Use of tryptophan and metabolites thereof as biomarkers for treatment methods

Abstract

Provided herein is the use of certain biomarkers, such as tryptophan and metabolites thereof for use in certain treatment methods. Suitable tryptophan metabolites that may serve as useful biomarkers include, for example, kynurenine (Kyn), kynurenic acid (KynA), and quinolinic acid. Such biomarkers may be used in identifying a subject who may benefit from treatment, predicting the responsiveness or monitoring the response of the subject to the treatment, or determining or adjusting the dosing or dosing regimen of the treatment to the subject.

Description

USE OF TRYPTOPHAN AND METABOLITES THEREOF AS BIOMARKERS FOR TREATMENT METHODSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 655,505, filed June 3, 2024, the contents of which are hereby incorporated by reference in their entirety.FIELD

[0002] The present disclosure relates to the discovery of certain biomarkers for use in certain treatment methods, including specifically the use of tryptophan and metabolites thereof for use in such methods.BACKGROUND

[0003] Exercise has a positive clinical effect on many diseases via several different mechanisms. Many of these effects are driven by an increase in signaling of the energy sensor AMPK (5' adenosine monophosphate-activated protein kinase), and the downstream transcriptional co-activator Pgcla. Changes in skeletal muscle function and changes elsewhere in the body secondary to skeletal muscle changes are important drivers of this positive clinical effect. Kynurenine, a metabolite of tryptophan, is further metabolized to kynurenic acid in muscle, reducing circulating Kyn levels that can be harmful to CNS function, and increasing circulating KynA levels that can have positive peripheral effects. Exercise exerts benefit in part by increasing Kyn metabolism to KynA by skeletal muscle via increase in AMPK and Pgcla signaling.

[0004] Pharmaceutical interventions to increase AMPK and Pgcla signaling to mimic and / or augment the positive effect of exercise are desirable in many diseases. However, it is difficult to measure AMPK and Pgcla signaling in tissues. Tissue biopsy is invasive and uncomfortable and AMPK and Pgcla complexes are unstable and difficult to measure. A marker in blood / plasma of an increase in AMPK and Pgcla signaling would allow identification of subjects with reduced AMPK / Pgcla signaling who would benefit from an intervention, to identify subjects who successfully respond to an intervention with an increase in AMPK / Pgcla signaling and toidentify an effective dose of a pharmaceutical intervention that increases AMPK / Pgcla signaling.BRIEF SUMMARY

[0005] Provided herein are methods that use tryptophan and metabolites thereof as biomarkers to address the technical problem known in the art described above.

[0006] In some aspects, provided herein are methods that use tryptophan and / or tryptophan metabolites thereof, including selected thresholds and / or ratios of the foregoing, for identifying a subject who may benefit from treatment, predicting the responsiveness or monitoring the response of the subject to the treatment, and determining or adjusting the dosing or dosing regimen of the treatment to a subject.

[0007] In some embodiments, the treatment comprises administering 4-chloro-N-[2-[(4- chlorophenyl)methyl]-3-oxo-l,2,4-thiadiazol-5- yl]benzamide, or a salt thereof. In one variation, the treatment comprises a sodium salt of 4-chloro-N-[2-[(4-chlorophenyl)methyl]-3-oxo-l,2,4- thiadiazol-5- yl]benzamide which is referred to herein as “Compound A”.

[0008] In certain aspects, tryptophan and / or its metabolites may be useful as biomarkers for identifying a subject who may benefit from such treatment, predicting the responsiveness or monitoring the response of the subject to such treatment, or determining or adjusting the dosing or dosing regimen of such treatment to the subject.

[0009] In other aspects, provided is a method of treating depression in a subject in need thereof, comprising administering to the subject 4-chloro-N-[2-[(4-chlorophenyl)methyl]-3-oxo- l,2,4-thiadiazol-5- yl]benzamide, or a salt thereof, or a prodrug thereof. In certain aspects, provided is use of 4-chloro-N-[2-[(4-chlorophenyl)methyl]-3-oxo-l,2,4-thiadiazol-5- yl]benzamide, or a salt thereof, or a prodrug thereof, for the manufacture of a medicament for the treatment the diseases, disorders, and conditions described herein. In certain aspects, provided is use of 4-chloro-N-[2-[(4-chlorophenyl)methyl]-3-oxo-l,2,4-thiadiazol-5- yl]benzamide, or a salt thereof, or a prodrug thereof, for treating the diseases, disorders, and conditions described herein.

[0010] In certain aspects, provided is a method of treating a disease, disorder, or condition ameliorated by activation of AMPK in a subject in need thereof, comprising administering to the subject (i) 4-chloro-N-[2-[(4-chlorophenyl)methyl]-3-oxo-l,2,4-thiadiazol-5- yl]benzamide, or a salt thereof (including a pharmaceutically acceptable salt thereof), or a prodrug thereof, and (ii) tryptophan supplement. In some embodiments, the method further comprises monitoring the subject’s plasma tryptophan levels. In certain embodiments, the tryptophan supplement is administered when the plasma level of tryptophan in the subject drops below 25 pmol / L after administering 4-chloro-N-[2-[(4-chlorophenyl)methyl]-3-oxo-l,2,4-thiadiazol-5- yl]benzamide, or a pharmaceutically acceptable salt thereof, or a prodrug thereof to the subject.

[0011] In some aspects, provided is a combination comprising: (i) 4-chloro-N-[2-[(4- chlorophenyl)methyl]-3-oxo-l,2,4-thiadiazol-5- yl]benzamide, or a salt thereof (including a pharmaceutically acceptable salt thereof), or a prodrug thereof, and (ii) tryptophan supplement. In some variations, provided is a mixture comprising the aforementioned combination. In other variations, provided is an admixture comprising the aforementioned combination.

[0012] In other aspects, provided are also kits and articles of manufacture related to the methods described herein and the use of the biomarkers described herein.DESCRIPTION OF THE FIGURES

[0013] The present application can be understood by reference to the following description taken in conjunction with the accompanying figures.

[0014] FIG. 1 depicts the kynurenine metabolic pathway, processing free tryptophan. The portion of the pathway occurring in the liver is circled.

[0015] FIG. 2 depicts kynurenine levels (on the vertical axis) across samples from three tissue types and three groups of diet conditions (on the horizontal axis). The horizontal axis’s tissue types are further subdivided into regular diet (RD), high-fat diet (HFD), or both HFD and Compound A-supplemented diet groups. Asterisks and brackets denote significant p-values in comparing levels between diet groups.

[0016] FIG. 3 depicts kynurenic acid levels (on the vertical axis) across samples from three tissue types and three groups of diet conditions (on the horizontal axis). The horizontal axis’s tissue types are further subdivided into regular diet (RD), high-fat diet (HFD), or both HFD and Compound A-supplemented diet groups. Asterisks and brackets denote significant p-values in comparing levels between diet groups.

[0017] FIG. 4 depicts the ratio of kynurenic acid levels to kynurenine levels (on the vertical axis) across samples from three tissue types and three groups of diet conditions (on the horizontal axis). The horizontal axis’s tissue types are further subdivided into regular diet (RD), high-fat diet (HFD), or both HFD and Compound A-supplemented diet groups. Asterisks and brackets denote significant p-values in comparing ratios between diet groups.

[0018] FIG. 5 depicts tryptophan levels (on the vertical axis) across samples from three tissue types and three groups of diet conditions (on the horizontal axis). The horizontal axis’s tissue types are further subdivided into regular diet (RD), high-fat diet (HFD), or both HFD and Compound A-supplemented diet groups. Asterisks and brackets denote significant p-values in comparing levels between diet groups.

[0019] FIG. 6 depicts the ratio of kynurenine levels to tryptophan levels (on the vertical axis) across samples from three tissue types and three groups of diet conditions (on the horizontal axis). The horizontal axis’s tissue types are further subdivided into regular diet (RD), high-fat diet (HFD), or both HFD and Compound A-supplemented diet groups. Asterisks and brackets denote significant p-values in comparing ratios between diet groups.

[0020] FIGS. 7A-7D depicts a series of initial results for exploring the relationship between kynurenic acid (KynA), kynurenine (Kyn), and health improvement in mice. FIG. 7A depicts the significant increase in KynA / Kyn ratio in the plasma of mice fed a combination of a high-fat diet (HFD) and Compound A. RD = regular diet. Asterisks and brackets denote significant p-values in comparing ratios between diet groups. FIG. 7B depicts increased qRT-PCR fold changes in levels of mRNA expression of Pgcla in mice (vertical axis) fed with various concentrations of Compound A (horizontal axis). FIG. 7C further depicts an increase of expression levels (vertical axis) in kynurenic aminotransferases 1 and 3 (horizontal axis) in skeletal muscle of mice fed with a mixture of HFD and Compound A compared to other dietary groups. FIG. 7D depicts thefrequency of selecting sugar water over non-sugar water (% = percentage of the time sugar water was selected, along the vertical axis) by mice treated with various diets (along the horizontal axis). LFD = low-fat diet.

[0021] FIG. 8 depicts a schematic outline of experimental changes in feeding patterns for mice study subjects.

[0022] FIG. 9 depicts a schematic outline of the experiments described in Example 2. For Experiment 1, 12-week-old Fl -mice were set up for 9 weeks of diet on low fat diet (LFD), high fat diet (HFD), or HFD supplemented with 0.25 mg / g of Compound A (HFD-Cmpd A). After 9 weeks of diet, mice were sacrificed and plasma, liver, and muscle extracts were isolated for metabolomic and gene expression analyses. For Experiment 2, 12-week-old Fl-mice were set up for 18 weeks of diet on LFD, HFD, or HFD-Cmpd A, where one group of mice was switched from HFD to HFD-Cmpd A at 9 weeks of diet, whereas another group of mice were switched from HFD-Cmpd A to HFD at 9 weeks of diet. After 18 weeks of diet, mice were sacrificed and plasma, liver, and muscle extracts were isolated for metabolomic and gene expression analyses.

[0023] FIGS. 10A-10G depict data from the experiments described in Example 2. The data demonstrate that Compound A averts and reverts plasma levels of tryptophan by promoting kynurenic acid production. FIGS. 10A-10D depict metabolic analyses of tryptophan metabolites in plasma extracts isolated from Fl -mice after 9 weeks of diet. FIGS. 10E-10G depict metabolic analyses of tryptophan metabolites in plasma extracts isolated from Fl -mice after 18 weeks of diet, where one group of mice was switched from HFD to Compound A at 9 weeks of diet, whereas another group of mice was switched from Compound A to HFD at 9 weeks of diet.

[0024] FIGS. 11A-11C depict how Compound A promotes gene expression of key regulatory enzymes of the kynurenic pathway of tryptophan metabolism. FIG. HA depicts a schematic diagram of tryptophan metabolism. Tryptophan is primarily metabolized in the liver to kynurenine which is secreted into circulation and transported to muscles where kynurenine is further metabolized to either 3 -hydroxy -kynurenine (3-HK) or kynurenic acid (KynA). FIG. 11B depicts qRT-PCR analyses of liver extracts of tryptophan-di oxygenase (TDO) and indol- dioxygenase (IDO), which are key regulatory enzymes converting tryptophan to kynurenine in liver. FIG. 11C depicts qRT-PCR analyses of muscles extracts of kynurenine amino-transferases (KAT1, KAT3 and KAT4) which promote conversion of kynurenine to KynA. KAT1, KAT3 and KAT4 are transcriptionally regulated by PGC-la in muscle. Also shown, is the expression of kynurenine mono-oxygenase (KMO) that converts kynurenine towards the “neurotoxic” arm of kynurenine metabolism through 3-HK.

[0025] FIGS. 12A-12H depict data demonstrating that HFD-induced obesity results in hyperglycemia, hyperinsulinemia, and insulin resistance which is averted and reverted by Compound A. FIG. 12A and FIG. 12B depict body weight and body fat after 9 weeks of diet. FIGS. 12C-12E depict fasted blood glucose and plasma insulin levels and calculated homeostatic model assessment of insulin resistance (HOMA-IR) after 9 weeks of diet. FIGS. 12F-12H depict fasted blood glucose and plasma insulin levels and HOMA-IR at diet-switch point 9 weeks of diet and at 18 weeks of diet.

[0026] FIGS. 13A-13C depict data showing that HFD-treated mice have reduced sucrose preference (anhedonic), spend less time in center zone (anxious), and have impaired bedding behavior, whereas Compound-A-treated mice mitigate obesity-associated depressive behavior. FIG. 13A shows data from the sucrose preference test after 9 weeks of diet. FIG. 13B shows data from the open field test after 9 weeks of diet. FIG. 13C shows data from the nesting test after 9 weeks of diet.DETAILED DESCRIPTION

[0027] The following description sets forth exemplary methods, parameters, compositions and the like. It should be recognized, however, that such description is not intended as a limitation on the scope of the present disclosure but is instead provided as a description of exemplary embodiments.Biomarker Criteria

[0028] In some aspects, provided herein are biomarkers that enable identification of subjects with reduced AMPK / Pgcla signaling who would benefit from and / or successfully respond to a suitable treatment with an increase in AMPK / Pgcla signaling, and the identification of an effective dose of a treatment that increases AMPK / Pgcla signaling. In some embodiments, the biomarkers provided herein are reliable indicators of the level of AMPK / Pgcla signaling whichcan be directly determined in, for example, a blood sample without biopsy. This presents technical advantages as compared to traditional methods of measuring the AMPK and Pgcla signaling, which typically require biopsy and freezing of the samples immediately after biopsy. In some embodiments of the methods herein, the biomarkers comprise tryptophan and / or metabolites thereof.

