 2 ) 0-2 OR ⁇ , -(CH 2 ) 0-2 CH(OR ⁇ ) 2 ; -O(haloR ⁇ ), -CN, -N 3 , -(CH 2 ) 0-2 C(O)R ⁇ , -(CH 2 ) 0-2 C(O)OH, -(CH 2 ) 0-2 C(O)OR ⁇ , -(CH 2 ) 0-2 SR ⁇ , -(CH 2 )O 2SH, -(CH 2 )O 2 NH 2 , -(CH 2 ) O 2 NHR ⁇ , -
  • Suitable divalent substituents that are bound to vicinal substitutable carbons of an “optionally substituted” group include: -O(CR * 2 ) 2 -3O-, wherein each independent occurrence of R * is selected from hydrogen, C 1-6 aliphatic which is optionally substituted as defined below, or an unsubstituted 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.
  • Suitable substituents on a substitutable nitrogen of an “optionally substituted” group include wherein each R : is independently hydrogen, C 1-6 aliphatic which is optionally substituted as defined below, unsubstituted -OPh, or an unsubstituted 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or, notwithstanding the definition above, two independent occurrences of taken together with their intervening atom(s) form an unsubstituted 3-12-membered saturated, partially unsaturated, or aryl mono- or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.
  • Suitable substituents on the aliphatic group of are independently halogen, - R ⁇ , -(haloR ⁇ ), -OH, -OR ⁇ , -O(haloR ⁇ ), -CN, -C(O)OH, -C(O)OR ⁇ , -NH 2 , -NHR ⁇ , -NR ⁇ 2 , or -NO 2 , wherein each R ⁇ is unsubstituted or where preceded by “halo” is substituted only with one or more halogens, and is independently C 1-4 aliphatic, -CH 2 Ph, -0(CH 2 ) 0 -1 Ph, or a 5-6- membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.
  • the terms “optionally substituted”, “optionally substituted alkyl,” “optionally substituted “optionally substituted alkenyl,” “optionally substituted alkynyl”, “optionally substituted carbocyclic,” “optionally substituted aryl”, “ optionally substituted heteroaryl,” “optionally substituted heterocyclic,” and any other optionally substituted group as used herein, refer to groups that are substituted or unsubstituted by independent replacement of one, two, or three or more of the hydrogen atoms thereon with typical substituents including, but not limited to:
  • -NH 2 protected amino, -NH alkyl, -NH alkenyl, -NH alkynyl, -NH cycloalkyl, -NH -aryl, -NH -heteroaryl, -NH -heterocyclic, -dialkylamino, -diarylamino, -diheteroarylamino,
  • -C(O)- alkyl -C(O)- alkenyl, -C(O)- alkynyl, -C(O)- carbocyclyl, -C(O)-aryl, -C(O)- heteroaryl, -C(O)-heterocyclyl, -CONH 2 , -CONH- alkyl, -CONH- alkenyl, -CONH- alkynyl, -CONH-carbocyclyl, - CONH-aryl, -CONH-heteroaryl, -CONH-heterocyclyl,
  • -OCO 2 - alkyl -OCO 2 - alkenyl, -OCO 2 - alkynyl, -OCO 2 - carbocyclyl, -OCO 2 -aryl, -OCO 2 - heteroaryl, -OCO 2 -heterocyclyl, -OCONH 2 , -OCONH- alkyl, -OCONH- alkenyl, -OCONH- alkynyl, -OCONH- carbocyclyl, -OCONH- aryl, -OCONH- heteroaryl, -OCONH- heterocyclyl,
  • -S(O)- alkyl - S(O)- alkenyl, - S(O)- alkynyl, - S(O)- carbocyclyl, - S(O)-aryl, - S(O)- heteroaryl, - S(O)-heterocyclyl -SO 2 NH 2 , -SO 2 NH- alkyl, -SO 2 NH- alkenyl, -SO 2 NH- alkynyl, - SO 2 NH- carbocyclyl, -SO 2 NH- aryl, -SO 2 NH- heteroaryl, -SO 2 NH- heterocyclyl,
  • -mono-, di-, or tri-alkyl silyl -alkyl, -alkenyl, -alkynyl, -aryl, -arylalkyl, -heteroaryl, -heteroarylalkyl, -heterocycloalkyl, -cycloalkyl, -carbocyclic, -heterocyclic, polyalkoxyalkyl, polyalkoxy, -methoxymethoxy, - methoxyethoxy, -SH, -S- alkyl, -S- alkenyl, -S- alkynyl, -S- carbocyclyl, -S-aryl, -S-heteroaryl, - S-heterocyclyl, or methylthiomethyl.
  • the term “pharmaceutically acceptable salt” refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio.
  • Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge et al., describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1-19.
  • Pharmaceutically acceptable salts of the compounds described herein include those derived from suitable inorganic and organic acids and bases.
  • Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art such as ion exchange.
  • inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid
  • organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art such as ion exchange.
  • salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2- hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate
  • Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and N + (C 1-4 alkyl) 4 salts.
  • Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like.
  • Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, loweralkyl sulfonate and aryl sulfonate.
  • compounds referred to herein are also meant to include all isomeric (e.g., enantiomeric, diastereomeric, and geometric (or conformational)) forms of the structure; for example, the R and S configurations for each asymmetric center, Z and E double bond isomers, and Z and E conformational isomers. Therefore, single stereochemical isomers as well as enantiomeric, diastereomeric, and geometric (or conformational) mixtures of the present compounds are within the scope of the invention. Unless otherwise stated, all tautomeric forms of the compounds described herein are within the scope of the invention.
  • compounds referred to herein are also meant to include compounds that differ only in the presence of one or more isotopically enriched atoms.
  • compounds having the present structures including the replacement of hydrogen by deuterium or tritium, or the replacement of a carbon by a 13 C- or 14 C-enriched carbon are within the scope of this invention.
  • the group comprises one or more deuterium atoms.
  • a compound which has multiple potential sites of attack for oxidative metabolism for example benzylic hydrogen atoms and hydrogen atoms bonded to a nitrogen atom, is prepared as a series of analogues in which various combinations of hydrogen atoms are replaced by deuterium atoms, so that some, most or all of these hydrogen atoms have been replaced by deuterium atoms.
  • Half-life determinations enable favorable and accurate determination of the extent of the extent to which the improvement in resistance to oxidative metabolism has improved. In this way, it is determined that the half-life of the parent compound can be extended by up to 100% as the result of deuterium-hydrogen exchange of this type.
  • Deuterium-hydrogen exchange in a compound can also be used to achieve a favorable modification of the metabolite spectrum of the starting compound in order to diminish or eliminate undesired toxic metabolites. For example, if a toxic metabolite arises through oxidative carbon- hydrogen (C-H) bond cleavage, it can reasonably be assumed that the deuterated analogue will greatly diminish or eliminate production of the unwanted metabolite, even if the particular oxidation is not a rate-determining step. Further information on the state of the art with respect to deuterium-hydrogen exchange may be found, for example in Hanzlik et al, J. Org. Chem. 55, 3992-3997, 1990, Reider et al. J. Org.
  • the biomarkers disclosed herein have predictive value for TLR inhibitor treatment.
  • Such biomarker information may be used, for instance, for predicting the therapeutic effectiveness of a TLR inhibitor, for predicting the suitability of an individual with a disease to initiate treatment with a TLR inhibitor, for predicting the suitability of an individual with a disease who is receiving treatment with a TLR inhibitor to continue the treatment or for selecting an individual with a disease for treatment with a TLR inhibitor.
  • a TLR inhibitor for predicting therapeutic effectiveness of a TLR inhibitor in an individual with a disease, for predicting the suitability of an individual with a disease to initiate treatment with a TLR inhibitor, for predicting the suitability of a patient to continue treatment with a TLR inhibitor and/or for treating indvi duals with a disease with a TLR inhibitor.
