Compositions and methods for treating hypothyroidism

Abstract

The present disclosure provides methods of determining the responsiveness of a subject to treatment for hypothyroidism. The present disclosure also provides methods of determining the risk that a subject will exhibit residual symptoms following treatment for hypothyroidism. The present disclosure also provides methods of treating hypothyroidism and residual symptoms thereof.

Description

COMPOSITIONS AND METHODS FOR TREATING HYPOTHYROIDISMRELATED APPLICATIONS

[0001] This application claims priority to, and the benefit of, United States Provisional Application No. 63 / 648,826, filed on May 17, 2024, the content of which is incorporated by reference herein in its entirety.BACKGROUND OF THE INVENTION

[0002] Hypothyroidism is one of the most prevalent diseases, influencing approximately 5% of the global population. Hypothyroidism leads to decreased levels of thyroxine (FT4) and thyroid-related metabolites, including free triiodothyronine (FT3). Thyroid hormone replacement therapy using levothyroxine is used to treat hypothyroidism and results in the normalization of FT4 and FT3. Despite adequate levothyroxine therapy (i.e., the FT4 and thyroid stimulating hormone (TSH) have been corrected to normal range), up to 15% of treated hypothyroidism patients still report decreased quality of life, persistent psychological symptoms, and cognitive impairment. Multiple studies have found that patients treated with levothyroxine report a decreased quality of life in standardized questionnaires. Furthermore, levothyroxine treated hypothyroidism patients also report impaired cognitive function and psychological well-being symptoms such as poor memory, fatigue, and anxiety when compared to the general population or matched healthy controls. The exact causes of these residual symptoms remain unknown. However, several potential mechanisms have been proposed such as low efficiency of T4-T3 conversion, different individual T4-TSH set-points, and coexistence of other autoimmune diseases.SUMMARY

[0003] The present disclosure provides methods of determining the responsiveness of a subject to a hypothyroidism treatment, the methods comprising: a) determining the concentration of at least one of six metabolites in a biological sample from the subject, wherein the six metabolites comprise dehydroepiandrosterone sulfate (DHEA-S), androstenediol (3beta,17beta) disulfate, androstenediol (3 alpha, 17alpha) monosulfate, androstenediol (3beta,17beta) monosulfate, pregnenediol sulfate, and pregnenolone sulfate; b) identifying that the subject is responsive or nonresponsive to the hypothyroidism treatment based on the concentration of the at least one metabolite measured in step (a). In someaspects, step (a) comprises: i) determining the concentration of at least two of the six metabolites; ii) determining the concentration of at least three of the six metabolites; iii) determining the concentration of at least four of the six metabolites; iv) determining the concentration of at least five of the six metabolites; or v) determining the concentration of each of the six metabolites.

[0004] In some aspects, identifying that the subject is responsive or nonresponsive to the hypothyroidism treatment based on concentration of the at least one metabolite measured in step (a) comprises: i) comparing the one or more concentrations measured in step (a) to one or more corresponding predetermined cutoff values; and ii) identifying the subject as being nonresponsive to the hypothyroidism treatment when one or more of the concentrations measured in step (a) is less than its corresponding predetermined cutoff value. In some aspects, a subject can be identified as being responsive to the hypothyroidism treatment when one or more of the concentrations measured in step (a) is equal to or greater than its corresponding predetermined cutoff value.

[0005] In some aspects, a corresponding predetermined cutoff value is determined through the analysis of the concentration of the at least one metabolite in a training set, wherein the training set comprises the concentration of the at least one metabolite measured in biological samples from one or more training subjects, wherein the one or more training subjects comprise a plurality of training subjects that respond to the hypothyroidism treatment and a plurality of training subjects that do not respond to the hypothyroidism treatment.

[0006] In some aspects, a subject who is identified as not responsive has a higher risk of exhibiting one or more residual symptoms of hypothyroidism.

[0007] The present disclosure provides methods of determining the responsiveness of a subject to a hypothyroidism treatment, the methods comprising: a) at a first time point, determining the concentration of at least one of six metabolites in a biological sample from the subject, wherein the six metabolites comprise dehydroepiandrosterone sulfate (DHEA-S), androstenediol (3beta,17beta) disulfate, androstenediol (3 alpha, 17alpha) monosulfate, androstenediol (3beta,17beta) monosulfate, pregnenediol sulfate, and pregnenolone sulfate; b) at a second time point, determining the concentration of the at least one metabolite in a biological sample from the subject; c) comparing the concentration of the at least one metabolite at the first time point and the concentration of the at least one metabolite at the second time point; d) identifying that the subject is responding to the hypothyroidism treatment when the concentration of the at least one metabolite at the second time point is greater than concentration of the at least one metabolite at the first time or identifying that thesubject is not responding to the hypothyroidism treatment when the concentration of the at least one metabolite at the second time point is equal to or less than the concentration of the at least one metabolite. In some aspects, the first time point is before the administration of the hypothyroidism treatment and the second time point is after the administration of at least one amount of the hypothyroidism treatment. In some aspects, the first time point is after the initial administration of the hypothyroidism treatment and the second time is after one or more additional doses of the hypothyroidism treatment has been administered to the subject.

[0008] In some aspects, the preceding methods can comprise changing the dosage of the hypothyroidism treatment based on the change in the concentration of the at least one of six metabolites between the first time point and the second time point.

[0009] In some aspects, the preceding methods can further comprise administering to a subject identified as being nonresponsive to the hypothyroidism treatment at least one amount of at least one of six therapeutic metabolites, wherein the six therapeutic metabolites comprise dehydroepiandrosterone sulfate (DHEA-S), androstenediol (3beta,17beta) disulfate, androstenediol (3alpha, 17alpha) monosulfate, androstenediol (3beta,17beta) monosulfate, pregnenediol sulfate, and pregnenolone sulfate. In some aspects, the methods comprise: i) administering to the subject at least one amount of at least two of the six therapeutic metabolites; ii) administering to the subject at least one amount of at least three of the six therapeutic metabolites; iii) administering to the subject at least one amount of at least four of the six therapeutic metabolites; iv) administering to the subject at least one amount of at least five of the six therapeutic metabolites; or v) administering to the subject at least one amount of each of the six therapeutic metabolites.

[0010] The present disclosure provides methods of determining the risk that a subject exhibits one or more residual symptoms after administration of a hypothyroidism treatment, the methods comprising: a) determining the concentration of at least one of six metabolites in a biological sample from the subject, wherein the six metabolites comprise dehydroepiandrosterone sulfate (DHEA-S), androstenediol (3beta, 17beta) disulfate, androstenediol (3alpha, 17alpha) monosulfate, androstenediol (3beta,17beta) monosulfate, pregnenediol sulfate, and pregnenolone sulfate; b) identifying the risk that the subject exhibits one or more residual symptoms based on the concentration of the at least one metabolite measured in step (a). In some aspects, step (a) comprises: i) determining the concentration of at least two of the six metabolites; ii) determining the concentration of at least three of the six metabolites; iii) determining the concentration of at least four of the sixmetabolites; iv) determining the concentration of at least five of the six metabolites; or v) determining the concentration of each of the six metabolites.

[0011] In some aspects, identifying the risk that the subject exhibits residual symptoms after administration of the hypothyroidism treatment based on the concentration of the at least one metabolite measured in step (a) comrpises: i) comparing the one or more concentrations measured in step (a) to one or more corresponding predetermined cutoff values; and ii) identifying that the subject is at increased risk of exhibiting residual symptoms when one or more of the concentrations measured in step (a) is less than its corresponding predetermined cutoff value. In some aspects, a subject can be identified as being at lower risk of exhibit residual symptoms when one or more of the concentrations measured in step (a) is equal to or greater than its corresponding predetermined cutoff value.

[0012] In some aspects, the preceding methods can further comprise administering to a subject identified as being at an increased risk of exhibiting residual symptoms at least one amount of at least one of six therapeutic metabolites, wherein the six therapeutic metabolites comprise dehydroepiandrosterone sulfate (DHEA-S), androstenediol (3beta,17beta) disulfate, androstenediol (3alpha, 17alpha) monosulfate, androstenediol (3beta,17beta) monosulfate, pregnenediol sulfate, and pregnenolone sulfate. In some aspects, the method comprises: i) administering to the subject at least one amount of at least two of the six therapeutic metabolites; ii) administering to the subject at least one amount of at least three of the six therapeutic metabolites; iii) administering to the subject at least one amount of at least four of the six therapeutic metabolites; iv) administering to the subject at least one amount of at least five of the six therapeutic metabolites; or v) administering to the subject at least one amount of each of the six therapeutic metabolites.

[0013] In some aspects, determining the concentration of the at least one metabolite comprises the use of mass spectrometry.

[0014] In some aspects, a biological sample comprises blood, plasma, serum, urine, breast milk, cerebrospinal fluid, mucus, gastric juice, peritoneal fluid, pleural fluid, saliva, sebum, semen, sweat, tears, vaginal secretion, vomit, endolymph, perilymph, neuronal tissue, or any combination thereof.

[0015] The present disclosure provides methods of treating hypothyroidism in a subject, the methods comprising administering to the subject least one amount of at least one of six therapeutic metabolites, wherein the six therapeutic metabolites comprise dehydroepiandrosterone sulfate (DHEA-S), androstenediol (3beta, 17beta) disulfate, androstenediol (3alpha, 17alpha) monosulfate, androstenediol (3beta,17beta) monosulfate,pregnenediol sulfate, and pregnenolone sulfate, or a pharmaceutically acceptable salt thereof. In some aspects, the at least one metabolite is dehydroepiandrosterone sulfate (DHEA-S), or a pharmaceutically acceptable salt thereof.

[0016] In some aspects, the at least one metabolite is androstenediol (3beta,17beta) disulfate, or a pharmaceutically acceptable salt thereof.

[0017] In some aspects, the at least one metabolite is androstenediol (3alpha, 17alpha) monosulfate, or a pharmaceutically acceptable salt thereof.

[0018] In some aspects, the at least one metabolite is androstenediol (3beta,17beta) monosulfate, or a pharmaceutically acceptable salt thereof.

[0019] In some aspects, the at least one metabolite is pregnenediol sulfate, or a pharmaceutically acceptable salt thereof.

[0020] In some aspects, the at least one metabolite is pregnenolone sulfate, or a pharmaceutically acceptable salt thereof.

[0021] In some aspects, the preceding methods comprise: i) administering to the subject at least one amount of at least two of the six therapeutic metabolites; ii) administering to the subject at least one amount of at least three of the six therapeutic metabolites; iii) administering to the subject at least one amount of at least four of the six therapeutic metabolites; iv) administering to the subject at least one amount of at least five of the six therapeutic metabolites; or v) administering to the subject at least one amount of each of the six therapeutic metabolites.

[0022] In some aspects, a subject has been identified to be nonresponsive to a hypothyroidism treatment.

[0023] In some aspects, a subject exhibits one or more residual symptoms following treatment with levothyroxine.

[0024] In some aspects, the preceding methods can further comprise administering at least one additional therapy to the subject.

[0025] In some aspects, an at least one additional therapy is levothyroxine.

[0026] In some aspects, residual symptoms can comprise reduced cognitive function, poor memory, fatigue, anxiety, reduced general health, or any combination thereof.

[0027] In some aspects, a subject has previously received one or more amounts of the hypothyroidism treatment.