[0029] In some aspects, provided herein is the use tryptophan and metabolites thereof as biomarkers for identifying a subject who may benefit from a suitable treatment, predicting the responsiveness or monitoring the response of the subject to a suitable treatment, and determining or adjusting the dosing or dosing regimen of a suitable treatment to a subject in need thereof. In some embodiments, the treatment is for treating disease, disorder, or condition associated with reduced level AMPK and Pgcla signaling and / or can be improved by increasing the level of AMPK and Pgcla signaling. Such diseases, disorders and conditions are further described herein.

[0030] In some embodiments, the effect of the treatment (e.g., a treatment compound) on a subject is determined based on levels of one or more of the following biomarkers or combination of biomarkers: Tryptophan (Trypt), Kynurenine (Kyn), Kynurenic acid (KynA), Quinolinic acid (QA), and Xanthurenic acid (XA). In some embodiments, the level is plasma level (e.g., based on a blood sample). In some embodiments, the effect of the treatment on a subject is determined based on the plasma level of Trypt. In some variations, the biomarkers are selected from Trypt, Kyn, KynA, and QA. In certain variations, the biomarker is KynA. In other variations, a plurality of biomarkers is used, including KynA in combination with Trypt, Kyn, and QA, such as determining a ratio between KynA, and one or more of Trypt, Kyn, and QA.

[0031] In other embodiments, the effect of the treatment (e.g., a treatment compound) on a subject is determined based one or more of the following ratios of Kyn to Trypt; KynA to Trypt; KynA to Kyn; QA to KynA; QA to Kyn; KynA to XA; and QA to XA. In some embodiments, the effect of the treatment (e.g., a treatment compound) on a subject is determined based one or more of the following ratios of Kyn to Trypt; KynA to Trypt; KynA to Kyn; QA to KynA; and QA to Kyn. In some embodiments, the effect of the treatment (e.g., a treatment compound) on asubject is determined based KynA to Trypt. In some embodiments, the effect of the treatment (e.g., a treatment compound) on a subject is determined based KynA to Kyn.

[0032] In some embodiments, the effect of the treatment (e.g., a treatment compound) on a subject is determined based on one or more of the following:(a) the level of tryptophan;(b) the level of KynA;(c) the ratio of Kyn to tryptophan;(d) the ratio of KynA to Kyn;(e) the ratio of KynA to tryptophan;(f) the ratio of Quinolinic acid to KynA; or(g) the ratio of Quinolinic acid to Kyn.

[0033] In some embodiments of the foregoing, the ratio is a molar ratio or equivalent thereof. It should be understood that one or ordinary skill in the art would be able to determine equivalent ratios of what has been provided, including for example the corresponding mass ratio. In some embodiments of the foregoing, the levels and / or ratios are determined in plasma.

[0034] In some embodiments, the levels and / or ratios of the biomarkers described herein are determined before the administration of a treatment (e.g., a treatment compound as described herein) as a baseline.

[0035] In certain embodiments, before the administration of the treatment, the biomarker criteria described herein may be used to: determine if administration of a treatment (e.g., a treatment compound as described herein) would be recommended or should be prescribed to a subject in need thereof; and / or predict the responsiveness of a subject with a disease, disorder, or condition to the administration of a treatment (e.g., a treatment compound as described herein); and / orpromote or advertise administration of a treatment (e.g., a treatment compound as described herein) to a target audience if one or more of the biomarker criteria provided herein are satisfied.

[0036] In some embodiments, before administration of the treatment, when one or more of the biomarker criteria provided herein are met, then one or more of the following may occur: the administration of the treatment (e.g., a treatment compound as described herein) is recommended or prescribed to a subject in need thereof; and / or the subject with a disease, disorder, or condition is likely to be responsive to the administration of a treatment (e.g., a treatment compound as described herein); and / or the administration of a treatment (e.g., a treatment compound as described herein) is advertised to a target audience.

[0037] In some embodiments of the methods provided herein, before administration of the treatment, when one or more of the following criteria are met in a subject in need thereof (e.g., a subject having one or more of the disease, disorders, or conditions set forth herein):(i) the plasma level of tryptophan is above 50 pmol / L (e.g., above any of 50 pmol / L, 55 pmol / L, 60 pmol / L, 65 pmol / L, 70 pmol / L, 75 pmol / L, 80 pmol / L, 85 pmol / L, and 90 pmol / L);(ii) the molar ratio of KynA to Kyn in plasma is below 0.4 (e.g., below any of 0.35, 0.3, 0.25, 0.2, 0.15, 0.1, or 0.05);(iii) the molar ratio of KynA to tryptophan in plasma is below 0.02 (e.g., below any of 0.015, 0.01, 0.005, 0.002, and 0.001);(iv) the molar ratio of Quinolinic acid to KynA in plasma is above 3 (e.g., above any of 4, 5, 6, 7, 8, or 9); or(v) the molar ratio of Quinolinic acid to Kyn in plasma below 0.2 (e.g., below any of 0.15, 0.1, 0.05, or 0.01); then the method may further comprise at least one of:recommending or prescribing administration of a treatment (e.g., a treatment compound as described herein) to the subject in need thereof; and / or identifying the subject with a disease, disorder, or condition as likely to be responsive to the administration of a treatment (e.g., a treatment compound as described herein); and / or selecting a treatment (e.g., a treatment compound as described herein) for the subject with a disease, disorder, or condition .

[0038] It should be understood that one or ordinary skill in the art would be able to determine equivalent ratios of what has been provided, including for example the corresponding mass ratio. In some embodiments, the levels and / or ratios of the biomarkers described herein are determined during or after administration of a treatment (e.g., a treatment compound as described herein). In some embodiments, the biomarker criteria provided herein also encompasses the change in the levels and / or ratios from the baseline to the levels and / or ratios during or after administration of a treatment (e.g., a treatment compound as described herein). In some embodiments, during and / or after the administration of the treatment, the biomarker criteria described herein may be used to: monitor whether a subject in need thereof administered a treatment (e.g., a treatment compound as described herein) is responding well to the treatment; and / or adjust the dose or dosing regimen of the treatment (e.g., a treatment compound as described herein) administered to the subject in need thereof; and / or prescribe a supplemental therapy or treatment to the subject in need thereof who is currently administered a treatment (e.g., a treatment compound as described herein).

[0039] In some embodiments, during and / or after the administration of the treatment, the biomarker criteria include at least one of the following:(i) the plasma level of tryptophan after the administration of the treatment is decreased by at least 2 pmol / mL (e.g., at least any of 5 pmol / mL, 10 pmol / mL, 15 pmol / mL, 20 pmol / mL, or 25 pmol / mL) compared to the baseline before the treatment;(ii) the increase in plasma level of KynA after the treatment compared to the baseline before the treatment is at least 0.005 pmol / mL (e.g., at least any of 0.01 pmol / mL, 0.015 pmol / mL, 0.02 pmol / mL, 0.025 pmol / mL, 0.03 pmol / mL, or 0.05 pmol / mL);(iii) the plasma level of KynA after the treatment is at least doubled (e.g., increased by at least any of 100%, 150%, 200%, 250%, or 300%) compared to the baseline before the treatment;(iv) the molar ratio of Kyn to tryptophan in plasma is increased;(v) the increase in the molar ratio of KynA to tryptophan in plasma after the treatment compared to the baseline before the treatment is at least 0.005 (e.g., at least any of 0.01, 0.015, or 0.02);(vi) the molar ratio of KynA to tryptophan in plasma is increased;(vii) the plasma level of KynA is increased and the plasma level of Tryptophan is decreased;(viii) the decrease in the molar ratio of Quinolinic acid (QA) to KynA in plasma after the treatment compared to the baseline before the treatment is at least 0.25 (e.g., at least any of 0.5, 0.75, or 1); or(ix) the increase in the molar ratio of Quinolinic acid (QA) to Kyn in plasma after the treatment compared to the baseline before the treatment is at least 0.005 (e.g., at least any of 0.01, 0.015, 0.02, or 0.025).

[0040] It should be understood that one or ordinary skill in the art would be able to determine equivalent ratios of what has been provided, including for example the corresponding mass ratio.

[0041] In some embodiments, during and / or after the administration of the treatment, satisfying at least one of the biomarker criteria discussed above suggests the subject is responsive to the treatment.

[0042] In some variations, the biomarker criteria are based on Trypt. In variations, provided is a method comprising determining the plasma level of Trypt before the administration of thetreatment. In some embodiments, the biomarker criteria comprise that before the administration of the treatment, the plasma level of tryptophan is above 50 pmol / L (e.g., above any of 50 pmol / L, 55 pmol / L, 60 pmol / L, 65 pmol / L, 70 pmol / L, 75 pmol / L, 80 pmol / L, 85 pmol / L, and 90 pmol / L). In some embodiments, provided is a method comprising determining the plasma level of Trypt during or after the administration of the treatment. In some embodiments, during or after the administration of the treatment, when the Trypt level is below 25 pmol / L (e.g., below any of 20 pmol / L, 15 pmol / L, or 10 pmol / L), the method provided herein further comprises administering tryptophan supplement or decreasing the dosing of the treatment. In some embodiments, during or after the administration of the treatment, when the Trypt level is below 25 pmol / L (e.g., below any of 20 pmol / L, 15 pmol / L, or 10 pmol / L), the method provided herein further comprises administering tryptophan supplement.

[0043] In some variations, the biomarker criteria are based on KynA. In variations, provided is a method comprising determining the plasma level of KynA before the administration of the treatment. In some embodiments, provided is a method comprising determining the plasma level of KynA during or after the administration of the treatment. In some embodiments, the biomarker criteria comprises that the increase in plasma level of KynA after the treatment compared to the baseline before the treatment is at least 0.005 pmol / mL (e.g., at least any of 0.01 pmol / mL, 0.015 pmol / mL, 0.02 pmol / mL, 0.025 pmol / mL, 0.03 pmol / mL, or 0.05 pmol / mL). In some embodiments, the biomarker criteria comprises that the plasma level of KynA after the treatment is at least doubled (e.g., increased by at least any of 100%, 150%, 200%, 250%, or 300%) compared to the baseline before the treatment. In some embodiments, if at least one of the biomarker criteria of the foregoing is satisfied, the subject is likely to be responsive to the treatment. In some embodiments, the biomarker criteria are based on the level of KynA in combination with Trypt, Kyn, and QA, such as the ratio between KynA, and one or more of Trypt, Kyn, and QA combination.

[0044] In some embodiments, the biomarker criteria are evaluated before the administration of the treatment to determine whether a subject would be responsive to the treatment and / or whether the treatment should be prescribed to the subject. In some embodiments, such biomarker criteria include the ratio of KynA to Kyn in plasma, the ratio of KynA to tryptophan in plasma, the ratio of Quinolinic acid to KynA in plasma, and the ratio of Quinolinic acid to Kyn inplasma. In some variations of the foregoing, the aforementioned ratios are molar ratios or equivalents thereof. In some embodiments, the biomarker criteria includes that the molar ratio of KynA to Kyn in plasma is below 0.4 (e.g., below any of 0.35, 0.3, 0.25, 0.2, 0.15, 0.1, or 0.05) before the administration of the treatment. In some embodiments, the biomarker criteria includes that the molar ratio of KynA to tryptophan in plasma is below 0.02 (e.g., below any of 0.015, 0.01, 0.005, 0.002, and 0.001) before the administration of the treatment. In some embodiments, the biomarker criteria includes that the molar ratio of Quinolinic acid to KynA in plasma is above 3 (e.g., above any of 4, 5, 6, 7, 8, or 9) before the administration of the treatment. In some embodiments, the biomarker criteria includes that the molar ratio of Quinolinic acid to Kyn in plasma below 0.2 (e.g., below any of 0.15, 0.1, 0.05, or 0.01) before the administration of the treatment.

[0045] In some embodiments, the biomarker criteria are evaluated during or after the administration of the treatment to determine whether a subject has been responsive to the treatment and / or whether the dosing or dosing regimen should be adjusted. In some embodiments, such biomarker criteria are based on the change of the ratio of KynA to Kyn in plasma, the ratio of KynA to tryptophan in plasma, the ratio of Quinolinic acid to KynA in plasma, and the ratio of Quinolinic acid to Kyn in plasma after the administration of the treatment compared to the baseline before the administration of the treatment. In some variations of the foregoing, the aforementioned ratios are molar ratios or equivalents thereof. In some embodiments, such biomarker criteria includes (i) the increase in plasma level of KynA after the treatment compared to the baseline before the treatment is at least 0.005 pmol / mL (e.g., at least any of 0.01 pmol / mL, 0.015 pmol / mL, 0.02 pmol / mL, 0.025 pmol / mL, 0.03 pmol / mL, or 0.05 pmol / mL); (ii) the plasma level of KynA after the treatment is at least doubled (e.g., increased by at least any of 100%, 150%, 200%, 250%, or 300%) compared to the baseline before the treatment; (iii) the increase in the molar ratio of KynA to tryptophan in plasma after the treatment compared to the baseline before the treatment is at least 0.005 (e.g., at least any of 0.01, 0.015, or 0.02); (iv) the molar ratio of KynA to tryptophan in plasma is increased; (v) the plasma level of KynA is increased and the plasma level of Tryptophan is decreased; (vi) the decrease in the molar ratio of Quinolinic acid (QA) to KynA in plasma after the treatment compared to the baseline before the treatment is at least 0.25 (e.g., at least any of 0.5, 0.75, or 1); and / or (vii) the increase in the molar ratio of Quinolinic acid (QA) to Kyn in plasma after thetreatment compared to the baseline before the treatment is at least 0.005 (e.g., at least any of 0.01, 0.015, 0.02, or 0.025).

[0046] In some variations of the foregoing, the biomarker criteria set forth above may be used for a treatment comprising 4-chloro-N-[2-[(4-chlorophenyl)methyl]-3-oxo-l,2,4-thiadiazol- 5-yl]benzamide, or a salt thereof, including specifically the sodium salt of the aforementioned compound (in which this sodium salt is referred to as “Compound A”). In some embodiments, the biomarker criteria set forth above may be used for a treatment comprising a prodrug of Compound A, such as Compound B, B-l, and B-2 provided herein.