  • the methods and uses disclosed herein comprise the step of determining the expression level of one or more biomarkers.
  • the method for predicting therapeutic effectiveness of a TLR inhibitor in an individual with a disease for predicting the suitability of an individual with a disease to initiate treatment with a TLR inhibitor or for predicting the suitability of a patient to continue treatment with a TLR inhibitor are practised outside the human or animal body.
  • the expression level of the one or more biomarkers may be determined in a sample obtained from the individual.
  • the sample is a blood sample or a tissue sample.
  • the disclosure provides the use of one or more biomarkers described herein as a predictive biomarker for the treatment of an individual with a disease with a TLR inhibitor.
  • the disclosure provides a method for predicting therapeutic effectiveness of a TLR inhibitor in an individual with a disease comprising determining the expression level of one or more biomarkers described herein in the individual, wherein the biomarker expression level indicates the therapeutic effectiveness of the TLR inhibitor in the individual.
  • the disclosure provides a method for predicting the suitability of an individual with a disease to initiate treatment with a TLR inhibitor comprising determining the expression level of one or more biomarkers described herein in the individual, wherein the biomarker expression level indicates the suitability of the individual to initiate the treatment.
  • the disclosure provides a method for predicting the suitability of an individual with a disease who is receiving treatment with a TLR inhibitor to continue the treatment comprising determining the expression level of one or more biomarkers described herein in the individual, wherein the biomarker expression level indicates the suitability of the individual to continue the treatment.
  • the disclosure provides a TLR inhibitor for use in a method of treating a disease in an individual comprising administering the TLR inhibitor to an individual that is likely to respond to such treatment as reflected by the expression level of one or more biomarkers described herein.
  • these methods and uses comprise the steps of determining the expression level of one or more biomarkers and comparing each determined expression level to a reference expression level.
  • the disclosure provides a method for predicting therapeutic effectiveness of a TLR inhibitor in an individual with a disease comprising determining the expression level of one or more biomarkers described herein in the individual, comparing each determined expression level to a reference expression level and predicting therapeutic effectiveness of the TLR inhibitor on the basis of this comparison.
  • the disclosure provides a method for predicting the suitability of an individual with a disease to initiate treatment with a TLR inhibitor comprising determining the expression level of one or more biomarkers described herein in the individual, comparing each determined expression level to a reference expression level and predicting the suitability of the individual to initiate the treatment on this basis.
  • the disclosure provides a method for predicting the suitability of an individual with a disease who is receiving treatment with a TLR inhibitor to continue the treatment comprising determining the expression level of one or more biomarkers described herein in the individual, comparing each determined expression level to a reference expression level and predicting the suitability of the individual to continue the treatment on this basis.
  • the disclosure provides a TLR inhibitor for use in a method of treating a disease in an individual comprising administering the TLR inhibitor to an individual that is likely to respond to such treatment as reflected by a comparison of the expression level of one or more biomarkers described herein in the individual to corresponding reference expression levels.
  • the step of comparing the determined expression level of a biomarker to a reference expression level may serve to determine if the individual is likely to benefit from the treatment with a TLR inhibitor or not.
  • these methods and uses comprise the steps of determining the expression level of a biomarker in an individual with a disease, comparing the determined expression level to a reference expression level and determining that the individual has a higher likelihood of therapeutic efficacy of a TLR inhibitor, and thus is predicted to benefit from TLR7 inhibitor treatment, if the expression of the biomarker is above the reference expression level and a lower likelihood of therapeutic efficacy of a TLR inhibitor, and thus is predicted not to benefit from TLR7 inhibitor treatment, if the expression of the biomarker is below the reference expression level.
  • these methods and uses comprise the steps of determining the expression level of a biomarker in an individual with a disease, comparing the determined expression level to a reference expression level and determining that the individual has a higher likelihood of therapeutic efficacy of a TLR inhibitor if the expression of the biomarker is below the reference expression level and a lower likelihood of therapeutic efficacy of a TLR inhibitor if the expression of the biomarker is above the reference expression level.
  • a determination of therapeutic efficacy of a TLR inhibitor then also translates into the suitability of an individual to initiate or continue treatment with a TLR inhibitor. For instance, an individual that is determined to have a higher likelihood of therapeutic efficacy of the TLR inhibitor, i.e. predicted to respond to TLR inhibitor treatment, would also be predicted to be suitable to initiate or continue treatment with the TLR inhibitor.
  • the reference expression level for a biomarker is derived from a second individual with or without the disease or from a population of individuals with or without the disease.
  • the reference expression level of a biomarker is determined retrospectively based on the biomarker expression levels of the patients in a patient population before treatment with the TLR inhibitor and the respective treatment outcome. The patient population may then be divided into two groups, one that showed a certain treatment efficacy and one that did not. In some embodiments, the expression level in between these two groups is defined as the reference expression level. In other embodiments, the expression level that is characteristic of one of the two groups is defined as the reference expression level.
  • the reference expression level is defined independently of a treatment outcome.
  • the reference expression level may be defined around the upper limit of the expression level range observed in this population or to correspond to the arithmetic mean or median of such range.
  • the biomarker expression level of a healthy individual may be defined as the reference expression level.
  • the reference expression level of a biomarker is defined as follows:
  • the reference expression level is defined as follows:
  • the reference expression level is defined as follows:
  • the expression level of the one or more biomarkers indicates the suitability of the individual to initiate the treatment.
  • a patient that is determined to have a higher likelihood of therapeutic efficacy of a TLR inhibitor or to benefit from treatment with a TLR inhibitor is predicted to be suitable to initiate treatment with a TLR inhibitor, whereas a patient that is determined to have a lower likelihood of therapeutic efficacy of a TLR inhibitor or to benefit from treatment with a TLR inhibitor is predicted to be unsuitable to initiate treatment with a TLR inhibitor.
  • the expression level of the one or more biomarkers indicates the suitability of the individual to continue the treatment.
  • a patient that is determined to have a higher likelihood of therapeutic efficacy of a TLR inhibitor or to benefit from treatment with a TLR inhibitor is predicted to be suitable to continue treatment with the TLR inhibitor, whereas a patient that is determined to have a lower likelihood of therapeutic efficacy of a TLR inhibitor or to benefit from treatment with a TLR inhibitor is predicted to be unsuitable to continue treatment with the TLR inhibitor.
  • the inididual is selected based on the expression level of the one or more biomarkers.
  • a patient that is determined to have a higher likelihood of therapeutic efficacy of a TLR inhibitor or to benefit from treatment with a TLR inhibitor is selected for treatment with a TLR inhibitor, whereas a patient that is determined to have a lower likelihood of therapeutic efficacy of a TLR inhibitor or to benefit from treatment with a TLR inhibitor is not selected for treatment with a TLR inhibitor.
  • the methods disclosed herein may also comprise the step of administering a TLR inhibitor to an indidividual that is likely to benefit from such treatment.
  • a TLR inhibitor for use in a method of treating a disease in an individual having high expression of one or more biomarkers, which method comprises administering the TLR inhibitor to the individual. Also provided herein is a TLR inhibitor for use in a method of treating a disease in an individual comprising determining the expression of one or more biomarkers in a sample from the individual and administering the TLR inhibitor to the individual if the expression level of the one or more biomarker is higher than the corresponding reference expression level.
  • Diseases for which the autoantibodies and RNAs disclosed herein may serve as a biomarker include any disease that is caused, mediated and/or propagated by TLR activity, such as TLR7 and/or TLR8 activity.
  • the disease is an autoimmune disease.
  • the disease is an idiopathic inflammatory myopathy, such as polymyositis or dermatomyositis, or a lupus disease, such as systemic lupus erythematosus, cutaneous lupus erythematosus or lupus nephritis.
  • the disease is selected from the group consisting of arthritis, pancreatitis, mixed connective tissue disease, lupus, myositis, antiphospholipid syndrome, systemic onset arthritis, and irritable bowel syndrome.