[0028] In some aspects, a hypothyroidism treatment comprises the administration of levothyroxine.

[0029] Any of the above aspects, or any other aspect described herein, can be combined with any other aspect described herein.

[0030] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In the specification, the singular forms also include the plural unless the context clearly dictates otherwise; as examples, the terms “a,” “an,” and “the” are understood to be singular or plural and the term “or” is understood to be inclusive. By way of example, “an element” means one or more element. Throughout the specification the word “comprising,” or variations such as “comprises” or “comprising,” will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from the context, all numerical values provided herein are modified by the term “about.” Unless specifically stated or obvious from context, as used herein, the term “or” is understood to be inclusive and covers both “or” and “and”.

[0031] Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The references cited herein are not admitted to be prior art to the claimed invention. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting. Other features and advantages of the disclosure will be apparent from the following detailed description and claim.BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The above and further features will be more clearly appreciated from the following detailed description when taken in conjunction with the accompanying drawings.

[0033] FIGs. 1A-1B show MR analyses assessing plasma metabolite levels and susceptibility to hypothyroidism. FIG. 1A shows a volcano plot illustrating the effect of each plasma metabolite and the susceptibility to hypothyroidism from the MR analyses using the inverse variance weighted method. The bottom dotted horizontal line represents an empirical p-value=0.05. The top doted horizontal line represents the p-value at FDR=0.05. A metabolite’s shape denotes whether the metabolite passed (triangle) or failed (circle) the sensitivity analyses. FIG. IB shows an MR scater plot showing the SNP effect on genetically predicted liability to hypothyroidism and the effect of the same SNP on plasma dehydroisoandrosterone sulfate and pregnanediol sulfate levels; FDR: false discovery rate; MR: Mendelian randomization; SNP: single nucleotide polymorphism.

[0034] FIGs. 2A-2J shows the MR scater plots for the 10 indicated metabolites. FIG. 2A shows the MR scater plot for 7-methylguanine. FIG. 2B shows the MR scater plot for 5- methyluridine (ribothymidine). FIG. 2C shows the MR scater plot for androstenediol (3beta, 17beta) disulfate (2). FIG. 2D shows the MR scater plot for androstenediol (3beta, 17beta) monosulfate (3). FIG. 2E shows the MR scatter plot for androstenediol (3beta, 17beta) monosulfate (1). FIG. 2F shows the MR scater plot for pregnenolone sulfate. FIG. 2G shows the MR scater plot for 2-aminobutyrate. FIG. 2H shows the MR scater plot for N- delta-acetylomithine. FIG. 21 shows the MR scater plot for glucose. FIG. 2J shows the MR scater plot for 2-hydroxybutyrate / 2-hydroxyisobutyrate. MR: Mendelian randomization.

[0035] FIGs. 3A-3L shows the MR funnel plots for the 12 indicated metabolites. FIG. 3A shows the MR funnel plot for dehydroepiandrosterone sulfate (DHEA-S). FIG. 3B shows the MR funnel plot for pregnanediol sulfate (C12H34O5S). FIG. 3C shows the MR funnel plot for 7-methylguanine. FIG. 3D shows the MR funnel plot for 5 -methyluridine (ribothymidine). FIG. 3E shows the MR funnel plot for androstenediol (3beta, 17beta) disulfate (2). FIG. 3F shows the MR funnel plot for androstenediol (3beta, 17beta) monosulfate (3). FIG. 3G shows the MR funnel plot for androstenediol (3beta, 17beta) monosulfate (1). FIG. 3H shows the MR funnel plot for pregnenolone sulfate. FIG. 31 shows the MR funnel plot for 2-aminobutyrate. FIG. 3J shows the MR funnel plot for N-delta- acetylomithine. FIG. 3K shows the MR funnel plot for glucose. FIG. 3L shows the MR funnel plot for 2-hydroxybutyrate / 2-hydroxyisobutyrate. MR: Mendelian randomization.

[0036] FIG. 4 shows an MR analysis showing the susceptibility to hypothyroidism based on the top 12 metabolites influenced by hypothyroidism using metabolite GWAS summary statistics from Surendran et al., 2022, or Chen et al., 2023. CI: confidence interval; s.d.: standard deviation; GWAS: genome-wide association study; MR: Mendelian randomization.

[0037] FIG. 5 shows an MR analysis depicting the effects of free thyroxine (FT4), free triiodothyronine (FT3), and thyroid stimulating hormone (TSH) on plasma levels. CI: confidence interval.

[0038] FIGs. 6A-6B show free T4 (FIG. 6A) and TSH (FIG. 6B) levels stratified between euthyroid controls (N=7,885) and individuals with adequately treated hypothyroidism (N=803) in the CLSA cohort. T4: thyroxine; TSH: thyroid stimulating hormone; CLSA: Canadian longitudinal study of aging.

[0039] FIG. 7 shows a comparison of hypothyroidism-influenced metabolite levels between cases of individuals with adequately treated hypothyroidism with levothyroxine and euthyroid controls. The Y-axis shows the mean and standard error of the mean for metabolite levels after an adjustment for age, sex, hour since last drink or meal, BMI, and recruitment center. P-values=two-sided t-test. MR: Mendelian randomization.

[0040] FIGs. 8A-8B shows associations of hypothyroidism-influenced metabolites with cognitive function and self-rated general health, identified by MR. FIG. 8A shows interval plots depicting the association between decreased metabolite levels and cognitive function compared to euthyroid controls. FIG. 8B shows the association between decreased metabolite levels and odds of self-reported general health being “fair or poor” as compared to those being “excellent” among thyroid controls. MR: Mendelian randomization.

[0041] FIGs. 9A-9B shows associations of MR-highlighted hypothyroidism-influenced metabolites with cognitive function in males and females. FIG. 9A shows interval plots showing the association between decreased metabolite levels and cognitive function compared to euthyroid controls in females. FIG. 9B shows interval plots showing the association between decreased metabolite levels and cognitive function compared to euthyroid controls in males. MR: Mendelian randomization.DETAILED DESCRIPTION

[0042] The present disclosure provides, inter alia, compositions and methods for the treatment of hypothyroidism and determining a subject’s responsiveness to treatment for hypothyroidism. For example, the present disclosure provides compositions and methods directed to the administration of dehydroepiandrosterone sulfate (DHEA-S), androstenediol (3beta,17beta) disulfate, androstenediol (3alpha, 17alpha) monosulfate, androstenediol (3beta,17beta) monosulfate, pregnenediol sulfate, and pregnenolone sulfate for the treatment of hypothyroidism. In another example, the present disclosure provides compositions and methods for determining the concentration of dehydroepiandrosterone sulfate, androstenediol (3beta,17beta) disulfate, androstenediol (3alpha,17alpha) monosulfate, androstenediol (3beta,17beta) monosulfate, pregnenediol sulfate, and pregnenolone sulfate in biological samples from a subject for use in methods of determining the responsiveness. Thecompositions and methods described herein are based on the surprising and unexpected finding, described in the experimental examples, that following treatment with levothyroxine, subjects who exhibit residual symptoms (e.g. reduced cognitive function poor memory, fatigue, anxiety, reduced general health, or any combination thereof) exhibit dysregulated levels of dehydroepiandrosterone sulfate, androstenediol (3beta,17beta) disulfate, androstenediol (3alpha, 17alpha) monosulfate, androstenediol (3beta,17beta) monosulfate, pregnenediol sulfate, and pregnenolone sulfate. More, specifically, Applicants have unexpectedly found that in these subjects exhibiting residual symptoms, the levels of these six metabolites remain decreased despite the administration of levothyroxine.

[0043] Methods of Treatment

[0044] The present disclosure provides methods of treating hypothyroidism in a subject, the method comprising administering to the subject least one amount of at least one of six therapeutic metabolites, wherein the six therapeutic metabolites comprise dehydroepiandrosterone sulfate (DHEA-S), androstenediol (3beta,17beta) disulfate, androstenediol (3alpha, 17alpha) monosulfate, androstenediol (3beta,17beta) monosulfate, pregnenediol sulfate, and pregnenolone sulfate, or a pharmaceutically acceptable salt thereof.

[0045] In some aspects, the preceding methods can comprise i) administering to the subject at least one amount of at least two of the six therapeutic metabolites; ii) administering to the subject at least one amount of at least three of the six therapeutic metabolites; iii) administering to the subject at least one amount of at least four of the six therapeutic metabolites; iv) administering to the subject at least one amount of at least five of the six therapeutic metabolites; or v) administering to the subject at least one amount of each of the six therapeutic metabolites.

[0046] Accordingly, the present disclosure provides methods of treating hypothyroidism in a subject, the methods comprising administering to the subject at least one amount of dehydroepiandrosterone sulfate, or a pharmaceutically acceptable salt thereof.

[0047] Accordingly, the present disclosure provides methods of treating hypothyroidism in a subject, the methods comprising administering to the subject at least one amount of androstenediol (3beta,17beta) disulfate, or a pharmaceutically acceptable salt thereof.

[0048] Accordingly, the present disclosure provides methods of treating hypothyroidism in a subject, the methods comprising administering to the subject at least one amount of androstenediol (3alpha, 17alpha) monosulfate, or a pharmaceutically acceptable salt thereof.

[0049] Accordingly, the present disclosure provides methods of treating hypothyroidism in a subject, the methods comprising administering to the subject at least one amount of androstenediol (3beta,17beta) monosulfate, or a pharmaceutically acceptable salt thereof.

[0050] Accordingly, the present disclosure provides methods of treating hypothyroidism in a subject, the methods comprising administering to the subject at least one amount of pregnenediol sulfate, or a pharmaceutically acceptable salt thereof.

[0051] Accordingly, the present disclosure provides methods of treating hypothyroidism in a subject, the methods comprising administering to the subject at least one amount of pregnenolone sulfate, or a pharmaceutically acceptable salt thereof.

[0052] In some aspects of the preceding methods, the dehydroepiandrosterone sulfate, androstenediol (3beta,17beta) disulfate, androstenediol (3alpha, 17alpha) monosulfate, androstenediol (3beta,17beta) monosulfate, pregnenediol sulfate, and / or pregnenolone sulfate is / are administered in an amount sufficient enough to increase the concentration of said metabolite(s) in the subject such that the concentration(s) is / are equal to or greater than one or more corresponding predetermined cutoff values.

[0053] In some aspects, the predetermined cutoff values described above can be determined through the analysis of the concentration of dehydroepiandrosterone sulfate, androstenediol (3beta,17beta) disulfate, androstenediol (3alpha, 17alpha) monosulfate, androstenediol (3beta,17beta) monosulfate, pregnenediol sulfate, and / or pregnenolone sulfate measured in biological samples from one or more training subjects. In some aspects, the one or more training subjects comprise a plurality of training subjects that do not have hypothyroidism. In some aspects, the one or more training subjects comprise a plurality of training subjects that have hypothyroidism. In some aspects, the one or more training subject comprise a plurality of training subj ects that have hypothyroidism and that respond to a hypothyroidism treatment (e. g. treatment with levothyroxine). In some aspects, the one or more training subject comprise a plurality of training subjects that have hypothyroidism and that do not respond to a hypothyroidism treatment (e.g. treatment with levothyroxine). In some aspects, analysis can comprise genome-wide association study (GWAS) analysis and / or mendelian randomization (MR) analysis.