[0047] It should be understood that the biomarkers herein may be measured by any suitable methods or techniques known in the art, including for example by chromatography (e.g., HPLC), mass spectroscopy, or ELISA.Methods Using Said Biomarkers

[0048] In some aspects, provided herein are methods using one or more of the biomarkers described above, at the levels and / or ratios set forth above, for identifying subjects who may benefit from, have an increased likelihood of being responsive to or exhibiting benefit from, a treatment comprising 4-chloro-N-[2-[(4-chlorophenyl)methyl]-3-oxo-l,2,4-thiadiazol-5- yl]benzamide, or a salt thereof. The treatment can be recommended or selected for subjects or monitored, and the dosing or the dosing regimen may be adjusted based on such methods.

[0049] In one aspect, provided is a method of identifying a subject who may benefit from a treatment comprising 4-chloro-N-[2-[(4-chlorophenyl)methyl]-3-oxo-l,2,4-thiadiazol-5- yl]benzamide, or a salt thereof. In another aspect, provided is a method of identifying a subject having increased PGC-1 alpha activity and / or increased expression of KAT enzymes in response to a treatment comprising 4-chloro-N-[2-[(4-chlorophenyl)methyl]-3-oxo-l,2,4-thiadiazol-5- yl]benzamide, or a salt thereof. In some embodiments of the foregoing aspects, the method comprises: determining the level of one or more biomarkers and / or the ratio of two or more biomarkers in a sample obtained from the subject; and assessing the subject based on one or more of the biomarker criteria described herein.

[0050] In certain embodiments of the foregoing, the method further comprises: providing a recommendation that the subject will be more likely to respond to the treatment. In yet other embodiments, the method further comprises: administering an effective amount of 4-chloro-N- [2-[(4-chlorophenyl)methyl]-3-oxo-l,2,4-thiadiazol-5- yl]benzamide, or a salt thereof, or a prodrug thereof, to the subject.

[0051] In some aspects, provided is a method for predicting responsiveness of a subject with a disease, disorder, or condition to a treatment comprising 4-chloro-N-[2-[(4- chlorophenyl)methyl]-3-oxo-l,2,4-thiadiazol-5- yl]benzamide, or a salt thereof. In other aspects, provided is a method for determining likelihood that a subject with a disease, disorder, or condition will exhibit benefit from a treatment comprising 4-chloro-N-[2-[(4- chlorophenyl)methyl]-3-oxo-l,2,4-thiadiazol-5- yl]benzamide, or a salt thereof. In some embodiments of the foregoing aspects, the method comprises: determining the level of one or more biomarkers and / or the ratio of two or more biomarkers in a sample obtained from the subject; and assessing the subject based on one or more of the biomarker criteria described herein. In certain embodiments of the foregoing, the method further comprises: informing the subject that they have an increased likelihood of being responsive to the treatment or an increased likelihood of exhibiting benefit from the treatment. In yet other embodiments, the method further comprises: administering an effective amount of 4-chloro-N-[2-[(4- chlorophenyl)methyl]-3-oxo-l,2,4-thiadiazol-5- yl]benzamide, or a salt thereof, or a prodrug thereof, to the subject.

[0052] In some aspects, provided is a method for selecting a treatment for a subject with a disease, disorder, or condition , wherein the treatment comprises 4-chloro-N-[2-[(4- chlorophenyl)methyl]-3-oxo-l,2,4-thiadiazol-5- yl]benzamide, or a salt thereof. In some embodiments of the foregoing, the method comprises: determining the level of one or more biomarkers and / or the ratio of two or more biomarkers in a sample obtained from the subject; and assessing the subject based on one or more of the biomarker criteria described herein.

[0053] In other embodiments, the method comprises: (a) determining the level of one or more biomarker and / or the ratio of two or more biomarkers in a sample obtained from the subject; (b) assessing the subject based on one or more of the biomarker criteria describedherein; (c) selecting the treatment if the subject is identified as likely to respond to the treatment; and (d) recommending to the subject the selected treatment.

[0054] In certain aspects, provided is a method of determining whether a subject having a disease, disorder, or condition is likely to respond to a treatment comprising 4-chloro-N-[2-[(4- chlorophenyl)methyl]-3-oxo-l,2,4-thiadiazol-5- yl]benzamide, or a salt thereof. In some embodiments, the method comprises: (a) determining the level of one or more biomarker and / or the ratio of two or more biomarkers in a sample obtained from the subject; (b) assessing the subject based on one or more of the biomarker criteria described herein; and (c) informing the subject that they have an increased likelihood of being responsive to the treatment.

[0055] In yet other embodiments, the method further comprises: administering an effective amount of 4-chloro-N-[2-[(4-chlorophenyl)methyl]-3-oxo-l,2,4-thiadiazol-5- yl]benzamide, or a salt thereof, or a prodrug thereof, to the subject.

[0056] In some aspects, provided is a method for treating a disease, disorder, or condition in a subject in need thereof. In some embodiments, the method comprises: determining the level of one or more biomarker and / or the ratio of two or more biomarkers in a sample obtained from the subject; assessing the subject based on one or more of the biomarker criteria described herein; and administering an effective amount of 4-chloro-N-[2-[(4-chlorophenyl)methyl]-3-oxo-l,2,4- thiadiazol-5- yl]benzamide, or a salt thereof, or a prodrug thereof, to the subject.

[0057] In some aspects, provided is a method for advertising a treatment comprising 4- chloro-N-[2-[(4-chlorophenyl)methyl]-3-oxo-l,2,4-thiadiazol-5- yl]benzamide, or a salt thereof, or a prodrug thereof, directed to treatment of a disease, disorder ,or condition . In some embodiments, the method comprises: promoting, to a target audience, the use of the 4-chloro-N- [2-[(4-chlorophenyl)methyl]-3-oxo-l,2,4-thiadiazol-5- yl]benzamide, or a salt thereof, or a prodrug thereof, for treating a subject with a disease, disorder, or condition based on determining the level of one or more biomarker and / or the ratio of two or more biomarkers in a sample obtained from the subject, and assessing the subject based on one or more of the biomarker criteria described herein.

[0058] In some aspects, provided is a method of determining the dosing or the dosing regimen of 4-chloro-N-[2-[(4-chlorophenyl)methyl]-3-oxo-l,2,4-thiadiazol-5- yl]benzamide, or a salt thereof, or a prodrug thereof, for treatment of a disease, disorder, or condition in a subject in need thereof. In some embodiments, the method comprises: identifying a minimally effective dose and a maximally effective dose of 4-chloro-N-[2-[(4-chlorophenyl)methyl]-3-oxo-l,2,4- thiadiazol-5- yl]benzamide, or a salt thereof, or a prodrug thereof, or the dose regimen of 4- chloro-N-[2-[(4-chlorophenyl)methyl]-3-oxo-l,2,4-thiadiazol-5- yl]benzamide, or a salt thereof, or a prodrug thereof, based on comparing an amount of at least one biomarker described herein, in a sample of the subject after or during treatment with a dose or dosing regimen of — chloro-N- [2-[(4-chlorophenyl)methyl]-3-oxo-l,2,4-thiadiazol-5- yl]benzamide, or a salt thereof, or a prodrug thereof, to an amount of the at least one biomarker in a sample obtained from the subject before the treatment, wherein a change in the amount of the at least one biomarker after or during the treatment as compared to before the treatment is indicative of the efficacy of or responsiveness to the dose or dosing regimen of 4-chloro-N-[2-[(4-chlorophenyl)methyl]-3-oxo- l,2,4-thiadiazol-5- yl]benzamide, or a salt thereof for treatment of the subject.

[0059] In some embodiments, the minimally effective dose is the lowest dose of 4-chloro-N- [2-[(4-chlorophenyl)methyl]-3-oxo-l,2,4-thiadiazol-5- yl]benzamide, or a salt thereof, or a prodrug thereof, that gives a positive change in the aforementioned biomarkers, and ratios thereof. In some embodiments, the maximally effective dose is the dose 4-chloro-N-[2-[(4- chlorophenyl)methyl]-3-oxo-l,2,4-thiadiazol-5- yl]benzamide, or a salt thereof that normalizes the aforementioned biomarker levels and ratios thereof.

[0060] In some aspects, provided is a method of monitoring the response of a subject having a treatment comprising 4-chloro-N-[2-[(4-chlorophenyl)methyl]-3-oxo-l,2,4-thiadiazol-5- yl]benzamide, or a salt thereof. In some embodiments, the method comprises: determining the level of one or more biomarker and / or the ratio of two or more biomarkers in a sample obtained from the individual; monitoring the response of the subject undergoing the treatment based on one or more of the biomarker criteria described herein; and adjusting the dose of 4-chloro-N-[2- [(4-chlorophenyl)methyl]-3-oxo-l,2,4-thiadiazol-5- yl]benzamide, or a salt thereof, or a prodrug thereof, or the dose regimen of 4-chloro-N-[2-[(4-chlorophenyl)methyl]-3-oxo-l,2,4-thiadiazol- 5- yl]benzamide, or a salt thereof, or a prodrug thereof, based on the comparison.

[0061] In certain embodiments, the method further comprises: determining tryptophan levels in a sample from the subject during or after administration of the treatment; and supplementing the subject with tryptophan if the subject’s tryptophan levels are below the clinically defined normal range. In one variation, the clinically defined normal range is less than 25 pmol / L (e.g., below any of 20 pmol / L, 15 pmol / L, or 10 pmol / L). In some embodiments, the method further comprises: adjusting the dosing of the treatment during or after administration of the treatment; and supplementing the subject with tryptophan if the subject’s tryptophan levels are below the clinically defined normal range. In one variation, the clinically defined normal range is less than 25 pmol / L (e.g., below any of 20 pmol / L, 15 pmol / L, or 10 pmol / L).

[0062] In some variations of the foregoing, the sampling is performed in vitro.Samples

[0063] In some embodiments of the foregoing methods, kits and articles of manufacture, the sample comprises tissue, whole blood, plasma, serum, or cerebrospinal fluid, or any combination thereof. In certain embodiments, the sample comprises whole blood, plasma, or serum, or any combination thereof. In certain embodiments, the sample comprises skeletal muscle tissue, or liver tissue.

[0064] In some embodiments, biopsy is not needed to obtain the sample described herein. In some embodiments, the level of one or more biomarker and / or the ratio of two or more biomarkers can be determined in a blood sample wherein no biopsy is needed to obtain the blood sample. In some embodiments, the level of one or more biomarker and / or the ratio of two or more biomarkers can be determined without freezing the sample. In some embodiments, unlike the traditional method of measuring AMPK and Pgcla signaling, which typically requires biopsy and the frozen of sample immediately after biopsy, the methods, kits, and articles of manufacture provided herein can reliably probe the reduced AMPK / Pgcla signaling in a subject with a blood sample, a plasma sample, or a serum sample, and no freezing-thaw process is needed.

[0065] In one embodiment, the sample comprises plasma, and the methods involve determining or detecting plasma levels, or muscle tissue levels, or liver tissue levels of the biomarkers herein.Diseases, Disorders, and Conditions

[0066] In some embodiments of the foregoing methods, kits and articles of manufacture, the disease, disorder or condition is ameliorated by the activation of AMPK. In certain embodiments, the disease, disorder, or condition is depression or a depression-related central nervous system (CNS) disorder; osteoporosis, including post-menopausal osteoporosis; acute kidney injury; Inflammatory Bowel Disease (or Inflammatory Bowel Syndrome); acute pancreatitis; muscle disorders, including mitochondrial myopathies; retinal disease; Primary Sjogren’s Syndrome; post-stroke cognitive decline; or systemic sclerosis. In some embodiments, the disease, disorder, or condition is associated with and / or affected by reduced AMPK and Pgcla signaling. In some embodiments, the disease, disorder, or condition is neurodegenerative disorders such as Huntington’s disease and post-stroke cognitive decline. In some embodiments, the disease, disorder, or condition is associated with dysfunctional states of the kynurenine pathway and includes, but are not limited to, HIV dementia, Tourette syndrome, Tic disorder, psychiatric disorders such as schizophrenia, bipolar disorder, major depression, and anxiety disorders, multiple sclerosis, encephalopathies, lipid metabolism, liver fat metabolism, systemic lupus erythematosus, glutaric aciduria, vitamin B6 deficiency, eosinophilia-myalgia syndrome, myalgic encephalomyelitis, chronic fatigue syndrome, depression, obesity, chronic obstructive pulmonary disease (COPD), and long COVID. In some embodiments, the disease, disorder, or condition is cancer, diabetes, hyperinsulinemia or an associated condition, a condition / disorder where fibrosis plays a role, sexual dysfunction, osteoporosis, a neurodegenerative disease, or progressive renal disease. In some embodiments, the disease, disorder, or condition is hyperinsulinemia or an associated condition. In some embodiments, the disease, disorder, or condition is a glycogen storage disorder. In some embodiments, the disease, disorder, or condition is McArdle’s disease. In some embodiments, the disease, disorder, or condition is type 2 diabetes, glucose intolerance, insulin resistance, metabolic syndrome, dyslipidemia, hyperinsulinism in childhood, hypercholesterolemia, high blood pressure, obesity, fatty liver conditions, diabetic nephropathy, diabetic neuropathy, diabetic retinopathy, cardiovascular disease, atherosclerosis, cerebrovascular conditions such as stroke, systemic lupus erythematosus, neurodegenerative diseases such as Alzheimer’s disease, or polycystic ovary syndrome. In some embodiments, the disease, disorder, or condition is type 2 diabetes. In some embodiments, the disease, disorder, or condition is obesity. In some embodiments, the disease,disorder, or condition is obesity-associated depression. In some embodiments, the disease, disorder, or condition is progressive renal disease. In some embodiments, the disease, disorder, or condition is chronic renal failure.