  • the disease is selected from the group consisting of rheumatoid arthritis, autoimmune pancreatitis, systemic lupus erythematosus, cutaneous lupus erythematosus, lupus nephritis, type I diabetes mellitus, multiple sclerosis, antiphospholipid syndrome, sclerosing cholangitis, systemic onset arthritis, irritable bowel disease, scleroderma, Sjogren’s disease, vitiligo, polymyositis, dermatomyositis, pemphigus vulgaris, pemphigus foliaceus, inflammatory bowel disease including Crohn's disease and ulcerative colitis, autoimmune hepatitis, hypopit
  • the disease is selected from the group consisting of polyangiitis overlap syndrome, Kawasaki's disease, sarcoidosis, glomerulonephritis, and cryopathies.
  • the disease is selected from the group consisting of systemic lupus erythematosus, rheumatoid arthritis, autoimmune skin disease, and multiple sclerosis.
  • the disease is selected from the group consisting of pancreatitis, glomerulonephritis, pyelitis, sclerosing cholangitis, and type I diabetes.
  • the disease is diabetes and/or diabetic-related disease or disorder.
  • the disease is an inflammatory disease.
  • the disease is associated with chronic pathogen stimulation.
  • the disease is a viral disease, e.g., resulting from infection with HIV or SARS-CoV-2, such as CO VID-19.
  • the disease is selected from rheumatoid arthritis, psoriatic arthritis, osteoarthritis, systemic lupus erythematosus, lupus nephritis, ankylosing spondylitis, osteoporosis, systemic sclerosis, multiple sclerosis, polymyositis, dermatomyositis, psoriasis, type I diabetes, type II diabetes, inflammatory bowel disease, Crohn’s disease, ulcerative colitis, hyperimmunoglobulinemia D, periodic fever syndrome, cryopyrin-associated periodic syndromes, Schnitzler's syndrome, systemic juvenile idiopathic arthritis, adult-onset Still's disease, gout, pseudogout, SAPHO syndrome, Castleman's disease
  • the biomarker is an autoantibody.
  • the biomarker is an autoantibody having reactivity against a protein selected from the group consisting of TROVE2, SSB, TRIM21, IL36RN, HIST1H4A, ZMYND8, CEP290, CENPJ, ZDHHC16, FARSA, STAT2, HARS, ITGB7, S100A11, KRT8 and IL4R.
  • the biomarker is an autoantibody having reactivity against a protein selected from the group consisting of TROVE2, SSB, TRIM21, IL36RN, HIST1H4A, ZMYND8, CENPJ, FARSA, STAT2, HARS, ITGB7, S100A11, KRT8 and IL4R and patients, e.g., having a lupus disease, such as systemic lupus erythematosus or lupus nephritis, or an idiopathic inflammatory myopathy, such as dermatomyositis or polymyositis, that have a high expression for one or more of such biomarkers are predicted to benefit from treatment with a TER inhibitor, e.g., a TLR7 and/or TLR8 inhibitor, whereas patients having a low expression of these biomarkers are predicted not benefit from such treatment.
  • a TER inhibitor e.g., a TLR7 and/or TLR8 inhibitor
  • the biomarker is an autoantibody having reactivity against a protein selected from CEP290 andZDHHC I 6 and patients, e.g., having a lupus disease, such as systemic lupus erythematosus or lupus nephritis, or an idiopathic inflammatory myopathy, such as dermatomyositis or polymyositis, that have a low expression for one or both of such biomarkers are predicted to benefit from treatment with a TLR inhibitor, e.g., a TLR7 and/or TLR8 inhibitor, whereas patients having a high expression of these biomarkers are predicted not benefit from such treatment.
  • a lupus disease such as systemic lupus erythematosus or lupus nephritis
  • an idiopathic inflammatory myopathy such as dermatomyositis or polymyositis
  • biomarkers that positively correlate with TLR inhibitor treatment outcome include any of the autoantibodies having reactivity against a protein selected from the group consisting of TROVE2, SSB, TRIM21, IL36RN, HIST1H4A, ZMYND8, CENPJ, FARSA, STAT2, HARS, ITGB7, S100A11, KRT8 and IL4R.
  • biomarkers that negatively correlate with TLR inhibitor treatment outcome include any of the autoantibodies having reactivity against a protein selected from the group consisting of CEP290 and ZDHHC16.
  • the biomarker is an autoantibody having reactivity against a protein selected from the group consisting of TROVE2, SSB, TRIM21, IL36RN, HIST1H4A, ZMYND8, CEP290, CENPJ, ZDHHC16 and FARSA and it has predictive value for TLR inhibitor treatment of patients having a lupus disease, such as systemic lupus erythematosus or lupus nephritis.
  • a protein selected from the group consisting of TROVE2, SSB, TRIM21, IL36RN, HIST1H4A, ZMYND8, CEP290, CENPJ, ZDHHC16 and FARSA and it has predictive value for TLR inhibitor treatment of patients having a lupus disease, such as systemic lupus erythematosus or lupus nephritis.
  • the biomarker is an autoantibody having reactivity against a protein selected from the group consisting of TROVE2, SSB, TRIM21, IL36RN, HIST1H4A, ZMYND8, CENPJ and FARSA and patients, e.g., patients having a lupus disease, such as systemic lupus erythematosus or lupus nephritis, that have a high expression for one or more of such biomarkers are predicted to benefit from treatment with a TLR inhibitor, e.g., a TLR7 and/or TLR8 inhibitor, whereas patients having a low expression of these biomarkers are predicted not benefit from such treatment.
  • a TLR inhibitor e.g., a TLR7 and/or TLR8 inhibitor
  • the biomarker is an autoantibody having reactivity against a protein selected from CEP290 and ZDHHC16 and patients, e.g., patients having a lupus disease, such as systemic lupus erythematosus or lupus nephritis, that have a low expression of one or both of such biomarkers are predicted to benefit from treatment with a TLR inhibitor, e.g., a TLR7 and/or TLR8 inhibitor, whereas patients having a high expression of these biomarkers are predicted not benefit from such treatment.
  • a TLR inhibitor e.g., a TLR7 and/or TLR8 inhibitor
  • the biomarker is an autoantibody having reactivity against a protein selected from the group consisting of TRIM21, IL36RN, HIST1H4A, CENPJ, STAT2, HARS, ITGB7, S100A11, KRT8 and IL4R and it has predictive value for TLR inhibitor treatment of patients having an idiopathic inflammatory myopathy, such as dermatomyositis or polymyositis
  • the biomarker is an autoantibody having reactivity against a protein selected from the group consisting of TRIM21, IL36RN, HIST1H4A, CENPJ, STAT2, HARS, ITGB7, S100A11, KRT8 and IL4R and patients, e.g., patients having an idiopathic inflammatory myopathy, such as dermatomyositis or polymyositis, that have a high expression for one or more of such biomarkers are predicted to benefit from treatment with a
  • the biomarker is an autoantibody having reactivity against TROVE2 and it has predictive value for TLR inhibitor treatment of patients having a lupus disease or an idiopathic inflammatory myopathy, such as dermatomyositis, systemic lupus erythematosus or lupus nephritis.
  • the biomarker is an autoantibody having reactivity against TROVE2 and patients having a lupus disease or an idiopathic inflammatory myopathy, such as dermatomyositis, systemic lupus erythematosus or lupus nephritis, that have a high expression of such biomarker are predicted to benefit from treatment with a TLR inhibitor, e.g., a TLR7 and/or TLR8 inhibitor, whereas patients having a low expression of this biomarker are predicted not benefit from such treatment.
  • a TLR inhibitor e.g., a TLR7 and/or TLR8 inhibitor
  • the biomarker is an autoantibody having reactivity against TRIM21 and it has predictive value for TLR inhibitor treatment of patients having a lupus disease or an idiopathic inflammatory myopathy, such as dermatomyositis, polymyositis, systemic lupus erythematosus or lupus nephritis.