[0054] In some aspects, the subject to be treated has been identified to be nonresponsive to a hypothyroidism treatment. In some aspects, the hypothyroidism treatment is levothyroxine.

[0055] In some aspects, the subject to be treated exhibits one or more residual symptoms following treatment with levothyroxine. In some aspects, the one or more residual symptomscomprise reduced cognitive function, poor memory, fatigue, anxiety, reduced general health, or any combination thereof.

[0056] In some aspects, the preceding treatment methods can further comprise administering at least one additional therapy to the subject. In some aspects, the at least one additional therapy is levothyroxine.

[0057] In some aspects, the at least one additional therapy and the at least one therapeutic metabolite can be administered in temporal proximity. As used herein, the term “temporal proximity” refers to that administration of one therapeutic agent occurs within a time period before or after the administration of another therapeutic agent, such that the therapeutic effect of the one therapeutic agent overlaps with the therapeutic effect of the other therapeutic agent. In some embodiments, the therapeutic effect of the one therapeutic agent completely overlaps with the therapeutic effect of the other therapeutic agent. In some embodiments, “temporal proximity” means that administration of one therapeutic agent occurs within a time period before or after the administration of another therapeutic agent, such that there is a synergistic effect between the one therapeutic agent and the other therapeutic agent.“Temporal proximity” may vary according to various factors, including but not limited to, the age, gender, weight, genetic background, medical condition, disease history, and treatment history of the subject to which the therapeutic agents are to be administered; the disease or condition to be treated or ameliorated; the therapeutic outcome to be achieved; the dosage, dosing frequency, and dosing duration of the therapeutic agents; the pharmacokinetics and pharmacodynamics of the therapeutic agents; and the route(s) through which the therapeutic agents are administered. In some embodiments, “temporal proximity” means within 15 minutes, within 30 minutes, within an hour, within two hours, within four hours, within six hours, within eight hours, within 12 hours, within 18 hours, within 24 hours, within 36 hours, within 2 days, within 3 days, within 4 days, within 5 days, within 6 days, within a week, within 2 weeks, within 3 weeks, within 4 weeks, with 6 weeks, or within 8 weeks. In some embodiments, multiple administration of one therapeutic agent can occur in temporal proximity to a single administration of another therapeutic agent. In some embodiments, temporal proximity may change during a treatment cycle or within a dosing regimen.

[0058] The therapeutic metabolites of the present disclosure can be administered via a variety of routes, including, but not limited to, oral, pulmonary, rectal, parenteral, transdermal, subcutaneous, intravenous, intramuscular, intraperitoneal, inhalational, buccal, sublingual, intrapleural, intrathecal, intranasal, or any combination thereof.

[0059] Methods of Determining Responsiveness to Hypothyroidism Treatments and Modifying Dosages

[0060] The present disclosure provides methods of determining the responsiveness of a subject to a hypothyroidism treatment, the methods comprising: a) determining the concentration of at least one of six metabolites in a biological sample from the subject, wherein the six metabolites comprise dehydroepiandrosterone sulfate, androstenediol (3beta,17beta) disulfate, androstenediol (3alpha, 17alpha) monosulfate, androstenediol (3beta,17beta) monosulfate, pregnenediol sulfate, and pregnenolone sulfate; b) identifying that the subject is responsive or nonresponsive to the hypothyroidism treatment based on the concentration of the at least one metabolite measured in step (a).

[0061] In some aspects of the preceding methods, step (a) can comprise: i) determining the concentration of at least two of the six metabolites; ii) determining the concentration of at least three of the six metabolites; iii) determining the concentration of at least four of the six metabolites; iv) determining the concentration of at least five of the six metabolites; or v) determining the concentration of each of the six metabolites.

[0062] In some aspects of the preceding methods, identifying that the subject is responsive or nonresponsive to the hypothyroidism treatment based on concentration of the at least one metabolite measured in step (a) comprises: i) comparing the one or more concentrations measured in step (a) to one or more corresponding predetermined cutoff values; and ii) identifying the subject as being nonresponsive to the hypothyroidism treatment when one or more of the concentrations measured in step (a) is less than its corresponding predetermined cutoff value. In some aspects, a subject can be identified as being responsive to the hypothyroidism treatment when one or more of the concentrations measured in step (a) is equal to or greater than its corresponding predetermined cutoff value.

[0063] In some aspects, the predetermined cutoff values described above can be determined through the analysis of the concentration of dehydroepiandrosterone sulfate, androstenediol (3beta,17beta) disulfate, androstenediol (3alpha, 17alpha) monosulfate, androstenediol (3beta,17beta) monosulfate, pregnenediol sulfate, and / or pregnenolone sulfate measured in biological samples from one or more training subjects. In some aspects, the one or more training subjects comprise a plurality of training subjects that do not have hypothyroidism. In some aspects, the one or more training subjects comprise a plurality of training subjects that have hypothyroidism. In some aspects, the one or more training subject comprise a plurality of training subj ects that have hypothyroidism and that respond to a hypothyroidism treatment (e. g. treatment with levothyroxine). In some aspects, the one or more training subject comprise aplurality of training subjects that have hypothyroidism and that do not respond to a hypothyroidism treatment (e.g. treatment with levothyroxine). In some aspects, analysis can comprise genome-wide association study (GWAS) analysis and / or mendelian randomization (MR) analysis.

[0064] In some aspects of the preceding methods, the subject has previously received one or more amounts of the hypothyroidism treatment. In some aspects, the hypothyroidism treatment comprises the administration of levothyroxine.

[0065] In some aspects, any of the preceding methods can comprise changing the dosage of the hypothyroidism treatment based on whether the subject is identified as being responsive to the treatment or not responsive to the treatment.

[0066] In some aspects of the preceding methods, a subject who is identified as not responsive has a higher risk of exhibiting one or more residual symptoms of hypothyroidism. In some aspects, the one or more residual symptoms can comprise reduced cognitive function, poor memory, fatigue, anxiety, reduced general health, or any combination thereof.

[0067] The present disclosure also provides methods of determining the responsiveness of a subject to a hypothyroidism treatment, the methods comprising: a) at a first time point, determining the concentration of at least one of six metabolites in a biological sample from the subject, wherein the six metabolites comprise dehydroepiandrosterone sulfate (DHEA-S), androstenediol (3beta,17beta) disulfate, androstenediol (3 alpha, 17alpha) monosulfate, androstenediol (3beta,17beta) monosulfate, pregnenediol sulfate, and pregnenolone sulfate; b) at a second time point, determining the concentration of the at least one metabolite in a biological sample from the subject; c) comparing the concentration of the at least one metabolite at the first time point and the concentration of the at least one metabolite at the second time point; d) identifying that the subject is responding to the hypothyroidism treatment when the concentration of the at least one metabolite at the second time point is greater than concentration of the at least one metabolite at the first time or identifying that the subject is not responding to the hypothyroidism treatment when the concentration of the at least one metabolite at the second time point is equal to or less than the concentration of the at least one metabolite.

[0068] In some aspects of the preceding methods, steps (a) and (b) can comprise: i) determining the concentration of at least two of the six metabolites; ii) determining the concentration of at least three of the six metabolites; iii) determining the concentration of at least four of the six metabolites; iv) determining the concentration of five of the six metabolites; or v) determining the concentration of each of the six metabolites.

[0069] In some aspects of the preceding methods, the first time point is before the administration of the hypothyroidism treatment and the second time point is after the administration of at least one amount of the hypothyroidism treatment.

[0070] In some aspects of the preceding methods, the first time point is after the initial administration of the hypothyroidism treatment and the second time is after one or more additional doses of the hypothyroidism treatment has been administered to the subject.

[0071] In some aspects, any of the preceding methods can comprise changing the dosage of the hypothyroidism treatment based on the change in the concentration of the at least one of six metabolites between the first time point and the second time point.

[0072] In some aspects, any of the preceding methods can further comprise administering to a subject identified as being nonresponsive to the hypothyroidism treatment at least one amount of at least one of six therapeutic metabolites, wherein the six therapeutic metabolites comprise dehydroepiandrosterone sulfate, androstenediol (3beta,17beta) disulfate, androstenediol (3alpha, 17alpha) monosulfate, androstenediol (3beta,17beta) monosulfate, pregnenediol sulfate, and pregnenolone sulfate. In some aspects, the methods can further comprise i) administering to the subject at least one amount of at least two of the six therapeutic metabolites; ii) administering to the subject at least one amount of at least three of the six therapeutic metabolites; iii) administering to the subject at least one amount of at least four of the six therapeutic metabolites; iv) administering to the subject at least one amount of at least five of the six therapeutic metabolites; or v) administering to the subject at least one amount of each of the six therapeutic metabolites.

[0073] In some aspects of the preceding methods, the hypothyroidism treatment comprises the administration of levothyroxine.

[0074] Methods of Determining Risk of Residual Symptoms Following Hypothyroidism Treatments

[0075] The present disclosure provides methods of determining the risk that a subject exhibits one or more residual symptoms after administration of a hypothyroidism treatment, the method comprising: a) determining the concentration of at least one of six metabolites in a biological sample from the subject, wherein the six metabolites comprise dehydroepiandrosterone sulfate, androstenediol (3beta,17beta) disulfate, androstenediol (3alpha, 17alpha) monosulfate, androstenediol (3beta,17beta) monosulfate, pregnenediol sulfate, and pregnenolone sulfate; b) identifying the risk that the subject exhibits one or more residual symptoms based on the concentration of the at least one metabolite measured in step (a).

[0076] In some aspects of the preceding methods, step (a) can comprise: i) determining the concentration of at least two of the six metabolites; ii) determining the concentration of at least three of the six metabolites; iii) determining the concentration of at least four of the six metabolites; iv) determining the concentration of at least five of the six metabolites; or v) determining the concentration of each of the six metabolites.

[0077] In some aspects of the preceding methods, identifying the risk that the subject exhibits residual symptoms after administration of the hypothyroidism treatment based on the concentration of the at least one metabolite measured in step (a) comprises: i) comparing the one or more concentrations measured in step (a) to one or more corresponding predetermined cutoff values; and ii) identifying that the subject is at increased risk of exhibiting residual symptoms when one or more of the concentrations measured in step (a) is less than its corresponding predetermined cutoff value. In some aspects, a subject can be identified as being at lower risk of exhibit residual symptoms when one or more of the concentrations measured in step (a) is equal to or greater than its corresponding predetermined cutoff value.

[0078] In some aspects, the predetermined cutoff values described above can be determined through the analysis of the concentration of dehydroepiandrosterone sulfate, androstenediol (3beta,17beta) disulfate, androstenediol (3alpha, 17alpha) monosulfate, androstenediol (3beta,17beta) monosulfate, pregnenediol sulfate, and / or pregnenolone sulfate measured in biological samples from one or more training subjects. In some aspects, the one or more training subjects comprise a plurality of training subjects that do not have hypothyroidism. In some aspects, the one or more training subjects comprise a plurality of training subjects that have hypothyroidism. In some aspects, the one or more training subject comprise a plurality of training subj ects that have hypothyroidism and that respond to a hypothyroidism treatment (e. g. treatment with levothyroxine). In some aspects, the one or more training subject comprise a plurality of training subjects that have hypothyroidism and that do not respond to a hypothyroidism treatment (e.g. treatment with levothyroxine). In some aspects, analysis can comprise genome-wide association study (GWAS) analysis and / or mendelian randomization (MR) analysis.