[0067] In one aspect, provided is a method of treating depression in a subject in need thereof, comprising administering to the subject 4-chloro-N-[2-[(4-chlorophenyl)methyl]-3-oxo-l,2,4- thiadiazol-5- yl]benzamide, or a salt thereof, or a prodrug thereof. In certain aspects, provided is use of 4-chloro-N-[2-[(4-chlorophenyl)methyl]-3-oxo-l,2,4-thiadiazol-5- yl]benzamide, or a salt thereof, or a prodrug thereof, for the manufacture of a medicament for the treatment the diseases, disorders, and conditions described herein, including depression. In certain aspects, provided is use of 4-chloro-N-[2-[(4-chlorophenyl)methyl]-3-oxo-l,2,4-thiadiazol-5- yl]benzamide, or a salt thereof, or a prodrug thereof, for treating the diseases, disorders, and conditions described herein including depression. In one variation, the depression is obesity-associated depression. In certain variations, the subject is obese. In some variations, the subject is an adult with an initial body mass index (BMI) of 30 kg / m2or greater (obesity) or 27 kg / m2or greater (overweight). In other variations, the combination therapies are administered to a pediatric subject. In certain variations, the subject is a pediatric subject aged 12 years and older. In certain variations, the pediatric subject has an initial BMI at the 95thpercentile or greater for age and sex (obesity).

[0068] In some variations of the foregoing, the treatment compound is in salt form. In certain variations, the salt of the compound is a pharmaceutically acceptable salt, including those described herein. In other variations, the treatment compound is 4-chloro-N-[2-[(4- chlorophenyl)methyl]-3-oxo-l,2,4-thiadiazol-5- yl]benzamide (i.e., in non-salt form).Tryptophan Supplement

[0069] In certain aspects, provided is a method of treating a disease, disorder, or condition ameliorated by activation of AMPK in a subject in need thereof, comprising administering to the subject (i) 4-chloro-N-[2-[(4-chlorophenyl)methyl]-3-oxo-l,2,4-thiadiazol-5- yl]benzamide, or a salt thereof, or a prodrug thereof, and (ii) tryptophan supplement.

[0070] In some embodiments, the method further comprises: monitoring the subject’s plasma tryptophan levels. In certain embodiments, the method further comprises: determiningtryptophan levels in a sample from the subject during or after administration of the treatment; and supplementing the subject with tryptophan if the subject’s tryptophan levels are below the clinically defined normal range. In one variation, the clinically defined normal range is less than 25 pmol / L (e.g., below any of 20 pmol / L, 15 pmol / L, or 10 pmol / L).

[0071] In some embodiments, the method further comprises: adjusting the dosing of the treatment, as described herein, during or after administration of the treatment; and supplementing the subject with tryptophan if the subject’s tryptophan levels are below the clinically defined normal range. In one variation, the clinically defined normal range is less than 25 pmol / L (e.g., below any of 20 pmol / L, 15 pmol / L, or 10 pmol / L)

[0072] In some aspects, provided is a combination comprising: (i) 4-chloro-N-[2-[(4- chlorophenyl)methyl]-3-oxo-l,2,4-thiadiazol-5- yl]benzamide, or a salt thereof, or a prodrug thereof, and (ii) tryptophan supplement. In some variations of the foregoing, the combination requires both active agents, but that does not necessarily have to be in a fixed dose format. In certain variations, the two components in the combination are sequentially administered. In other variations, the two components in the combination are simultaneously administered.

[0073] In some variations, provided is a mixture comprising the aforementioned combination. In other variations, provided is an admixture comprising the aforementioned combination. In one variation, an admixture is a mixture of two or more drug preparations which are combined without any limitation on dosage of one compared to the other and it is understood that both need not be administered in the same formulation or same dosage form.

[0074] In some variations of the foregoing, the salt of the compound is a pharmaceutically acceptable salt, including those described herein.

[0075] In some variations of the foregoing, the disease, disorder, or condition is as described herein.Kits and Articles of Manufacture

[0076] In some aspects, provided is a diagnostic kit, comprising: one or more reagents for determining the level of one or more biomarker and / or the ratio of two or more biomarkers in asample obtained from the subject; and assessing the subject based on one or more of the biomarker criteria described herein, indicating whether or not the subject may benefit from the treatment.

[0077] In some embodiments, the kit further comprises: instructions to use the kit to select a treatment comprising 4-chloro-N-[2-[(4-chlorophenyl)methyl]-3-oxo-l,2,4-thiadiazol-5- yl]benzamide, or a salt thereof for treating the disease, disorder, or condition based on one or more of the biomarker criteria described herein .

[0078] In some embodiments, the instructions are to use the kit to select a medicament other than treatment comprising 4-chloro-N-[2-[(4-chlorophenyl)methyl]-3-oxo-l,2,4-thiadiazol-5- yl]benzamide, or a salt thereof based on one or more of the biomarker criteria described herein.

[0079] In other aspects, provided is an article of manufacture, comprising: packaged together, 4-chloro-N-[2-[(4-chlorophenyl)methyl]-3-oxo-l,2,4-thiadiazol-5- yl]benzamide, or a salt thereof, or a prodrug thereof, in a pharmaceutically acceptable carrier, and a label or package insert indicating that 4-chloro-N-[2-[(4-chlorophenyl)methyl]-3-oxo-l,2,4-thiadiazol-5- yl]benzamide, or a salt thereof, or a prodrug thereof, is for treating an individiaul with a disease, disorder, or condition based on one or more of the biomarker criteria described herein. In some embodiments, the article of manufacture further comprises: a container.

[0080] In some variations of the foregoing, the kit or the article of manufacture further comprises a tryptophan supplement as described herein. In certain variations, the instructions, label or package insert provided to use the kit or the article of manufacture (as the case may be) further comprises instructions regarding co-admini strati on of the tryptophan supplement in accordance to the methods described herein.Treatment Compounds

[0081] In some embodiments of the foregoing, the treatment compound may be any suitable (AMPK activator). Certain exemplary treatment compounds are described in further detail below.Compound A

[0082] In some embodiments, the treatment compound is 4-chloro-N-[2-[(4- chlorophenyl)methyl]-3-oxo-l,2,4-thiadiazol-5-yl]benzamide, or a salt thereof, or a prodrug thereof. In certain embodiments, the salt is a pharmaceutically acceptable salt. In some variations, “pharmaceutically acceptable” generally refers to a material that is not biologically or otherwise undesirable, e.g., the material may be incorporated into a pharmaceutical composition administered to a patient without causing any significant undesirable biological effects or interacting in a deleterious manner with any of the other components of the composition in which it is contained. In one variation, a pharmaceutically acceptable salt is a salt which retains at least some of the biological activity of the free (non-salt) compound and which can be administered as drugs or pharmaceuticals to a subject (e.g., a human).

[0083] In certain embodiments, the treatment compound is an alkali metal salt of 4-chloro-N- [2-[(4-chlorophenyl)methyl]-3-oxo-l,2,4-thiadiazol-5-yl]benzamide. “Alkali metals” are metals found, along with hydrogen, in group I of the periodic table. The alkali metals are lithium, sodium, potassium, rubidium, caesium and francium. It will therefore be understood that an “alkali metal salt” is a chemical compound consisting of an assembly of cations of one or more alkali metals and associated anions. Accordingly, the term “an alkali metal salt of 4-chloro-N- [2-[(4-chlorophenyl)methyl]-3-oxo-l,2,4-thiadiazol-5-yl]benzamide” refers to a compound comprising alkali metal cations (e.g. lithium, rubidium, caesium and, particularly, sodium and potassium) and anions of 4-chloro-N-[2-[(4-chlorophenyl)methyl]-3-oxo-l,2,4-thiadiazol-5- yl]benzamide. For example, alkali metal salts of 4-chloro-N-[2-[(4-chlorophenyl)methyl]-3-oxo- l,2,4-thiadiazol-5-yl]benzamide may be referred to as follows:wherein X+represents the alkali metal (e.g. lithium, rubidium, caesium or, particularly, sodium or potassium) cation.

[0084] It will be understood that a “sodium salt” is a chemical compound consisting of an assembly of cations of sodium and associated anions. Accordingly, the term “a sodium salt of 4- chloro-N-[2-[(4-chlorophenyl)methyl]-3-oxo-l,2,4-thiadiazol-5-yl]benzamide” refers to a compound comprising sodium cations and anions of 4-chloro-N-[2-[(4-chlorophenyl)methyl]-3- oxo- 1 ,2,4-thiadiazol-5-yl]benzamide.wherein Na+represents the sodium cation (also referred to herein as “Compound A”).

[0085] The skilled person will recognize that, when dissolved in a suitable solvent (e.g. water) the alkali metal salt of 4-chloro-N-[2-[(4-chlorophenyl)methyl]-3-oxo-l,2,4-thiadiazol-5- yl]benzamide may dissociate into its anionic and cationic components.

[0086] The compound name 4-chloro-N-[2-[(4-chlorophenyl)methyl]-3-oxo-l,2,4-thiadiazol- 5-yl]benzamide was derived using the commercially available software package Autonom (brand of nomenclature software provided as an add-on for use in the Symyx Draw 2.1 (TM) office suite marketed by MDL Information Systems).

[0087] Throughout this specification, structures may or may not be presented with chemical names. Where any question arises as to nomenclature, the structure prevails. Where it is possible for the compound to exist as a tautomer (e.g. in an alternative resonance form) the depicted structure represents one of the possible tautomeric forms, wherein the actual tautomeric form(s) observed may vary depending on environmental factors such as solvent, temperature or pH. All tautomeric (and resonance) forms and mixtures thereof are included within the scope ofthe invention. For example, the following tautomers are included within the scope of the invention:

[0088] For the avoidance of doubt, alkali metal salts of 4-chloro-N-[2-[(4- chlorophenyl)methyl]-3-oxo-l,2,4-thiadiazol-5-yl]benzamide are solid under ambient conditions, and thus the scope of the invention includes all amorphous, crystalline and part crystalline forms thereof.

[0089] Alkali metal salts of 4-chloro-N-[2-[(4-chlorophenyl)methyl]-3-oxo-l,2,4-thiadiazol- 5-yl]benzamide may be prepared in accordance with techniques that are well known to those skilled in the art. For example, 4-chloro-N-[2-[(4-chlorophenyl)methyl]-3-oxo-l,2,4-thiadiazol- 5-yl]benzamide may be reacted with the appropriate alkali metal hydroxide, or an alternative alkali metal base compound. Salt switching techniques may also be used to convert one salt into another salt.

[0090] Sodium salts of 4-chloro-N-[2-[(4-chlorophenyl)methyl]-3-oxo-l,2,4-thiadiazol-5- yl]benzamide may be prepared in accordance with techniques that are well known to those skilled in the art. For example, 4-chloro-N-[2-[(4-chlorophenyl)methyl]-3-oxo-l,2,4-thiadiazol- 5-yl]benzamide may be reacted with sodium hydroxide, or an alternative sodium base compound. Salt switching techniques may also be used to convert one salt into another salt.

[0091] Where the salt is prepared from 4-chloro-N-[2-[(4-chlorophenyl)methyl]-3-oxo-l,2,4- thiadiazol-5-yl]benzamide, 4-chloro-N-[2-[(4-chlorophenyl)methyl]-3-oxo-l,2,4-thiadiazol-5- yl]benzamide may be prepared in accordance with techniques that are well known to those skilled in the art. For example, 4-chloro-N-[2-[(4-chlorophenyl)methyl]-3-oxo-l,2,4-thiadiazol- 5-yl]benzamide may be made in accordance with the techniques described in international patent application WO 2011 / 004162, and all of its content is hereby incorporated by reference.

[0092] Unless indicated otherwise, all technical and scientific terms used herein will have their common meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0093] In particular embodiments, the alkali metal salt of 4-chloro-N-[2-[(4- chlorophenyl)methyl]-3-oxo-l,2,4-thiadiazol-5-yl]benzamide is a sodium or potassium salt of 4- chloro-N-[2-[(4-chlorophenyl)methyl]-3-oxo-l,2,4-thiadiazol-5-yl]benzamide. Preferably, the salt is a sodium salt of 4-chloro-N-[2-[(4-chlorophenyl)methyl]-3-oxo-l,2,4-thiadiazol-5- yl]benzamide.Compound B

[0094] In some embodiments, the treatment compound is a compound of Formula (I)

[0095] , or a salt thereof, wherein R1is selected from the group consisting of -C(O)-C2H4-CO2H and -PO3H2, or a salt or solvate thereof (hereinafter “Compound B”).

[0096] In some embodiments, the Compound B is able to metabolise in vivo to form 4- chloro-N-[2-[(4-chlorophenyl)methyl]-3-oxo-l,2,4-thiadiazol-5-yl] benzamide. In certain embodiments, the compound is a salt of Compound B. For example, salts of Compound B may be referred to as follows:(hereinafter “Compound B-l”), and2X®(hereinafter “Compound B-2”), wherein X represents an alkali metal, alkaline earth metal or quaternary ammonium (e.g. lithium, magnesium, calcium, ammonium, tetramethylammonium and, particularly, sodium and potassium) cation, with appropriate stoichiometric adjustments being made in view of charges of the ions. In certain embodiments, X+represents an alkali metal (e.g. lithium, rubidium, caesium or, particularly, sodium or potassium) cation.Pharmaceutical Dosage Forms

[0097] The treatment compound is administered to a subject (e.g., a human) in need thereof in the form of a pharmaceutical formulation, which is also referred to herein as a pharmaceutical dosage form.

[0098] In one embodiment, the treatment compound is the sole active pharmaceutical ingredient present in the dosage form. In a further embodiment, treatment compound is present in the dosage form alongside one or more other active pharmaceutical ingredients, or may be administered as part of a combination therapy with one or more other active pharmaceutical ingredients.