  • a lupus disease or an idiopathic inflammatory myopathy such as dermatomyositis, polymyositis, systemic lupus erythematosus or lupus nephritis.
  • the biomarker is an autoantibody having reactivity against TRIM21 and patients having a lupus disease or an idiopathic inflammatory myopathy, such as dermatomyositis, polymyositis, systemic lupus erythematosus or lupus nephritis, that have a high expression of such biomarker are predicted to benefit from treatment with a TLR inhibitor, e.g., a TLR7 and/or TLR8 inhibitor, whereas patients having a low expression of this biomarker are predicted not benefit from such treatment.
  • a TLR inhibitor e.g., a TLR7 and/or TLR8 inhibitor
  • the biomarker is an autoantibody having reactivity against SSB and it has predictive value for TLR inhibitor treatment of patients having a lupus disease or an idiopathic inflammatory myopathy, such as dermatomyositis, polymyositis, systemic lupus erythematosus or lupus nephritis.
  • a lupus disease or an idiopathic inflammatory myopathy such as dermatomyositis, polymyositis, systemic lupus erythematosus or lupus nephritis.
  • the biomarker is an autoantibody having reactivity against SSB and patients having a lupus disease or an idiopathic inflammatory myopathy, such as dermatomyositis, polymyositis, systemic lupus erythematosus or lupus nephritis, that have a high expression of such biomarker are predicted to benefit from treatment with a TLR inhibitor, e.g., a TLR7 and/or TLR8 inhibitor, whereas patients having a low expression of this biomarker are predicted not benefit from such treatment.
  • a TLR inhibitor e.g., a TLR7 and/or TLR8 inhibitor
  • the biomarker is an autoantibody having reactivity against IL36RN and it has predictive value for TLR inhibitor treatment of patients having a lupus disease or an idiopathic inflammatory myopathy, such as dermatomyositis, polymyositis, systemic lupus erythematosus or lupus nephritis.
  • a lupus disease or an idiopathic inflammatory myopathy such as dermatomyositis, polymyositis, systemic lupus erythematosus or lupus nephritis.
  • the biomarker is an autoantibody having reactivity against IL36RN and patients having a lupus disease or an idiopathic inflammatory myopathy, such as dermatomyositis, polymyositis, systemic lupus erythematosus or lupus nephritis, that have a high expression of such biomarker are predicted to benefit from treatment with a TLR inhibitor, e.g., a TLR7 and/or TLR8 inhibitor, whereas patients having a low expression of this biomarker are predicted not benefit from such treatment.
  • a TLR inhibitor e.g., a TLR7 and/or TLR8 inhibitor
  • the biomarker is an autoantibody having reactivity against HIST1H4A and it has predictive value for TLR inhibitor treatment of patients having a lupus disease or an idiopathic inflammatory myopathy, such as dermatomyositis, polymyositis, systemic lupus erythematosus or lupus nephritis.
  • a lupus disease or an idiopathic inflammatory myopathy such as dermatomyositis, polymyositis, systemic lupus erythematosus or lupus nephritis.
  • the biomarker is an autoantibody having reactivity against HIST1H4A and patients having a lupus disease or an idiopathic inflammatory myopathy, such as dermatomyositis, polymyositis, systemic lupus erythematosus or lupus nephritis, that have a high expression of such biomarker are predicted to benefit from treatment with a TLR inhibitor, e.g., a TLR7 and/or TLR8 inhibitor, whereas patients having a low expression of this biomarker are predicted not benefit from such treatment.
  • a TLR inhibitor e.g., a TLR7 and/or TLR8 inhibitor
  • the biomarker is an autoantibody having reactivity against CENPJ and it has predictive value for TLR inhibitor treatment of patients having a lupus disease or an idiopathic inflammatory myopathy, such as dermatomyositis, polymyositis, systemic lupus erythematosus or lupus nephritis.
  • a lupus disease or an idiopathic inflammatory myopathy such as dermatomyositis, polymyositis, systemic lupus erythematosus or lupus nephritis.
  • the biomarker is an autoantibody having reactivity against CENPJ and patients having a lupus disease or an idiopathic inflammatory myopathy, such as dermatomyositis, polymyositis, systemic lupus erythematosus or lupus nephritis, that have a high expression of such biomarker are predicted to benefit from treatment with a TLR inhibitor, e.g., a TLR7 and/or TLR8 inhibitor, whereas patients having a low expression of this biomarker are predicted not benefit from such treatment.
  • a TLR inhibitor e.g., a TLR7 and/or TLR8 inhibitor
  • the biomarker is an autoantibody having reactivity against STAT2 and it has predictive value for TLR inhibitor treatment of patients having a lupus disease or an idiopathic inflammatory myopathy, such as dermatomyositis, polymyositis, systemic lupus erythematosus or lupus nephritis.
  • a lupus disease or an idiopathic inflammatory myopathy such as dermatomyositis, polymyositis, systemic lupus erythematosus or lupus nephritis.
  • the biomarker is an autoantibody having reactivity against STAT2 and patients having a lupus disease or an idiopathic inflammatory myopathy, such as dermatomyositis, polymyositis, systemic lupus erythematosus or lupus nephritis, that have a high expression of such biomarker are predicted to benefit from treatment with a TLR inhibitor, e.g., a TLR7 and/or TLR8 inhibitor, whereas patients having a low expression of this biomarker are predicted not benefit from such treatment.
  • a TLR inhibitor e.g., a TLR7 and/or TLR8 inhibitor
  • the biomarker is an autoantibody having reactivity against TRIM21 or HARS and it has predictive value for TLR inhibitor treatment of patients having dermatomyositis.
  • the biomarker is an autoantibody having reactivity against TRIM21 or HARS and patients having dermatomyositis that have a high expression of one or both of these biomarkers are predicted to benefit from treatment with a a TLR inhibitor, e.g., a TLR7 and/or TLR8 inhibitor, whereas patients having a low expression of one or both of these biomarkers are predicted not benefit from such treatment.
  • a TLR inhibitor e.g., a TLR7 and/or TLR8 inhibitor
  • the biomarker is an autoantibody having reactivity against SAE1 and it has predictive value for TLR inhibitor treatment of patients having a lupus disease or an idiopathic inflammatory myopathy, such as dermatomyositis, polymyositis, systemic lupus erythematosus or lupus nephritis.
  • a lupus disease or an idiopathic inflammatory myopathy such as dermatomyositis, polymyositis, systemic lupus erythematosus or lupus nephritis.
  • the biomarker is an autoantibody having reactivity against SAE1 and patients having dermatomyositis that have a high expression of such biomarker are predicted to benefit from treatment with a TLR inhibitor, e.g., a TLR7 and/or TLR8 inhibitor, whereas patients having a low expression of this biomarker are predicted not benefit from such treatment.
  • a TLR inhibitor e.g., a TLR7 and/or TLR8 inhibitor
  • the biomarker is an RNA.
  • the biomarker is an RNA selected from the group consisting of His-tRNA, Y1, Y4 and Ul. In some embodiments, the biomarker is an RNA selected from the group consisting of His-tRNA, Y1, Y4 and Ul and patients having a lupus disease or an idiopathic inflammatory myopathy, such as dermatomyositis, polymyositis, systemic lupus erythematosus or lupus nephritis, that have a high expression of one or more of such biomarkers are predicted to benefit from treatment with a TLR inhibitor, e.g., a TLR7 and/or TLR8 inhibitor, whereas patients having a low expression of these biomarkers are predicted not benefit from such treatment.
  • a TLR inhibitor e.g., a TLR7 and/or TLR8 inhibitor
  • biomarkers that positively correlate with TLR inhibitor treatment outcome include an RNA selected from the group consisting of His-tRNA, Y1, Y4 and UL
  • the uses and methods of the invention involve the administration of a TLR inhibitor.
  • the TLR inhibitor is a TLR7 and/or TLR8 inhibitor.