[0079] In some aspects of the preceding methods, the one or more residual symptoms can comprise reduced cognitive function, reduced general health, or any combination thereof.

[0080] In some aspects, the preceding methods can further comprise administering to a subject identified as being at an increased risk of exhibiting residual symptoms at least one amount of at least one of six therapeutic metabolites, wherein the six therapeutic metabolites comprise dehydroepiandrosterone sulfate, androstenediol (3beta,17beta) disulfate,androstenediol (3alpha, 17alpha) monosulfate, androstenediol (3beta,17beta) monosulfate, pregnenediol sulfate, and pregnenolone sulfate. In some aspects, the methods comprise i) administering to the subject at least one amount of at least two of the six therapeutic metabolites; ii) administering to the subject at least one amount of at least three of the six therapeutic metabolites; iii) administering to the subject at least one amount of at least four of the six therapeutic metabolites; or iv) administering to the subject at least one amount of each of the six therapeutic metabolites.

[0081] In some aspects of the preceding methods, the hypothyroidism treatment comprises the administration of levothyroxine.

[0082] Hypothyroidism

[0083] Hypothyroidism is a condition that results from a failure of the thyroid gland to secrete a physiologically sufficient amount of thyroid hormone. Thus, hypothyroidism is characterized by an elevated level of thyroid-stimulating hormone (TSH). There are two common causes of hypothyroidism. The first involves inflammation or autoimmunity to the thyroid gland, resulting in damage to the hormone-secreting cells and failure of thyroid hormone secretion. A common form of thyroid inflammation results from the autoimmune disease Hashimoto's thyroiditis. A second common cause of hypothyroidism results from surgical treatment of other conditions that require removal of all or part of the thyroid gland, for example after surgical removal of a cancerous thyroid gland. A less common cause of hypothyroidism results from secondary effects produced on a normal thyroid gland that causes a decrease in thyroid hormone secretion. For example, if the pituitary gland fails to produce enough thyroid stimulating hormone (TSH) then the result is a lack of stimulation of the thyroid to produce thyroid hormone.

[0084] Hypothyroidism is the most common thyroid disorder and manifests through the thyroid gland’s inability to produce sufficient thyroid hormones, such as triiodothyronine (T3) and thyroxine (T4). Symptoms associated with hypothyroidism include cold intolerance, lethargy, fatigue, chronic constipation, and a variety of hair and skin changes. While none of these conditions are life-threatening, the disease, if left untreated, could result in myxedema, coma, or death.

[0085] Early symptoms of hypothyroidism include weakness, fatigue, cold intolerance, constipation, weight gain, depression, joint or muscle pain, thin or brittle fingernails and / or hair, or paleness. Late symptoms of hypothyroidism include slow speech, dry, flaky skin, thickening of the skin, puffy face, hands and / or feet, decreased taste and smell, thinning of the eyebrows, hoarseness, or abnormal menstrual periods.

[0086] Additionally, symptoms of hypothyroidism may include overall swelling, muscle spasms or cramps, muscle pain, muscle atrophy, uncoordinated movement, absent menstruation, joint stiffness, dry hair, hair loss, facial swelling, drowsiness, appetite loss, separated sutures, or loss of teeth.

[0087] Levothyroxine Treatment

[0088] A hypothyroidism treatment as disclosed herein can include treatment with levothyroxine. As would be appreciated by the skilled artisan, levothyroxine is a synthetic version of the body’s natural thyroid hormone, thyroxine (T4). Levothyroxine is a hormone compound that exert their physiologic actions through control of DNA transcription and protein synthesis. Levothyroxine diffuses into the cell nucleus and binds to thyroid receptor proteins attached to DNA. The hormone nuclear receptor complex activates gene transcription and synthesis of mRNA and cytoplasmic proteins. In some aspects, levothyroxine directly influences DNA transcription to increase body metabolism by, for example, increasing gluconeogenesis, protein synthesis, or the mobilization of glycogen stores. In some aspects, a levothyroxine treatment can be part of a hypothyroidism treatment. In some aspects, a levothyroxine treatment can be, for example, Levothrioid®, Levo-T®, Levoxyl®, Synthroid®, Tirosint®, Unithroid®.

[0089] In some aspects, a hypothyroidism treatment comprises administration of a thyroid hormone. A thyroid hormone can be levothyroxine:, or a pharmaceutically acceptable salt, analog, derivative, salt or ester thereof. As would be appreciated by the skilled artisan, it is understood that levothyroxine may be identified by any one of the following names: O-(4-Hydroxy-3,5- diiodophenyl)-3,5-diiodo-L-tyrosine, thyroxine, tetraiodothyronine, L-thyroxine, thyroxin, or (S)-2-Amino-3-[4-(4-hydroxy-3,5-diiodophenoxy)-3,5-diiodophenyl]propanoic acid. As would be appreciated by the skilled artisan, levothyroxine may be identified as CAS No. 51- 48-9.

[0090] In some aspects, levothyroxine can be administered orally.

[0091] In some aspects, levothyroxine can be administered in an amount of about 25 pg, or about 50 pg, or about 75 pg, or about 88 pg, or about 100 pg, or about 112 pg, or about 125 pg, or about 137 pg, or about 150 pg, or about 175 pg, or about 200 pg, or about 300 pg.

[0092] In some aspects, a therapeutically effective amount of levothyroxine can be any of the levothyroxine amounts described herein.

[0093] Therapeutic Metabolites of the Present Disclosure

[0094] In some aspects, a therapeutic metabolite can be dehydroepiandrosterone sulfateor an analog or derivative thereof. As would be appreciated by the skilled artisan, it is understood that dehydroepiandrosterone sulfate may be identified by any one of the following names: DHEA-S, DHEA sulfate, androstenolone sulfate, prasterone sulfate, androst-5-en-3f>- ol-17-one 3 -sulfate, 17-Oxoandrost-5-en-3[3-yl hydrogen sulfate, or (3aS,3bR,7S,9aR,9bS,l laS)-9a,lla-Dimethyl-l-oxo-2,3,3a,3b,4,6,7,8,9,9a,9b,10,ll,l la- tetradecahydro-lH-cyclopenta[a]phenanthren-7-yl hydrogen sulfate. As would be appreciated by the skilled artisan, dehydroepiandrosterone sulfate may be identified as CAS No. 651-48-9.

[0095] In some aspects, a therapeutic metabolite can be androstenediol (3beta,17beta) disulfate:or an analog or derivative thereof. As would be appreciated by the skilled artisan, it is understood that androstenediol (3beta,17beta) disulfate may be identified by any one of thefollowing names: 4-androsten-3beta,17beta-diol disulfate, 4-androstenediol disulfate, 4- androstene-3beta,17beta-diol disulfate, androst-4-ene-3beta,17beta-diyl bis(hydrogen sulfate), (3beta,17beta)-androst-4-ene-3,17-diyl bis(hydrogen sulfate), or[(3S,8R,9S,10R,13S,14S,17S)-10,13-dimethyl-3-sulfooxy-2,3,6,7,8,9,ll,12,14,15,16,17- dodecahydro-lH-cyclopenta[a]phenanthren-17-yl] hydrogen sulfate. As would be appreciated by the skilled artisan, androstenediol (3beta,17beta) disulfate (2) may be identified as CAS No. 1357147-18-2.

[0096] In some aspects, a therapeutic metabolite can be androstenediol (3alpha,17alpha) monosulfate:or an analog or derivative thereof. As would be appreciated by the skilled artisan, it is understood that androstenediol (3alpha, 17alpha) monosulfate may be identified by any one of the following names: 4-androsten-3 alpha, 17alpha-diol 3-sulfate, 4-androsten- 3a,17a-diol monosulfate, androstenediol-3-sulfuric acid, Androstenediol monosulfate, or [(lS,2R,5S,10R,HS,14S,15S)-14-hydroxy-2,15- dimethyltetracyclo[8.7.0.0A{2,7}.0A{l l,15}]heptadec-6-en-5-yl]oxidanesulfonic acid.

[0097] In some aspects, a therapeutic metabolite can be androstenediol (3beta,17beta) monosulfate:, or an analog or derivative thereof. As would be appreciated by the skilled artisan, it is understood that androstenediol (3beta, 17beta)monosulfate may be identified by any one of the following names: 4-androsten-3beta, 17beta- diol 3-sulfate, 4-androsten-3p,17P-diol monosulfate, androstenediol-3-sulfuric acid, Androstenediol monosulfate, androstenediol-3-sulfate, or [(3S,8R,9S,10R,13S,14S,17S)-17- hydroxy-10,13-dimethyl-2,3,6,7,8,9,l l,12,14,15,16,17-dodecahydro-lH- cyclopenta[a]phenanthren-3-yl] hydrogen sulfate. As would be appreciated by the skilled artisan, androstenediol (3beta, 17beta) monosulfate may be identified as CAS No. 26704-15- 4.

[0098] In some aspects, a therapeutic metabolite can be pregnenediol sulfate:or an analog or derivative thereof. As would be appreciated by the skilled artisan, it is understood that pregnenediol sulfate may be identified by any one of the following names: [(lS,3aS,3bR,5aS,7R,9aS,9bS,llaS)-l-acetyl-9a,l la-dimethyl-hexadecahydro-lH- cyclopenta[a]phenanthren-7-yl]oxidanesulfonic acid.

[0099] In some aspects, a therapeutic metabolite can be pregnenolone sulfate:or an analog or derivative thereof. As would be appreciated by the skilled artisan, it is understood that pregnenolone sulfate may be identified by any one of the following names: pregnenolone monosulfate, pregnenolone hydrogen sulfate, Pregnenolone 3P-sulfate, 5- Pregnen-3P-ol-20-one sulfate, (3P)-3-(Sulfooxy)pregn-5-en-20-one, 5-Pregnen-3P-sulfate-20- one, 20-Oxo-5-pregnen-3P-yl sulfate, 20-Oxopregn-5-en-3P-yl hydrogen sulfate, or(lS,3aS,3bS,7S,9aR,9bS,l laS)-l-Acetyl-9a,l la-dimethyl- 2,3,3a,3b,4,6,7,8,9,9a,9b,10,ll,l la-tetradecahydro-lH-cyclopenta[a]phenanthren-7-yl hydrogen sulfate. As would be appreciated by the skilled artisan, pregnenolone sulfate may be identified as CAS No. 1247-64-9.

[0100] Any of the therapeutic metabolites described herein can be administered as a pharmaceutically acceptable salt.

[0101] Kits

[0102] The present disclosure provides kits for use in the methods described herein.

[0103] Accordingly, the present disclosure provides kits comprising at least one of dehydroepiandrosterone sulfate (DHEA-S), androstenediol (3beta, 17beta) disulfate, androstenediol (3alpha, 17alpha) monosulfate, androstenediol (3beta,17beta) monosulfate, pregnenediol sulfate, and pregnenolone sulfate, or a pharmaceutically acceptable salt thereof.

[0104] The present disclosure also provides kits comprising at least one reagent that allows for the determination of the concentration of at least one dehydroepiandrosterone sulfate (DHEA-S), androstenediol (3beta,17beta) disulfate, androstenediol (3alpha, 17alpha) monosulfate, androstenediol (3beta,17beta) monosulfate, pregnenediol sulfate, and pregnenolone sulfate.

[0105] The kits of the present disclosure can further comprise instructions for performing one or more of the methods described herein.