[0099] In particular embodiments, the treatment compound is provided in the form of particles having a particle size distribution defined by a D90 of less than about 10 pm (e.g. as measured using laser diffraction). In one embodiment, the particles containing the treatment compound may have a particle size distribution defined by a D90 of less than about 10 pm (e.g. from about 5 pm to about 10 pm) (e.g. as measured using laser diffraction). The particle size distribution may alternatively be defined by a D90 of less than about 8 pm (e.g. from about 5 pm to about 8 pm). In a further embodiment, the particles consisting of the treatment compound may have a particle size distribution defined by a D50 of less than about 6 pm (e.g. from about 0.5 pm to about 6 pm). In a yet further embodiment, the particle size distribution of the particlesconsisting of the treatment compound may further be a defined by a DIO of less than about 2 pm (e.g. from about 0.2 pm to about 2 pm). The particle size distribution parameters mentioned above may be applicable, individually or in combination. For example, in particular embodiments, the dosage form comprises particles containing the treatment compound, said particles having a particle size distribution defined by a D90 of less than about 10 pm and a D50 of less than about 6 pm. Still further, said particles may have a particle size distribution defined by a D90 of less than 9 pm; a D50 of less than 6 pm or less than 5 pm; and a DIO of less than 2 pm or less than 1.5 pm. The particle size distribution of particles containing the treatment compound may be measured by laser diffraction, using, for example a commercially available particle size analyser.Dosage

[0100] The skilled person will understand that the pharmaceutical dosage forms described herein may act systemically, and may therefore be administered accordingly using suitable techniques known to those skilled in the art. The pharmaceutical dosage form as described herein will normally be administered orally, e.g., as an oral pharmaceutical dosage form. Thus, in some variations, provided is an oral pharmaceutical dosage form comprising from about 200 to about 1000 mg of the treatment compound, such as Treatment Compounds A, B, B-l, and B-2 In one variation, provided is an oral pharmaceutical dosage form comprising from about 200 to about 1000 mg of a sodium salt of 4-chloro-N-[2-[(4-chlorophenyl)methyl]-3-oxo-l,2,4- thi adi azol - 5 -y 1 ]b enzami de .

[0101] In particular embodiments, the pharmaceutical dosage form referred to in the first and second aspects of the invention may comprise, for example, from about 200 mg to about 800 mg, from about 200 mg to about 600 mg, or from about 200 mg to about 400 mg) of the treatment compound. In some variations, the pharmaceutical dosage form of the comprises from about 200 to about 400 mg of the treatment compound, such as the sodium salt of 4-chloro-N-[2-[(4- chlorophenyl)methyl]-3-oxo-l,2,4-thiadiazol-5-yl]benzamide.Enteric Coating

[0102] Dosage forms intended for oral administration may further comprise an enteric coating in order to prevent or minimise dissolution or disintegration in the gastric environment. As such, oral preparations (e.g. capsules or tablets) coated by an enteric coating may provide targeted release of the treatment compound in the small intestine. For example, the enteric coating may be present on surface of the formulation (e.g. on the surface of a tablet or a capsule), or each of the particles containing the treatment compound may be coated with the enteric coating. Thus, in particular embodiments, the pharmaceutical dosage form used in the method of the invention further comprises an enteric coating.

[0103] In certain embodiments, the enteric coating is present on the pharmaceutical dosage form, and in some variations, said coating may be provided as an outer layer on the pharmaceutical dosage form.

[0104] Alternatively, particles containing the treatment compound may be individually coated with the enteric coating, and said coated particles may be prepared into the pharmaceutical dosage form. Thus, in particular embodiments, the pharmaceutical dosage form contains particles comprising the treatment compound and each particle is coated with the enteric coating.

[0105] The term “enteric coating” refers to a substance (e.g. a polymer) that is incorporated into an oral medication (e.g. applied onto the surface of a tablet, a capsule, particles or pellets) and that inhibits dissolution or disintegration of the medication in the gastric environment. Enteric coatings are typically stable at the highly acidic pH found in the stomach, but break down rapidly in the relatively basic pH of the small intestine. Therefore, enteric coatings prevent release of the active ingredient in the medication until it reaches the small intestine.

[0106] Any enteric coating known to the skilled person may be used in the present invention. Particular enteric coating materials that may be mentioned include those which comprise beeswax, shellac, an alkylcellulose polymer resin (e.g. ethylcellulose polymers, carboxymethylethylcellulose, or hydroxypropyl methylcellulose phthalate) or an acrylic polymer resin (e.g. acrylic acid and methacrylic acid copolymers, methyl methacrylate copolymers,ethoxyethyl methacrylates, cyanoethyl methacrylate, methacrylate copolymers, methacrylic acid copolymer, aminoalkyl methacrylate copolymer, poly(acrylic acid), poly(methacrylic acid), methacrylic acid alkylamide copolymer, poly(methyl methacrylate), poly(methacrylic acid) (anhydride), polymethacrylate, methyl methacrylate copolymer, poly(methyl methacrylate) copolymer, polyacrylamide, poly(methacrylic acid anhydride), and glycidyl methacrylate copolymers), cellulose acetate phthalate and polyvinyl acetate phthalate.Dosage Forms

[0107] In some variations, the treatment compound may be provided in the form of a tablet or particularly a capsule. For example, capsules such as soft gelatin capsules may be prepared containing the treatment compound alone, or together with a suitable vehicle, e.g. vegetable oil, fat etc. Similarly, hard gelatin capsules may contain the treatment compound alone, or in combination with solid powdered ingredients such as a disaccharide (e.g. lactose or saccharose), an alcohol sugar (e.g. sorbitol or mannitol), a vegetable starch (e.g. potato starch or corn starch), a polysaccharide (e.g. amylopectin or cellulose derivatives) or gelling agent (e.g. gelatin).

[0108] The pharmaceutical dosage forms described herein may be prepared in accordance with standard and / or accepted pharmaceutical practice. The pharmaceutical dosage forms of the first and second aspects of the invention will generally be provided as a mixture comprising the treatment compound and one or more pharmaceutically acceptable excipients. The one or more pharmaceutically acceptable excipients may be selected with due regard to the intended route of administration in accordance with standard pharmaceutical practice. Such pharmaceutically acceptable excipients are preferably chemically inert to the active compound and are preferably have no detrimental side effects or toxicity under the conditions of use. Suitable pharmaceutical formulations may be found in, for example, Remington The Science and Practice of Pharmacy, 19th ed., Mack Printing Company, Easton, Pennsylvania (1995). A brief review of methods of drug delivery may also be found in e.g. Langer, Science 249, 1527 (1990).Excipients

[0109] In some variations, the pharmaceutical compositions comprise the treatment compound, and at least one pharmaceutically acceptable excipient. In particular, the at least onepharmaceutically acceptable excipient may be a lubricant, a binder, a filler, a surfactant, a diluent, an anti -adherent, a coating, a flavouring, a colourant, a glidant, a preservative, a sweetener, a disintegrant, an adsorbent, a buffering agent, an antioxidant, a chelating agent, a dissolution enhancer, a dissolution retardant or a wetting agent.

[0110] Particular pharmaceutically acceptable excipients that may be mentioned include mannitol, PVP (polyvinylpyrrolidone) K30, lactose, saccharose, sorbitol, starch, amylopectin, cellulose derivatives, gelatin, or another suitable ingredients, as well as disintegrating agents and lubricating agents such as sodium lauryl sulfate, Na-docusate, magnesium stearate, calcium stearate, sodium stearyl fumarate and polyethylene glycol waxes. In the preparation of a pharmaceutical dosage form of the treatment compound for oral administration, particles containing the treatment compound (preferably milled) may be mixed, either together or separately, with mannitol, PVP (polyvinylpyrrolidone) K30 and sodium lauryl sulfate.[OHl] In the preparation of a pharmaceutical dosage form, the treatment compound may be mixed, either together or separately, with one or more of the pharmaceutical excipients (including basic excipients) listed above.

[0112] Mixtures of the treatment compound and one or more pharmaceutically acceptable excipients may be processed into pellets or granules, or compressed into tablets. Thus, pharmaceutical dosage form of the method of the inventions may be a tablet, mini-tablets, blocks, pellets, particles, granules or a powder for oral administration.

[0113] Pharmaceutical formulations that may be mentioned include those in which the treatment compound is present in a total amount that is at least 1% (or at least 10%, at least 30% or at least 50%) by weight of the formulation. That is, the weight ratio of the treatment compound to the totality of the components (i.e. the treatment compound and all pharmaceutical excipients, e.g. adjuvants, diluents and carriers) of the pharmaceutical formulation is at least 1 :99 (or at least 10:90, at least 30:70 or at least 50:50).

[0114] A “therapeutically effective amount”, an “effective amount” or a “dosage” as used herein refers to an amount of a treatment compound that is sufficient to produce a desired effect, which can be a therapeutic and / or beneficial effect. The effective amount or dosage will varywith the age or general condition of the individual or subject (e.g. a human), the severity of the condition being treated, the particular agents administered, the duration of the treatment, the nature of any concurrent treatment, the pharmaceutically acceptable carrier used, and like factors within the knowledge and expertise of those skilled in the art. As appropriate, a “therapeutically effective amount”, “effective amount” or “dosage” in any individual case can be determined by one of skill in the art by reference to the pertinent texts and literature and / or by using routine experimentation. Those skilled in the art will appreciate that the therapeutic effects need not be complete or curative, as long as some benefit is provided to the subject.

[0115] The skilled person will understand that suitable formulations may be administered (for example, by way of one or more preparations as described herein) at varying doses, with suitable doses being readily determined by one of skill in the art. The total dosage of the treatment compound that is to be administered to a subject in need thereof may range from about 0.01 to about 2000 mg / kg of body weight per day (mg / kg / day), about 0.1 to about 500 mg / kg / day, or about 1 to about 100 mg / kg / day. Such dosages may be, for example, oral dosages of the formulations of the second aspect of the invention.

[0116] When administered orally, treatment with such formulations (including capsules containing such formulations) may comprise administration of a unit dose formulation containing from about 0.01 mg to about 3000 mg of the treatment compound, for example from about 0.1 mg to about 2000 mg, or from about 1 mg to about 1000 mg (e.g. from about 10 mg to about 500 mg), of the treatment compound. Advantageously, treatment may comprise administration of the treatment compound (including capsules containing said formulation) using a single daily dose. Alternatively, the total daily dosage of the treatment compound may be administered in divided doses two, three or four times daily (e.g. twice daily with reference to the doses described herein, such as a dose of 100 mg, 250 mg, 500 mg or 1000 mg twice daily). The skilled physician will recognise that the dosage will vary from subject to subject.

[0117] In particular embodiments, the daily dose of the treatment compound administered to a subject is in the range of from about 1 mg to about 3000 mg, from about 1 mg to about 1000 mg, from about 200 mg to about 800 mg, from about 200 mg to about 600 mg, or from about 200 mg to about 400 mg).

[0118] The term "about," as used herein when referring to a measurable value such as an amount of a compound, dose, time, temperature, and the like, refers to variations of 20%, 10%, 5%, 1%, 0.5%, or even 0.1% of the specified amount. It is contemplated that, at each instance, such terms may be replaced with the notation “±10%”, or the like (or by indicating a variance of a specific amount calculated based on the relevant value). It is also contemplated that, at each instance, such terms may be deleted.

[0119] For the avoidance of doubt, the dose administered to a subject, particularly a huma subject, in the context of the present invention should be sufficient to effect a therapeutic response in the subject over a reasonable timeframe. One skilled in the art will recognize that the selection of the exact dose and composition and the most appropriate delivery regimen will also be influenced by inter alia the pharmacological properties of the formulation, the nature and severity of the condition being treated, and the physical condition and mental acuity of the recipient, as well as the potency of the specific compound, the age, condition, body weight, sex and response of the subject to be treated, and the stage / severity of the disease.