  • the TLR inhibitor is a TLR7 and TLR8 inhibitor.
  • the TLR inhibitor is a small molecule, such as a small molecule inhibitor of TLR7 and/or TLR8.
  • the TLR inhibitor is selected from the group consisting of 5- [(3R, 5 S)-3 -amino-5-(trifluoromethyl)piperidin- 1 -yl] quinoline-8-carbonitrile; (3R, 5 S)- 1 -(8- methoxy-1 ,7-naphthyridin-5-yl)-5-methylpiperidin-3-amine; 2- ⁇ 4-[2-(7,8- dimethyl[l,2,4]triazolo[l,5-a]pyridin-6-yl)-3-(propan-2-yl)-lH-indol-5-yl]piperidin-l- yl ⁇ acetamide; rel-(2R,6R)-4-(8-cyanoquinolin-5yl)-N-((3R,4S)-4-fluoropyrrolidin-3-yl)-6- methylmorpholine-2-carboxamide hydrochloride; (S)-N-(
  • the TLR7 and/or TLR8 inhibitor is a quinoline derivative.
  • the TLR7 and/or TLR8 inhibitor is a compound of formula I, or a pharmaceutically acceptable salt thereof, wherein:
  • Ring B is aryl or heteroaryl having 1 -4 heteroatoms independently selected from nitrogen, oxygen, or sulfur; each of which is optionally substituted;
  • R 1 is -Me, -CF 3 , -OMe, -OEt, or -CN; each R 2 IS independently -R, halogen, -haloalkyl, -OR, -SR, -CN, - NO 2 , -SO 2 R, -SOR, -C(O)R, -CO 2 R, -C(O)N(R) 2 , -NRC(O)R, -NRC(O)N(R) 2 , -NRSO 2 R, or -N(R) 2 ; each R 3 IS independently -R, halogen, -haloalkyl, -OR, -SR, -CN, - NO 2 , -SO 2 R, -SOR, -C(O)R, -CO 2 R, -C(O)N(R) 2 , -NRC(O)R, -NRC(O)N(R) 2 , -NRSO 2 R, or
  • X is C(R 4 ) 2 , O, NR 4 , S, S(R 4 ), or S(R 4 ) 2 ; each R 4 is independently -R, halogen, -haloalkyl, -OR, -SR, -CN, - NO 2 , -SO 2 R, -SOR, -C(O)R, -CO 2 R, -C(O)N(R) 2 , -NRC(O)R, -NRC(O)N(R) 2 , -NRSO 2 R, or -N(R) 2 ; each R 5 is independently -R, halogen, -haloalkyl, -OR, -SR, -CN, - NO 2 , -SO 2 R, -SOR, -C(O)R, -CO 2 R, -C(O)N(R) 2 , -NRC(O)R, -NRC(O)N(R) 2 ,
  • Ring A is G> aryl or a 6 membered monocyclic heteroaryl having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur; each of which is optionally substituted.
  • Ring A is phenyl, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, or triazinyl; each of which is optionally substituted.
  • Ring A is phenyl, pyridyl, or pyrimidinyl; each of which is optionally substituted.
  • Ring B is Cr> aryl or a 5-6 membered monocyclic heteroaryl having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur; each of which is optionally substituted.
  • Ring B is phenyl, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazinyl, pyrrole, imidazole, isoxazole, oxazole, or thiazole; each of which is optionally substituted.
  • Ring A and Ring B is
  • Ring A and Ring B is
  • Ring A and Ring B is
  • Ring A and Ring B is
  • Ring A and Ring B is
  • R 1 is -CN.
  • each R 2 is independently C 1-6 aliphatic, C 3-10 aryl, a 3-8 membered saturated or partially unsaturated carbocyclic ring, a 3-7 membered heterocylic ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or a 5-6 membered monocyclic heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur; each of which is optionally substituted.
  • each R 2 is independently methyl, ethyl, ethyl, propyl, i- propyl, butyl, s-butyl, t-butyl, straight or branched pentyl, or straight or branched hexyl; each of which is optionally substituted.
  • each R 2 is independently phenyl, naphthyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, adamantyl, cyclooctyl, [3.3.0]bicyclooctanyl, [4.3.0]bicyclononanyl, [4.4.0]bicyclodecanyl, [2.2.2]bicyclooctanyl, fluorenyl, indanyl, tetrahydronaphthyl, acridinyl, azocinyl, benzimidazolyl, benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl, benzthiazolyl, benztriazolyl, benztetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazolinyl
  • each R 2 is independently halogen, -haloalkyl, -OR, -SR, - CN, -NO 2 , -SO 2 R, -SOR, -C(O)R, -CO 2 R, -C(O)N(R) 2 , -NRC(O)R, -NRC(O)N(R) 2 , -NRSO 2 R, or -N(R) 2 .
  • each R 2 is independently -F.
  • each R 3 is independently C 1-6 aliphatic, C 3-10 aryl, a 3-8 membered saturated or partially unsaturated carbocyclic ring, a 3-7 membered heterocylic ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or a 5-6 membered monocyclic heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur; each of which is optionally substituted.
  • each R 3 is independently methyl, ethyl, ethyl, propyl, i- propyl, butyl, s-butyl, t-butyl, straight or branched pentyl, or straight or branched hexyl; each of which is optionally substituted.
  • each R 3 is independently methyl.
  • each R 3 is independently phenyl, naphthyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, adamantyl, cyclooctyl, [3.3.0]bicyclooctanyl, [4.3.0]bicyclononanyl, [4.4.0]bicyclodecanyl, [2.2.2]bicyclooctanyl, fluorenyl, indanyl, tetrahydronaphthyl, acridinyl, azocinyl, benzimidazolyl, benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl, benzthiazolyl, benztriazolyl, benztetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazolinyl
  • each R 3 is independently halogen, -haloalkyl, -OR, -SR, - CN, -NO 2 , -SO 2 R, -SOR, -C(O)R, -CO 2 R, -C(O)N(R) 2 , -NRC(O)R, -NRC(O)N(R) 2 , -NRSO 2 R, or -N(R) 2 .
  • each R 3 is independently -F.
  • X is C(R 4 ) 2 . In certain embodiments, X is CH 2 .
  • each R 4 is independently C 1-6 aliphatic, C 3-10 aryl, a 3-8 membered saturated or partially unsaturated carbocyclic ring, a 3-7 membered heterocylic ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or a 5-6 membered monocyclic heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur; each of which is optionally substituted.
  • each R 4 is independently methyl, ethyl, ethyl, propyl, i- propyl, butyl, s-butyl, t-butyl, straight or branched pentyl, or straight or branched hexyl; each of which is optionally substituted.
  • each R 4 is independently phenyl, naphthyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, adamantyl, cyclooctyl, [3.3.0]bicyclooctanyl, [4.3.0]bicyclononanyl, [4.4.0]bicyclodecanyl, [2.2.2]bicyclooctanyl, fluorenyl, indanyl, tetrahydronaphthyl, acridinyl, azocinyl, benzimidazolyl, benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl, benzthiazolyl, benztriazolyl, benztetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazolinyl
  • each R 4 is independently halogen, -haloalkyl, -OR, -SR, - CN, -NO 2 , -SO 2 R, -SOR, -C(O)R, -CO 2 R, -C(0)N(R) 2 , -NRC(0)R, -NRC(0)N(R) 2 , -NRSO 2 R, or -N(R) 2 .
  • each R 4 4 I iSs independently -H, C 1-6 aliphatic, - OR, -C(O)R, -CO 2 R, -C(O)N(R) 2 , -NRC(O)R, -NRC(O)N(R) 2 , -NRSO 2 R, or -N(R) 2 ; each of which is optionally substituted.
  • each R 4 is independently -H, C 1-6 aliphatic, -C(O)N(R) 2 , - NRC(O)R, or -N(R) 2 ; each of which is optionally substituted.