[0106] General Methods and Definitions

[0107] The term "biological sample" as used herein refers to any sample of biological origin potentially containing one or more biomarkers. A biological sample can comprise blood, plasma, serum, urine, breast milk, cerebrospinal fluid, mucus, gastric juice, peritoneal fluid, pleural fluid, saliva, sebum, semen, sweat, tears, vaginal secretion, vomit, endolymph, perilymph or any combination thereof. A biological sample can be a plasma sample. A biological sample can be a blood sample. A biological sample can be a cerebrospinal fluid sample.

[0108] The terms “subject” and “patient” are used interchangeably herein. In some embodiments, the subject treated in accordance with the methods described herein is a human patient. In some aspects, the subject is male. In some aspects, the subject is female.

[0109] In some aspects of the methods of the present disclosure, the methods can further comprise administering to a subject who is identified as being nonresponsive to a hypothyroidism treatment at least one alternative hypothyroidism treatment either in lieu of the hypothyroidism treatment or in combination with the hypothyroidism treatment. In a non-limiting example, a subject who is determined to be non-responsive to treatment with levothyroxine using the methods of the present disclosure can be administered an alternative hypothyroidism treatment alone or a combination of the levothyroxine and an alternative hypothyroidism treatment.

[0110] In some aspects of the methods of the present disclosure, the methods can further comprise administering to a subject who is identified as being at risk of exhibiting one or more residual symptoms after administration of a hypothyroidism treatment at least one alternative hypothyroidism treatment either in lieu of the hypothyroidism treatment or in combination with the hypothyroidism treatment. In a non-limiting example, a subject who is determined to be at risk of one or more residual symptoms after treatment with levothyroxine using the methods of the present disclosure can be administered an alternative hypothyroidism treatment alone or a combination of the levothyroxine and an alternative hypothyroidism treatment.

[0111] In some aspects of the methods of the present disclosure, the methods can further comprise modifying the dosage of a hypothyroidism treatment for a subject who is identified as being non-responsive to that hypothyroidism treatment. In a non-limiting example, if a subject is identified as being non-responsive to levothyroxine using the methods of the present disclosure, the methods can further comprise modifying the dosage (e.g. increasing the dosage) of levothyroxine.

[0112] In some aspects of the methods of the present disclosure, the methods can further comprise modifying the dosage of a hypothyroidism treatment for a subject who is identified as being at risk of having one or more residual symptoms following that hypothyroidism treatment. In a non-limiting example, if a subject is identified as being at risk of having one or more residual symptoms following levothyroxine treatment using the methods of the present disclosure, the methods can further comprise modifying the dosage (e.g. increasing the dosage) of levothyroxine.

[0113] Alternative hypothyroidism treatments can be any hypothyroidism treatment known in the art.

[0114] Experimental Examples

[0115] General Methods

[0116] Mendelian Randomization and Sensitivity Analysis Assessing the Effect of Genetic Susceptibility to Hypothyroidism on Metabolite Levels

[0117] Applicants followed the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE)-MR guidelines for reporting MR results (Skrivankova VW et al. (2021) BMJ (375):n2233, Skrivankova VW et al. (2021) JAMA (16): 1614-1621).

[0118] Hypothyroidism Genome-Wide Association Studies (GWAS)

[0119] Applicants used the largest hypothyroidism GWAS meta-analysis comprising 51,194 cases and 443,383 controls of European ancestry from the FinnGen and UK Biobank (Mathieu S et al. (2022) 25(9): 104992) to identify the genetic determinants of hypothyroidism. Hypothyroid cases were determined based on clinical diagnosis and treatment with levothyroxine. Using the UK Biobank data, age, sex, and the first 20 ancestry-based principal components (PC) were included in the model. Using the FinnGen data, age, sex, genotyping batch, and first 10 PCs were included in the model. The genetic effects on hypothyroidism risk were meta-analyzed in a fixed-effect model. Further details of the demographics of the cohorts and GWAS analyses can be found in Table 1.

[0120] Table 1: Cohort Characteristics for the Three GWAS Used for Two-SampleMendelian Randomization\ \ \ \Melabolom.ics Genome-Wide Association Studies (GWAS)

[0122] Applicants used genetic associations identified for plasma metabolite levels from two recent large metabolomics GWAS studies on individuals of European ancestry (the total sample size was up to 22,595) (Surendran P et al. (2022) Nat Med. 28(22):2321-2332, Chen Y et al. (2023) Nat Genet. 55(l):44-53). Specifically, Applicants focused on 458 unambiguously identified non-xenobiotic metabolites that have known chemical identities and were investigated in both studies. In the INTERVAL and EPIC-Norfolk studies, metabolite values were natural -log-transformed, winsorized to 5 standard deviations, and then adjusted the metabolite levels for age, sex, other study-specific variables, and PCs in a multivariable linear regression model. The residuals of the regression model were then standardized (mean = 0, standard deviation = 1) and used as the phenotype in GWAS analyses (Surendran P et al. (2022) Nat Med. 28(22):2321-2332). In the Canadian Longitudinal Study on Aging (CLSA) study, metabolite values were natural-log-transformed, trimmed to remove outliers larger than 3 standard deviations from the mean, and then standardized (mean = 0, standard deviation = 1). The standardized metabolites measures were then tested for association with genetic variants, while including sex, age, hour since last meal or drink, genotyping batch, and first 10 PCs as covariates (Chen Y et al. (2023) Nat Genet. 55(l):44-53). Considering that the normalization and transformation approaches were similar across studies and both GWAS beta values have the same unit (i.e., 1 standard deviation), fixed-effects meta-analyses were performed to obtain inverse variance weighted (IVW) estimates for genetic effects on the levels of the metabolites. As part of the sensitivity analysis, Applicants undertook a separate GWAS of ULPC-MS / MS- measured thyroxine using European ancestry individuals (excluding treated hypothyroidism cases) from the CLSA (Chen Y et al. (2023) Nat Genet. 55(l):44-53, Forgetta V et al. (2022) BMJ Open. 12(3)e059021).

[0123] Hypothyroidism Instrumental Variables Selection

[0124] LD-independent genome-wide significant genetic variants for hypothyroidism were selected using PLINK 1.9 clumping function (clump-kb 1Mb, clump-r2 0.001, clump-pl 5x10"8) (Chang CC et al. (2015) Gigascience. 4:7). The European ancestry of individuals in the 1000 Genomes phase 3 dataset was used to derive the LD reference panel. Proxy SNPs were identified using the SNAPPY program when a selected genetic instrument was not available in the metabolomics GWAS meta-analysis. Genetic variants in the major histocompatibility complex (MHC) region were removed to reduce the risk of horizontal pleiotropy and confounding effects arising from its complex LD structure (chr6:27977797-33948354 in build GRCh37). A total of 143 independent genetic variants were used as instrumental variables for susceptibility to hypothyroidism. F-statistics were calculated to assess the suitability of the]V— fc— 1 R2genetic variants as instrumental variables using the following formula F = ( — - — ) (— ) where N is the effective sample size, k is the number of genetic variants, and the variance in exposure explained by variant is estimated using GWAS -estimated per-SNP effect size ( / ?) and minor allele frequency (MAF) by R2= 2 X ft2x MAF x (1 — MAF) (Lawlor DA et al., (2008) Stat Med. 27(8): 1133-1163, Staley JR et al. (2016) Bioinformatics. 32(20):3207-3209). Since the allele frequencies of genetic variants were not provided by the original GWAS meta-analysis for hypothyroidism (Mathieu S et al. (2022) 25(9): 104992), the allele frequencies from the UK Biobank European ancestral population were used to estimate the variance in exposure explained by variant. As a sensitivity analysis for the MR independence assumption, Applicants checked if any of these variants are associated with potential confounders of the hypothyroidism and metabolite level associations by querying the PhenoScanner database (Staley JR et al. (2016) Bioinformatics. 32(20):3207-3209, Kamat MA et al. (2019) Bioinformatics. 32(22)). A P-value threshold of 5 / 108was used to select variant-trait associations and proxies were defined as having an r2of 0.8 in individuals with European ancestry.

[0125] Two-Sample MR and Sensitivity Analyses

[0126] To estimate the causal effect of exposure (i.e., susceptibility to hypothyroidism) on the outcome (i.e., circulating plasma metabolite levels), an IVW random effects model was used with sensitivity analyses using MR Egger, MR weighted median, and MR pleiotropy residual sum and outlier (MR-PRESSO) analyses (Verbanck M et al. (2018) Nat Genet. 50(5):693-698). IVW estimates were obtained by meta-analyzing the SNP-specific Wald ratio estimates. The IVW random effects model was chosen because it is more robust to heterogeneity among instruments (Bowden J et al. (2017) Stat Med. 36(11): 1783-1802). Cochran’s Q statistic was calculated to check for possible heterogeneity among SNPs (Cochran WG et al. (1954) Biometrics. 10(1): 101). The MR-Egger method assumes No Measurement Error (NOME) for SNP effects and also makes the instrument strength independent of the direct effects (InSIDE) assumption (Bowden J et al. (2015) Int J Epidemiol. 44(2):512-525). Applicants estimated MR- Egger intercepts to detect possible directional horizontal pleiotropy (Bowden J et al. (2015) Int J Epidemiol. 44(2):512-525). The weighted median method assumes that at least 50% of the total weight of instruments comes from valid genetic variants (Bowden J et al. (2016) Genet Epidemiol. 40(4):304-314). Compared to IVW, the causal effect estimate from weighted median estimation is more robust to more invalid instruments (Bowden J et al. (2016) Genet Epidemiol. 40(4):304-314). MR-PRESSO test was performed to detect possible outlierinstruments and obtain corrected effect estimates after removing outliers (Verbanck M et al. (2018) Nat Genet. 50(5):693-698). Leave-one-out analyses were also performed to verify if the observed causal relationships were driven by a single variant.

[0127] MR analyses were performed using functions in the TwoSampleMR package (version 0.5.6) and MRPRESSO (version 1.0) in R (version 4.1.2). Metabolites with an IVW false discovery rate (FDR) <0.05 (accounting for 458 tests) were retained. FDR was used since metabolite levels can be highly correlated. Next, only metabolites with an MR-weighted median P <0.05, MR-Egger intercept P >0.05, and MR PRES SO outlier-corrected test P <0.05 (if outliers were detected) were retained.

[0128] Bidirectional MR

[0129] To check if hypothyroidism-influenced metabolites can also modify the risk of hypothyroidism, MR was performed using metabolites as exposures and susceptibility to hypothyroidism as the outcome. Specifically, the metabolite quantitative trait loci (mQTL) supported by biological and expression evidence (z.e., with effector genes) were used as instrumental variables for metabolite exposures (Chen Y et al. (2023) Nat Genet. 55(l):44-53). Metabolites that had a P <0.05 based on Wald ratio or IVW estimates were removed from further analyses to avoid potential bias from reverse causation.

[0130] MR Analyses Testing the Effect of Thyroid Hormone Levels on Hypothyroidism-Driven Metabolites

[0131] To assess whether metabolite levels were also affected by blood levels of FT4, FT3, or TSH, even when they are in normal ranges, two-sample MR analyses were performed using FT4, FT3, and TSH as the exposures and the 11 hypothyroidism-influenced metabolites that were identified in the above MR analysis as outcomes. This study included up to 119,120, 59,061, and 271,040 adults of European ancestry for FT4, FT3, and TSH analysis (Sterenborg R et al. (2023) Nat Commun.) Individuals who had TSH levels outside the cohort-specific reference range were taking thyroid medication, or with a history of thyroid surgery were excluded from the analysis. The FT4, FT3, and TSH levels were inverse normal transformed in the participating studies. A fixed-effect model with inverse-variance weighting was applied to meta-analyze the GWAS results.

