Companion diagnostics and strategies for treatment with aryl hydrocarbon receptor agonist compounds

Abstract

Described is a method for improving therapeutic response and / or preventing adverse events that may occur during or during treatment with a compound that has activity as an aryl hydrocarbon receptor (AhR) agonist.An exemplary method includes, prior to treating the subject with an AhR agonist compound, testing or instructing to test a sample from the subject to be treated for diagnostic markers related to aryl hydrocarbon receptor biology.Based on the results from the testing step, one or more of the following are performed: (i) administering the AhR agonist compound; (ii) not administering the AhR agonist compound; and (iii) changing the dose or dosing protocol of the AhR agonist compound.

Description

[Technical field]

[0001]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 320,900, filed March 17, 2022, which is incorporated by reference in its entirety.

[0002]

[0002] TECHNICAL FIELD The subject matter described herein relates to methods for improving clinical response rates and for preventing or reducing adverse events that may result from treatment with aryl hydrocarbon receptor (AhR) agonists. [Background technology]

[0003]

[0003] Background Aryl hydrocarbon receptor (AhR) agonists are a diverse set of both naturally occurring and synthetic small molecules that can bind to and activate the AhR. Activation results in a conformational change that exposes a nuclear localization signal, thereby allowing translocation to the nucleus and binding to aryl hydrocarbon nuclear translocator (ARNT), also known as hypoxia inducible factor 1β (HIF-1β), forming a heterodimer that can regulate the transcription of numerous genes that express a consensus deoxyribonucleic acid (DNA) sequence known as the xenobiotic response element (XRE). Binding to the XRE can alter the expression of numerous genes that affect inflammation, development, and metabolism.

[0004]

[0004] Many of the signaling proteins that interact with AhR pathway are shared by other signaling pathways. This is reflected by both canonical AhR signaling, which requires ARNT as described above, and non-canonical signaling that can occur in the absence of ARNT, where agonized AhR can interact with alternative proteins and provide alternative signals in response to AhR agonists. Signaling proteins involved in AhR pathway are also utilized in other pathways, as highlighted by how ARNT, also known as hypoxia-inducible factor 1β (HIF-1β), is required for HIF signaling, which allows survival during oxygen stress. These complex interactions can lead to unexpected adverse events (AEs) under some conditions of AhR signaling.

[0005]

[0005] When activated by AhR agonists, some XRE-responsive genes control aspects of metabolism through the expression of cytochrome P450 (CYP) enzymes such as CYP1A1, CYP1A2, CYP1B1 and CYP19A1 or other enzymes such as UDP-glucuronosyltransferase 1A6 (UGT1A6) and glutathione S-transferase A1 (GSTA1). Some of these same CYPs are involved in the formation and metabolism of estradiol (E2). In particular, CYP1A1, CYP1A2 and CYP1B1 are involved in the production of various hydroxyestradiols, including 6α, 6β, 7α, 12β, 15α, 15β, 16α and 16β hydroxyestradiol from estradiol (E2), making these metabolites potential biomarkers of AhR activity.

[0006]

[0006] Some of these CYPs can metabolize AhR agonists, creating a regulatory feedback loop. AhR agonists switch on AhR signaling, which increases the production of CYPs. This process can then metabolize AhR agonists into non-bioactive metabolites. Other regulation of the AhR itself occurs through the production of inhibitory proteins, including aryl hydrocarbon receptor repressor (AhRR). Activated AhR can be detected by nuclear colocalization of AhR itself, but this is difficult to do in a clinical setting, since it requires both a biopsy and the ability to perform immunohistochemistry to find nuclear colocalization or to determine the amount of AhR protein in the cytosolic versus nuclear fractions. Even when this can be done, it provides a relatively weak signal, since AhR is a protein that constantly shuttles between the cytosol and the nucleus.

[0007]

[0007] Therefore, other more sensitive means for detecting AhR signaling can be used. CYP1A1 is routinely used as a surrogate for AhR signaling because it is highly conserved across species, has a very low level of basal expression, and can be rapidly and dramatically induced by AhR agonists.

[0008]

[0008] Naturally occurring AhR agonists are derived from many sources, including food (eating cruciferous vegetables with high indole content), environmental sources (ultraviolet light-mediated generation of tryptophan dimers), metabolic sources (kynurenine metabolites) or microbial sources (tryptophan-derived catabolic products).Many of these AhR agonists derived from tryptophan can be found in the skin and intestine.For example, bacterial catabolism of tryptophan into molecules such as indole-3-acetic acid (IAA) in the intestine. Reports have demonstrated an association between dysregulated tryptophan metabolism and autoinflammatory diseases such as psoriasis (Harden et al., Journal of Allergy and Clinical Immunology 137(6):1830-1840 (2016)), hidradenitis suppurativa (HS) (Guenin-Mace et al., JCI Insight 5(20):1-14 (2020)), and ulcerative colitis (Lamas et al., Nature Medicine 22(6):598-605 (2016)); Sofia et al., Inflammatory Bowel Diseases 24(7):1471-1480 (2018) suggests that dysregulated tryptophan metabolism and AhR signaling are associated with autoinflammatory diseases.

[0009] AhR agonists have been used in the clinic to treat autoinflammatory diseases such as multiple sclerosis, ulcerative colitis, Crohn's disease, uveitis, psoriasis, nail psoriasis, and atopic dermatitis.However, not all patients respond to AhR therapy, and response is difficult to predict for any given patient.

[0010] AhR agonists have been and continue to be investigated in pharmaceutical settings.There are many approved drugs with low levels of AhR activity, including itraconazole, ketoconazole, omeprazole, 4-hydroxytamoxifen, raloxifene, tranilast, flutamide and leflunomide.Other more potent AhR agonists have been developed or are currently being developed, such as tapinarof, linomide (roquinimex), indirubin, 6-formyl indolo[3,2-b]carbazole (FICZ) derivatives, and laquinimod.Additional AhR agonists may be in development, such as semaxanib, urolithin A, 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE), AQ-312, JNJ-2482272, DMI-9523. Still other such synthetic and natural products containing indigo and indigo derivatives, including but not limited to isoindigo, meisoindigo, and natura alpha or their prodrugs, have been investigated as therapeutic agents. Derivatives of any of the above compounds or related compounds may be investigated as therapeutic agents.

[0011]

[0011] In clinical settings, not all patients respond to AhR therapy, and the magnitude of response and safety profile are often suboptimal. In a phase 3 study of tapinarof for the treatment of psoriasis vulgaris, only 35.2%-40.2% had a meaningful clinical response as measured by physician global assessment. More than 20% of patients developed inflammation of hair follicles (folliculitis) (Lebwohl et al., New England Journal of Medicine 385(24):2219-2229(2021)).

[0012]

[0012] Additionally, in various clinical and non-clinical studies, chronic use or exposure to AhR agonists has resulted in reports of pulmonary arterial hypertension, folliculitis, pneumonia, respiratory failure, myocardial ischemia, myocardial infarction, pericarditis, pleuritis, peritonitis, pneumonia, chest pain, abdominal pain, diarrhea, vomiting, intussusception, joint pain, chest pain, back pain, liver damage, changes in liver enzymes, infections, peripheral neuropathy, headache, pancytopenia or leukopenia.

[0013]

[0013] Clinically, pulmonary arterial hypertension has been reported in published case reports following long-term use of Indigo naturalis, a plant-derived traditional medicine that contains multiple AhR agonists.

[0014] Pulmonary arterial hypertension (PAH) is a progressive disease characterized by elevated pulmonary vascular resistance, which causes a chronic increase in pulmonary arterial pressure resulting from restricted flow through the pulmonary arterial circulation. These pathobiological features can lead to right heart failure and premature death. PAH can be idiopathic, hereditary, or associated with a number of conditions (connective tissue disease, human immunodeficiency virus, or congenital heart disease). In some cases, PAH is associated with drugs or toxins. Drugs that induce AhR, including serotin, have been linked to the development of PAH. In histological sections of lungs from patients with PAH, CYP1B1 and CYP19A1 were highly expressed, suggesting a possible link between PAH, CYP expression, and pulmonary exposure to AhR agonists (White 2012).

[0015]

[0015] Other severe side effects have occurred in late-stage trials of long-acting systemic AhR agonists for the treatment of multiple sclerosis. In a Phase 3 trial of laquinimod, there were seven reported cases of nonfatal myocardial ischemia in the high-dose (1.2 mg / day) group but none in the low-dose group (0.6 mg / day) during treatment of relapsing-remitting multiple sclerosis. Ultimately, the high dose of laquinimod was dropped, and the low dose failed to meet the primary endpoint of the trial. Linomide (Roquinimex) was in Phase 3 trials but was discontinued due to cardiovascular toxicity, specifically pleurisy, pericarditis, and chest pain. Summary of the Invention [Problem to be solved by the invention]

[0016]

[0016] Although there is considerable hope for the therapeutic use of AhR agonists, the pharmacological causes that lead to the occurrence of these reported side effects and the methods for reducing or preventing them are lacking.There is a need for methods for enhancing response rates and preventing and reducing the adverse effects that result from the administration of AhR agonists. [Means for solving the problem]

[0017]

[0017] The following aspects and embodiments thereof described and illustrated below are intended to be exemplary and illustrative, and not limiting in scope.

[0018]

[0018] In one aspect, a method is provided for increasing the response and / or success rate of treatment with an aryl hydrocarbon receptor (AhR) agonist.In another aspect, a method is provided for preventing or reducing adverse events prior to or during treatment with a compound that has activity as an aryl hydrocarbon receptor (AhR) agonist.In another aspect, a method is provided for preventing or reducing adverse events prior to treatment with a compound that has activity as an aryl hydrocarbon receptor (AhR) agonist.

[0019] In an embodiment, a method for treatment with an aryl hydrocarbon receptor (AhR) agonist compound includes, prior to treating the subject with an AhR agonist compound, providing or directing the providing of a sample from the subject and testing or directing the testing of the sample for diagnostic markers related to aryl hydrocarbon receptor biology. Based on the results from the testing or directing the testing, one or more of the following are performed: (i) administering the AhR agonist compound; (ii) not administering the AhR agonist compound; and (iii) modifying the dose or dosing protocol of the AhR agonist compound.

[0020]

[0020] In one embodiment, the step of testing or the step of instructing to test includes: a. an AhR agonist compound in a skin, feces, urine, or plasma sample obtained from a subject; b. Tryptophan in a skin, feces, urine or plasma sample obtained from the subject; c. a cytochrome P450 (CYP) enzyme selected from CYP1A1, CYP1A2, CYP1B1, and CYP19A1 in a colon sample or biopsy, intestinal biopsy, skin biopsy, or peripheral blood mononuclear cells (PBMCs) obtained from the subject; d. Aryl hydrocarbon receptor inhibitor (AhRR) mRNA or protein in a colon sample or biopsy, intestinal biopsy, skin biopsy, or peripheral blood mononuclear cells (PBMCs) obtained from the subject; e. Nuclear-localized AhR in a colon sample or biopsy, intestinal biopsy, or skin biopsy obtained from a subject; f. IL-22 mRNA or protein levels in a sample of plasma, a colon biopsy, an intestinal biopsy, or a skin biopsy obtained from the subject; and g. A polymorphism in caspase recruitment domain 9 (CARD9) in a sample obtained from a subject. The present invention relates to a diagnostic marker selected from the group consisting of:

[0021] In an embodiment, the AhR agonist compound in a sample from a subject is the level of AhR agonist compound in the sample having activity and / or potential to agonize AhR, to give a total level of AhR agonist compound in the sample prior to treatment with the AhR agonist compound. In an embodiment, the tryptophan in a sample from a subject is the level of tryptophan in the sample. In an embodiment, the sample is from a subject prior to treatment with the AhR agonist compound. In an embodiment, the diagnostic marker is tested and quantified from a sample obtained from the subject prior to treatment with the AhR agonist compound, to give a baseline level of the diagnostic marker prior to treatment with the AhR agonist compound.

[0022]

[0022] In one embodiment, the method achieves an improvement in response to treatment with an AhR agonist compound that is increased by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% or 75% compared to subjects treated with the same AhR agonist compound who were not tested for the diagnostic marker.

[0023]

[0023] In an aspect, a method for preventing or reducing adverse events prior to or during treatment with a compound having activity as an aryl hydrocarbon receptor (AhR) agonist includes providing or directing the providing of a sample from a subject prior to or during treatment of the subject with an AhR agonist compound and testing or directing the testing of the sample for diagnostic markers related to aryl hydrocarbon receptor biology. Based on the results from the testing or directing the testing, one or more of the following are performed: i. administering the AhR agonist compound according to a schedule selected to reduce the likelihood or severity of adverse events; ii. administering an AhR agonist compound at a dose selected to reduce the likelihood or severity of an adverse event; iii. no AhR agonist compounds are administered; iv. administering a treatment other than an AhR agonist; and v. Administering an AhR antagonist or a compound that induces CYP.

[0024]

[0024] In one embodiment, the step of testing or the step of instructing to test includes: a. Polymorphisms in AhR, ARNT, HIF-1α or HIF-2α, AhRR, BMPR2, CYP1A1, CYP1A2, CYP1B1 and CYP19A1 or their promoters; b. the ability of microsomes or cells from the subject to metabolize AhR agonists in vitro or in vivo; and c. The ability of an AhR agonist to induce AhR translocation to the nucleus or to induce CYP1A1, CYP1A2, CYP19A1 and / or CYP1B1 expression in vitro or in vivo. The present invention relates to a diagnostic marker selected from the group consisting of:

[0025]

[0025] In one embodiment, the step of administering includes (i), (ii) or (v), and the administering step according to (i), (ii) or (v) reduces the occurrence or severity of an adverse event in a subject compared to a subject not administered an AhR agonist compound or a compound that induces a CYP enzyme.