  • each R 4 is independently
  • each R 4 is independently [00157] In certain embodiments, each R 4 is independently
  • each R 5 is independently C 1-6 aliphatic, C 3-10 aryl, a 3-8 membered saturated or partially unsaturated carbocyclic ring, a 3-7 membered heterocylic ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or a 5-6 membered monocyclic heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur; each of which is optionally substituted.
  • each R 5 is independently methyl, ethyl, ethyl, propyl, i- propyl, butyl, s-butyl, t-butyl, straight or branched pentyl, or straight or branched hexyl; each of which is optionally substituted.
  • each R 5 is independently phenyl, naphthyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, adamantyl, cyclooctyl, [3.3.0]bicyclooctanyl, [4.3.0]bicyclononanyl, [4.4.0]bicyclodecanyl, [2.2.2]bicyclooctanyl, fluorenyl, indanyl, tetrahydronaphthyl, acridinyl, azocinyl, benzimidazolyl, benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl, benzthiazolyl, benztriazolyl, benztetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazolinyl
  • each R 5 is independently halogen, -haloalkyl, -OR, -SR, - CN, -NO 2 , -SO 2 R, -SOR, -C(O)R, -CO 2 R, -C(O)N(R) 2 , -NRC(O)R, -NRC(O)N(R) 2 , -NRSO 2 R, or -N(R) 2 .
  • each R 5 is independently methyl, cyclopropyl, -F, or -CF 3 .
  • each R 5 is independently -F, or -CF 3 .
  • each of X, Ring A, Ring B, R 1 , R 2 , R 3 , R 4 , R 5 , k, m, n, p, r, and t is as defined above and described in embodiments, classes and subclasses above and herein, singly or in combination.
  • the TLR7 and/or TLR8 inhibitor is a compound of formula I- a,
  • the TLR inhibitor is a TLR7 and/or TLR8 inhibitor selected from or a pharmaceutically acceptable salt of either of these.
  • the TLR inhibitor is enpatoran, afimetoran or E6742.
  • the TLR inhibitor and other therapeutic agents disclosed herein are administered as such or in a pharmaceutically acceptable composition. They may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir.
  • the TLR inhibitor is a small molecule and administered orally.
  • the oral formulation is a tablet or capsule. In another embodiment, the oral formulation is a solution or suspension which may be given to a subject in need thereof via mouth or nasogastric tube. Any oral formulations of the invention may be administered with or without food. In some embodiments, pharmaceutically acceptable compositions of this invention are administered without food. In other embodiments, pharmaceutically acceptable compositions of this invention are administered with food.
  • compositions of this invention are orally administered in any orally acceptable dosage form.
  • exemplary oral dosage forms are capsules, tablets, aqueous suspensions or solutions.
  • carriers commonly used include lactose and corn starch.
  • Lubricating agents such as magnesium stearate, are also typically added.
  • useful diluents include lactose and dried cornstarch.
  • aqueous suspensions are required for oral use, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening, flavoring or coloring agents are optionally also added.
  • compositions of the present invention that are optionally combined with the carrier materials to produce a composition in a single dosage form will vary depending upon the host treated, the particular mode of administration.
  • provided compositions should be formulated so that a dosage of between 0.01 - 100 mg/kg body weight/day of the compound can be administered to a patient receiving these compositions.
  • the total amount of TLR inhibitor administered to the subject in need thereof is between about 10 mg to about 500 mg per day. In one aspect of this embodiment, the total amount of TLR inhibitor administered is between about 50 mg and about 300 mg per day. In another aspect, the total amount of TLR inhibitor administered is between about 100 mg and about 200 mg per day.
  • the TLR inhibitor is administered once a day. In another aspect of this embodiment, the TLR inhibitor is administered twice a day. [00174] In one embodiment, the amount of TLR inhibitor administered to the subject in need thereof is about 50 mg twice a day. In another embodiment, the amount of TLR inhibitor administered to the subject in need thereof is about 100 mg twice a day. In another embodiment, the amount of TLR inhibitor administered to the subject in need thereof is about 200 mg twice a day.
  • the TLR inhibitor is administered for a period of about 7 days to about 21 days. In one aspect of any of the above embodiments, the TLR inhibitor is administered for about 14 days. In one embodiment, the TLR inhibitor is administered for longer periods of time, e.g., for months or years. In one embodiment, the TLR inhibitor is administered for an indefinite period of time.
  • 50 mg of the TLR inhibitor of the invention is administered twice a day for about 14 days. In one embodiment of the invention, 50 mg of the TLR inhibitor of the invention is administered twice a day for an indefinite period of time. In another embodiment of the invention, 100 mg of the TLR inhibitor of the invention is administered twice a day for about 14 days. In one embodiment of the invention, 100 mg of the TLR inhibitor of the invention is administered twice a day for an indefinite period of time. In another embodiment of the invention, 200 mg of the TLR inhibitor of the invention is administered twice a day for about 14 days. In one embodiment of the invention, 200 mg of the TLR inhibitor of the invention is administered twice a day for an indefinite period of time.
  • the TLR inhibitor can be administered in combination with other known therapeutic agents.
  • the one or more additional therapeutic agents is selected from anti-inflammatories, antibiotics, anti-coagulants, antiparasitic agent, antiplatelet agents and dual antiplatelet therapy, angiotensin converting enzyme (ACE) inhibitors, angiotensin II receptor blockers, beta-blockers, statins and other combination cholesterol lowering agents, specific cytokine inhibitors, complement inhibitors, anti-VEGF treatments, JAK inhibitors, immunomodulators, anti-inflammasone therapies, sphingosine-1 phosphate receptors binders, N- methyl-d-aspartate (NDMA) receptor glutamate receptor antagonists, corticosteroids, Granulocyte-macrophage colony-stimulating factor (GM-CSF), anti-GM-CSF, interferons, angiotensin receptor-neprilysin inhibitors, calcium channel blockers, vasodilators, diuretics, muscle relaxants, and antiviral medications.
  • ACE angiotensin converting enzyme
  • the TLR inhibitor is administered in combination with an antiviral agent.
  • the antiviral agent is remdesivir.
  • the antiviral agent is lopinavir-ritonavir, alone or in combination with ribavirin and interferon-beta.
  • the TLR inhibitor is administrated in combination with a broad- spectrum antibiotic.
  • a TLR inhibitor as described herein is administered in combination with a corticosteroid.
  • the corticosteroid is a glucocorticosteroid.
  • the corticosteroid is a mineralocorticoid.
  • Corticosteroids include, but are not limited to, corticosterone and derivatives, prodrugs, isomers and analogs thereof, cortisone and derivatives, prodrugs, isomers and analogs thereof (i.e., Cortone), aldosterone and derivatives, prodrugs, isomers and analogs thereof, dexamethasone and derivatives, prodrugs, isomers and analogs thereof (i.e., Decadron), prednisone and derivatives, prodrugs, isomers and analogs thereof (i.e., Prelone), fludrocortisones and derivatives, prodrugs, isomers and analogs thereof, hydrocortisone and derivatives, prodrugs, isomers and analogs thereof (i.e., cortisol or Cortef), hydroxycortisone and derivatives, prodrugs, isomers and analogs thereof, betamethasone and derivatives, prodrugs, isomers and analogs thereof (i.e., Celestone),
  • the corticosteroid is fludrocortisone or a derivative, prodrug, isomer or analog thereof. In some embodiments, the corticosteroid is fludrocortisone. In some embodiments, the corticosteroid is hydroxycortisone or a derivative, prodrug, isomer or analog thereof. In some embodiments, the corticosteroid is hydroxycortisone.
  • the TLR inhibitor is administered in combination with chloroquine or hydroxychloroquine. In one aspect of this embodiment, the TLR inhibitor is further combined with azithromycin.
  • the TLR inhibitor is administered in combination with interferon- 1-beta (Rebif®).