[0132] The selected independent genome-wide significant genetic associations were selected by removing variants in the MHC region. A total of 187 genetic instruments for TSH, 65 for FT4, and seven for FT3 were used. MR and sensitivity analyses were also performed using functions from the TwoSampleMR package (version 0.5.6) and MRPRESSO (version 1.0) in R (version 4.1.2).

[0133] CLSA Cohort

[0134] CLSA is a longitudinal cohort study of 50,000 individuals aged between 45 and 85 at baseline, recruited from multiple locations across Canada (Raina P et al. (2019) Int J Epidemiol. 48(6)). For the observational analyses, 9,651 individuals were identified with metabolomic profiles and covariates measurements obtained at baseline in the CLSA study. Individuals with thyroid cancer, hyperthyroidism, or inconsistent self-reported sex and biological sex at baseline were excluded. Individuals with abnormal T4 or TSH levels (i.e., T4: < 5pmol / L or TSH: <0.1 mIU / L or > 5mIU / L) were also excluded to retain only individuals in a euthyroid state. Next, those with self-reported, doctor-diagnosed hypothyroidism, and are receiving hypothyroidism medications were identified. In total, 803 hypothyroidism cases who self-reported taking hypothyroidism drugs at baseline with normal TSH levels (adequately treated hypothyroidism cases), and 7,885 individuals with normal TSH levels and no reported thyroid diseases (euthyroid controls) were identified.

[0135] Metabolite Profiling

[0136] Metabolite measurements were assessed of the 11 hypothyroidism-influenced metabolites identified by MR in the CLSA cohort. Metabolomics profiling of the plasma samples of CLSA participants was performed by Metabolon, Inc. (Durham, NC, USA) using their UPLC-MS / MS platform (i.e., Metabolon HD4 platform) (Chen Y et al. (2023) Nat Genet. 55(l):44-53). Batch-normalized levels of metabolites generated by Metabolon, Inc. were used.

[0137] Covariates in Observational Association Testing

[0138] Age (years), biological sex, body mass index (BMI, kg / m2), hour since last meal or drink, smoking status, and recruitment centers at baseline in CLSA as covariates were included for observational association studies. Smoking status was categorized as “Yes (I currently smoke)”, “No (I don’t smoke and I never have)”, and “Former (I don’t smoke now but I have in the past)”. Recruitment centers included the 10 CLSA recruiting sites across Canada.

[0139] Comparing Adjusted Metabolite Levels Using Student’s T-Test

[0140] Applicants next tested whether the 11 hypothyroidism-driven metabolites were different amongst individuals with hypothyroidism in a euthyroid state, compared to euthyroid controls in the CLSA cohort. Metabolite levels were natural log-transformed, trimmed to remove outliers 5 standard deviations away from the mean, and then standardized to have a mean of 0 and a standard deviation of 1. For each metabolite, the standardized metabolite levels were regressed on age, sex, hour since the last meal or drink, BMI, and recruitment centers. Student’s t-tests were performed to assess whether the residualized metabolite levels differed between euthyroid controls and treated hypothyroidism cases in a euthyroid state. Adjustedmetabolite levels between euthyroid controls and adequately treated hypothyroidism cases were also compared within males and females separately.(00141] Association of Metabolites with Cognitive Function and Self-Rated General Health in Euthyroid Treated Hypothyroidism Cases and Euthyroid Controls In CLSA

[0142] To understand the associations between hypothyroidism, hypothyroidism-influenced metabolites, and cognitive function, Applicants first tested the association of hypothyroidism status (i.e., individuals with adequately treated hypothyroidism vs. euthyroid controls) with cognitive function measurements, including animal fluency tests (AFT) score 1 and 2, Mental Alternation Tests (MAT) score (Teng E. (1995) Clin Nerophy col. 9(3):287), The Rey Auditory Verbal Learning Tests - Trial 1 (REYI) and five-minute delayed recall (REYII), and set switching time (i.e., Stroop time) (COLTIME) (Bayard S et al. (2011) Archives of Clinical Neuropshycology. 26(7):653-661, Troyer AK et al. (2006) Again, Neuropsychology, and Cognition. 13(l):20-35) at baseline in the CLSA. To do so, Applicants inverse normalized the cognitive measures and then applied linear regression adjusting for biological sex, age, hour since last meal or drink, BMI, smoking status, language used for cognitive function test (English / French), education levels (less than secondary education, secondary education, no post-secondary education, some post-secondary education, and post-secondary degree / diploma), and recruitment centers.

[0143] Applicants then investigated the association between hypothyroidism-influenced metabolites and cognitive function within the subsets of individuals with adequately treated hypothyroidism or euthyroid controls separately. Linear regression models were fitted with cognitive function measures as outcomes and metabolite levels as predictors. The cognitive function measures were inverse normal transformed. Biological sex, age, hour since last meal or drink, BMI, smoking status, language used for cognitive function test, education levels, and recruitment centers were included as covariates in the models. The same association analyses were also performed in a sex-specific manner.

[0144] Since decreased quality of life has been often reported by hypothyroidism patients, Applicants tested if hypothyroidism-influenced metabolites were associated with health perception using the “Self-rated general health” measure which is an ordinal variable with 5 levels: 1 as “Excellent”, 2 as “Very good”, 3 as “Good”, 4 as “Fair”, and 5 as “Poor”. Specifically, Applicants compared the individuals who reported “Excellent” vs “Fair or poor” (i.e., reported “Fair” or “Poor”) for their general health. Applicants performed logistic regression using binarized self-rated general health (“Excellent” as 0 and “Fair or poor” as 1) as the outcome and standardized levels of the hypothyroidism-influenced metabolites as thepredictor. Analyses were performed within the subsets of euthyroid individuals with adequately treated hypothyroidism or euthyroid controls separately. Biological sex, age, hour since last meal or drink, BMI, smoking status, and recruitment centers were included as covariates.

[0145] Association between Hypothyroidism and Adrenal Insufficiency

[0146] Considering that the identified hypothyroidism-influenced metabolites that differed, despite adequate levothyroxine therapy, were steroids mainly synthesized by the adrenal gland (Chakraborty S et al. (2021) Genes Immun. 22(3): 125-140), Applicants next assessed the genetic and observational correlation between hypothyroidism and adrenal insufficiency to understand if these diseases shared similar causal pathways.

[0147] Genetic Correlation between Hypothyroidism and Autoimmune Adrenal Insufficiency

[0148] The same hypothyroidism GWAS meta-analysis used in MR was used to compute the genetic correlation between hypothyroidism and autoimmune adrenal insufficiency (Mathieu S et al. (2022) 25(9): 104992). An autoimmune adrenal insufficiency (z.e., Addison’s disease) GWAS which comprised 1,223 cases and 4,097 healthy controls of European ancestry from Sweden and Norway was used (Eriksson D et al. (2021) Nat Commun. 12(1):959). LD score regression was applied to infer the genetic correlation between hypothyroidism and adrenal insufficiency (Bulik-Sullivan BK et al. (2015) Nat Genet. 47(11): 1236-1241, Bulik-Sullivan BK et al. (2015) Nat Genet. 47(3):291-295).

[0149] Observational Correlation between Hypothyroidism and Adrenal Insufficiency

[0150] From 2006 to 2010, the UK Biobank recruited over 500,000 participants between 40 and 69 years old at multiple assessment centers across the United Kingdom and collected a large set of phenotypes and biological samples (By croft C et al. (2018) Nature. 562(7726):203- 209). Hypothyroidism and adrenal insufficiency cases from the UK Biobank cohort were identified. Individuals who had received a diagnosis of Hashimoto’s thyroiditis (ICD-10 code E06.3), hypothyroidism (ICD-10 code E03.9) and / or had received thyroxine treatment (field 20003, drug names: levothyroxine sodium, dextrothyroxine sodium, thyroxine sodium, thyroxine product, sodium thyroxine) were considered as cases of hypothyroidism. Individuals who had received a diagnosis of primary adrenocortical insufficiency (ICD-10 code E27.1) and / or other unspecified adrenocortical insufficiency (ICD-10 code E27.4) were considered as cases of adrenal insufficiency. Individuals without hypothyroidism or adrenal insufficiency were considered as controls for the corresponding disease. In total, 36,461 cases and 466,155 controls for hypothyroidism, and 681 cases and 501,935 controls for adrenal insufficiency were identified. The odds ratio was calculated to assess the extent of co-occurrence betweenhypothyroidism and adrenal insufficiency. / 2test was used to test whether these two traits cooccur more frequently than is expected by chance.

[0152] A total of eight genetic variants associated with adrenal insufficiency were used. IVW estimated the causal effect of adrenal insufficiency on the metabolite levels were calculated using functions from TwoSampleMR R package (version 0.5.6).

[0153] Statistics

[0154] All beta and odds ratio estimates from MR and association analyses are presented with a 95% confidence interval. Benjamini -Hochberg method was applied to control the FDR for multiple hypothesis testing for the MR, and the Student’s t-test comparing the metabolites between hypothyroidism patients and controls. A multiple-testing correction was not applied when checking the associations between hypothyroidism-influenced metabolites and cognitive function as well as self-rated general health because these metabolites are interrelated, and similarly, the cognitive measures are also not independent from one another. A nominal p- value less than 0.05 was considered significant for the association analysis.

[0155] Example 1. Study Design and Highlighted Results.

[0156] Applicants tested which metabolites were perturbed by hypothyroidism by performing a two-sample Mendelian randomization (MR) assessment. It was found that 11 metabolites in the blood plasma were influenced by hypothyroidism.

[0157] Applicants next tested if these 11 hypothyroidism-driven metabolites were normalized by levothyroxine treatment by comparing 803 cases of patients adequately treated with levothyroxine for hypothyroidism with 7885 cases of Euthyroid controls, which were individuals free of thyroid disease. Applicants found that 4 androstane and 2 pregnane steroids were not normalized by adequate replacement with levothyroxine.

[0158] Next, Applicants assessed whether these metabolites are associated with cognitive function and self-reported general health. Cross-sectional associations between metabolites and cognitive function were measured as well as self-rated general health in the CLSA cohort. Applicants tested genetic and epidemiological associations between hypothyroidism with adrenal insufficiency.

[0159] Example 2. Metabolites Influenced by Susceptibility to Hypothyroidism.

[0160] To estimate the causal effect of susceptibility to hypothyroidism on plasma metabolite levels, Applicants performed a two-sample MR using hypothyroidism as the exposure and 458 circulating metabolite levels as the outcomes. These metabolites included 235 lipids, 145amino acids, 20 nucleotides, 18 carbohydrates, 18 cofactors and vitamins, 16 peptides, and 6 energy substrates. The specifics of the cohorts employed in the genome-wide association studies (GWAS) for selecting genetic instruments and for use as outcomes in MR are detailed in Table 1. Applicants selected 143 genetic instruments for hypothyroidism risk with a minimum F-statistic of 79.6, suggesting that associations between genetic variants and the risk of hypothyroidism are robust (Lawlor DA et al. (2008) Stat Med. 27(8): 1133-1163, Burgess S et al. (2011) Int J Epidemiol. 40(3):755-764). The associations between these genetic variants and potential confounding traits were checked using the PhenoScanner database (Staley JR et al. (2016) Bioinformatics. 32(20):3207-3209, Kamat MA et al. (2019) Bioinformatics. 35(22)).