[0026]

[0026] In one embodiment, the administering step according to (i), (ii) or (v) reduces the occurrence or severity of adverse events in a subject by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% or 75%.

[0027]

[0027] In an embodiment, a method for preventing or reducing adverse events during treatment with a compound having activity as an aryl hydrocarbon receptor (AhR) agonist includes providing or directing the providing of a sample from a subject during treatment of the subject with an AhR agonist compound and testing or directing the testing of the sample for diagnostic markers related to aryl hydrocarbon receptor biology. Based on the results from the testing or directing the testing, one or more of the following are performed: i. administering an AhR agonist at a dosing frequency that is reduced compared to a clinically approved or previously planned dosing frequency; ii. administering an AhR agonist at a dose that is reduced compared to the clinically approved or previously planned dose; iii. administering an initial starting dose of an AhR agonist followed by subsequent doses that are greater than the initial starting dose; iv. Discontinuing use of AhR agonists; v. Continued use of AhR agonists with safety monitoring; vi. administering treatments other than AhR agonists; vii. Concomitant administration of AhR antagonists or compounds that induce CYP1A, CYP1A2, CYP1B1, or CYP19A1; viii. Concomitant administration of concomitant medications; ix. discontinuation of concomitant medications, foods, drugs or activities; and x. No change in treatment.

[0028]

[0028] In one embodiment, the step of testing or the step of instructing to test includes: a. Plasma, urine, colon or skin biopsy levels of the administered AhR agonist or its metabolites; b. Plasma liver enzymes such as alanine aminotransferase (ALT) or aspartate aminotransferase (AST); c. PBMC expression of nuclear AhR or increased AhRR, CYP1A1, CYP1A2, CYP1B1 or CYP19A1 and / or CYP1B1 expression compared to healthy subjects; d. systolic blood pressure, mean pulmonary artery pressure (mPAP), pulmonary artery systolic pressure (PASP), left atrial dimension (LAD), left ventricular end diastolic dimension (LVEDD), left ventricular ejection fraction (LVEF), inferior vena cava diameter, right atrial area (RA area), tricuspid annular systolic excursion (TAPSE), tricuspid regurgitation pressure gap (TRPG), aortic regurgitation, mitral regurgitation, valve thickening, valve motion restriction, shortness of breath and / or fatigue; e. hematocrit or plasma erythropoietin (EPO) level; and f. Plasma or urinary levels of serotonin, melatonin, 17B-estradiol (E2), or 16α-hydroxyestrone The present invention relates to a diagnostic marker selected from the group consisting of:

[0029]

[0029] In one embodiment, the steps include (i), (ii), (iii), (v), (vii), (viii), or (ix), and administration according to (i), (ii) or (v), continuation according to (v), co-administration according to (vii) or (viii), and / or discontinuation according to (ix) reduces the occurrence or severity of an adverse event in the subject compared to a subject not administered the AhR agonist compound, not administered a compound that induces a CYP enzyme or a concomitant medication, and / or continuing the concomitant medication, food, drug or activity.

[0030]

[0030] In one embodiment, the method reduces the occurrence or severity of an adverse event in a subject by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% or 75%.

[0031]

[0031] In one embodiment, continuing use of the AhR agonist in conjunction with safety monitoring includes continuing use with a safety monitoring program selected from an echocardiogram, a timed walking test and a questionnaire regarding shortness of breath and / or the ability to exercise without fatigue.

[0032] In an embodiment, a method is provided for preventing or reducing adverse events prior to or during treatment with a compound that has activity as an aryl hydrocarbon receptor (AhR) agonist.The method includes: referring to product labeling that prohibits certain concomitant drugs, drugs, foods, dietary supplements and / or activities that can increase AhR signaling, increase AhR agonist absorption or hinder the ability to metabolize AhR agonists, and based on the referring step, decide whether to administer, not administer, or administer with the dose range or frequency of AhR agonist adjusted to the subject based on the possibility of adverse events for the subject.

[0033]

[0033] In one embodiment, a method for preventing or reducing adverse events prior to or during treatment with a compound having activity as an aryl hydrocarbon receptor (AhR) agonist includes referring to product labeling that contraindicates the use of AhR agonists in patients with known medical conditions, predispositions or family history that may increase the likelihood of developing cardiovascular disease, including pulmonary arterial hypertension and valvular heart disease.

[0034] In an embodiment, systemic exposure to AhR agonists or C max The method for reducing AhR agonist comprises administering a compound that induces a CYP enzyme capable of metabolizing a to-be-administered AhR agonist, wherein the administering step precedes administration of an oral dosage form containing the AhR agonist.

[0035] In one embodiment, the compound that induces CYP is an AhR agonist. In one embodiment, the AhR agonist that induces CYP is the same as the AhR agonist in the oral dosage form, and the AhR agonist that induces CYP is administered at a lower dose than the AhR agonist in the oral dosage form. In one embodiment, the administering step reduces the occurrence or severity of adverse events in the subject compared to a subject that is not administered the compound that induces CYP enzymes. In one embodiment, the administering step reduces the occurrence or severity of adverse events in the subject by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% or 75%.

[0036]

[0036] In one embodiment, the adverse event for any of the methods described herein is selected from the group consisting of folliculitis, essential hypertension, pulmonary hypertension, pulmonary arterial hypertension, pneumonia, respiratory failure, myocardial ischemia, myocardial infarction, pericarditis, pleuritis, peritonitis, pneumonia, chest pain, abdominal pain, diarrhea, vomiting, intussusception, joint pain, back pain, liver damage, liver enzyme changes, infection, peripheral neuropathy, headache, pancytopenia, and leukopenia.

[0037] In an embodiment, a method of treating one or more patients with an AhR agonist includes providing a data storage facility including a database of patient records, each patient record having a medication treatment approval field for inputting a first prescription for an AhR agonist for treating the patient; a central control unit having one or more processors coupled to a communications network, the central control unit being coupled to the data storage facility for reading and writing data to the data storage facility via the network. The central control unit controls the transmission of data to and the reception of data from the data storage facility via the network, and the central control unit is programmed to output via the network a first approval of a first prescription of an AhR agonist for a patient who has previously been subjected to one or more initial medical tests, each of which provides an initial medical test result. The initial medical tests are selected from the group consisting of a physician's consultation, a genetic test, a physiological function test, and a medical imaging test. The output of the first approval depends on the satisfactory results of one or more of the initial medical tests entered into each patient's record, and is further programmed to schedule one or more subsequent tests for each patient prior to the step of allowing the entry of the prescription in the medication approval field. At least one of the subsequent medical tests is an echocardiographic imaging test performed in a manner that provides measurements of one or more internal cardiac structure dimensions and cardiac flow velocities, and the patient receives or continues to receive the medication only upon the entry of a satisfactory echocardiographic evaluation result. The central controller inhibits the approval output of the first or subsequent prescription upon the entry of an unsatisfactory test result. The central controller manages one or more aspects of the approved prescription for the patient selected from the group consisting of dosage; dosing regimen; and intended duration of use, thereby inhibiting abuse or misuse of the AhR agonist, and reports the integrated and analyzed data to a regulatory agency.

[0038]

[0038] In certain embodiments, the diagnostic value used in the method is determined by a step of comparing with a reference value, standard or sample. In other embodiments, the diagnostic value is determined by the amount that changes or differs from the reference value, standard or sample, and the amount that changes is a decrease or increase in the amount of the diagnostic value compared to the reference. In one embodiment, the change is a statistically significant change. In one embodiment, the change is a statistically significant reduction in the diagnostic value compared to the reference (the reference can be, for example, a baseline value, a standard, a historical control, a disease-matched cohort or a healthy control cohort).

[0039] In other embodiments, the diagnostic value is the plasma concentration of a biomarker or compound.

[0040] In some cases, the diagnostic value or biomarker is present in an amount that is different from the amount of a reference sample, value or standard by about 10%, about 15%, about 20%, about 25% or about 30%. In one embodiment, the amount in the sample is a decreased amount compared to the reference. In another embodiment, the different amount in the sample is an increased amount compared to the reference. For example, biomarkers that are polymorphisms, markers can be determined to be present, absent or heterozygous. Markers that are metabolites can be evaluated for changes from a standard group or reference sample.

[0041] In an embodiment, the reference sample, standard or value is a standard test value or range of values ​​for the marker in humans. In another embodiment, the reference sample, standard or value is a reference genome. In another embodiment, the reference sample, standard or value is a baseline concentration value or range for the marker in humans. In another embodiment, the reference sample, standard or value is a tissue or fluid sample from a human subject or patient prior to treatment with an AhR agonist compound (baseline). In another embodiment, the reference sample, standard or value is a tissue or fluid sample from a human subject or patient during the course of treatment with an AhR agonist compound. In another embodiment, the reference sample, standard or value is a collection or population of biological samples from healthy subjects or similarly disease-matched subjects, each sample is evaluated for the marker, and the average value for the marker in the collection or population is determined and taken as the reference sample, standard or value.

[0042] In one embodiment, the diagnostic test involves detecting the concentration of the administered AhR agonist compound or a metabolite thereof in a sample.

[0043]

[0043] In the above embodiments, the diagnostic test is performed on a blood, serum or plasma sample from a subject.

[0044] In another embodiment, the diagnostic test involves evaluating DNA in a sample for polymorphisms in AhR, AhRR, CYP1A1, CYP1A2, CYP1B1 or their respective promoters or other genes or gene products.

[0045]

[0045] In another embodiment, the test includes evaluating (i) DNA in the sample for polymorphisms in CYP450 enzymes, (ii) xenobiotic metabolizing enzymes regulated by xenobiotic response elements, (iii) polymorphisms in xenobiotic response elements that alter AhR function, and / or (iv) polymorphisms in the gene BMPR2.

[0046] In an embodiment, the xenobiotic metabolizing enzyme is CYP1A1, CYP1A2 or CYP1B1.

[0047] In another embodiment, the test evaluates two or more polymorphisms to assign a polymorphism risk score.

[0048] In another embodiment, the testing involves genotyping via whole genome sequencing, targeted sequencing, exome sequencing or detection via PCR for specific polymorphisms.

[0049] In yet another embodiment, the test comprises detecting the ability to metabolize an AhR agonist in microsomes or cells derived from the sample.

[0050] In an embodiment, the cells or microsomes are derived from a sample which is blood, skin, vasculature, liver, intestine, colon or lung.

[0051] In yet another embodiment, the assay comprises detecting binding of an AhR agonist to a recombinant polymorphic AhR.

[0052] In another embodiment, the test involves detecting CYP1A1, CYP1A2 or CYP1B1 mRNA expression or protein levels.

[0053]

[0053] In another embodiment, the test involves detecting the ability of a xenobiotic metabolizing enzyme to mediate the deethylation of a 7-ethoxyresorufin substrate to resorufin (EROD assay) in microsomes or cells derived from the sample.

[0054] In another embodiment, the test involves detecting the level of an enzyme in a blood or plasma sample that is indicative of AhR activation or liver toxicity.

[0055] In one embodiment, the enzyme is alanine aminotransferase or aspartate aminotransferase.

[0056] In another embodiment, the test involves detecting the presence or absence in the sample of a gene product that is regulated by a xenobiotic response element.

[0057]

[0057] In one embodiment, the gene is selected from the group consisting of CYP1A1, CYP1A2, CYP1B1, IGFBP10, and GSTP1.

[0058]

[0058] In embodiments, the tests include polymerase chain reaction, quantitative polymerase chain reaction, loop-mediated isothermal amplification (LAMP), genotyping including whole genome sequencing, targeted sequencing, exome sequencing, microarrays, RNA sequencing, mass spectrometry, immunohistochemistry, or immunofluorescence.

[0059] In yet another embodiment, the test comprises detecting in the sample the presence or absence of a metabolic or catabolic product regulated by an AhR-regulated enzyme, in an embodiment, the product is selected from the group consisting of serotonin, tryptophan, melatonin, 5-hydroxy-L-tryptophan (5-HTP), 5-hydroxyindoleacetic acid (5-HIAA), 17β-estradiol (E2) or 16α-hydroxyestrone, a steroid, or a hormone.

[0060] In one embodiment, the diagnostic test involves analyzing or investigating for deficient proteins or cells induced by activated hypoxia inducible factor.

[0061] In an embodiment, the protein is erythropoietin (EPO).

[0062] In another embodiment, the cell deficiencies are red blood cells and are detected by analyzing or investigating the relative volume or concentration of red blood cells via hematocrit or complete blood count.

[0063] In some embodiments, the method further comprises administering to the subject sample a compound that is metabolized by the AhR agonist-induced enzyme to produce a metabolite, and detecting the metabolite.In an embodiment, the compound is 7-ethoxyresorfin, the AhR agonist-induced enzyme is CYP1A1, and the metabolite is resorufin.

[0064] In yet another embodiment, the subject is a subject who experiences lifestyle activities that may activate aryl hydrocarbon receptor.Lifestyle activities may be smoking, pollutant exposure, hypoxic or hyperoxic conditions, and / or taking food, supplements or drugs that have AhR activation potential.

[0065] In other embodiments, the subject is a subject who has lifestyle activities that inhibit the metabolism of AhR agonists.The activities can inhibit the activity of CYP enzymes.In some embodiments, the lifestyle activities can be bergamottin, grapefruit intake, inhalation of marijuana or its components such as tetrahydrocannabinol or cannabidiol, or use of concomitant drugs, drugs or alcohol.

[0066]

[0066] In other embodiments, the subject is a subject who has a lifestyle activity that increases the absorption of the AhR agonist. The lifestyle activity can be a high-fat or high-alcohol diet.