  • the TLR inhibitor is administered in combination with dexamethasone.
  • the TLR inhibitor is administered in combination with one or more additional therapeutic agents selected from hydroxychloroquine, chloroquine, ivermectin, tranexamic acid, nafamostat, virazole, ribavirin, lopinavir/ritonavir, favipiravir, arbidol, leronlimab, interferon beta- la, interferon beta- lb, beta-interferon, azithromycin, nitrazoxamide, lovastatin, clazakizumab, adalimumab, etanercept, golimumab, infliximab, sarilumab, tocilizumab, anakinra, emapalumab, pirfenidone, belimumab, rituximab, ocrelizumab, anifrolumab, ravulizumab-cwvz
  • the TLR inhibitor is administered in combination with one or more anti-inflammatory agent.
  • the anti-inflammatory agent is selected from corticosteroids, steroids, COX-2 inhibitors, and non-steroidal anti-inflammatory drugs (NSAID).
  • the anti-inflammatory agent is diclofenac, etodolac, fenoprofen, flurbirprofen, ibuprofen, indomethacin, meclofenamate, mefenamic acid, meloxicam, nabumetone, naproxen, oxaprozin, piroxicam, sulindac, tolmetin, celecoxib, prednisone, hydrocortisone, fludocortisone, bethamethasone, prednisolone, triamcinolone, methylprednisone, dexamethasone, fluticasone, and budesonide (alone or in combination with formoterol, salmeterol, or vilanterol).
  • the TLR inhibitor is administered in combination with one or more immune modulators.
  • the immune modulator is a calcineurin inhibitor, antimetabolite, or alkylating agent.
  • the immune modulator is selected from azathioprine, mycophenolate mofetil, methotrexate, dapson, cyclosporine, cyclophosphamide, and the like.
  • the TLR inhibitor is administered in combination with one or more antibiotics.
  • the antibiotic is a broad-spectrum antibiotic.
  • the antibiotic is a pencillin, anti-straphylococcal penicillin, cephalosporin, aminopenicillin (commonly administered with a betalactamase inhibitor), monobactam, quinoline, aminoglycoside, lincosamide, macrolide, tetracycline, glycopeptide, antimetabolite or nitroimidazole.
  • the antibiotic is selected from penicillin G, oxacillin, amoxicillin, cefazolin, cephalexin, cephotetan, cefoxitin, ceftriazone, augmentin, amoxicillin, ampicillin (plus sulbactam), piperacillin (plus tazobactam), ertapenem, ciprofloxacin, imipenem, meropenem, levofloxacin, moxifloxacin, amikacin, clindamycin, azithromycin, doxycycline, vancomycin, Bactrim, and metronidazole.
  • the TLR inhibitor is administered in combination with one or more anti-coagulants.
  • the anti-coagulant is selected from apixaban, dabigatran, edoxaban, heparin, rivaroxaban, and warfarin.
  • the TLR inhibitor is administered in combination with one or more antip latlet agents and/or dual antiplatelet therapy.
  • the antiplatelet agent and/or dual antiplatelet therapy is selected from aspirin, clopidogrel, dipyridamole, prasugrel, and ticagrelor.
  • the TLR inhibitor is administered in combination with one or more ACE inhibitors.
  • the ACE inhibitor is selected from benazepril, captopril, enalapril, fosinopril, lisinopril, moexipril, perindopril, quinapril, ramipril and trandoliapril.
  • the TLR inhibitor is administered in combination with one or more angiotensin II receptor blockers.
  • the angiotensin II receptor blocker is selected from azilsartan, candesartan, eprosartan, irbesartan, losartan, Olmesartan, telmisartan, and valsartan.
  • the TLR inhibitor is administered in combination with one or more beta-blockers.
  • the beta-blocker is selected from acebutolol, atenolol, betaxolol, bisoprolol/hydrochlorothiazide, bisoprolol, metoprolol, nadolol, propranolol, and sotalol.
  • the TLR inhibitor is administered in combination with one or more alpha and beta-blocker.
  • the alpha and beta-blocker is carvedilol or labetalol hydrochloride.
  • the TLR inhibitor is administered in combination with one or more interferons.
  • the TLR inhibitor is administered in combination with one or more angiotensin receptor-neprilysin inhibitors.
  • the angiotensin receptor-neprilysin inhibitor is is sacubitril/valsartan.
  • the TLR inhibitor is administered in combination with one or more calcium channel blockers.
  • the calcium channel blocker is selected from amlodipine, diltiazem, felodipine, nifedipine, nimodipine, nisoldipine, and verapamil.
  • the TLR inhibitor is administered in combination with one or more vasodilators.
  • the one or more vasodilator is selected from isosorbide dinitrate, isosorbide mononitrate, nitroglycerin, and minoxidil.
  • the TLR inhibitor is administered in combination with one or more diuretics.
  • the one or more diuretics is selected from acetazolamide, amiloride, bumetanide, chlorothiazide, chlorthalidone, furosemide, hydrochlorothiazide, indapamide, metalozone, spironolactone, and torsemide.
  • the TLR inhibitor is administered in combination with one or more muscle relaxants.
  • the muscle relaxant is an antispasmodic or antispastic.
  • the one or more muscle relaxants is selected from casisoprodol, chlorzoxazone, cyclobenzaprine, metaxalone, methocarbamol, orphenadrine, tizanidine, baclofen, dantrolene, and diazepam.
  • the TLR inhibitor is administered in combination with one or more antiviral medications.
  • the antiviral medication is remdesivir.
  • the TLR inhibitor is administered in combination with one or more additional therapeutic agents selected from antiparasitic drugs (including, but not limited to, hydroxychloroquine, chloroquine, ivermectin), antivirals (including, but not limited to, tranexamic acid, nafamostat, virazole [ribavirin], lopinavir/ritonavir, favipiravir, leronlimab, interferon beta-la, interferon beta-lb, beta-interferon), antibiotics with intracellular activities (including, but not limited to azithromycin, nitrazoxamide), statins and other combination cholesterol lowering and anti-inflammatory drugs (including, but not limited to, lovastatin), specific cytokine inhibitors (including, but not limited to, clazakizumab, adalimumab, etanercept, golimumab, infliximab, sarilumab,
  • the combination of a TLR inhibitor with one or more additional therapeutic agents reduces the effective amount (including, but not limited to, dosage volume, dosage concentration, and/or total drug dose administered) of the TLR inhibitor and/or the one or more additional therapeutic agents administered to achieve the same result as compared to the effective amount administered when the TLR inhibitor or the additional therapeutic agent is administered alone.
  • the combination of a TLR inhibitor with the additional therapeutic agent reduces the total duration of treatment compared to administration of the additional therapeutic agent alone.
  • the combination of a TLR inhibitor with the additional therapeutic agent reduces the side effects associated with administration of the additional therapeutic agent alone.
  • the combination of an effective amount of the TLR inhibitor with the additional therapeutic agent is more efficacious compared to an effective amount of the TLR inhibitor or the additional therapeutic agent alone. In one embodiment, the combination of an effective amount of the TLR inhibitor with the one or more additional therapeutic agent results in one or more additional clinical benefits than administration of either agent alone.
  • the invention provides a method for advertising a TLR inhibitor comprising promoting, to a target audience, the use of the TLR inhibitor for treating a disease in an individual that has a higher likelihood of therapeutic efficacy as determined by the use of one or more biomarkers described herein.
  • the invention provides a method for advertising a TLR inhibitor comprising promoting, to a target audience, the use of the TLR inhibitor for treating a disease in an individual having high or low expression of one or more biomarkers described herein. Promotion may be conducted by any means available. In some embodiments, the promotion is by a package insert accompanying the TLR inhibitor.
  • the promotion may also be by a package insert accompanying another therapeutic agent, such as the therapeutic agents mentioned herein that the TLR inhibitor can be combined with.