[0161] As a positive control, Applicants assessed whether genetic susceptibility to hypothyroidism influenced FT4 levels using two-sample MR. To do so, genetic instruments were used for susceptibility to hypothyroidism as exposure and the GWAS summary statistics for UPLC-MS / MS -measured thyroxine from the INTERVAL and EPIC-Norfolk cohorts (Surendran P et al. (2022) Nat Med. 28(22):2321-2332) (N = 14,249) and from the CLSA cohort (N = 7,294, removed individuals receiving thyroxine therapy) as outcomes. Genetic susceptibility to hypothyroidism decreased thyroxine levels in both studies (inverse variance weighted (IVW) Beta: -0.04, 95%CI (-0.07, -0.01), IVW P=4.5xl0’3for the INTERVAL and EPIC-Norfolk cohorts, and IVW Beta: -0.05, 95%CI (-0.09, -0.01), IVW P=0.02 for the CLSA cohort). This finding supports the validity of the study design since hypothyroidism is known to reduce FT4 levels.

[0162] Out of 458 metabolites tested, genetic susceptibility to hypothyroidism had an estimated causal effect on 16 metabolite levels, after accounting for multiple testing (IVW false discovery rate (FDR) < 0.05) (FIG. 1A). After removing the metabolites that were at risk of horizontal pleiotropy (MR-Egger intercept P <0.05), and those with inconsistent causal effects in sensitivity testing (MR weighted median P >0.05 or MR PRES SO corrected P >0.05, if an outlier was detected), 12 metabolites were retained for further investigation (Table 2). For example, a 1 standard deviation increase in genetic susceptibility to hypothyroidism was associated with 0.05 standard deviation decrease in dehydroisoandrosterone sulfate (DHEA-S) level (IVW Beta: -0.05, 95%CI (-0.08, -0.03), IVW P=l.lxl0'5) and a 0.06 standard deviation decrease in pregnenediol sulfate level (IVW Beta: -0.06, 95%CI (-0.09, -0.03), IVW P=7.2xl0‘5) (FIG. IB)

[0163] From the results of MR leave-one-out sensitivity analyses, as well as MR scatter and funnel plots, it was concluded that these estimated causal relationships were not driven by a single variant and there was little evidence of horizontal pleiotropy (FIG. IB, FIGs. 2A-2J,FIGs. 3A-3L). Lastly, using bidirectional MR analysis, one metabolite (2-aminobutyrate) was identified that had a potential causal effect on hypothyroidism- / , e., showed potential for reverse causation — and this metabolite was then removed from subsequent analyses (Table 3).

[0164] Additionally, MR estimates of all 11 hypothyroidism-influenced metabolites were consistent using the two constituent studies of the GWAS meta-analysis, although the magnitude of the effect was different for some of the metabolites (FIG. 4). Overall, using the two-sample MR approach, 11 metabolites were identified to be influenced by susceptibility to hypothyroidism, which was subsequently utilized in further analyses.

[0165] Table 2: MR Sensitivity Analysis Result of 16 MR-Identified HypothyroidismMetabolites

[0166] Table 3: Reverse MR Assessing Effects of Metabolites on Hypothyroidism

[0167] Example 3. Thyroid Hormone Levels within the Normal Range show Little Effect on Hypothyroidism-Influenced Metabolites.

[0168] Applicants then asked why these 11 metabolites are influenced by hypothyroidism. Whether the levels of these 11 hypothyroidism-influenced metabolites were affected by FT4, FT3, and TSH levels in individuals not known to have thyroid disease was explored. Two- sample MR analyses were performed using genetic determinants of FT4, FT3, and TSH as exposures, which were derived from the latest thyroid hormone GWAS meta-analysis. The sample sizes of the FT4, FT3, and TSH GWAS were up to 119,120, 59,061, and 271,040, respectively. Using MR, it was found that TSH had suggestive effects on two of these 11 hypothyroidism-influenced metabolites while FT4 and FT3 levels showed no clear effect on any (FIG. 5). One standard deviation increased in genetically predicted TSH levels suggested corresponding decrease in 2 of the hypothyroidism-influenced metabolites, pregnenolone sulfate (IVW Beta = -0.06, 95%CI (-0.1, -0.02), IVW FDR = 0.2, IVW P=6xl0’3), and pregnenediol sulfate (IVW Beta = -0.06, 95%CI (-0.1, -0.01), IVW FDR = 0.2, IVW P=lxl0’2), with low risks of horizontal pleiotropy in either case (MR Egger intercept P >0.05) and consistent causal effects estimated from MR weighted median estimation (P <0.05). Thus, two of the 11 metabolites may be marginally influenced by TSH levels, while none appeared to be influenced by FT4 or FT3 levels. Taken together, these findings suggest that most of the hypothyroidism-influenced metabolites are influenced by the factors that cause hypothyroidism, rather than direct perturbations of FT4, FT3, and TSH.

[0169] Example 4. Androstane and Pregnane Steroids Remained Perturbed after Thyroxine Treatment.

[0170] The over-arching purpose of this study was to identify metabolites influenced by susceptibility to hypothyroidism but were not corrected by adequate levothyroxine therapy. Therefore, after identifying metabolites influenced by hypothyroidism, it was next investigated whether the levels of such metabolites, after levothyroxine treatment, remained different from the normal levels. Metabolite levels were compared between individuals with adequately treated hypothyroidism and euthyroid controls from the CLSA cohort. A total of 8,688 individuals were included in the analysis, including 803 individuals with hypothyroidism who were taking hypothyroidism medication and had TSH lower than 5mIU / L and higher than O.lmlU / L at baseline (i.e., adequately treated individuals with hypothyroidism). As expected, it was found that the individuals with adequately treated hypothyroidism were more likely to be females (75.1% vs. 46.1%) and older (66.3 years vs. 62.4 years) when compared to the euthyroid controls (Table 4). Higher levels of FT4 were observed in the individuals withadequately treated hypothyroidism than in euthyroid controls without reported thyroid diseases (17.8 vs 14.8 pmol / L), which was likely due to levothyroxine treatment (FIG. 6A). Nevertheless, the TSH levels were similar between these two groups (2.0 vs 2.1 mIU / L) (FIG. 6B). Similar patterns of difference in FT4 and TSH between individuals with adequately treated hypothyroidism and euthyroid controls were observed within males and females (Table 4).

[0171] As shown in FIG. 7, it was found that six of 11 metabolites that were identified to be perturbed by susceptibility to hypothyroidism remained lower (FDR<0.05) in the individuals with adequately treated hypothyroidism compared to euthyroid controls. These six metabolites were: androstenediol (3alpha, 17alpha) monosulfate (P=2xl0‘5), pregnenediol sulfate (P=4xl0‘4), DHEA-S (P=lxl0‘3), androstenediol (3beta,17beta) disulfate (P=lxl0‘3), pregnenolone sulfate (P=8xl0‘3), and androstenediol (3beta,17beta) monosulfate (P=8xl0‘3), which all belong to a category of steroids. When comparing metabolites within males and females, five out of six steroids showed were consistently lower in cases with adequately treated hypothyroidism compared to euthyroid controls across both sexes. Meanwhile, the reduction in androstenediol (3beta, 17beta) monosulfate in cases with adequately treated hypothyroidism was only evident in males (P=3xl0‘4), with no significant observation in females (P=0.3). In sum, five of 11 hypothyroidism-influenced metabolites were corrected by thyroid hormone therapy while six adrenal steroid metabolites that were decreased by susceptibility to hypothyroidism remained perturbed after adequate treatment.

[0172] Table 4: CLSA Cohort Characteristics

[0173] Example 5. Hypothyroidism-Influenced Steroid Levels were Associated with Cognitive Function and Self-Rated General Health.

[0174] Having identified metabolites influenced by hypothyroidism that remained different despite adequate thyroid hormone replacement, the association of hypothyroidism as well as the six hypothyroidism-influenced steroids was next assessed with six measures of cognitive function in the CLSA cohort using regression analyses.

[0175] To begin with, minor differences were found between adequately treated hypothyroidism cases and euthyroid controls for all cognitive measures (P value ranges: 0.24- 0.96). Interestingly, among individuals with adequately treated hypothyroidism, lower values for any of the six hypothyroidism-influenced steroids were associated with reduced performance in verbal fluency (AFT1 and AFT2) and set switching time (COLTIME) (P<0.05 for six metabolites with all three measures of cognitive performance). Furthermore, reduction in DHEA-S was associated with reduced performance in five of the six cognitive tests, except for the mental alternation test (MAT) score amongst cases with adequately treated hypothyroidism. (FIG. 8A). Consistent, but less profound, the same positive associations were observed between DHEA-S and cognitive function measures in euthyroid controls. Moving to sex-stratified association tests, the positive association between hypothyroid-influenced steroids and cognitive measures could be seen in both treated male and female hypothyroidism cases, though the associations are weaker in males due to much smaller sample sizes. Interestingly, the observed associations between DHEA-S and the three measurements in euthyroid controls, AFT1, AFT2, and COLTIME, from the sex-combined analysis were predominantly driven by male participants (FIGs. 9A-9B).

[0176] Applicants next used logistic regression to test the association between hypothyroidism-influenced steroids and a measure of self-rated general health that is available in CLSA (“Excellent” vs “Fair or poor”). It was found that decreased levels of all six steroids were associated with increased odds of worse self-reported general health among euthyroid controls (FIG. 8B, Table 5). For cases with adequately treated hypothyroidism, similar trends were observed in some metabolites (e.g., DHEA-S and androstenediol (3beta,17beta) disulfate) but with wider confidence intervals, which is likely due to much smaller sample sizes (FIG.8B)

[0177] Table 5: Association between Metabolites and Self-Rated General Health using Logistic Regression

[0178] Example 6. Association between Hypothyroidism and Adrenal Insufficiency.

[0179] The six hypothyroidism-influenced steroids, which differed despite levothyroxine treatment, are all part of the steroidogenesis pathways active in the adrenal gland (Chakraborty S et al. (2021) Genes Immun. 22(3): 125-140). Therefore, the genetic and observational correlation between hypothyroidism and adrenal insufficiency was assessed using available GWAS summary statistics and UK Biobank data. Using LD score regression (Bulik-Sullivan B et al. (2015) Nat Genet. 47(3):291-295), a positive genetic correlation (rg=0.53, 95% CI:(0.35, 0.71), P=l.lxl0'8) was found between hypothyroidism and autoimmune adrenal insufficiency, indicating that a significant proportion of the genetic predisposition is shared between these two conditions. It was also found that hypothyroidism and adrenal insufficiency co-occurred more frequently than is expected by chance (odds ratio = 6.9, 95% CI:5.9-9.1; P=5xl0'172) (Table 6) using the UK Biobank data. Lastly, whether genetic susceptibility to adrenal insufficiency affects the six hypothyroidism-influenced steroids was checked using two-sample MR. Increased susceptibility to adrenal insufficiency showed negligible effect on these steroids and all of the 95% confidence intervals included zero (P value range: 0.12-0.61) (Table 7). Taken together, these findings suggest that hypothyroidism and adrenal insufficiency share some of the genetic determinants, but changes in our six metabolites of interest here may not be entirely driven by adrenal insufficiency.