[0067] In yet other embodiments, the subject is one at risk for reduced or altered metabolism of the AhR agonist compound.

[0068] In embodiments, a genetic polymorphism in CYP1A1, CYP1A2, or CYP1B1 results in reduced metabolism of an AhR agonist.

[0069] In other embodiments, the method further comprises treating or recommending treatment of the subject with a drug that increases the metabolism or clearance of the AhR agonist.

[0070] In the above embodiments, a drug that increases AhR metabolism or clearance is given prior to initiating therapy with an AhR agonist without an explicit need for a diagnosis.

[0071]

[0071] The drug that increases AhR metabolism or clearance can be a CYP inducer. The drug that increases AhR metabolism or clearance can be an AhR agonist itself. Alternatively, the drug that increases AhR metabolism or clearance can be not an AhR agonist. The drug that increases AhR metabolism or clearance can be the same AhR agonist that is the subject that is administered therapeutically. The drug that increases AhR metabolism or clearance can be a lower dose or a less bioavailable form of the AhR agonist that is the subject that is administered for therapeutic use. This lower dose or less bioavailable form of the AhR agonist can result in the induction of CYP at a high level locally without inducing a high level of systemic CYP expression. The local expression of CYP can occur in the gastrointestinal tract and / or liver, and not in the lungs.

[0072] This strategy of pre-administration of a compound prior to therapeutic dosing with an AhR agonist without the need for diagnostic testing may be referred to as "lead in dosing", "run in dosing", "priming dosing", "starting dose", "initial dosing", "metabolic preactivation dosing" or even "reverse additive dosing". No approved drug is known to date that utilizes this strategy to induce local metabolic activation with the aim of reducing subsequent systemic exposure. Strategies are known in which the dose is increased or decreased until intolerance is not tolerated and / or when the desired therapeutic outcome is observed. The relatively low initial dose intended for metabolic preactivation of the subsequently administered drug is reasonable for an orally administered drug intended for local activity in the gut, which may be metabolized by local first-pass metabolism when systemic exposure is undesirable, encompassing a small and novel pharmacological space, and therefore this is why it has not been disclosed in the art before.

[0073]

[0073] The disclosures herein relating to the use of oral AhR agonists and withdrawal doses for use in the gastrointestinal tract may additionally be used to limit systemic exposure using other forms of local administration, such as to the eye, ear, skin, vagina, or lungs.

[0074]

[0074] In other embodiments, the method further comprises treating or recommending treatment of the subject with a drug that increases activity of CYP1A1, CYP1A2, or CYP1B1.

[0075]

[0075] In other embodiments, the adverse event of interest is selected from the group consisting of folliculitis, essential hypertension, pulmonary arterial hypertension, pneumonia, respiratory failure, myocardial ischemia, myocardial infarction, pericarditis, pleuritis, peritonitis, pneumonia, chest pain, abdominal pain, diarrhea, vomiting, intussusception, joint pain, back pain, liver damage, liver enzyme changes, infection, peripheral neuropathy, headache, pancytopenia, and leukopenia.

[0076] In other embodiments, the AhR agonist compound is selected from the group consisting of itraconazole, FICZ, ITE, urolithin A, semaxanib, ketoconazole, omeprazole, 4-hydroxytamoxifen, raloxifene, tranilast, flutamide, leflunomide, tapinarof and tapinarof derivatives, linomide (loquinimex), laquinimod, Aquilion AQ-312, DMI Bioscience DMI-9523, Resolvex RLV-102, 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE), JNJ-2482272, FICZ derivatives such as Nogra NPD-0614-02 and NPD-0614-24, Nexys NEX003, Artus The indigo derivative is selected from the group consisting of ARTX-28, Seidai, indigo, indirubin, or any derivative thereof. Specific examples of indigo derivatives include, but are not limited to, isoindigo, meisoindigo, and natura alpha.

[0077]

[0077] In some aspects, the methods of the present invention involve the use of diagnostic tests that rely on genetic, enzymatic and / or pharmacokinetic parameters associated with AhR agonists, activation of AhR and / or activation or inhibition and / or increase or decrease of metabolic enzymes, transporters, downstream messengers, metabolites, hormones or enzymes.

[0078]

[0078] Once one or more diagnostic values ​​have been obtained, in some embodiments, the diagnostic values ​​can be used by the patient or clinician in a number of ways, including maintaining treatment, stopping treatment, changing treatment, co-administering treatment, altering safety monitoring, changing dosage, altering dosing schedule, determining a starting dose, or some combination of the above, if appropriate.

[0079]

[0079] In addition to the exemplary aspects and embodiments described above, further aspects and embodiments will become apparent upon study of the following description.

[0080]

[0080] Additional embodiments of the method of the present invention will be apparent from the following description and claims. As can be understood from the preceding and following descriptions, each and every feature described herein, and each and every combination of two or more of such features, are included within the scope of the present disclosure, unless the features included in such combinations are mutually inconsistent. In addition, any feature or combination of features can be specifically excluded from any embodiment of the present disclosure. Additional aspects and advantages of the present disclosure are described in the following description and claims, especially when considered in conjunction with the accompanying examples. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0081]

[0081] I. Definition Various aspects are now more fully described below. Such aspects may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey its scope to those skilled in the art.

[0082]

[0082] When a range of values ​​is provided, it is intended that each intervening value between the upper and lower limits of the range and any other stated or intervening value within the stated range is encompassed within the present disclosure.For example, if a range of 1 μm to 8 μm is stated, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, and 7 μm, as well as ranges of values ​​equal to or greater than 1 μm and equal to or less than 8 μm, are also expressly intended to be disclosed.

[0083]

[0083] The singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to a "polymer" includes a single polymer as well as two or more of the same or different polymers, a reference to an "excipient" includes a single excipient as well as two or more of the same or different excipients, etc.

[0084]

[0084] The word "about", when immediately preceding a numerical value, means a range of plus or minus 10% of that value, e.g., "about 50" means 45-55, "about 25,000" means 22,500-27,500, etc., unless the context of this disclosure indicates otherwise or is inconsistent with such an interpretation. For example, in a list of numerical values ​​such as "about 49, about 50, about 55", "about 50" means a range extending to less than half of the interval between the preceding value and the following value, e.g., from more than 49.5 to less than 52.5. Furthermore, the phrase "less than about" or "more than about" should be understood in light of the definition of the term "about" provided herein.

[0085]

[0085] The methods of the present disclosure can comprise, consist essentially of, or consist of the disclosed components.

[0086]

[0086] Less than the entire disclosure may be claimed for any reason, while reserving the right to proviso out or exclude any individual member of any such group, including any subrange or combination of subranges within a group, which may be claimed according to a range or in any similar manner. Furthermore, less than the entire disclosure may be claimed for any reason, while reserving the right to proviso out or exclude any individual substituent, analog, compound, ligand, structure, or group thereof, or any element of a claimed group.

[0087] Throughout this disclosure, various patents, patent applications and publications are referred to.The disclosures of these patents, patent applications and publications are incorporated by reference in their entirety into this disclosure in order to more fully describe the state of the art as known to those skilled in the art at the date of this disclosure.If there is any inconsistency between the cited patents, patent applications and publications and this disclosure, this disclosure shall control.

[0088] For convenience, some terms used in this specification, examples and claims are collected here.Unless otherwise defined, all technical and scientific terms used in this disclosure have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs.

[0089]

[0089] The methods described herein may be used with any biological sample including, but not limited to, skin, sebum, mucus, sperm, whole blood, plasma, white blood cells, peripheral blood mononuclear cells, red blood cells, colon, intestine, feces, urine, tears, liver, lung, endothelium, sputum, saliva, oral swabs, and / or nasal swabs.

[0090] The results from the diagnosis described herein can be used alone or in combination.The results of diagnosis can be combined with other patient or historical control information to inform treatment decisions.These treatment decisions can be to administer, not administer, or change the dose, duration or route of administration of AhR agonist or AhR antagonist for the treatment of human disease.

[0091] Throughout this disclosure, reference is made to measuring the activity of various gene products. In the examples and claims, gene products measured as mRNA, protein or enzyme activity can be substituted in any example. For example, CYP1A1 can be measured at the mRNA level via qPCR, at the protein level via Western blot, or at the enzyme level via EROD assay. A specific method of measurement can be replaced by another means of measurement in any example.

[0092]

[0092] Similarly, the examples given are not intended to be limiting. When measuring AhR-regulated gene activity in one tissue, other AhR-regulated genes can be substituted in different tissues.For example, CYP1A1 in skin can be replaced by CYP1A2 in colonic epithelium.

[0093] The magnitude of change in the examples is not intended to be proportionally limiting. Meaningful change depends on the specific diagnostic assay. Meaningful change to inform treatment decisions can be change from baseline, change from matched control, change from reference value from healthy individual, change from unrelated region or some other change. Change can be more than or less than 1%, 10%, 25%, 50%, 75%, 90% or 99%. Change can be based on the mean, standard deviation, cutoff, interquartile range or some other meaningful change. Change can be based on meaningful change in database, clinical trial, prior experiment or intra-experimental control. Change can be based on high or low value. Change may or may not be statistically significant.

[0094] The term simultaneous administration can be administration at the same time or immediately before or after. In some cases, the term can mean within one minute, ten minutes, one hour, or one day of administration of something else.

[0095]

[0095] II. Method Data from patients with autoimmune inflammatory disease often show a correlation between the level of AhR agonist and / or AhR signaling and disease.In some cases, reduced AhR agonist in plasma or tissues compared to healthy patients as controls correlates with disease severity.It is not known whether low levels of AhR agonist and / or AhR signaling at baseline prior to treatment with AhR agonist predict the clinical response of patients to AhR agonist for treating inflammatory disease.If the level of AhR agonist or AhR signaling correlates with response, response rate, or response magnitude to administration of AhR agonist, a companion diagnostic that measures this will allow more efficient clinical trials, enhanced clinical response and response rate, and / or greater response magnitude to administration of AhR agonist. This could also be used to define target patient populations and minimize the number of non-responders to save costs and healthcare resources, and could allow other interventions that may have greater benefit to be administered to AhR agonist non-responders.

[0096]

[0096] In some embodiments, the method of the present invention relates to determining, analyzing and / or correlating a subject's response to AhR agonist therapy with the amount of baseline AhR signaling and / or expression level of endogenous AhR agonist. The method also relates to enhancing or improving a subject's response to treatment with AhR agonist compounds. In other embodiments, the method of the present invention relates to classifying patients based on their level of AhR signaling, the presence of certain or all AhR agonists, the activity or abundance of certain metabolic enzymes induced by AhR signaling and / or the level of AhR agonist precursor molecules. The method has utility as a diagnostic, which can enhance or improve the response or success rate to therapy with AhR agonists, for example, to treat inflammatory diseases.

[0097] Some gene mutations cause the reduced ability to produce or respond to AhR agonists.For example, inflammatory bowel disease (IBD)-related risk alleles in caspase recruitment domain 9 (CARD9) cannot properly metabolize tryptophan and related molecules, thereby resulting in the defective production of AhR agonists.These patients may be more responsive to the administration of AhR agonists than CARD9-normal IBD patients.

[0098] Some tryptophan-derived AhR agonists can be produced by microflora.For example, certain species of bacteria and fungi are known to be capable of producing indigo and indirubin.Patients who lack or have low abundance of these microorganisms may be more responsive to the administration of AhR agonists.

[0099] In response to AhR agonist administration, intestinal epithelial cells produce the anti-inflammatory cytokine interleukin 22 (IL-22) (Lamas 2018). Baseline levels of IL-22 may be predictive of response to AhR therapy. Low levels of IL-22 may predict higher response rates to AhR therapy.

[0100]

[0100] Other baseline measurements related to AhR biology may be correlated with response to AhR agonists. Diagnostics for clinical use that may enhance or improve response or success rates to AhR agonist therapy are described and contemplated.

[0101]

[0101] In a first aspect, methods are provided and described herein for preventing, reducing or ameliorating adverse events prior to or during treatment of a subject with an AhR agonist compound.

[0102] Exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin, also known as TCDD or dioxin, can result in systemic, constitutive, chronic AhR activation. TCDD is unusual in its high potency and long half-life. TCDD is one of the most potent known AhR agonists and cannot be easily metabolized in humans. A single large exposure can result in the constitutive activation of AhR for several years, leading to a number of possible toxicities, including chloracne, gastritis, keratosis, chronic obstructive pulmonary disease (COPD) and sebaceous gland involution.

[0103] Further evidence that constitutive activation of AhR can be problematic comes from mouse studies using chronic dosing of TCDD, which increased mean arterial blood pressure, but only after 15 days of exposure. Adverse events occurred after relatively long exposures, suggesting that AhR signaling itself is not toxic, but that constitutive systemic activation over more than a few weeks can result in toxicity.

[0104]

[0104] Although the AhR is highly conserved across species, it has been shown to be highly conserved across species, with a 50% lethal dose (LD ) for a group of animals exposed to TCDD. 50 ) values ​​vary over 5000-fold between species, from 1 μg / kg in guinea pigs to over 5000 μg / kg in hamsters. The metabolism of specific AhR agonists can be highly variable and species-dependent. In mice, the magnitude of the response to TCDD depends on the AhR allele that has an Ala to Val substitution at codon 375 that reduces affinity for TCDD and subsequent xenobiotic metabolizing enzymes (Mimura 2003).

[0105] Polymorphisms in genes or regulatory elements in AhR pathway or interacting pathways may change the response to AhR agonist or its metabolism.A number of known polymorphisms in AhR have been shown to cause loss of CYP1A1 induction (Wong 2001).Other polymorphisms in either AhR or HIF signaling pathway may contribute to the adverse events that result from the administration of AhR agonist.