[0120] In any event, the medical practitioner, or other skilled person, will be able to determine routinely the actual dosage which will be most suitable for a subject. The above- mentioned dosages are exemplary of the average case; there can, of course, be individual instances where higher or lower dosage ranges are merited, and such are within the scope of this invention.ENUMERATED EMBODIMENTS

[0121] The following enumerated embodiments are representative of some aspects of the invention.1. A method of identifying a subject who may benefit from a treatment comprising 4- chloro-N-[2-[(4-chlorophenyl)methyl]-3-oxo-l,2,4-thiadiazol-5- yl]benzamide, or a salt thereof, or a prodrug thereof, or identifying a subject having increased PGC-lalpha activity and / or increased expression of KAT enzymes in response to the treatment, the method comprising: determining (i) a level of at least one biomarker, wherein the at least one biomarker is selected from tryptophan and metabolites thereof, and / or (ii) a ratio of two or more of theforegoing biomarkers, in a sample obtained from the subject, wherein (i) the level of the at least one biomarker, and / or (ii) the ratio of two of more of the foregoing biomarkers as compared to a reference level / ratio indicates that the subject may benefit from the treatment, optionally, providing a recommendation that the subject will be more likely to respond to the treatment.2. The method of embodiment 1, wherein the method comprises determining at least one of the following:(a) the level of tryptophan;(b) the level of KynA;(c) the ratio of Kyn to tryptophan;(d) the ratio of KynA to Kyn;(e) the ratio of KynA to tryptophan;(f) the ratio of Quinolinic acid to KynA; and / or(g) the ratio of Quinolinic acid to Kyn.3. The method of embodiment 1 or 2, wherein the sample comprises a plasma sample.4. The method of any one of embodiments 1-3, further comprising providing a recommendation that the subject will be more likely to respond to the treatment when at least one of the following occurs:(i) the plasma level of tryptophan is above 70 pmol / L;(ii) the molar ratio of KynA to Kyn in plasma is below 0.3;(iii) the molar ratio of KynA to tryptophan in plasma is below 0.01;(iv) the molar ratio of Quinolinic acid to KynA in plasma is above 5; and / or(v) the molar ratio of Quinolinic acid to Kyn in plasma below 0.1.5. A method for predicting responsiveness of a subject with a disease, disorder, or condition to a treatment comprising 4-chloro-N-[2-[(4-chlorophenyl)methyl]-3-oxo-l,2,4-thiadiazol-5- yl]benzamide, or a salt thereof, or a prodrug thereof, the method comprising: determining (i) a level of at least one biomarker, wherein the at least one biomarker is selected from tryptophan and metabolites thereof, and / or (ii) a ratio of two or more of the foregoing biomarkers, in a sample obtained from the subject, wherein (i) the level of the at least one biomarker, and / or (ii) the ratio of two of more of the foregoing biomarkers as compared to a reference level / ratio indicates that the subject is more likely to respond to the treatmentt; and optionally, informing the subject that they have an increased likelihood of being responsive to the treatment or an increased likelihood of exhibiting benefit from the treatment.6. The method of embodiment 5, wherein the method comprises determining at least one of the following in the subject:(a) the level of tryptophan;(b) the level of KynA;(c) the ratio of Kyn to tryptophan;(d) the ratio of KynA to Kyn;(e) the ratio of KynA to tryptophan;(f) the ratio of Quinolinic acid to KynA; and / or(g) the ratio of Quinolinic acid to Kyn.7. The method of embodiment 5 or 6, wherein the sample comprises a plasma sample.8. The method of any one of embodiments 5-7, further comprising informing the subject that they have an increased likelihood of being responsive to the treatment or an increasedlikelihood of exhibiting benefit from the treatment, when at least one of the following occurs in the subject:(i) the plasma level of tryptophan is above 70 pmol / L;(ii) the molar ratio of KynA to Kyn in plasma is below 0.3;(iii) the molar ratio of KynA to tryptophan in plasma is below 0.01;(iv) the molar ratio of Quinolinic acid to KynA in plasma is above 5; and / or(v) the molar ratio of Quinolinic acid to Kyn in plasma below 0.1.9. A method for selecting a treatment for a subject with a disease, disorder, or condition, wherein the treatment comprises 4-chloro-N-[2-[(4-chlorophenyl)methyl]-3-oxo-l,2,4- thiadiazol-5- yl]benzamide, or a salt thereof, or a prodrug thereof, the method comprising:(a) detecting (i) a level of at least one biomarker, wherein the at least one biomarker is selected from tryptophan and metabolites thereof, and / or (ii) a ratio of two or more of the foregoing biomarkers, in a sample obtained from the subject prior to administration of the treatment to the subject;(b) comparing (i) the level of the at least one biomarker, and / or (ii) the ratio of two or more of the foregoing biomarkers to a reference level / ratio which identifies a subject who is likely to respond to the treatment;(c) selecting the treatment if the subject is identified as likely to respond to the treatment; and(d) recommending to the subject the selected treatment.10. The method of embodiment 9, wherein the method comprises detecting at least one of the following:(a) the level of tryptophan;(b) the level of KynA;(c) the ratio of Kyn to tryptophan;(d) the ratio of Kyn A to Kyn;(e) the ratio of KynA to tryptophan;(f) the ratio of Quinolinic acid to KynA; and / or(g) the ratio of Quinolinic acid to Kyn.11. The method of embodiment 9 or 10, wherein the sample comprises a plasma sample.12. The method of any one of the embodiments 9-11, wherein the subject is identified as likely to respond to the treatment when at least one of the following occurs:(i) the plasma level of tryptophan is above 70 pmol / L;(ii) the molar ratio of KynA to Kyn in plasma is below 0.3;(iii) the molar ratio of KynA to tryptophan in plasma is below 0.01;(iv) the molar ratio of Quinolinic acid to KynA in plasma is above 5; and / or(v) the molar ratio of Quinolinic acid to Kyn in plasma below 0.1.13. The method of any of the foregoing embodiments, further comprising: administering an effective amount of 4-chloro-N-[2-[(4-chlorophenyl)methyl]-3-oxo- l,2,4-thiadiazol-5- yl]benzamide, or a salt thereof, or a prodrug thereof, to the subject.14. A method for advertising a treatment comprising 4-chloro-N-[2-[(4- chlorophenyl)methyl]-3-oxo-l,2,4-thiadiazol-5- yl]benzamide, or a salt thereof, or a prodrug thereof, directed to treating a disease, disorder, or condition, the method comprising: promoting, to a target audience, the use of the treatment based on (i) a level of at least one biomarker, wherein the at least one biomarker is selected from tryptophan and metabolites thereof, and / or (ii) a ratio of two or more of the foregoing biomarkers, in a sample of the subject.15. The method of embodiment 14, wherein the treatment is promoted based on at least one of the following:(i) the plasma level of tryptophan is above 70 pmol / L;(ii) the molar ratio of KynA to Kyn in plasma is below 0.3;(iii) the molar ratio of KynA to tryptophan in plasma is below 0.01;(iv) the molar ratio of Quinolinic acid to KynA in plasma is above 5; and / or(v) the molar ratio of Quinolinic acid to Kyn in plasma below 0.1.16. A method of determining the dosing or the dosing regimen of 4-chloro-N-[2-[(4- chlorophenyl)methyl]-3-oxo-l,2,4-thiadiazol-5- yl]benzamide, or a salt thereof, or a prodrug thereof, for treatment of a disease, disorder, or condition in a subject in need thereof, the method comprising: identifying a minimally effective dose and a maximally effective dose of 4-chloro-N-[2- [(4-chlorophenyl)methyl]-3-oxo-l,2,4-thiadiazol-5- yl]benzamide, or a salt thereof, or a prodrug thereof, or the dose regimen of 4-chloro-N-[2-[(4-chlorophenyl)methyl]-3-oxo-l,2,4-thiadiazol- 5- yl]benzamide, or a salt thereof, or a prodrug thereof, based on comparing (i) a level of the at least one biomarker, and / or (ii) a ratio of two or more of the foregoing biomarkers, wherein the at least one biomarker is selected from tryptophan and metabolites thereof, in a sample of the subject after or during treatment with a dose or dosing regimen of 4-chloro-N-[2-[(4- chlorophenyl)methyl]-3-oxo-l,2,4-thiadiazol-5- yl]benzamide, or a salt thereof, or a prodrug thereof, to (i) the level of the at least one biomarker, and / or (ii) the ratio of two or more of the foregoing biomarkers in a sample obtained from the subject before the treatment, wherein a change in (i) the level of the at least one biomarker, and / or (ii) the ratio of two or more of the foregoing biomarkers after or during the treatment as compared to before the treatment is indicative of the efficacy of or responsiveness to the dose or dosing regimen of 4-chloro-N-[2- [(4-chlorophenyl)methyl]-3-oxo-l,2,4-thiadiazol-5- yl]benzamide, or a salt thereof, or a prodrug thereof, for treatment of the subject.17. The method of embodiment 16, wherein:the minimally effective dose is the lowest dose of 4-chloro-N-[2-[(4- chlorophenyl)methyl]-3-oxo-l,2,4-thiadiazol-5- yl]benzamide, or a salt thereof, or a prodrug thereof, that gives a positive change in the aforementioned biomarkers, and ratios thereof; and the maximally effective dose is the dose of 4-chloro-N-[2-[(4-chlorophenyl)methyl]-3- oxo-l,2,4-thiadiazol-5- yl]benzamide, or a salt thereof, or a prodrug thereof, that normalizes the aforementioned biomarker levels and ratios thereof.18. The method of embodiment 16 or 17, wherein the level of the at least one biomarker, and / or (ii) the ratio of two or more of the foregoing biomarkers comprises at least one of the following:(a) the level of tryptophan;(b) the level of KynA;(c) the ratio of Kyn to tryptophan;(d) the ratio of KynA to Kyn;(e) the ratio of KynA to tryptophan;(f) the ratio of Quinolinic acid to KynA; and / or(g) the ratio of Quinolinic acid to Kyn.19. The method of any one of embodiments 16-18, wherein the sample comprises a plasma sample.20. The method of any one of embodiments 16-19, wherein before the treatment, the subject has at least one of the following:(i) the plasma level of tryptophan is above 70 pmol / L;(ii) the molar ratio of KynA to Kyn in plasma is below 0.3;(iii) the molar ratio of KynA to tryptophan in plasma is below 0.01;(iv) the molar ratio of Quinolinic acid to KynA in plasma is above 5; and / or(v) the molar ratio of Quinolinic acid to Kyn in plasma below 0.1.21. The method of embodiment 17, wherein the positive change in the aforementioned biomarkers, and ratios thereof, comprises at least one of the following:(i) the plasma level of tryptophan after the administration of the treatment is decreased by at least 5 pmol / mL compared to the baseline before the treatment;(ii) the increase in plasma level of KynA after the treatment compared to the baseline before the treatment is at least 0.01 pmol / mL;(iii) the plasma level of KynA after the treatment is at least doubled compared to the baseline before the treatment;(iv) the molar ratio of Kyn to tryptophan in plasma is increased;(v) the increase in the molar ratio of KynA to tryptophan in plasma after the treatment compared to the baseline before the treatment is at least 0.01;(vi) the molar ratio of KynA to tryptophan in plasma is increased;(vii) the plasma level of KynA is increased and the plasma level of Tryptophan is decreased;(viii) the decrease in the molar ratio of Quinolinic acid (QA) to KynA in plasma after the treatment compared to the baseline before the treatment is at least 0.5; and / or(ix) the increase in the molar ratio of Quinolinic acid (QA) to Kyn in plasma after the treatment compared to the baseline before the treatment is at least 0.01.22. The method of any one of embodiments 16-21, wherein the efficacy of or responsiveness to the dose or dosing regimen of 4-chloro-N-[2-[(4-chlorophenyl)methyl]-3-oxo-l,2,4-thiadiazol- 5- yl]benzamide, or a salt thereof, or a prodrug thereof, is determined when the plasma level of4-chloro-N-[2-[(4-chlorophenyl)methyl]-3-oxo-l,2,4-thiadiazol-5- yl]benzamide reaches a steady state.23. The method of any one of embodiments 16-22, wherein the efficacy of or responsiveness to the dose or dosing regimen of 4-chloro-N-[2-[(4-chlorophenyl)methyl]-3-oxo-l,2,4-thiadiazol- 5- yl]benzamide, or a salt thereof, or a prodrug thereof, is determined at least two weeks after the treatment.24. A method of monitoring the response of a subject in thereof having a treatment comprising 4-chloro-N-[2-[(4-chlorophenyl)methyl]-3-oxo-l,2,4-thiadiazol-5- yl]benzamide, or a salt thereof, or a prodrug thereof, the method comprising: determining (i) a level of at least one biomarker, wherein the at least one biomarker is selected from tryptophan and metabolites thereof, and / or (ii) a ratio of two or more of the foregoing biomarkers in a sample from the subject before administration of the treatment; administering the treatment; determining (i) a level of at least one biomarker, wherein the at least one biomarker is selected from tryptophan and metabolites thereof, and / or (ii) a ratio of two or more of the foregoing biomarkers in a sample from the subject during or after administration of the treatment; comparing (i) the level of the at least one biomarker, and / or (ii) the ratio of two or more of the foregoing biomarkers in the sample from the subject before the administration of the treatment and during or after the administration of the treatment; and optionally, adjusting the dose of 4-chloro-N-[2-[(4-chlorophenyl)methyl]-3-oxo-l,2,4- thiadiazol-5- yl]benzamide, or a salt thereof, or a prodrug thereof, or the dose regimen of 4- chloro-N-[2-[(4-chlorophenyl)methyl]-3-oxo-l,2,4-thiadiazol-5- yl]benzamide, or a salt thereof, or a prodrug thereof, based on the comparison.25. The method of embodiment 24, wherein the level of the at least one biomarker, and / or (ii) the ratio of two or more of the foregoing biomarkers comprises at least one of the following:(a) the level of tryptophan;(b) the level of KynA;(c) the ratio of Kyn to tryptophan;(d) the ratio of KynA to Kyn;(e) the ratio of KynA to tryptophan;(f) the ratio of Quinolinic acid to KynA; and / or(g) the ratio of Quinolinic acid to Kyn.26. The method of embodiment 24 or 25, wherein the sample comprises a plasma sample.27. The method of any one of embodiments 24-26, wherein before the treatment, at least one of the following occurs in the subject:(i) the plasma level of tryptophan is above 70 pmol / L;(ii) the molar ratio of KynA to Kyn in plasma is below 0.3;(iii) the molar ratio of KynA to tryptophan in plasma is below 0.01;(iv) the molar ratio of Quinolinic acid to KynA in plasma is above 5; or(v) the molar ratio of Quinolinic acid to Kyn in plasma below 0.1.28. The method of any one of embodiments 24-26, wherein the subject is responsive to the treatment if at least one of the following occurs:(i) the plasma level of tryptophan after the administration of the treatment is decreased by at least 5 pmol / mL compared to the baseline before the treatment;(ii) the increase in plasma level of KynA after the treatment compared to the baseline before the treatment is at least 0.01 pmol / mL;(iii) the plasma level of KynA after the treatment is at least doubled compared to the baseline before the treatment;(iv) the molar ratio of Kyn to tryptophan in plasma is increased;(v) the increase in the molar ratio of KynA to tryptophan in plasma after the treatment compared to the baseline before the treatment is at least 0.01;(vi) the molar ratio of KynA to tryptophan in plasma is increased;(vii) the plasma level of KynA is increased and the plasma level of Tryptophan is decreased;(viii) the decrease in the molar ratio of Quinolinic acid (QA) to KynA in plasma after the treatment compared to the baseline before the treatment is at least 0.5; or(ix) the increase in the molar ratio of Quinolinic acid (QA) to Kyn in plasma after the treatment compared to the baseline before the treatment is at least 0.01.29. The method of any one of embodiments 24-28, wherein the dose of 4-chloro-N-[2-[(4- chlorophenyl)methyl]-3-oxo-l,2,4-thiadiazol-5-yl]benzamide, or a salt thereof, or a prodrug thereof, is adjusted when the plasma level of tryptophan drops below 25 pmol / L after the treatment.30. The method of any one of embodiments 24-28, wherein the method further comprises administering tryptophan supplement when the plasma level of tryptophan drops below 25 pmol / L after the treatment.31. The method of embodiment 30, wherein the tryptophan supplement comprises an NAD+ precursor.32. The method of embodiment 30, wherein the tryptophan supplement comprises nicotinamide riboside.33. The method of any one of embodiments 24-32, wherein the time point during or after administration of the treatment is after the plasma level of 4-chloro-N-[2-[(4-chlorophenyl)methyl]-3-oxo-l,2,4-thiadiazol-5- yl]benzamide, or a salt thereof, reaches a steady state.34. The method of any one of embodiments 20-26, wherein the time point during or after administration of the treatment is at least two weeks after the administration of the treatment.35. The method of any one of embodiments 1, 5, 9, 14, 16, or 24, wherein the level of the at least one biomarker, and / or the ratio of two of more of the foregoing biomarkers is plasma level, or muscle tissue level, or liver tissue level.36. The method of any one of embodiments 1, 5, 9, 14, 16, 24, or 35, wherein the sample comprises tissue, whole blood, plasma, serum, or cerebrospinal fluid, or any combination thereof.37. A method of treating a disease, disorder, or condition ameliorated by the activation of AMPK in a subject in need thereof, comprising:(a) administering to the subject 4-chloro-N-[2-[(4-chlorophenyl)methyl]-3-oxo-l,2,4- thiadiazol-5- yl]benzamide, or a salt thereof, or a prodrug thereof;(b) monitoring the subject’s plasma levels of tryptophan after step (a); and(c) administering to the subject a tryptophan supplement if the subject’s plasma tryptophan levels are below 25 pmol / L.38. The method of embodiment 37, wherein the tryptophan supplement comprises an NAD+ precursor.39. The method of embodiment 37, wherein the tryptophan supplement comprises nicotinamide riboside.40. The method of any one of the preceding embodiments, wherein the 4-chloro-N-[2-[(4- chlorophenyl)methyl]-3-oxo-l,2,4-thiadiazol-5- yl]benzamide, or a salt thereof, or a prodrug thereof, is a sodium salt of 4-chloro-N-[2-[(4-chlorophenyl)methyl]-3-oxo-l,2,4-thiadiazol-5- yl]benzamide.41. The method of any one of embodiments 5-40, wherein the disease, disorder, or condition is associated with and / or affected by reduced AMPK and Pgcla signaling.42. The method of any one of embodiments 5-40, wherein the disease, disorder, or condition is depression or a depression-related central nervous system disorder, osteoporosis, acute kidney injury, inflammatory bowel disease, acute pancreatitis, a muscle disorder, retinal disease, Primary Sjogren’s Syndrome, post-stroke cognitive decline, or systemic sclerosis, or any combination thereof.43. The method of embodiment 42, wherein the disease, disorder, or condition is postmenopausal osteoporosis.44. The method of embodiment 42, wherein the disease, disorder, or condition is a mitochondrial myopathy.45. The method of any one of embodiments 5-40, wherein the disease, disorder, or condition is a glycogen storage disorder.46. The method of any one of embodiments 5-40, wherein the disease, disorder, or condition is McArdle’s disease.47. The method of any one of embodiments 5-40, wherein the disease, disorder, or condition is neurodegenerative disorders.48. The method of any one of embodiments 5-40, wherein the disease, disorder, or condition is Huntington’s disease or post-stroke cognitive decline.49. The method of any one of embodiments 5-40, wherein the disease, disorder, or condition is associated with dysfunctional states of the kynurenine pathway.50. The method of any one of embodiments 5-40, wherein the disease, disorder, or condition is HIV dementia, Tourette syndrome, Tic disorder, a psychiatric disorder, multiple sclerosis, encephalopathies, lipid metabolism, liver fat metabolism, systemic lupus erythematosus, glutaric aciduria, vitamin B6 deficiency, eosinophilia-myalgia syndrome, myalgic encephalomyelitis,chronic fatigue syndrome, depression, obesity, chronic obstructive pulmonary disease (COPD), or long COVID.51. The method of any one of embodiments 5-40, wherein the disease, disorder, or condition is schizophrenia, bipolar disorder, major depression, or an anxiety disorder.52. The method of any one of embodiments 5-40, wherein the disease, disorder, or condition is cancer, diabetes, hyperinsulinemia or an associated condition, a condition / disorder where fibrosis plays a role, sexual dysfunction, osteoporosis, a neurodegenerative disease, or progressive renal disease.53. The method of any one of embodiments 5-40, wherein the disease, disorder, or condition is hyperinsulinemia or an associated condition.54. The method of any one of embodiments 5-40, wherein the disease, disorder, or condition is type 2 diabetes, glucose intolerance, insulin resistance, metabolic syndrome, dyslipidemia, hyperinsulinism in childhood, hypercholesterolemia, high blood pressure, obesity, fatty liver conditions, diabetic nephropathy, diabetic neuropathy, diabetic retinopathy, cardiovascular disease, atherosclerosis, cerebrovascular conditions such as stroke, systemic lupus erythematosus, neurodegenerative diseases such as Alzheimer’s disease, or polycystic ovary syndrome.55. The method of any one of embodiments 5-40, wherein the disease, disorder, or condition is type 2 diabetes.56. The method of any one of embodiments 5-40, wherein the disease, disorder, or condition is obesity.57. The method of any one of embodiments 5-40, wherein the disease, disorder, or condition is progressive renal disease.58. The method of any one of embodiments 5-40, wherein the disease, disorder, or condition is chronic renal failure.59. A method of treating depression in a subject in need thereof, comprising administering to the subject 4-chloro-N-[2-[(4-chlorophenyl)methyl]-3-oxo-l,2,4-thiadiazol-5- yl]benzamide, or a salt thereof, or a prodrug thereof.60. The method of embodiment 59, wherein the depression is obesity-associated depression.61. The method of embodiment 59 or 60, wherein the subject is obese.62. The method of any one of embodiments 59-61, further comprising: monitoring the subject’s plasma levels of tryptophan after administration of the 4-chloro- N-[2-[(4-chlorophenyl)methyl]-3-oxo-l,2,4-thiadiazol-5- yl]benzamide, or a salt thereof, or a prodrug thereof; and administering to the subject a tryptophan supplement if the subject’s plasma tryptophan levels are below 25 pmol / L.63. The method of embodiment 62, wherein the tryptophan supplement comprises an NAD+ precursor.64. The method of embodiment 62, wherein the tryptophan supplement comprises nicotinamide riboside.65. A combination comprising: (i) 4-chloro-N-[2-[(4-chlorophenyl)methyl]-3-oxo-l,2,4- thiadiazol-5- yl]benzamide, or a salt thereof, or a prodrug thereof, and (ii) tryptophan supplement.66. The combination of embodiment 65, wherein the tryptophan supplement comprises an NAD+ precursor.67. The combination of embodiment 65, wherein the tryptophan supplement comprises nicotinamide riboside.EXAMPLES