[0180] Table 6: Contingency Table showing Counts of Individuals with Hypothyroidism and Adrenal Insufficiency from the UK Biobank Cohort

[0181] Table 7: Estimated Causal Effect of Susceptibility to Addison’s Disease on Hypothyroidism-Influenced Metabolite Levels

[0182] Without wishing to be bound by theory, the results described in this example demonstrate that subjects having hypothyroidism who exhibit residual symptoms aftertreatment with levothyroxine exhibit decreased levels of one or more of dehydroepiandrosterone sulfate (DHEA-S), androstenediol (3beta,17beta) disulfate, androstenediol (3alpha, 17alpha) monosulfate, androstenediol (3beta,17beta) monosulfate, pregnenediol sulfate, and pregnenolone sulfate. Accordingly, the levels of these metabolites can be used to determine responsive to levothyroxine treatment, as well as to determine the risk of a subject to develop residual symptoms. Finally, these metabolites can also be administered to the subject in order to treat the hypothyroidism and alleviate any residual symptoms following levothyroxine therapy.

Claims

What is claimed is:

1. A method of determining the responsiveness of a subject to a hypothyroidism treatment, the method comprising: a) determining the concentration of at least one of six metabolites in a biological sample from the subject, wherein the six metabolites comprise dehydroepiandrosterone sulfate (DHEA-S), androstenediol (3beta,17beta) disulfate, androstenediol (3alpha, 17alpha) monosulfate, androstenediol (3beta,17beta) monosulfate, pregnenediol sulfate, and pregnenolone sulfate; b) identifying that the subject is responsive or nonresponsive to the hypothyroidism treatment based on the concentration of the at least one metabolite measured in step (a).

2. The method of claim 1, wherein step (a) comprises: i) determining the concentration of at least two of the six metabolites; ii) determining the concentration of at least three of the six metabolites; iii) determining the concentration of at least four of the six metabolites; iv) determining the concentration of at least five of the six metabolites; or v) determining the concentration of each of the six metabolites.

3. The method of claim 1 or claim 2, wherein identifying that the subject is responsive or nonresponsive to the hypothyroidism treatment based on concentration of the at least one metabolite measured in step (a) comprises: i) comparing the one or more concentrations measured in step (a) to one or more corresponding predetermined cutoff values; and ii) identifying the subject as being nonresponsive to the hypothyroidism treatment when one or more of the concentrations measured in step (a) is less than its corresponding predetermined cutoff value.

4. The method of claim 3, wherein the corresponding predetermined cutoff value is determined through the analysis of the concentration of the at least one metabolite in a training set, wherein the training set comprises the concentration of the at least one metabolite measured in biological samples from one or more training subjects,wherein the one or more training subjects comprise a plurality of training subjects that respond to the hypothyroidism treatment and a plurality of training subjects that do not respond to the hypothyroidism treatment.

5. The method of any one of the preceding claims, wherein the subject has previously received one or more amounts of the hypothyroidism treatment.

6. The method of claim 5, wherein the hypothyroidism treatment comprises the administration of levothyroxine.

7. The method of any one of the preceding claims, wherein a subject who is identified as not responsive has a higher risk of exhibiting one or more residual symptoms of hypothyroidism.

8. The method of claim 7, wherein the one or more residual symptoms comprise reduced cognitive function, poor memory, fatigue, anxiety, reduced general health, or any combination thereof.

9. A method of determining the responsiveness of a subject to a hypothyroidism treatment, the method comprising: a) at a first time point, determining the concentration of at least one of six metabolites in a biological sample from the subject, wherein the six metabolites comprise dehydroepiandrosterone sulfate (DHEA-S), androstenediol (3beta, 17beta) disulfate, androstenediol (3alpha, 17alpha) monosulfate, androstenediol (3beta,17beta) monosulfate, pregnenediol sulfate, and pregnenolone sulfate; b) at a second time point, determining the concentration of the at least one metabolite in a biological sample from the subject; c) comparing the concentration of the at least one metabolite at the first time point and the concentration of the at least one metabolite at the second time point; d) identifying that the subject is responding to the hypothyroidism treatment when the concentration of the at least one metabolite at the second time point is greater than concentration of the at least one metabolite at the first time or identifying that the subject is not responding to the hypothyroidism treatment when the concentration of the at least one metabolite at the second time point is equal to or less than the concentration of the at least one metabolite.

10. The method of claim 9, wherein the first time point is before the administration of the hypothyroidism treatment and the second time point is after the administration of at least one amount of the hypothyroidism treatment.

11. The method of claim 9, wherein the first time point is after the initial administration of the hypothyroidism treatment and the second time is after one or more additional doses of the hypothyroidism treatment has been administered to the subject.

12. The method of any one of claims 9-11, further comprising changing the dosage of the hypothyroidism treatment based on the change in the concentration of the at least one of six metabolites between the first time point and the second time point.

13. The method of any one of the preceding claims, wherein the method further comprises administering to a subject identified as being nonresponsive to the hypothyroidism treatment at least one amount of at least one of six therapeutic metabolites, wherein the six therapeutic metabolites comprise dehydroepiandrosterone sulfate (DHEA-S), androstenediol (3beta,17beta) disulfate, androstenediol (3alpha, 17alpha) monosulfate, androstenediol (3beta,17beta) monosulfate, pregnenediol sulfate, and pregnenolone sulfate.

14. The method of claim 13, wherein the method comprises: i) administering to the subject at least one amount of at least two of the six therapeutic metabolites; ii) administering to the subject at least one amount of at least three of the six therapeutic metabolites; iii) administering to the subject at least one amount of at least four of the six therapeutic metabolites; iv) administering to the subject at least one amount of at least five of the six therapeutic metabolites; or v) administering to the subject at least one amount of each of the six therapeutic metabolites.

15. A method of determining the risk that a subject exhibits one or more residual symptoms after administration of a hypothyroidism treatment, the method comprising: a) determining the concentration of at least one of six metabolites in a biological sample from the subject, wherein the six metabolites comprise dehydroepiandrosterone sulfate (DHEA-S), androstenediol (3beta,17beta) disulfate, androstenediol (3alpha, 17alpha) monosulfate, androstenediol (3beta,17beta) monosulfate, pregnenediol sulfate, and pregnenolone sulfate; b) identifying the risk that the subject exhibits one or more residual symptoms based on the concentration of the at least one metabolite measured in step (a).

16. The method of claim 15, wherein step (a) comprises: i) determining the concentration of at least two of the six metabolites; ii) determining the concentration of at least three of the six metabolites; iii) determining the concentration of at least four of the six metabolites; iv) determining the concentration of at least five of the six metabolites; or v) determining the concentration of each of the six metabolites.

17. The method of claim 16, wherein identifying the risk that the subject exhibits residual symptoms after administration of the hypothyroidism treatment based on the concentration of the at least one metabolite measured in step (a) comprises: i) comparing the one or more concentrations measured in step (a) to one or more corresponding predetermined cutoff values; and ii) identifying that the subject is at increased risk of exhibiting residual symptoms when one or more of the concentrations measured in step (a) is less than its corresponding predetermined cutoff value.

18. The method of any one of claims 15-17, wherein the one or more residual symptoms comprise reduced cognitive function, reduced general health, or any combination thereof.

19. The method of any one of claims 15-18, wherein the method further comprises administering to a subject identified as being at an increased risk of exhibiting residual symptoms at least one amount of at least one of six therapeutic metabolites, wherein the six therapeutic metabolites comprise dehydroepiandrosterone sulfate (DHEA-S), androstenediol (3beta,17beta) disulfate, androstenediol (3alpha, 17alpha)monosulfate, androstenediol (3beta,17beta) monosulfate, pregnenediol sulfate, and pregnenolone sulfate.

20. The method of claim 19, wherein the method comprises: i) administering to the subject at least one amount of at least two of the six therapeutic metabolites; ii) administering to the subject at least one amount of at least three of the six therapeutic metabolites; iii) administering to the subject at least one amount of at least four of the six therapeutic metabolites; iv) administering to the subject at least one amount of at least five of the six therapeutic metabolites; or v) administering to the subject at least one amount of each of the six therapeutic metabolites.

21. The method of any one of the preceding claims, wherein determining the concentration of the at least one metabolite comprises the use of mass spectrometry.

22. The method of any one of the preceding claims, wherein the biological sample comprises blood, plasma, serum, urine, breast milk, cerebrospinal fluid, mucus, gastric juice, peritoneal fluid, pleural fluid, saliva, sebum, semen, sweat, tears, vaginal secretion, vomit, endolymph, perilymph, neuronal tissue, or any combination thereof.

23. The method of any one of claims 1-22, wherein the hypothyroidism treatment is levothyroxine.

24. A method of treating hypothyroidism in a subject, the method comprising administering to the subject least one amount of at least one of six therapeutic metabolites, wherein the six therapeutic metabolites comprise dehydroepiandrosterone sulfate (DHEA-S), androstenediol (3beta,17beta) disulfate, androstenediol (3alpha, 17alpha) monosulfate, androstenediol (3beta,17beta) monosulfate, pregnenediol sulfate, and pregnenolone sulfate, or a pharmaceutically acceptable salt thereof.

25. The method of claim 24, wherein the at least one metabolite is dehydroepiandrosterone sulfate (DHEA-S), or a pharmaceutically acceptable salt thereof.

26. The method of claim 24, wherein the at least one metabolite is androstenediol (3beta,17beta) disulfate, or a pharmaceutically acceptable salt thereof.

27. The method of claim 24, wherein the at least one metabolite is androstenediol (3alpha, 17alpha) monosulfate, or a pharmaceutically acceptable salt thereof.

28. The method of claim 24, wherein the at least one metabolite is androstenediol (3beta,17beta) monosulfate, or a pharmaceutically acceptable salt thereof.

29. The method of claim 24, wherein the at least one metabolite is pregnenediol sulfate, or a pharmaceutically acceptable salt thereof.

30. The method of claim 24, wherein the at least one metabolite is pregnenolone sulfate, or a pharmaceutically acceptable salt thereof.

31. The method of claim 24, wherein the method comprises: i) administering to the subject at least one amount of at least two of the six therapeutic metabolites; ii) administering to the subject at least one amount of at least three of the six therapeutic metabolites; iii) administering to the subject at least one amount of at least four of the six therapeutic metabolites; iv) administering to the subject at least one amount of at least five of the six therapeutic metabolites; or v) administering to the subject at least one amount of each of the six therapeutic metabolites.

32. The method of any one of claims 24-31, wherein the subject has been identified to be nonresponsive to a hypothyroidism treatment.

33. The method of claim 32, wherein the hypothyroidism treatment is levothyroxine.

34. The method of any one of claim 24-33, wherein the subject exhibits one or more residual symptoms following treatment with levothyroxine.

35. The method of claim 34, wherein the one or more residual symptoms comprise reduced cognitive function, poor memory, fatigue, anxiety, reduced general health, or any combination thereof.

36. The method of any one of claims 24-35, wherein the method further comprises administering at least one additional therapy to the subject.

37. The method of claim 36, wherein the at least one additional therapy is levothyroxine.

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