[0106]

[0106] Continuous activation of AhR, either due to gene polymorphism, continuous administration of AhR agonist or defective metabolism of AhR agonist, can cause sequestration of ARNT (also known as HIF-1β). ARNT plays an important physiological function from its role as a sensor of hypoxia, and deletion of the gene encoding ARNT (HIF-1β) is lethal. ARNT (HIF-1β) is the canonical binding partner for AhR and HIF-1α and HIF-2α, and sequestration of ARNT by one pathway can reduce the functionality of other pathways (Vorrink et al., International Journal of Molecular Sciences 15(8):13916-13931(2014)). ARNT (HIF-1β) binds to HIF-1α and HIF-2α to form activated hypoxia-inducible factors HIF1 and HIF2, respectively, which are crucial transcription factors mediating the response to hypoxia. It is possible that constitutive activation of AhR can result in sequestration of ARNT (HIF-1β) and impaired responses to hypoxic conditions, which may lead to adverse events.

[0107] A recent publication (Masaki 2021) demonstrates the interplay between AhR and HIF signaling, showing that plasma levels of AhR agonists correlate with the severity of pulmonary arterial hypertension (PAH). In PAH, it is unknown whether AhR agonists in plasma are causative or correlated. The paper shows that in rats, both hypoxia and strong systemic AhR activation followed by normoxic conditions are required in bone marrow and non-bone marrow derived cells to cause PAH-like disease. However, many patients with PAH have areas of the lung that are hypoxic due to inflammation and / or vasoconstriction. In these lung areas, HIF signaling may interfere with AhR signaling and subsequent CYP1A production by sequestering ARNT (HIF-1β), leading to impaired local metabolism of AhR agonists and therefore relatively high levels of these agonists.

[0108] Alternatively, the overflowing AhR activation may itself overwhelm the available ARNT. In the absence of ARNT (HIF-1β), agonized AhR (and to a lesser extent unagonized AhR) may interact with RelA and RelB and other proteins in the non-canonical AhR signaling pathway, leading to the expression of IL-8, IL-6 and various chemokines (Vogel 2007b). Other non-canonical signaling that does not require ARNT (HIF-1β) may lead to the degradation of estrogen receptors, downregulation of CYP1A1 and induction of various chemoattractants (Lamas 2018). This non-canonical signaling may produce very different physiological effects compared to typical AhR signaling.

[0109] The association between AhR activation, endothelial / vascular expression of CYP1A1, hypertension, and observed adverse clinical events such as reversible pulmonary arterial hypertension and myocardial ischemia suggests a possible causal relationship. Too much oxygen (hyperoxia) induces CYP1A1 in the lungs and liver of wild-type B6 mice but not in AhR knockout mice (AhR- / -), while too little oxygen (hypoxia) inhibits the induction of CYP1A1 by TCDD (Gradin et al., Molecular and Cellular Biology 16(10):5221-5231(1996)); Vorrink et al., International Journal of Molecular Sciences 15(8):13916-13931(2014)).

[0110] The mechanism for the increase in arterial pressure after TCDD exposure was dependent on the presence of CYP1A1, which was shown to be expressed on the aortic endothelium (Kopf 2010). It is not known whether CYP1A1 has an active role in the process by increasing the metabolism of substrates or by some other mechanism. It is possible that CYP1A1 KO leads to an increase in the amount of AhR agonists, which may inhibit HIF signaling by sequestering ARNT (HIF-1β).

[0111]

[0111] In some embodiments, the methods of the present invention relate to a clinically advantageous companion diagnostic employed prior to the use of AhR agonists as a means to prevent adverse events, including but not limited to PAH.

[0112] In some embodiments, the method for preventing or reducing adverse events may not require a companion diagnostic.In these cases, previous research or clinical knowledge can inform strategies that are expected to result in relatively safe treatment with AhR agonists without the need to administer diagnostics prior to the start of therapy.One such example is a strategy that allows for the reduction of systemic exposure to AhR agonists.This can be achieved by administering a compound that increases the metabolism of AhR agonists prior to the start of therapy.

[0113]

[0113] In other embodiments, methods of monitoring chronic, constitutive and / or systemic AhR signaling during AhR agonist therapy to reduce the risk of developing adverse events and enable intervention to prevent or mitigate adverse events in order to make this class of drugs safer or more tolerable.

[0114]

[0114] Methods for monitoring for early signs / symptoms of PAH or other cardiovascular AEs during AhR therapy are also desirable.

[0115]

[0115] In some embodiments, methods for monitoring for chronic, constitutive and / or systemic activation of AhR or other early signs of the earliest onset of adverse events are provided to prevent and alleviate adverse events during AhR agonist therapy.

[0116] Tryptophan and related indoles are important starting materials for the final metabolism / catabolism and microbial production of AhR agonists. The reduced levels of these indole-derived AhR agonists result in relatively weak AhR activity. The changes in AhR biology that can be detected in patient biological samples are predictive of response to therapy using AhR agonists or even AhR antagonists. These diagnostics can be used to predict response to AhR agonist or AhR antagonist therapy.

[0117] In one embodiment, the method for enhancing the response to administration of AhR agonist involves determining the baseline total AhR activity of a patient.The patient with low baseline total AhR activity is most likely to respond to AhR agonist therapy.The patient with maximum baseline AhR activity is relatively less likely to respond to AhR agonist therapy.

[0118] For example, plasma and fecal analysis of ulcerative colitis patients treated with Aodai can be used diagnostically. Baseline fecal samples can be treated, for example, as reported in Lamas (Lamas et al., Nature Medicine 22(6):598-605(2016)) and tested in the AhR luciferase assay (sold by Indigo Bioscience). Plasma samples can be treated, for example, as reported in Masaki 2021, and tested at a dilution of 10% in the AhR luciferase assay. Total baseline AhR activity can be compared to positive control and healthy control samples.

[0119]

[0119] Patients with the lowest quartile of baseline AhR agonist activity may be most responsive to therapy with the AhR agonist mixture, Aodaizu.Specifically, these patients may respond more quickly, more continuously and with greater magnitude in terms of reduction of calprotectin, complete and partial Mayo score, endoscopic activity score and histological scoring than patients in the highest quartile of baseline AhR activity.That is, diagnostics are envisioned to obtain baseline feces or plasma from patients prior to treatment with AhR agonists as a means to evaluate whether AhR agonist therapy is a good treatment option.

[0120] In another embodiment, the method for enhancing the response to administration of an AhR agonist involves measuring a patient sample for specific AhR agonists that may be endogenously produced by the body, naturally produced by the microbiota, or diet.

[0121] AhR agonists can be measured in baseline patient samples via HPLC. Agonists include one or more of the following substances or derivatives thereof: 3-hydroxyindole, 3-methylindole, astaxanthin, canthaxanthin, cinnabarinic acid, 3,3'diindolylmethane (DIM), diosmin, 6-formylindolo[3,2-b]carbazole (FICZ), indigo, indirubin, indole, indole-3-acetic acid (IAA), indole-3-aldehyde (IAld), indole-3-acetaldehyde (IA). Ald), indole[3,2-b]carbazole (ICZ), indoxy-3-sulfate (I3S), 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid (ITE), indole-3-acetonitrile (I3AC), indole-3-carbinol (I3C), kynurenine, kynurenic acid, luteolin, malassedin, myricetin, quercitin, resveratrol, tamarixetin, tangeritin and / or xanthurenic acid.

[0122]

[0122] Patients with relatively low levels of one or more of the AhR agonists are more likely to respond to subsequent administration of the AhR agonist. The therapeutically administered AhR agonist can be the same or different from the AhR agonist detected in the sample.

[0123] In another embodiment, the method for enhancing the response to administration of an AhR agonist involves measuring the patient for baseline plasma tryptophan levels. It has been recognized that increased tryptophan metabolism is associated with activity in inflammatory bowel disease (Nikolaus et al., Gastroenterology 153(6):1504-1546 (2017)). Since tryptophan is the starting material for many endogenous AhR agonists, low tryptophan levels are considered a proxy for relatively low AhR activity. Tryptophan plasma levels have been shown to correlate with disease severity in Crohn's disease and ulcerative colitis.

[0124]

[0124] Plasma tryptophan levels have been investigated as a biomarker for response to infliximab and vedolizumab (Nikolaus et al., Gastroenterology 153(6):1504-1546 (2017)). Infliximab responders were shown to have a sustained increase in plasma tryptophan levels compared to non-responders. This effect was not seen with vedolizumab. Plasma tryptophan levels have not been investigated as a predictive marker for AhR therapy.

[0125]

[0125] Without being bound to any particular theory, baseline plasma tryptophan levels in IBD patients, particularly UC patients, may predict response to AhR therapy.By differentiating IBD patients based on plasma tryptophan levels, predictive diagnostics can be developed that improve the probability and / or magnitude of response to AhR therapy.

[0126]

[0126] Specifically, tryptophan and similar small molecules can be measured via ELISA, HPLC, UPLC, bioluminescence, coulometry or fluorescence assays.

[0127]

[0127] It has also been shown that the plasma kynurenic acid / tryptophan ratio, together with the expression of various metabolic enzymes in the tryptophan metabolic pathway, correlates with endoscopic subscores in UC (Sofia 2018). Specifically, plasma levels of kynurenic acid and picolinic acid correlated with the mayo endoscopic subscore. When the kynurenic acid / tryptophan ratio is normalized to tryptophan, this ratio provides a more standardized measure of the kynurenine biosynthetic pathway than kynurenic acid alone. Downstream KYN metabolites such as kynurenic acid, quinaldic acid, anthranilic acid, xanthurenic acid, 3-hydroxykynurenine, 3-hydroxyanthranilic acid, a-amino-B-carboxymuconic acid-E-semialdehyde, quinolinic acid, A-aminomuconic acid-E-semialdehyde, picolinic acid and / or glutaryl CoA can also be measured alone or as a ratio to tryptophan or other related molecules.

[0128] In addition to cellular biosynthesis and catabolic processes, the human microbiota produces numerous AhR agonists, many of which are produced from tryptophan or indole precursors. It has been demonstrated that high bacterial species diversity correlates with high serum tryptophan levels (Nikolaus 2017). Patients with relatively low species richness or relatively low levels of specific or all microorganisms known to produce AhR agonists may have reduced AhR signaling and may be more responsive to AhR agonist therapy. Thus, methods for detecting microbial richness and / or specific microorganisms known to produce AhR agonists can be utilized as diagnostics prior to administration of AhR agonists.

[0129]

[0129] Specifically, DNA or RNA can be extracted from patient samples such as feces, colon, intestine, or skin, and 16S rRNA amplicon sequencing can be performed on intestinal / skin samples to identify, classify and quantify bacterial species.In addition to the overall species richness that can be described by the number of different amplicons, specific bacterial species and strains that are known to produce AhR agonists can be identified. These species include, but are not limited to, the following species: Bacillus alvei, Bacteroides sp., Bacteroides thetaiotaomicron, Citrobacter sp., Clostridium novyi, Clostridium limosum, Clostridium tetani, Corynebacterium acnes, E. coli, Flavobacterium sp., Fusobacterium sp., Haemophilus influenza, Kleibsella planticola, and others. planticola, Kleibsella pneumonia, L. reuteri, Lactobacillus taiwanensis, Lactobacillus bulgaricus OLL1181, Providencia stuartii, Shigella flexneri and / or Vibrio cholera or any other related species or other species capable of producing AhR agonists. Similar approaches can be used separately or in combination to identify, classify and quantify fungi or viruses that produce AhR agonists.Specific examples of fungi include subspecies of the yeast genus Malassezia, such as M. globosa CBS7966 and M. restricta, which are known to produce indirubin. In one embodiment, relatively low levels of M. globosa strains on the skin would predict an enhanced response to AhR therapy.

[0130]

[0100] The enzymes that metabolize tryptophan or related metabolic / catabolic products can be predictive of response to AhR agonists. These enzymes can be involved in the kynurenine pathway starting from tryptophan. The differential activity of these enzymes can result in the reduction of certain AhR agonists and can be predictive of response to AhR agonist therapy. These enzymes can be measured via mRNA expression, protein level or enzyme activity. Specifically, these enzymes include: tryptophan 2,3-dioxygenase-2 (TDO) and / or indoleamine 2,3-dioxygenase-1 (IDO) or any other enzyme or combination of enzymes in the kynurenine pathway.

[0131]

[0130] Tryptophan may also be used in the body to produce serotonin and melatonin, meaning that levels of AhR activity may correlate with serotonin or melatonin, which can be measured in plasma or other biological samples and used as predictors of response to AhR agonist therapy.

[0132] CARD9 single base mutations are associated with inflammatory bowel disease.Specifically, patients with the following CARD9 mutations rs10870077, rs4077515, rs10781499 have increased risk of IBD.Since CARD9 plays a role in AhR biology, these patients may have differential responsiveness to AhR agonists compared to patients with more common CARD9 alleles.

[0133]

[0132] Compared to healthy controls, IBD patients have reduced levels of plasma IL-22 (Sakemi 2020). Baseline cytokine expression may be predictive of response to AhR agonists. Specifically, IL-6, IL-17A, IL-23, IL-10, and IL-22, which are involved in CARD9 biology and can be regulated by AhR signaling, can be measured alone or in combination with other metrics. For example, tryptophan / IL-22 ratio may be predictive of response to AhR therapy.

[0134] Other AhR-regulated genes include antimicrobial proteins or peptides.Specifically, these include the following proteins: S100 proteins (S100A7, S100A8, S100A9), late cornified envelope proteins (LCE1A, LCE3A, LCE3E), SLP1, LL37, SKALP, hBD2, regenerative islet-derived protein 3 gamma (REG3G), regenerative islet-derived protein 3 beta (REG3B).Low levels of these proteins indicate low AhR activity and can be suitable biomarkers for predicting response to AhR agonist therapy.