  • the promotion is by a package insert where the package insert provides instructions to receive therapy with the TLR inhibitor after measuring the expression level of one or more biomarkers described herein.
  • the promotion is followed by the treatment of the patient with the TLR inhibitor with or without another therapeutic agent.
  • the package insert indicates that the TLR inhibitor is to be used to treat the patient if the patient is determined to be likely to benefit from the treatment with the TLR inhibitor based on measuring the expression level of one or more biomarkers described herein.
  • the package insert indicates that the TLR inhibitor is not to be used to treat the patient if the patient is determined to be unlikely to benefit from the treatment with the TLR inhibitor based on measuring the expression level of one or more biomarkers described herein.
  • IFN-I activity was measured by DxTerity Diagnostics (Rancho Dominguez, California, USA), using the IFN-1 test, a commercially available chemical ligation-dependent probe amplification and gene expression test with relative quantitative analysis by capillary electrophoresis. Sample testing and analysis was performed as previously described (Kim et al. , J Mol Diagn . 2015 Mar; 17(2): 118-27). The IFN-1 test measures the expression levels of four IFN response genes (HERC5, IFI27, IFIT1 and RSAD2), i.e.
  • BID Mean IFN-I signature scores (50 mg BID -0.84; 100 mg BID -0.74; placebo -0.91) and the proportion of patients with high IFN-I signature scores at baseline (50 mg BID 35%; 100 mg BID 43%; placebo 35%) were reasonably consistent across treatment groups, as reflected by
  • Table 2 Patient demographics and clinical characteristics
  • DMARDs were defined as any systemic immunosuppressive agent other than IVIG or prednisone.
  • CDASI Cutaneous Dermatomyositis Disease Area and Severity Index DM dermatomyositis, DMARD disease-modifying antirheumatic drug, IBM inclusion body myositis, IVIG intravenous immunoglobulin, LN lupus nephritis, MMT Manuel Muscle Test, PM polymyositis, SLE systemic lupus erythematosus, SLEDAI SLE Disease Activity Index.
  • necrotic cell lysates human embryonic kidney 293 cells were suspended at a concentration of 50 xlO 6 cells/mL in phosphate buffered saline (PBS; Gibco, Grand Island, NY). The cells were frozen at -80°C for 10 minutes followed by thawing at 37°C. Four freeze/thaw cycles were performed. The lysate was centrifuged at 400 g for 5 minutes to separate non-lysed cells and the supernatant was collected as necrotic cell lysate.
  • PBS phosphate buffered saline
  • PBMCs were isolated from leukopacks collected from healthy donors (New York Blood Center, New York, NY) in sodium heparin tubes using Ficoll-Paque Plus (Cytiva Life Sciences, Uppsala, Sweeden). The cells were plated in 96-well U-bottom plates (4 xlO 5 cells/well) in RPMI 1640 Medium (Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS; Corning, Woodland, CA). Before stimulation, PBMCs were pre-treated with 1 pM of enpatoran for 30 minutes.
  • necrotic cell lysate (10% vol/vol) and IgG (0.1 mg/mL) purified from patients' plasma were added to the PBMCs, which were incubated at 37°C for 24 hours.
  • the supernatants were collected and cytokine production was measured by AlphaLISA (PerkinElmer, Waltham, MA).
  • PBMCs were plated in 96-well U-bottom plates (5 xlO 5 cells/well) in RPMI 1640 Medium supplemented with 10% FBS.
  • RNA molecules were custom synthesized by Azenta Life Sciences (Chelmsford, MA; Table 3 below). Each RNA was reconstituted in deionized sterile water at 100 pg/mL and 10 pL was added to 100 pL of LyoVec (InvivoGen, San Diego, CA) at room temperature for 30 minutes.
  • PBMCs were pre-treated with 1 pM of enpatoran for 30 minutes at 37°C or left untreated and the RNA-LyoVec complexes were added at a final RNA concentration of 1 ⁇ g/mL for 24 hours.
  • the supernatants were collected and cytokine production was measured using the MSD U-PLEX biomarker group (hu) assay.
  • Table 3 Sequences of the RNA molecules that were custom synthesized and used to stimulate PBMCs
  • RNA samples were analyzed by NanoString. A 46-gene custom panel with markers of inflammation was used. A total of 500 ng of RNA per sample was run on the nCounter Pro Analysis System (NanoString, Seattle, WA). The data were processed using nSolver (NanoString) and Log2 fold changes were calculated for each sample relative to cells treated with supernatants from control PBMCs.
  • Example 2a Activation of TLR7 and/or TLR8 by patient-derived immune complexes
  • DM samples were grouped based on the presence of myositis-specific autoantibodies against Jo-1 (HARS), EJ, TIFl-y, NXP2, SAE, Mi-2, MDA5 and ZO.
  • the majority of the DM samples that stimulated IFN-a were positive for anti -Jo- 1 autoantibody and one DM sample that stimulated IFN-a was positive for anti-SAEl autoantibody (Figure 3B).
  • IFN-GS Score IFN-GS Score
  • IgG samples were profiled for reactivity ( Figure 5).
  • a Spearman correlation matrix analysis was performed in Spotfire using the results of the ex vivo IgG stimulation assay ( Figures 3 and 4) and the autoantibody profiling data to identify reactivities that may be associated with TLR7 and/or TLR8 stimulation (as reflected by IFN-a expression) for either lupus patients (SLE/LN) or IIM patients (DM/PM) and the two disease types were evaluated separately. Reactivities were identified that strongly correlated with TLR7 and/or TLR8 activation (top hits shown in Figure 6A and 6B).
  • Expression levels of highly associated reactivities were evaluated by patient group ( Figure 6C-I) to illustrate how expression correlates with ex vivo stimulation on an individual patient level.
  • the DM patients who were anti-HARS positive and stimulatory in the ex vivo assay were also positive for anti-TRIM21. Based on these results, it is likely that more than one reactivity activates TLR7 and/or TLR8 and may be predictive for the treatment outcome with a TLR7 and/or TLR8 inhibitor. No single reactivity was uniquely correlated with positivity in the ex vivo assay.
  • Example 2c RNAs associated with immune complexes stimulate TLR7 and/or TLR8
  • RNA-binding proteins Several autoantibodies that were associated with TLR7 and/or TLR8 activation are known to react with RNA-binding proteins and, consequently, are likely to have RNA present in their immune complexes. Notable examples are Y4 and Y1 RNA with anti-Ro60 (Boccitto M, Wolin SL. Ro60 and Y RNAs: structure, functions, and roles in autoimmunity. Crit Rev Biochem Mol Biol 2019;54(2): 133-52), and U1 RNA with anti-Smith and anti-RNP (Kattah NH, Kattah MG, Utz PJ. The Ul-snRNP complex: structural properties relating to autoimmune pathogenesis in rheumatic diseases.
  • PBMCs were treated with RNA molecules complexed with LyoVec in the presence or absence of enpatoran and cytokine production was measured (Figure 7).
  • Control PBMCs and PBMCs treated with LyoVec did not show any cytokine production, while R848, a TLR7/8 agonist, effectively induced cytokines, which was inhibited by enpatoran.
  • His-tRNA, Y1, Y4 and U1 complexed with LyoVec were added to PBMCs, IFN-a2, IL-6, IL-10, and IFN-y were all induced ( Figure 7).
  • RNAs stimulate PBMCs in a TLR7 and/or TLR8- dependent manner and that these RNAs may thus serve as a biomarker reflecting an activated TLR7 and/or TLR8 pathway and be predictive for the treatment outcome with a TLR7 and/or TLR8 inhibitor.

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Abstract

La présente invention concerne divers biomarqueurs pour identifier des patients qui sont susceptibles de répondre positivement à un traitement avec un inhibiteur de TLR.
PCT/EP2024/076541 2023-09-22 2024-09-20 Biomarqueurs pour le traitement par inhibiteur de tlr Pending WO2025062016A2 (fr)

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