[0122] The presently disclosed subject matter will be better understood by reference to the following Examples, which are provided as exemplary of the invention, and not by way of limitation.Example 1. Analyzing biomarker levels affected by Compound A

[0123] Kynurenine pathway of tryptophan metabolism is involved in the patho-physiology of depression where the metabolite kynurenic acid is implicated to have neuroprotective function. High fat diet induced obesity promote anxiety and depression in rodents whereas physical activation or muscle specific activation of PGC-la stimulates kynurenic acid production and prevent anxiety and depression. In this example, we investigate the effect of the exercise mimetic and AMPK-activator Compound A on depressive-like behavior and the kynurenic arm of tryptophan metabolism. Specifically, the following example explores whether Compound A can regulate the Trp-Kyn pathway in muscle and affect obesity-associated depression. Compound A is a sodium salt of 4-chloro-N-[2-[(4-chlorophenyl)methyl]-3-oxo-l,2,4-thiadiazol-5- yl]benzamide.

[0124] Tryptophan (Trp) is an essential amino acid that is a precursor for both serotonin and melatonin, known regulators of mood and anxiety. The majority of Trp, 90-95%, is however metabolized through the Kyn pathway. Trp is converted to Kyn in the liver and Kynuerine aminotransferases (KATs) can convert Kyn to Kynurenic acid (KynA) (FIG. 1). See e.g., Trends in Molecular Medicine, Vol. 27, No. 10, 2021. Exercise has been shown to induce the expression of KATs in muscle of mice and humans and exercise also increases plasma levels of (KynA) in both mice and humans. Thus, the induction of KATs in muscle following exercise will enhance the conversion of Kyn to KynA and consequently reduce circulating Kyn levels. In contrast to KynA, Kyn, which can be further converted to the neurotoxic metabolite Quinolinic acid (QA), can cross the blood brain barrier and accumulation of Kyn in the brain has been associated with depression. Thus, the positive effects of exercise in ameliorating depression have been attributed, at least partly, to the conversion of Kyn to KynA.Materials and MethodsAnimal rearing

[0125] 10 weeks old B6CBAF1 / J (Fl) mice were randomized into three groups and were fed either a control diet of standard chow (regular diet, or RD), high fat diet (HFD) or Compound A- HFD, respectively, for 9 weeks. Body weight and food intake was monitored weekly. Body composition was assessed by EchoMRI, and 6 hours fasted blood glucose and insulin concentrations were determined at start and at 9 weeks of diet. After 9 weeks of diet, mice were sacrificed and total plasma, liver and muscles (soleus and vastus) were isolated for further analyses.

[0126] Animals were housed at 12: 12 hour light / dark cycle in a temperature / humidity controlled (22°C / 50%humidity) room and ad libitum feeding with either standard chow (regular diet), high fat diet (HFD) () or HFD () custom formulated with Compound A at 0.8mg / g Compound A.Targeted amino acid analysis

[0127] Amino acid standards (alanine, arginine, aspartic acid, cysteine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tyrosine, valine, glutamine, asparagine, GABA, citrulline, ornithine, taurine, tryptophan, 5-HTP, kynurenine and norvaline) were obtained. Isotopically labeled amino acid standards (alanine (13Cs,15N), arginine (13Ce,15N4), aspartic acid (13C4,15N), cystine (13Ce,15N2), glutamic acid (13Cs,15N), glycine (13C2,15N), histidine (13Ce,15Ns), isoleucine (13Ce,15N), leucine (13Ce,15N), lysine (13Ce,15N2), methionine (13Cs,15N), phenylalanine (13C9,15N), proline (13Cs,15N), serine (13Cs,15N), threonine (13C4,15N), tyrosine (13C9,15N), valine (13Cs,15N), Citrulline (d4), GABA (13C4), glutamine (13Cs), asparagine(13C4), ornithine (d6), tryptophan (d8), kynurenine (d4) ) were obtained. Stock solutions of each compound were prepared at a concentration of 500 ng / pL and stored at -80°C. A 10-point calibration curve (0.01-100 pmol / pL) was prepared by serial dilutions. lOpL of each calibration point was evaporated to dryness. Mass spectrometry -grade formic acid and HPLC-grade acetonitrile were obtained.

[0128] Amino acids were extracted from plasma, liver, and skeletal muscle, via 90: 10 (v / v) Methanol: water solution containing norvaline at 2.2 pmol / pL as an internal standard. Each sample was extracted for 2 minutes using a mixer mill, incubated in the freezer, and centrifuged at 4°C for 10 min. 20pL of the supernatant was transferred.

[0129] Extracted samples were derivatized by diluting the 20pL of supernatant with 60 pL of a borate buffer spiked with all isotopically labelled internal standards at a final concentration of 0.833 pmol / pL per sample. Finally, 20 pL of freshly prepared derivatization solution was added and the samples were immediately vortexed. The dried calibration curves were prepared in a similar way using the same spiked Ultra borate buffer. Samples were kept at room temperature for 30 minutes followed by 10 minutes at 55°C. For each batch quality control samples and procedure, blanks were included.

[0130] Amino acids were quantified by liquid chromatography-electroionization spraytandem mass spectrometry (LC-ESI-MSMS). Derivatized samples were analyzed using an ultra- high performance liquid chromatography pump, which consists of a binary pump, thermostated column compartment, and autosampler coupled to atriple quadrupole mass spectrometer (QqQ) equipped with a jet stream electrospray source operating in positive ion mode. Jet-stream gas temperature was 290°C with a gas flow of 11 L / min, sheath gas temperature 325°C, sheath gas flow of 12 L / min. The nebulizer pressure was set to 20 psi and the capillary voltage was set at 4 kV. The QqQ was run in a dynamic multiple reaction monitoring (MRM) mode with 2 min retention time windows and 500 msec cycle scans, allowing the mass spectrometry to quantify analytes as well as identify analytes.

[0131] Separation was achieved by injecting 1 pL of each sample onto an ethylene bridged hybrid C is 2.1x100 mm, 1.7 gm column held at 50 C in a column oven. The gradient eluents used were H2O 0.1% formic acid (A) and acetonitrile 0.1% formic acid (B) with a flow rate of 500 pL / min. The initial conditions consisted of 0% B, and the following gradient was used with linear increments: 0.54-3.50 minutes (0.1-9.1% B), 3.50-7.0 (9.1-17.0% B), 7.0-8.0 (17.0- 19.70% B), 8.0-8.5 (19.7% B), 8.5-9.0 (19.7-21.2% B), 9.0-10.0 (21.2-59.6% B), 10.0-11.0 (59.6-95.0% B), 11.0-11.5 (95.0% B), 11.5-15.0 (0% B). From 13.0 minutes to 14.8 minutes the flow rate was set at 800 pL / min for a faster equilibration of the column. The MS parameters were optimized for each compound. MRM transitions for the derivatized amino acids were optimized using optimization software. The fragmentor voltage was set at 380 V, the cell accelerator voltage at 7 V and the collision energies from 14-45V; nitrogen was used as collision gas. The data was quantified using quantitation software, and the amount of each amino acid was calculated based on the calibration curves.