[0135]

[0134] Still other AhR-regulated genes include proteins involved in barrier function, including ZO-1, involucrin, occludin, tricelluin, claudin-1, and claudin-5. Relatively low expression of these genes or gene products may be suitable biomarkers predicting response to AhR agonist therapy.

[0136]

[0135] The methods herein enable diagnosis that can be used to enhance response rates for patients prior to initiating AhR therapy or, alternatively, not to initiate AhR therapy due to a low likelihood of clinical response.

[0137] Methods for preventing adverse events prior to administration of AhR agonists are also considered. While some AhR agonist-based compounds may have a favorable profile for treating disease, adverse events are more likely to occur if (1) the AhR pathway is constitutively and / or chronically turned on, (2) the AhR agonist has systemic bioavailability, (3) the AhR agonist is not easily metabolized, and / or (4) the agonist is more active than desired. Methods for preventing AE prior to or during AhR therapy entail diagnosing either genetic, epigenetic, or environmental factors that may lead to chronic, systemic, or overly robust activation of AhR. Other methods for preventing AE prior to AhR therapy may rely on careful selection of starting doses of drugs that can induce therapeutic AhR agonists and / or the use of metabolic induction doses, and may not require the use of diagnostics.

[0138]

[0137] Polymorphic genes associated with AhR signaling, HIF signaling, AhR agonist metabolic activity, or associated with the development of certain diseases, such as pulmonary arterial hypertension, may increase the risk of developing adverse events in combination with AhR agonist therapy.

[0139] In some embodiments, genetic alterations can be identified by polymerase chain reaction, mRNA expression, protein expression, or binding or enzyme activity. Alterations can be caused by single nucleotide polymorphisms (SNPs), rearrangements, translocations, deletions, repeats, or any other changes to DNA. Alterations can result in loss of function, altered function, increased function, altered expression, altered regulation, altered protein binding, or any other changes that may affect the development of adverse events.

[0140] Changes can also be epigenetic in nature, leading to changes in the way that a particular gene or group of genes is regulated.Some environmental effects are known to have long-term effects on how some genes are regulated.These changes can be detected by examining chromatin immunoprecipitation, or by evaluating the amount of histone modification, DNA methylation, or the expression of small non-coding RNA.

[0141] Environmental activity may also affect the final development of adverse events in combination with AhR agonist therapy.Activities that may lead to hypoxia or HIF signaling in the lungs in combination with AhR agonist may have negative consequences.Alternatively, environmental factors that change the metabolism of AhR agonist or increase the amount of alternative AhR agonist may have negative consequences in combination with AhR agonist therapy.

[0142]

[0141] The diagnosis of the above-mentioned can inform the treatment decision with AhR agonist before or after starting the therapy with AhR agonist. The information obtained has utility in that it can prevent or reduce possible adverse events or inform about the risk of their occurrence.

[0143]

[0142] Long-term, systemic, and constitutive activation of AhR can result in toxicity that can manifest as side effects or adverse events. This can occur due to unsafe drugs, unsafe doses, unsafe medication, combination therapy, concurrent activity, or other genetic, environmental, or metabolic factors. Unsafe drugs can be drugs that result in systemic exposure to AhR-stimulating metabolites that are not metabolized to parent compounds or inactive compounds. Unsafe doses or medications can result in systemic and / or chronic activation of AhR without the opportunity for this pathway to moderate.

[0144] Even limited exposure to high levels of AhR agonists can cause adverse events and / or toxicity. Many AhR agonists have other activities at high levels. For example, tapinarof is known to be cytotoxic at levels above 5 μM. Therefore, even acute high levels of AhR exposure can cause cell damage, which leads to acute inflammation. Other AhR agonists can induce the expression of multiple liver enzymes, which can be interpreted as a sign of liver damage. This can occur after the first dose when high levels are found in the liver.

[0145] In one embodiment, diagnosis will evaluate DNA polymorphisms in AhR or ARNT. Specifically, there are known polymorphisms at codons 517, 554 and 570 of AhR. Experiments have shown that Lys554+Ile570 and Lys554+Ile570+Ser517 variants cannot properly induce CYP1A1 upon exposure to TCDD (Wong et al., Biochemical and Biophysical Research Communications 288(4):990-996(2001)), and these individuals may also have impaired metabolism of some AhR agonists, specifically AhR agonists that are metabolized by CYP1A1. The ARNT SNP rs2228099 has been associated with essential hypertension and may have utility in predicting adverse events (AEs) in combination with AhR agonist use (Polonikov et al., Pharmacogenetics and Genomics 27(2):57-69 (2017)).

[0146] In another embodiment, diagnostics evaluating DNA polymorphisms in CYP450 enzymes, such as CYP1A1, CYP1B1 and / or CYP1A2, or xenobiotic metabolizing enzymes regulated by XREs are contemplated. For example, there is a known polymorphism in CYP1A1, such as a SNP: rs4646903, which maps to the 3' end of CYP1A1 (CYP1A1 *2A or 6235[T / C]), and rs1048943, a nonsynonymous SNP resulting in an Ile462Val substitution. Other known polymorphisms in CYP1A1 occur at codons 286 and 461. CYP1A1 polymorphism Val462 has been associated with an increased rate of lung cancer. This polymorphism is also found more frequently in Asians than in Caucasian populations. Known polymorphisms in CYP1A2 include the 163C>A polymorphism (rs762551). Known polymorphisms and mutations in CYP genes or associated XREs are incorporated herein by reference (Saito et al., Cytokine 136:155264 (2002)). Specifically, SNPs -1061, -1035, -1020 and -947 in the XRE for CYP1A1 and -1222, -376 and -265 in the XRE for CYP1B1.

[0147] In another embodiment, a diagnosis is provided that evaluates polymorphisms near the XRE that alter AhR function. For example, rs2470893 is a known SNP located 196 base pairs from the CYP1A1 promoter XRE (Liu et al., Drug Metabolism and Disposition 46(9):1372-1381(2018)).

[0148]

[0147] In another embodiment, a diagnostic is provided that assesses a polymorphism in an aryl hydrocarbon receptor inhibitor that alters AhR function, for example, the Ala185Pro polymorphism.

[0149]

[0148] In another embodiment, a diagnosis is provided that evaluates a polymorphism in the gene BMPR2, which has been associated with the development of PAH (Masaki et al., Proceedings of the National Academy of Sciences 118(11)(2004)). In a related embodiment, the CYP1B1 polymorphism N453S is evaluated in combination with a polymorphism in BMPR2.

[0150]

[0149] In another embodiment, a diagnosis evaluating a polymorphism in the gene HIF1α, specifically the AA genotype at rs12434438, is envisaged.

[0151] In one embodiment, the diagnosis generates a polymorphism risk score based on multiple polymorphisms that are indicative of the relative risk of taking an AhR agonist, which may or may not be combined with other clinical, medical, or diagnostic data to make a decision regarding the suitability of a particular AhR agonist or the dosage or dosing of such an agonist to treat a disease.

[0152] In another embodiment, the diagnosis evaluates recombinant AhR that directly binds to AhR agonist or has known polymorphisms of AhR that induce CYP1A1 expression. In the former case, recombinant AhR protein or simply cell lysate is bound to antibody against AhR, the solution of AhR agonist is washed away, and shift in light scattering is observed. Alternatively, a secondary antibody is used in an enzyme-linked immunosorbent assay (ELISA)-based assay. Alternatively, recombinant AhR is added to ARNT together with AhR agonist compound, and the binding to DNA response element is determined using electrophoretic mobility shift assay, or the ability to induce CYP1A1 is evaluated in Hepa-1 group B mutant cells that express low endogenous AhR levels and do not normally express CYP1A1 in response to AhR agonist.

[0153] In another embodiment, a diagnosis is provided that evaluates the ability of microsomes or cells biopsied from a patient to metabolize AhR agonists.In one example, microsomes or cells derived from blood, skin, vasculature, liver, intestine, colon or lung are envisaged.In this example, the activity of these cells or microsomes is detected by using a reporter cell line to evaluate their ability to metabolize AhR agonists.Other similar types of readouts could be used, including but not limited to colorimetric or enzymatic or light scattering.

[0154] In another embodiment, the chromosomal structure or DNA methylation status of CYP1A1 promoter is investigated.In one case, the DNA structure of CA1 and CA2 CYP1A1 promoter regions is evaluated through nuclease accessibility assay as described in Vorrink 2014.More closed promoter regions are expected to be less inducible to activated AhR, which may result in less CYP1A1 induction and longer exposure to AhR agonist therapy, which may lead to adverse events.

[0155] Concomitant drugs or activities may function to activate AhR or enhance the action of AhR agonists.Concomitant drugs that activate AhR include ketoconazole or omeprazole.Specific activities of concern that may lead to AhR activation include AhR agonist intake through diet, such as indole-3-carbinol-containing vegetables such as broccoli, smoking, which leads to exposure to AhR agonist polycyclic aromatic hydrocarbons, exposure to AhR agonists in the form of pollutants, or exposure to hyperoxic conditions, such as when scuba diving.

[0156] Similarly, concomitant drugs, drugs or foods that inhibit the metabolism of AhR agonists may cause unsafe long-term exposure to AhR agonists.For example, indirubin is metabolized by CYP1A1, and known inhibitors of CYP1A1, such as grapefruit juice (bergamottin) and marijuana, including the isolated compounds from marijuana, such as tetrahydrocannabinol (THC) or cannabidiol (CBD), may cause the expression of CYP1A1 to be inhibited and higher and / or longer exposure to indirubin after administration.

[0157]

[0156] Ingestion of foods, drugs or supplements that increase the absorption or degradation of AhR agonists may increase systemic exposure. Specifically, high-fat or high-alcohol diets may increase the absorption of some AhR agonists and may be recommended not to be consumed.

[0158] In another embodiment, the patient who is judged to have increased risk for developing pulmonary arterial hypertension will be excluded from treatment with AhR agonist or will be treated with a relatively low dose and / or dosage of AhR agonist.Examples of patients with increased risk include those with family history of hereditary PAH, connective tissue disease, human immunodeficiency virus (HIV) infection, portal hypertension, congenital heart disease, schistosomiasis, or those with drugs or toxins associated with PAH, including but not limited to methamphetamine, fenfluramine, aminorex, dexfenfluramine, benfluorex, dasatinib, and toxic rapeseed oil.

[0159] In another embodiment, laboratory values ​​or clinical diagnostic values ​​related to cardiovascular function may form the basis of diagnosis. These include systolic blood pressure (systolic BP) or mean pulmonary artery pressure (mPAP). In other examples, the laboratory values ​​are pulmonary artery systolic pressure (PASP), left atrial diameter (LAD), left ventricular end diastolic diameter (LVEDD), left ventricular ejection fraction (LVEF), inferior vena cava diameter, right atrium area (RA area), tricuspid annular systolic excursion (TAPSE) or tricuspid regurgitation pressure difference (TRPG). In other cases, cardiovascular function will be focused on valve abnormalities or valvular heart disease and will be evaluated via echocardiograms looking for aortic regurgitation, mitral regurgitation, valve thickening or valve movement restriction. In some embodiments, the reference test value is a measurement of PASP as estimated via echocardiography or measured via right heart catheter prior to initiation of therapy or after 1, 3, 6, 12, 18, 20, or 24 months of continuous or intermittent therapy with an AhR agonist.

[0160]

[0159] In one embodiment, the drug labeling for AhR agonists will specifically exclude the concomitant use of CYP1A1, CYP1B1 and / or CYP1A2 inhibitors or medicines or drugs with AhR agonist activity.

[0161]

[0160] In another embodiment, the drug label for the AhR agonist would specifically exclude concomitant use of activities or environmental factors that may result in hypoxia, such as scuba diving, mountain climbing, high altitude flying or prolonged residence above a certain altitude, such as above 7,000, 8,000, 9,000, 10,000 or 11,000 feet or more.

[0162]

[0161] In another embodiment, the drug label for the AhR agonist would specifically exclude patients with medical conditions that lead to hypoxia, such as sleep apnea.

[0163]

[0162] In another embodiment, the drug label for the AhR agonist would specifically exclude consumption of a high fat meal or consumption of alcohol.

[0164]

[0163] Any of the above methods, alone or in combination, may form the basis of a diagnosis to prevent or reduce the onset of an adverse event prior to or during the use of AhR agonist therapy.

[0165] In some cases, it may not be possible to adequately reduce or eliminate the occurrence of adverse events prior to the step of starting AhR agonist therapy, and in these cases, the diagnosis of monitoring for the occurrence of adverse events before they occur or become severe is justified.Provided is a method for monitoring, reducing, and / or treating adverse events during the administration of AhR agonist.

[0166] In one embodiment, the diagnosis for AhR systemic activation is the EC of an AhR agonist required for activity against the human AhR. 50or another other reasonable amount and depending on the pharmacokinetic profile of the AhR agonist in plasma, blood or tissue. Diagnosis is based on the concentration (e.g., EC 50 ) can be used to ensure that the patient does not exceed the threshold concentration. For example, in the case of indigo, plasma levels of more than 1 nM, 10 nM, 50 nM, or 100 nM can serve as cutoffs. In the case of indirubin, plasma levels of 0.1 nM, 1 nM, 10 nM, or 25 nM could serve as cutoffs. The period during which the patient should not exceed this threshold concentration can be one day, one week, one month, three months, or some other period.

[0167] Constitutive activation of AhR can result in the reduction of free ARNT and therefore the reduction of the heterodimeric transcription factor hypoxia-inducible factor (HIF). In these circumstances, genes or proteins regulated by HIF and the processes regulated by these proteins may be inhibited. For example, there may be a reduction in erythropoietin in blood. Alternatively, there may be a reduction in red blood cell count, a process regulated by erythropoietin, which could be assessed by hematocrit. Measurement of the hematocrit of white blood cells, hemoglobin, and platelets can be correlated with AhR activity. In one embodiment, a 10% decrease in hematocrit from baseline may indicate excessive or constitutive systemic AhR exposure.

[0168] In another embodiment, the diagnostic test measures the capacity of cells or microsomes from patients using ethoxyresorufin-O-deethylase (EROD) assay, which is an assay for measuring the activity of xenobiotic metabolizing enzymes such as CYP1A1 and CYP1A2 in their ability to mediate the deethylation of 7-ethoxyresorufin substrate to resorufin (https: / / protocolexchange.researchsquare.com / article / nprot-3473 / v1). Increased activity in this assay indicates greater expression of these enzymes and greater exposure to AhR agonists. For example, measuring significant EROD activity in PBMCs with AhR expected to be locally acting would indicate systemic exposure.

[0169] In another embodiment, diagnostics are provided that measure blood levels of enzymes that indicate systemic AhR activation and / or liver toxicity.For example, diagnostics are provided that measure blood levels of liver enzymes such as alanine aminotransferase (ALT) or aspartate aminotransferase (AST).In an embodiment, ALT levels that are elevated more than 2 times above baseline for more than one week indicate the possibility of developing adverse events.

[0170] In another embodiment, a diagnosis is provided that evaluates genes or gene products regulated by XRE.The genes can be, for example, CYP1A1, CYP1A2, CYP1A2, CYP19A1, IGFBP10, GSTP1 or any of the genes listed in Adachi et al., Toxicological Sciences.80,161-169 (2004).These genes or gene product combinations can function as biomarkers with predictive value for the development of adverse events of AhR therapy.For example, a 50% increase in both CYP1A1 and AhRR from baseline in PBMCs can signal too much systemic AhR agonist exposure.

[0171]

[0170] The expression of metabolic enzymes such as CYP1A1 and CYP1A2 can increase the expression of metabolic products or decrease the expression of known CYP1A1 and CYP1A2 substrates. For example, CYP1A1 metabolism of tryptophan substrates can increase the level of serotonin. The level of serotonin in blood is normally very low. Therefore, the diagnosis of evaluating serotonin or tryptophan, 5-hydroxy-L-tryptophan (5-HTP) or 5-hydroxyindoleacetuc acid (5-HIAA) levels in blood or tissues may have diagnostic properties.

[0172]

[0171] In a relevant example, the substrate of CYP1A2 is melatonin. Thus, finding lower than expected levels of melatonin in blood, urine or saliva could be diagnostic of systemic AhR activation.

[0173] Cardiovascular monitoring can identify adverse events during treatment with AhR agonists before the patient becomes symptomatic. Findings from continuous cardiovascular monitoring can form the basis of a risk evaluation and mitigation strategy (REMS). In the case of an AhR agonist drug REMS program, patients must participate in and comply with ongoing cardiac monitoring requirements, which may include echocardiogram monitoring and / or periodic safety questionnaires. In one embodiment of the AhR REMS program, patients receive counseling regarding the possible risks of AhR agonists, must consent to echocardiograms every six months, and can only obtain prescriptions filled by pharmacies participating in the AhR REMS program in order to continue receiving the prescription. A pre-approved prescribing physician will evaluate the echocardiogram every six months to look for adverse signals such as changes in pulmonary artery pressure or valvular heart disease or new valvular abnormalities. The physician will assess the risk and pass a recommendation to dispense or not dispense the patient's prescription accordingly to a central computerized REMS program that relays the decision to the patient and the associated pharmacy.

[0174] Further information regarding the REMS program can be found in U.S. Patent Nos. 6,045,501, 6,315,720, 6,561,976, 6,561,977, 6,755,784, 6,869,399, 6,908,432, 7,141,018, 7,874,984, 7,959,566, 8,204,763, 8,315,886, 8,589,18 REMS programs have been described using thalidomide (U.S. Pat. Nos. 7,668,730, 7,765,106, 7,765,107, 779,717, 7,895,059, 8,263,650, 8,457,988, 8,589,182, and 8,731,963) for the treatment of multiple myeloma, and fenfluramine (U.S. Pat. No. 10,950,331) for the treatment of epilepsy. The disclosures relevant to REMS programs in these publications are incorporated herein by reference.

[0175]

[0174] AhR agonist REMS programs may rely on the use of a central controller, which is a pre-programmed centralized database linked to the Internet or other modes of electronic communication that can analyze data and communicate findings to physicians, pharmacies, and patients. In some cases, the central controller will not allow an initial or renewed prescription for an AhR agonist to be filled unless a set of conditions are met by a user authorized to enter the appropriate data. For example, one condition may include the timely entry of data from a regular echocardiogram demonstrating that cardiovascular risk is not increased.

[0176]

[0175] Any of the above methods, alone or in combination, may form the basis of a diagnosis to prevent or reduce the onset of adverse events after initiating the use of AhR agonist therapy.

[0177] In any of the above aspects and embodiments, the diagnostic information can be utilized by the patient, treatment protocol, or healthcare provider to change the dosage or dosing schedule, or can be used to restrict the potential patient from taking a particular AhR agonist compound, or from providing a different AhR agonist compound that was originally considered. In some embodiments, the patient may be recommended to reduce the dosage of the AhR agonist compound to three times a day, twice a day, or once a day, once every five days followed by two days off, every other day, one week followed by one week off, one month followed by one month off, or any other intermittent dosing regimen that may prevent or reduce the occurrence of adverse events. Alternatively, the patient may be recommended to reduce the daily dose by 25%, 50%, 75%, or any other amount.

[0178] In other embodiments, subjects are given concomitant medications that can lead to increased metabolism of AhR agonists, or are given medicines that act as local AhR antagonists.For example, patients can be given drugs that are known to increase the activation of CYP1A1 in the lungs to prevent the development of PAH during AhR agonist therapy.Alternatively, patients may be recommended diet or intake mechanisms to reduce systemic exposure, such as by taking AhR agonists with food, a glass of water, a glass of milk, or in the absence of high-fat or high-alcohol meals.

[0179]

[0178] In any of the above embodiments, the diagnosis has predictive value for detecting, preventing or reducing the onset of adverse events or side effects including, but not limited to, folliculitis, essential hypertension, pulmonary arterial hypertension, pneumonia, respiratory failure, myocardial ischemia, myocardial infarction, pericarditis, pleuritis, peritonitis, pneumonia, chest pain, abdominal pain, diarrhea, vomiting, intussusception, joint pain, back pain, liver damage, liver enzyme changes, infection, peripheral neuropathy, headache, pancytopenia, and / or leukopenia.

[0180] In any of the above embodiments, various interventions can be performed after the diagnosis is obtained to minimize the severity of possible adverse events or side effects.For example, the doctor may administer antioxidants, angiotensin II receptor antagonists, prostacyclin analogs or receptor agonists, endothelin receptor antagonists, PDE-5 inhibitors or soluble guanylate cyclase stimulants to minimize or prevent the onset of PAH.

[0181] In any of the above embodiments, the diagnostic information can be used to adjust the dose or administration of the drug containing the AhR agonist. The information can be used to select the patient for additional monitoring or testing to minimize the possibility of adverse events. The information can be used to include or exclude the patient from clinical trials or from eligibility to receive the drug. The information can be used on the label of the drug product. The information can be used to adjust the concomitant medication, supplements, food or beverage, diet, habits or activities that the patient should or should not participate in, such as exposure to hypoxic conditions, the intake or inhalation of tobacco or marijuana or cannabinoid-based products, or the use of hormonal contraception.

[0182] When high exposure to AhR agonist or systemic activation of AhR pathway or insufficient metabolism of AhR agonist is detected, the method herein considers intervention to prevent or reduce toxicity.If a method is known to determine which patient or activity is problematic with a given AhR agonist, then a treatment strategy with modified dosage or dosing can be implemented.Alternatively, such patients can be recommended not to take AhR agonist.

[0183] The methods described herein provide the basis for a set of diagnostics that inform treatment decisions prior to or during AhR agonist therapy. These methods have utility in increasing response to therapy, as well as in preventing and mitigating adverse events.

[0184] Strategies for reducing possible adverse events prior to administration of AhR agonists are also contemplated, and the strategies or methods do not require diagnosis.For example, the strategy of pre-administration of a compound prior to therapeutic dosing with AhR agonists, without the need for diagnostic testing, can reduce and / or prevent the occurrence of adverse events.

[0185] In one embodiment, the strategy uses sub-therapeutic doses of therapeutic AhR agonists as a means of turning on local metabolism. This strategy serves as a means of increasing the rate of first-pass metabolism, minimizing systemic exposure to subsequent therapeutic doses of AhR agonists.

[0186]

[0185] Metabolic enzymes such as CYP1A1 have half-lives measured in hours or days, and therefore this strategy can be utilized with AhR agonists that are to be given as once-daily therapy. The same strategy would have an impact on AhR agonists administered every other day, twice daily, or three times daily.

[0187] This strategy can utilize a dose that is 0.1%, 1%, 5%, 10%, 20%, 30% or 50% of the therapeutic dose.It can be administered 1 week, 3 days, 2 days, 1 day, 12 hours, 8 hours, 4 hours, 2 hours, 1 hour, immediately before or simultaneously with the therapeutic dose.In an embodiment, the administering step increases the local AhR metabolic rate.

[0188] This strategy can result in low or no systemic exposure to the withdrawal dose, and by definition will induce one or more enzymes that can metabolize the AhR agonist administered thereafter locally.The withdrawal dose may or may not have any therapeutic effect by itself.The withdrawal dose can be the AhR agonist itself or itself.

[0189] This strategy can result in low or no systemic exposure of the therapeutically administered AhR agonist. This strategy is useful for measuring the C that would be measured without utilizing this strategy. max Lower than C max The C of therapeutic AhR agonists in plasma or tissues may result in max can be reduced by 100%, 99%, 90%, 75%, 50%, 25% or 10% or any other clinically meaningful amount.

[0190] This strategy may result in a lower area under the curve (AUC) of a therapeutically administered AhR agonist in plasma or tissue than would be measured without utilizing this strategy. The reduction in AUC may be assessed from 0 to 24 hours and may be reduced by 100%, 99%, 90%, 75%, 50%, 25% or 10% or any other clinically meaningful amount.

[0191] This strategy can result in lower liver exposure and therefore lower production of liver enzymes.Some liver enzymes, such as ALT and AST, can be produced when the liver is damaged or stressed, and high levels of these and related biomarkers can be considered as adverse events.This strategy can result in a reduction in plasma ALT and / or AST by 100%, 99%, 90%, 75%, 50%, 25% or 10% or any other clinically meaningful amount compared to a strategy that does not utilize a run-in dose.This reduction can be observed 1 day, 2 days, 3 days, 1 week, 1 month, 2 months, 3 months, 6 months, 9 months or 1 year after the start of AhR agonist therapy.

[0192]

[0191] This strategy can be resumed if necessary if the patient misses a dose. This strategy could be resumed if the patient misses 1, 2, 3, 5 or 10 doses or has not taken a dose for 24, 48, 74, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks. The strategy could be resumed based on the results of any of the enclosed diagnostic tests. EXAMPLES

[0193] III. Examples The following examples are illustrative in nature and are not intended to be limiting in any way.

[0194] Example 1 Total baseline AhR agonist levels in plasma Prior to the initiation of therapy with derivatives of indole-3-acetic acid for the treatment of moderate to severe Crohn's disease, plasma samples are collected in the morning after an overnight fast. Plasma is tested using the AhR luciferase assay in 10% plasma as described in Masaki 2021. Patients have 95% reduced AhR luciferase activity compared to the mean activity level of healthy volunteers. Compared to matched plasma from moderate to severe Crohn's disease patients, the plasma has just 25% of the mean activity. Thus, patients have dramatically reduced plasma AhR agonist activity levels compared to healthy control plasma and compared to matched moderate to severe Crohn's disease patients. Based on clinical biomarker data, patients with less than 50% of the relative AhR luciferase activity in plasma compared to moderate to severe Crohn's disease plasma are more likely to meet the primary clinical endpoint of clinical remission (defined as a Crohn's Disease Activity Index of less than 150) by week 4 when treated with derivatives of indole-3-acetic acid. The gastroenterologist writes a prescription for indole-3-acetic acid derivative therapy based on these data, as the patient is more likely to respond to the therapy than other similar patients.

[0195] Example 2 Total baseline AhR agonist levels in feces Prior to the initiation of therapy with delayed release tablets containing indirubin prodrugs for the treatment of refractory moderate to severe ulcerative colitis, a set of three fecal samples is collected at the same time on three consecutive days at home and frozen. The samples are homogenized in saline solution, processed to remove any particulate matter, sterile filtered, and then frozen. The samples are then added to the AhR luciferase assay at 10% of the total volume. The average relative AhR luciferase activity compared to refractory moderate to severe ulcerative colitis matched with Mayo score is 800%. Clinical data from previous studies have suggested that patients with the highest relative AhR luciferase activity in feces are least likely to respond, since the colon is likely already stimulated with AhR agonists. This means that the patient or health care provider decides to try alternative therapies.

[0196] Example 3 Urinary AhR agonist levels Prior to the initiation of therapy with delayed release gel capsules containing a suspension of indigo for the treatment of ulcerative colitis, the total amount of indirubin and indigo in urine is determined from six frozen urine samples taken on awakening and before going to bed over a period of three days (Adachi et al., Toxicological Sciences 80(1):161-169 (2001)). Healthy urine contains an average of 0.22 μM indigo and 0.19 nM indirubin. However, this patient contains less than 0.01 μM indigo and indirubin combined. Previous studies suggest that less than 0.10 μM of either indigo or indirubin makes a candidate more likely to respond to therapy. Thus, the patient is started on indigo therapy.

[0197] Example 4 Serum cytokine levels Prior to the study of the use of oral delayed release dosage forms containing solid dispersions of indigo for the treatment of ulcerative colitis, a baseline blood sample is obtained. The level of IL-22 in serum is determined via ELISA. The patient has a level of less than 1 pg / mL. The average level of IL-22 for UC patients was determined to be 16 pg / mL (Sakemi et al., Journal of Human Genetics 47(8):419-444(2020)). That is, the patient has a lower level of IL-22 than a typical UC patient, and this low level of IL-22 suggests that a drug that induces IL-22 expression is relatively likely to be effective. That is, the patient may respond better to AhR therapy for UC and is treated with an oral indigo product.

[0198] Example 5 Levels of specific bacteria and abundance of bacterial species in feces Prior to the initiation of therapy with suppositories containing indirubin for the treatment of proctitis, fecal samples were obtained and the levels of specific organism species and the predominant bacterial phyla were determined by 16s rRNA sequencing. The levels of Lactobacillus, a phylum known to produce AhR agonists, were 100 cfu / g feces compared to healthy controls with an average of 30,000 cfu / g. Specifically, Lactobacillus bulgaricus OLL1181, a bacterium known to produce AhR agonists, was not detected in the feces compared to healthy controls with an average of 1,000 cfu / g. In addition, bacterial species richness, as measured by unique amplicon sequence variants, was determined. Patients had an average organism species richness of 10% compared to typical UC patients. Based on the data, patients with reduced levels of bacterial species and reduced levels of specific bacterial species capable of producing AhR agonists are relatively likely to respond to therapy with indirubin suppositories and to begin therapy.

[0199] Example 6 Levels of CYP1A enzyme activity in colonic biopsies During endoscopy to assess the severity of ulcerative colitis in a patient, a biopsy of the inflamed colon is collected. The biopsy is frozen and then processed for EROD assay to assess the level of CYP1A1 activity. The patient has a very low level of EROD activity (less than 2 standard deviations from the mean) in the colon biopsy compared to matched UC control, suggesting that the patient has a low level of AhR activity and is more soluble and responsive to therapy with AhR agonists than patients with high EROD activity. Thus, the patient's gastroenterologist decides to treat the patient with AhR agonists.

[0200] Example 7 PBMC response to AhR agonists Prior to the start of therapy with topical AhR agonist for the treatment of pustular psoriasis, blood samples are collected. PBMCs are processed and shipped fresh for stimulation with and without AhR agonist FICZ. After 24 hours, EROD activity is measured. Patient samples are highly responsive to FICZ as measured by high EROD activity compared to EROD activity from healthy normal PBMCs. The ratio of stimulated EROD activity to unstimulated can be indicative of baseline AhR activity and responsiveness to AhR therapy. In other words, a high ratio of stimulated EROD activity to unstimulated suggests that the patient may be responsive to therapy with AhR agonist. Based on the data, the patient's dermatologist prescribes a topical AhR agonist for the treatment of pustular psoriasis.

[0201] Example 8 Polymorphisms associated with AhR agonist deficiency Prior to initiation of therapy with indirubin ointment for the treatment of hidradenitis suppurativa, a cheek swab is obtained and the patient is screened for inflammatory disease risk alleles associated with AhR signaling, including a mutation in CARD9. The results show that the patient has a mutation in CARD9 (mutation rs10870077). Based on this result, the patient has impaired baseline tryptophan metabolism leading to impaired AhR signaling contributing to their disease and may be relatively responsive to AhR agonist therapy. Thus, the patient is initiated on indirubin therapy.

[0202] Example 9 mRNA levels of AhR-induced transcripts Prior to the start of therapy with FICZ derivative cream for the treatment of hidradenitis suppurativa, a skin biopsy of active nodule is obtained.The biopsy has 5% of the normal level of mRNA expression from matched HS patients for S100A7, a protein induced by AhR signaling.In other words, the patient has an abnormally low level of S100A7, which indicates a reduced level of AhR signaling that may be related to a low level of AhR agonist in skin.In other words, the patient is more likely to respond to therapy with AhR agonist therapy, and therefore the patient is treated with FICZ derivative cream.

[0203] Example 10 Tryptophan levels in the skin Prior to the start of therapy with tapinarof gel for the treatment of psoriasis vulgaris, a skin biopsy is obtained from plaque.The biopsy has 2% of the normal tryptophan level from matched patients.That is, the patient has abnormally low tryptophan, which suggests that it may be related to low levels of AhR agonist in skin and low baseline AhR signaling.That is, the patient is relatively likely to respond to therapy with AhR agonist therapy, and the patient is treated accordingly.

[0204]

[0101] Examples of how to prevent adverse events prior to initiation of therapy with AhR agonists that require diagnosis:

[0205] Example 11 Examination of AhR agonist metabolism in microsomes prior to therapy Prior to the start of oral Meisoindigo therapy for the treatment of rheumatoid arthritis, a physician requests a diagnostic to evaluate the subject's ability to properly metabolize Meisoindigo. Blood microsomes are extracted from the subject and incubated in a medium containing 10 nM Meisoindigo for 24 hours. The microsomes are removed via ultracentrifugation, and the remaining medium is added to a reporter cell line (such as the product IB06001 sold by Indigo Biosciences) that expresses luciferase behind the promoter for human CYP1A1 and can detect AhR agonist activity. After 12 hours of incubation and the addition of luciferin, the cell line produces the same amount of light as the control medium containing 10 nM Meisoindigo, suggesting that little to no Meisoindigo is metabolized in the presence of microsomes over 24 hours. Control samples of microsomes from healthy normal individuals typically emit the same amount of light after 24 hours as a control solution containing 0.1 nM Meisoindigo, suggesting that healthy normal individuals are able to metabolize 99% of the added Meisoindigo over a 24-hour period. Based on the results of this diagnosis, the physician believes that the patient has a defect that makes him or her a poor metabolizer of Meisoindigo and recommends that the patient not use Meisoindigo due to the potential for adverse events.

[0206] Example 12 Testing for induction of CYP1A1 in test spots prior to therapy Prior to the start of therapy with 0.02% indirubin ointment for psoriasis vulgaris, the doctor applies the ointment to a 20 cm diameter test spot. The next day, the doctor obtains biopsies of treated skin from these treated spots and control biopsies from untreated adjacent skin, and performs immunofluorescence staining for CYP1A1 and AhR proteins and for DNA via DAPI co-staining. Compared to control skin, no more CYP1A1 protein is detected in treated skin. In the same biopsy, AhR nuclear translocation is seen in the majority of epidermal cells in treated skin, but not in control skin. The doctor is concerned that this patient has a defect in CYP1A1 expression, which may lead to an inability to metabolize indirubin, leading to indirubin bioaccumulation. Therefore, the doctor writes a prescription for 0.002% indirubin ointment rather than 0.02% indirubin ointment.

[0207] Example 13 Testing the ability to metabolize a test dose of AhR agonist in vivo Prior to the start of therapy with sappan, urine and plasma samples are collected. The patient is given a single capsule containing 1.5 g of sappan, which is swallowed in the examination room. After 24 hours, the patient returns to the examination room and new blood urine and plasma samples are collected. Normally, a patient taking a test dose of 1.5 g of sappan can metabolize the indirubin contained in sappan to metabolites such as indigo carmine, so that after 24 hours, less than 0.1 nM indirubin remains in the plasma, and therefore a concomitant increase in indigo carmine (>5 nM) is observed in the urine. At baseline, the results show 0.01 nM indirubin in the plasma and 0.2 nM indigo carmine in the urine. After 24 hours, the level of indirubin in the plasma is 10 nM, and the level of indigo carmine is still 0.2 nM in the urine. Thus, the patient has a defective ability to metabolize indirubin to indigo carmine and is initiated on long-term indigo therapy.

[0208] Example 14 Sequencing of polymorphisms associated with insufficient AhR metabolism Prior to the initiation of indigo therapy for the treatment of ulcerative colitis, the physician performs DNA testing on the patient sample to sequence CYP1A1 and search for SNPs associated with defects in indigo metabolism.The subject has a polymorphism (T3801C) in CYP1A1, which is associated with poor indigo metabolism, and is therefore recommended not to use any AhR agonist that is metabolized by CYP1A1.

[0209] Example 15 PAH risk score prior to initiation of AhR therapy Prior to initiation of therapy of an amorphous solid dispersion of indigo designed to provide systemic exposure for the treatment of rheumatoid arthritis, a comprehensive PAH risk score is performed. If the risk score is >2, AhR therapy will not be initiated, if it is 1-2, it will be initiated with additional monitoring, and if it is <1, it will be initiated with standard monitoring. The score is the average of multiple weighted components, all scored 0-2. The components include genetic risk (BMPR2, AhR, CYP haplotypes), environmental components (dietary AhR intake, dietary CYP inhibitor intake, smoking status), age, sex, hypoxic exposure (air travel, home altitude, scuba diving, mountain climbing, sleep apnea, COPD, interstitial lung disease, exposure to environmental pollutants that are hypoxia mimics) and current PAH risk factors (relatives with PAH, mean pulmonary artery pressure and pulmonary artery systolic pressure, total plasma AhR agonist levels). In this case, the patient has a risk score of 0.55 and is considered to be at relatively low risk for developing PAH, and therefore AhR therapy for rheumatoid arthritis is initiated.

[0210] Example 16 Cardiac monitoring prior to AhR agonist administration Prior to initiating therapy with an oral suspension containing ITE for the treatment of lupus, physicians must perform pretreatment cardiac monitoring in accordance with the drug label. In this patient's case, the echocardiogram identifies evidence of valvular heart disease as indicated by additional features of valvular heart disease, namely moderate mitral regurgitation with valvular thickening. Based on these findings, the patient is considered to be at high risk for the development of adverse cardiac events and is not eligible for treatment with an AhR agonist.

[0211] Example 17 Pull-in dose to minimize changes in liver enzymes During clinical development, it was noted that standard dosing with 50 mg indigo twice daily in a modified release tablet formulation led to a 5-fold increase in ALT and AST after 2 days of dosing in 50% of patients. Therefore, the product label specified a clinical regimen whereby a withdrawal dose of 5 mg indigo was administered once daily for 2 consecutive days prior to the initiation of a therapeutic dose of 50 mg. This dosing regimen did not result in any significant changes in ALT or AST levels after 2 days of therapeutic dosing.

[0212] Example 18 Withdrawal dose after discontinuing therapy In clinical development, it was noted that once-daily dosing of delayed-release indirubin led to headaches during the first week of therapy. The entrainment dose of the CYP1A1 inducer, in this case omeprazole, a weak AhR agonist, in a standard omeprazole delayed-release capsule formulation containing 20 mg of drug, acts as a metabolic inducer and increases the C of indirubin. maxIt has been shown that reducing the incidence of headaches during the first week of therapy reduces the incidence of headaches. The label on delayed release indirubin indicates that patients should take a delayed release capsule containing 20 mg omeprazole 12 hours before taking the first dose of delayed release indirubin. The label also instructs that patients should take a delayed release capsule containing 20 mg omeprazole 12 hours before taking a subsequent dose of delayed release indirubin as a means to reduce the incidence of adverse events such as headaches in the event that the patient misses or stops taking daily delayed release indirubin for two or more doses.

[0213]

[0211] Examples relating to methods for minimizing or reducing adverse events while administering therapy with AhR agonists:

[0214] Example 19 Dose reduction due to systemic detection of liver enzymes A patient with resistant ulcerative colitis is treated with a delayed release capsule containing 1 gram of ALT twice daily. Prior to the start of therapy, a blood sample is obtained and the blood level of ALT is determined using standard laboratory techniques. Weekly blood draws are scheduled for the first six weeks. After two weeks of therapy, the blood level of ALT is five times the normal level. The diagnosis dictates a half-reduction in the dose to 0.5 g per day. After two weeks, the blood level of ALT has returned to normal and the patient is maintained on 0.5 g per day.

[0215] Example 20 Reduced dose and dosing due to accumulation of AhR agonists in plasma A patient with pouchitis is being treated with an oral suspension containing 10 mg indirubin twice daily. Plasma samples are obtained weekly during the first 6 weeks of therapy and sent for PK analysis of indirubin levels. At baseline, indirubin is undetectable. At week 1, indirubin level is 5 ng / mL. At week 2, indirubin level is 25 ng / mL. The physician reduces the dose to 2.5 mg once daily. At week 3, indirubin level is 2 ng / mL. At week 4, indirubin level is 3 ng / mL. At week 5, indirubin level is 2 ng / mL. At week 6, indirubin level is 3 ng / mL. The patient is responding adequately to therapy. Based on the plateauing of plasma indirubin levels at levels not expected to systemically activate the AhR, the physician maintains the dose at 2.5 mg / day to prevent the development of adverse events.

[0216] Example 21 Dose reduction and subsequent patient discontinuation due to elevated levels of systemic AhR signaling by detecting CYP1A1 mRNA in PBMCs A patient with Crohn's disease is administered 50 mg of indirubin in an immediate release tablet once a day. A diagnostic is set up to assess both CYP1A1 and CYP1A2 mRNA levels in the blood using RT-PCR of peripheral blood mononuclear cell mRNA every 2 weeks. After 2 weeks of therapy, the level of CYP1A1 mRNA increases 12-fold and the level of CYP1A2 mRNA increases 3-fold. The physician reduces the dose to 10 mg per day and the medication to once every other day. After 2 weeks, compared to baseline levels, the level of CYP1A1 mRNA increases 15-fold and the level of CYP1A2 mRNA increases 6-fold. Based on the continued increase in both biomarkers despite the dose and medication reduction, the physician immediately instructs the patient to stop therapy.

[0217] Example 22 Monitoring of plasma biomarkers associated with the development of PAH During the course of 3 months of treatment for atopic dermatitis with oral laquinimod, the patient comes in for regular follow-up visits, complaining of shortness of breath. The patient has responded well to therapy and has had no other adverse events. The physician orders a blood test for serotonin. A blood serotonin of 950 ng / mL is found (normal range is 101-283 ng / mL). In light of the findings, the physician is concerned that systemic CYP1A1 signaling is too high and recommends interrupting therapy and regular echocardiograms to evaluate for the possible development of PAH.

[0218] Example 23 Administration of AhR antagonists in combination with AhR agonists During the course of therapy with a nanoparticle formulation of synthetic indirubin for the treatment of gastritis, the patient complains of ad adverse events, in this case shortness of breath and difficulty performing physical exercise. Although the patient responds well to the therapy, the physician is concerned about the possible development of additional PAH-like symptoms. The physician maintains the indirubin therapy but prescribes the patient an inhalable AhR antagonist, in this case inhaled 1-methyl-N-[2-methyl-4-[2-(2-methylphenyl)diazenyl]phenyl-1H-pyrazole-5-carboxamide (CH223191), which is designed to reduce AhR signaling in the pulmonary epithelium. After two weeks of combined therapy, the patient no longer has shortness of breath, can exercise without problems, and is still responding well to the therapy, and both drugs are maintained.

[0219] Example 24 Label warning related to the risk of hypoxic conditions in combination with AhR agonists A patient visits the office prior to initiation of therapy with FICZ derivative eye drops for the treatment of severe refractory uveitis. The patient is a candidate for therapy based on the drug's label, but lives in Denver and is an avid climber with sleep apnea, and is therefore regularly exposed to hypoxic conditions. The drug's label warns of the possibility of development of PAH-like symptoms if the product is used in patients who frequently experience hypoxia, and therefore the physician prescribes the drug but requests that the patient return to the office monthly for an echocardiogram to monitor for the development of PAH symptoms.

[0220] Example 25 Cardiac risk reduction program during AhR agonist therapy Based on the labeling of the oral tablets containing linomide, the patient must participate in a Risk Evaluation and Mitigation Strategy (REMS). In the case of this drug, the patient must comply with ongoing echocardiogram monitoring requirements. Although the patient was not considered at high risk for the development of adverse cardiac events at baseline, after 6 months, data from the echocardiogram show signs of valvular heart disease as indicated by moderate aortic regurgitation and valvular motion restriction. The prescribing physician evaluates the echocardiogram and determines that the patient should be discontinued. The physician communicates this information to the REMS program by recording the data on a secure server. The server then alerts the patient and the prescribing pharmacy that the patient is no longer eligible for a refill of the linomide tablet prescription.

Claims

1. 1. A method for treatment with an aryl hydrocarbon receptor (AhR) agonist compound comprising: providing, or instructing the providing of, a sample from the subject prior to treating the subject with an AhR agonist compound; testing or directing the testing of the sample for diagnostic markers related to aryl hydrocarbon receptor biology; and Based on the results from the testing or instructing testing step, doing one or more of: (i) administering the AhR agonist compound; (ii) not administering the AhR agonist compound; and (iii) modifying the dose or dosing protocol of the AhR agonist compound. A method comprising:

2. The step of testing or the step of instructing testing, a. an AhR agonist compound in a skin, feces, urine, or plasma sample obtained from a subject; b. tryptophan in a skin, feces, urine or plasma sample obtained from the subject; c. a cytochrome P450 (CYP) enzyme selected from CYP1A1, CYP1A2, CYP1B1, and CYP19A1 in a colon sample or biopsy, intestinal biopsy, skin biopsy, or peripheral blood mononuclear cells (PBMCs) obtained from the subject; d. Aryl hydrocarbon receptor inhibitor (AhRR) mRNA or protein in a colon sample or biopsy, intestinal biopsy, skin biopsy, or peripheral blood mononuclear cells (PBMCs) obtained from the subject; e. Nuclear localized AhR in a colon sample or biopsy, intestinal biopsy, or skin biopsy obtained from the subject; f. IL-22 mRNA or protein levels in a plasma sample, colon biopsy sample, intestinal biopsy or skin biopsy obtained from the subject; and g. A polymorphism in caspase recruitment domain 9 (CARD9) in a sample obtained from a subject The method of claim 1 , wherein the diagnostic marker is selected from the group consisting of:

3. The method of claim 1 or 2, which achieves an improvement in response to treatment with an AhR agonist compound that is increased by at least about 25% compared to subjects treated with the same AhR agonist compound who were not tested for the diagnostic marker.

4. 1. A method for preventing or reducing an adverse event prior to or during treatment with a compound having activity as an aryl hydrocarbon receptor (AhR) agonist, comprising: providing, or instructing the providing of, a sample from the subject prior to or during treatment of the subject with an AhR agonist compound; testing or directing the testing of the sample for diagnostic markers related to aryl hydrocarbon receptor biology; and Based on the results from the step of testing or the step of directing testing, vi. administering the AhR agonist compound according to a schedule selected to reduce the likelihood or severity of adverse events; vii. administering the AhR agonist compound at a dose selected to reduce the likelihood or severity of an adverse event; viii. no administration of AhR agonist compounds; ix. administering a treatment other than an AhR agonist; and x. Administering an AhR antagonist or a compound that induces CYP performing one or more of the steps: A method comprising:

5. The step of testing or the step of instructing testing, a. Polymorphisms in AhR, ARNT, HIF-1α or HIF-2α, AhRR, BMPR2, CYP1A1, CYP1A2, CYP1B1 and CYP19A1 or their promoters; b. the ability of microsomes or cells from the subject to metabolize AhR agonists in vitro or in vivo; and c. The ability of AhR agonists to induce AhR translocation to the nucleus or induce CYP1A1, CYP1A2, CYP19A1 and / or CYP1B1 expression in vitro or in vivo. The method of claim 4, wherein the diagnostic marker is selected from the group consisting of:

6. The method of claim 4 or 5, wherein the performing step comprises (i), (ii) or (v), and the administering step according to (i), (ii) or (v) reduces the occurrence or severity of an adverse event in the subject compared to a subject not administered an AhR agonist compound or a compound that induces a CYP enzyme.

7. 7. The method of claim 6, wherein the administering step according to (i), (ii) or (v) reduces the incidence or severity of adverse events in the subject by at least about 20%.

8. 1. A method for preventing or reducing adverse events during treatment with a compound having activity as an aryl hydrocarbon receptor (AhR) agonist, comprising: providing, or instructing the providing of, a sample from the subject during treatment of the subject with an AhR agonist compound; testing or directing the testing of the sample for diagnostic markers related to aryl hydrocarbon receptor biology; and Based on the results from the step of testing or the step of directing testing, xi. administering an AhR agonist at a dosing frequency that is reduced compared to the clinically approved or previously planned dosing frequency; xii. administering an AhR agonist at a dose that is reduced compared to the clinically approved or previously planned dose; xiii. administering an initial starting dose of an AhR agonist followed by subsequent doses that are greater than the initial starting dose; xiv. discontinuing use of AhR agonists; xv. Continue use of AhR agonists with safety monitoring; xvi. administering a treatment other than an AhR agonist; xvii. co-administration of an AhR antagonist or a compound that induces CYP1A, CYP1A2, CYP1B1 or CYP19A1; xviii. simultaneous administration of concomitant medications; xix. discontinuation of concomitant medications, foods, drugs or activities; and xx. No change in treatment performing one or more of the steps: A method comprising:

9. The step of testing or the step of instructing testing, a. Plasma, urine, colon or skin biopsy levels of the administered AhR agonist or its metabolites; b. Plasma liver enzymes such as alanine aminotransferase (ALT) or aspartate aminotransferase (AST); c. PBMC expression of nuclear AhR or increased AhRR, CYP1A1, CYP1A2, CYP1B1 or CYP19A1 and / or CYP1B1 expression compared to healthy subjects; d. systolic blood pressure, mean pulmonary artery pressure (mPAP), pulmonary artery systolic pressure (PASP), left atrial dimension (LAD), left ventricular end diastolic dimension (LVEDD), left ventricular ejection fraction (LVEF), inferior vena cava diameter, right atrial area (RA area), tricuspid annular systolic excursion (TAPSE), tricuspid regurgitation pressure gap (TRPG), aortic regurgitation, mitral regurgitation, valve thickening, valve motion restriction, shortness of breath and / or fatigue; e. hematocrit or plasma erythropoietin (EPO) levels; and f. Plasma or urinary levels of serotonin, melatonin, 17B-estradiol (E2) or 16α-hydroxyestrone The method of claim 4 or 8, wherein the diagnostic marker is selected from the group consisting of:

10. 9. The method of claim 8, wherein the performing step comprises (i), (ii), (iii), (v), (vii), (viii), or (ix), and wherein the administering according to (i), (ii) or (v), the continuing according to (v), the co-administration according to (vii) or (viii), or the discontinuing according to (ix) reduces the occurrence or severity of adverse events in the subject compared to a subject not receiving an AhR agonist compound, not receiving a compound or concomitant medication that induces a CYP enzyme, and / or continuing the concomitant medication, food, drug, or activity.

11. 11. The method of claim 10, wherein the reduction in the occurrence or severity of an adverse event in a subject is at least about 20%.

12. The method of claim 8, wherein the step of continuing use of the AhR agonist in conjunction with safety monitoring includes continuing use with a safety monitoring program selected from an echocardiogram, a timed walking test and a questionnaire regarding shortness of breath, and / or the ability to exercise without fatigue.

13. 1. A method for preventing or reducing an adverse event prior to or during treatment with a compound having activity as an aryl hydrocarbon receptor (AhR) agonist, comprising: Referencing product labels that prohibit certain concomitant medications, drugs, foods, diets, supplements and / or activities that may increase AhR signaling, increase AhR agonist absorption, or interfere with the ability to metabolize AhR agonists; and Based on said referencing step, determining whether to administer, not administer, or administer with a dose range or frequency of the AhR agonist adjusted to the subject based on the likelihood of an adverse event for the subject. A method comprising:

14. 1. A method for preventing or reducing an adverse event prior to or during treatment with a compound having activity as an aryl hydrocarbon receptor (AhR) agonist, comprising: Referencing product labeling to contraindicate use of AhR agonists in patients with known medical conditions, predispositions, or family histories that may increase the likelihood of developing cardiovascular disease, including pulmonary arterial hypertension and valvular heart disease. A method comprising:

15. administering a compound that induces a CYP enzyme capable of metabolizing the AhR agonist to be administered, prior to administration of an oral dosage form containing the AhR agonist. Systemic exposure to AhR agonists or C ｍａｘ A method for reducing

16. 16. The method of claim 15, wherein the compound that induces CYP is an AhR agonist.

17. 17. The method of claim 16, wherein the CYP inducing AhR agonist is the same as the AhR agonist in the oral dosage form, and the CYP inducing AhR agonist is administered at a lower dose than the dose of the AhR agonist in the oral dosage form.

18. 18. The method of any one of claims 15 to 17, wherein the administering step reduces the occurrence or severity of an adverse event in the subject compared to a subject not administered a compound that induces a CYP enzyme.

19. 20. The method of claim 18, wherein the administering step reduces the occurrence or severity of an adverse event in the subject by at least about 20%.

20. 20. The method of any one of claims 1 to 19, wherein the adverse event is selected from the group consisting of folliculitis, essential hypertension, pulmonary hypertension, pulmonary arterial hypertension, pneumonia, respiratory failure, myocardial ischemia, myocardial infarction, pericarditis, pleuritis, peritonitis, pneumonia, chest pain, abdominal pain, diarrhea, vomiting, intussusception, joint pain, back pain, liver damage, liver enzyme changes, infection, peripheral neuropathy, headache, pancytopenia, and leukopenia.

21. 1. A method of treating one or more patients with an AhR agonist, comprising: a data storage facility including a database of patient records, each patient record having a medication authorization field for entering a first prescription for an AhR agonist for treating the patient; a central control unit having one or more processors coupled to a communications network, the central control unit being coupled to a data storage facility for reading and writing data to the data storage facility over the network; A process for providing Including, a central controller for controlling transmission of data to and reception of data from the data storage facility over the network; a central controller programmed to output, via the network, a first approval of a first prescription of an AhR agonist for a patient who has previously been subjected to one or more initial medical tests, each of which provides an initial medical test result; the initial medical testing is selected from the group consisting of a physician examination, a genetic test, a physiological test, and a medical imaging test; the output of the first approval is dependent on the satisfactory outcome of one or more of the initial medical tests entered into each patient's record; further programmed to schedule one or more subsequent tests for each patient prior to enabling entry of the prescription in the medication authorization field; at least one of said subsequent medical tests is an echocardiographic imaging test performed in a manner that provides measurements of one or more internal cardiac structure dimensions and cardiac flow velocities, and the patient receives or continues to receive medical therapy only upon entry of a satisfactory echocardiographic evaluation result; the central controller inhibits approval output of the first or subsequent prescription upon input of an unsatisfactory test result; The central control unit manages one or more aspects of the approved prescription for the patient selected from the group consisting of: dosage; dosing regimen; and intended duration of use, thereby preventing abuse or misuse of the AhR agonist, and the consolidated and analyzed data is reported to a regulatory agency.

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