[0132] For each tissue from which the samples were sourced, the average abundance of each amino acid analyte was compared between diet groups. The data was statistically analyzed to establish whether two diet groups had significantly different levels of each amino acid (where p>0.05 for the test statistic’s p-value).Results

[0133] Amino acid levels for kynurenine (Kyn) are depicted in FIG 2. The levels were significantly (p<0.05) higher in the plasma group treated with a HFD and Compound A than the group treated with the HFD alone. This was also true for samples taken from the liver. In samples taken from skeletal muscle, Kyn levels were significantly higher in the group treated with a HFD and Compound A than in each of the other two diet groups (with p<0.01 when compared to the RD group and p<0.001 when compared to the HFD group).

[0134] Amino acid levels for kynurenic acid (KynA) are depicted in FIG 3. The levels were significantly higher in the plasma samples from the group treated with a HFD and Compound A than those treated with a RD (p<0.01) or a HFD (p<0.001) alone. The RD and HFD groups’ KynA amino acid levels were also significantly (p<0.05) different from each other in the plasma samples. For liver samples, KynA levels were also significantly increased in the group treated with a HFD and Compound A than each of the RD group (p<0.01) and the HFD group (p<0.001). In samples taken from the skeletal muscle, KynA levels were significantly (p<0.01) higher in the group treated with a HFD and Compound A then the group treated with a HFD alone.

[0135] Compared ratios for the amino acid levels of kynurenic acid:kyrurenine (KynA:Kyn) are depicted in FIG 4. The ratio of KynA:Kyn was significantly higher in the plasma samples from the group treated with a HFD and Compound A than those treated with a RD (p<0.001) or a HFD (p<0.001) alone. In the liver samples, the KynA:Kyn ratio was significantly higher in the group treated with a HFD and Compound A than in the group treated with a RD (p<0.001) or a HFD (p<0.01) alone. In samples taken from the skeletal muscle, the ratio of KynA:Kyn was significantly different between the group treated with Compound A and a HFD or a HFD alone (p<0.01).

[0136] Additionally, amino acid levels of tryptophan are depicted in FIG. 5. In samples taken from plasma, the tryptophan levels were significantly lower in the group treated with a Compound A and a HFD than in the groups treated with a RD (p<0.001) or a HFD alone (p<0.001).

[0137] Further ratios are depicted in FIG. 6. In samples taken from the plasma, the ratio of KyrnTrp was significantly higher in the group given Compound A and a HFD than in the group given a RD (p<0.001) or a HFD (p<0.001) alone. The ratio of KyrnTrp was also significantly different between the samples of the group given a RD or a HFD (p<0.001).

[0138] In samples taken from the liver, the ratio of KyrnTrp was significantly higher in the group given Compound A and a HFD than in the group given a RD (p<0.01) or a HFD (p<0.001) alone. The ratio of KyrnTrp was also significantly different between the samples of the group given a RD or a HFD (p<0.05).

[0139] In samples taken from the skeletal muscle, the ratio of KyrnTrp was significantly higher in the group given Compound A and a HFD than in the group given a RD (p<0.001) or a HFD (p<0.001) alone.Conclusions

[0140] Compound A has been found to act as an exercise mimetic. Metabolic profiling of a wide variety of isolated metabolic tissues (islets, liver, muscle) and plasma of high fat diet fed (HFD) and diabetic mice was initiated. Preliminary data from analyses of plasma of mice fed a HFD + / - Compound A for 9 weeks identified metabolites of the tryptophan-kynurenin (Trp-Kyn) pathway to be substantially affected by Compound treatment. Notably, a significant increase in the plasma KynA / Kyn ratio was observed in mice on HFD formulated with Compound A (0.8mg / g) as compared with that of mice on both regular diet (RD) and HFD (FIG. 7A). PGCla expression is induced in skeletal muscle following exercise and gain- and loss-of-function expression of PGCla expression in muscle of mice show that PGCla positively regulates expression of KATs in skeletal muscle, conversion of Kyn to KynA, and resilience to stress6. Compound A increases the expression of PGCla in skeletal myotubes (FIG. 7B) and in muscle of diabetic mice (data not shown). In a separate cohort of mice have started to analyze skeletalmuscle expression of KATs and preliminary data show an increase in the expression of Katl and 3 in skeletal muscle of mice on HFD+ Compound A as compared with that of HFD and CD fed mice (FIG. 7C). Also initiated were sucrose preference tests, which assesses anhedonia, which is a core symptom of depression. These tests show 1) that mice on HFD for 9w show signs of depression compared with RD fed mice as evidence by the reduced preference for sucrose water, and 2) that Compound A ameliorates depression like behavior in mice on HFD (FIG. 7D). Together these findings suggest that Compound A functions as an exercise mimetic by enhancing the conversion of Kyn to KynA and averting HFD induced depression.Example 2: Analyzing biomarker effect on depression

[0141] This example studied the association between KynA or Kyn and known indicators of depression, such as anhedonia and certain nest-building behaviors, while accounting for other influences on depression, such as serotonin and PGCla. FIG. 9 presents a schematic outline of the experiment described in this example.

[0142] Diet regimens: 8-10 weeks old mice were put on RD, HFD, and HFD+Compound A, the latter at a concentration (0.8mg / g) that prevented increase in glucose and insulin levels but did not negate weight gain. The mice were exposed for the respective diets for 9 and 18w, respectively, where the 18w cohort included switching mice from HFD to HFD+Compound A, and vice versa, after 9 weeks (FIG. 8). Isolation of tissues / plasma and behavioural tests were performed at both 9 and 18w.

[0143] Behavioural tests: at 9 and 18 weeks of diet, low stress tests were performed, such as the sucrose preference test that measures anhedonic behaviour (a key sign of depression), and the nest building test that monitors general well-being and can be used to assess depression. Anxiety was assessed by open field and novel cage tests.

[0144] Metabolomics were performed on the following tissues: muscle, liver, brain, and plasma isolated from naive mice that had not previously been exposed to the sucrose preference test. The metabolic analyses included analyses of Kyn pathway metabolites including Trp, Kyn, and KynA, and QA. Since Trp is also the precursor of serotonin and low serotonin levels in the brain is associated with depression and anxiety, serotonin brain levels were also analysed.

[0145] In addition to metabolomics, mRNA levels of the rate limiting enzymes; IDO 1 and 2, TDO 1 and 2, KMO and KAT 1-4, in the Trp-Kyn pathway (FIG. 1) were assessed by qRTPCR on cDNA prepared from liver, muscle, and hippocampus. In addition, markers of neuroinflammation associated with depression, e.g. Mcpl Ccl2, rfc Il-lb zx^Il-6, were analysed by qRTPCR on cDNA prepared from hippocampus.

[0146] The role for Pgcla in mediating the Compound A-mediated effects on muscle KAT expression, rise in KynA levels, and amelioration of depression were assessed in mice with conditional inactivation of Pgcla in muscle. Mice with loxP sites flanking Pgcla exons 3-5 are available at JAX mice (strain# 009666) and are crossed with Mck-Cre mice to inactivate Pgcla in muscle.

[0147] Similarly, a role for AMPK in mediating the Compound A effects on muscle KAT expression, rise in KynA levels, and amelioration of depression was assessed in mice with conditional inactivation of the AMPK al / a2 subunits in muscle.

[0148] Methodology

[0149] Sucrose preference test: Mice were individually habituated to the sucrose test chambers with two bottles, one with drinking water and one with a 1% sucrose solution, overnight on 3 consecutive days occasions. At the day of the test the mice were deprived of food and water overnight and then individually placed in the sucrose chambers for 24 hrs. Nest building test', mice were individually housed in cages containing bedding and one square of nesting material, e.g. pressed cotton. The following morning the manipulation of the nestlet and the quality of the built nest are assessed manually according to a five-point scale.

[0150] Novel cage test: Mice were individually placed in a novel cage and filmed for 5 min. Locomotor activity (moving horizontally, rearing etc) was analysed.

[0151] Open field test: Mice were habituated to the test room for 1 hour on 5 consecutive days (in their conventional cages). The day before the test mice were transferred together from the cage to the test box for 10 min. For the test mice were individually placed in the test box and filmed for 10 min. Movements were tracked and analysed.

[0152] Metabolomics: Plasma and tissues samples were prepared and subjected to both GC- MS metabolomics, LC-MS metabolomics, and lipidomics analyses. In addition to mass spectrometry -based metabolomics, tryptophan metabolites were quantified via targeted LC-QqQ- MS analysis.

[0153] Results: The results from this example are provided in FIGS. 10A-13C.

[0154] Observations & Conclusions: Compound A was observed to avert and revert plasma levels of tryptophan by promoting kynurenic acid production. Liver and muscle expression of key regulatory enzymes of kynurenine and kynurenic acid synthesis were increased in Compound-A-treated mice compared to HFD-treated mice. HFD-induced obesity results in hyperglycemia, hyperinsulinemia, and insulin resistance, which was observed to be averted and reverted by Compound A. HFD-treated mice had reduced sucrose preference (anhedonic), spent less time in center zone (anxious), and had impaired bedding behavior, whereas Compound-A- treated mice mitigated obesity associated depressive behavior.

Claims

CLAIMSWhat is claimed is:

1. A method of identifying a subject who may benefit from a treatment comprising 4- chloro-N-[2-[(4-chlorophenyl)methyl]-3-oxo-l,2,4-thiadiazol-5- yl]benzamide, or a salt thereof, or a prodrug thereof, or identifying a subject having increased PGC-lalpha activity and / or increased expression of KAT enzymes in response to the treatment, the method comprising: determining (i) a level of at least one biomarker, wherein the at least one biomarker is selected from tryptophan and metabolites thereof, and / or (ii) a ratio of two or more of the foregoing biomarkers, in a sample obtained from the subject, wherein (i) the level of the at least one biomarker, and / or (ii) the ratio of two of more of the foregoing biomarkers as compared to a reference level / ratio indicates that the subject may benefit from the treatment, optionally, providing a recommendation that the subject will be more likely to respond to the treatment.

2. The method of claim 1, wherein the method comprises determining at least one of the following:(a) the level of tryptophan;(b) the level of KynA;(c) the ratio of Kyn to tryptophan;(d) the ratio of KynA to Kyn;(e) the ratio of KynA to tryptophan;(f) the ratio of Quinolinic acid to KynA; and / or(g) the ratio of Quinolinic acid to Kyn.

3. The method of claim 1 or claim 2, wherein the sample comprises a plasma sample.

4. The method of any one of claims 1-3, further comprising providing a recommendation that the subject will be more likely to respond to the treatment when at least one of the following occurs:(i) the plasma level of tryptophan is above 70 pmol / L;(ii) the molar ratio of KynA to Kyn in plasma is below 0.3;(iii) the molar ratio of KynA to tryptophan in plasma is below 0.01;(iv) the molar ratio of Quinolinic acid to KynA in plasma is above 5; and / or(v) the molar ratio of Quinolinic acid to Kyn in plasma below 0.1.

5. A method of determining the dosing or the dosing regimen of 4-chloro-N-[2-[(4- chlorophenyl)methyl]-3-oxo-l,2,4-thiadiazol-5- yl]benzamide, or a salt thereof, or a prodrug thereof, for treatment of a disease, disorder, or condition in a subject in need thereof, the method comprising: identifying a minimally effective dose and a maximally effective dose of 4-chloro-N-[2- [(4-chlorophenyl)methyl]-3-oxo-l,2,4-thiadiazol-5- yl]benzamide, or a salt thereof, or a prodrug thereof, or the dose regimen of 4-chloro-N-[2-[(4-chlorophenyl)methyl]-3-oxo-l,2,4-thiadiazol- 5- yl]benzamide, or a salt thereof, or a prodrug thereof, based on comparing (i) a level of the at least one biomarker, and / or (ii) a ratio of two or more of the foregoing biomarkers, wherein the at least one biomarker is selected from tryptophan and metabolites thereof, in a sample of the subject after or during treatment with a dose or dosing regimen of 4-chloro-N-[2-[(4- chlorophenyl)methyl]-3-oxo-l,2,4-thiadiazol-5- yl]benzamide, or a salt thereof, or a prodrug thereof, to (i) the level of the at least one biomarker, and / or (ii) the ratio of two or more of the foregoing biomarkers in a sample obtained from the subject before the treatment, wherein a change in (i) the level of the at least one biomarker, and / or (ii) the ratio of two or more of the foregoing biomarkers after or during the treatment as compared to before the treatment is indicative of the efficacy of or responsiveness to the dose or dosing regimen of 4-chloro-N-[2- [(4-chlorophenyl)methyl]-3-oxo-l,2,4-thiadiazol-5- yl]benzamide, or a salt thereof, or a prodrug thereof, for treatment of the subject.

6. The method of claim 5, wherein: the minimally effective dose is the lowest dose of 4-chloro-N-[2-[(4- chlorophenyl)methyl]-3-oxo-l,2,4-thiadiazol-5- yl]benzamide, or a salt thereof, or a prodrug thereof, that gives a positive change in the aforementioned biomarkers, and ratios thereof; and the maximally effective dose is the dose of 4-chloro-N-[2-[(4-chlorophenyl)methyl]-3- oxo-l,2,4-thiadiazol-5- yl]benzamide, or a salt thereof, or a prodrug thereof, that normalizes the aforementioned biomarker levels and ratios thereof.

7. The method of claim 5 or claim 6, wherein the level of the at least one biomarker, and / or (ii) the ratio of two or more of the foregoing biomarkers comprises at least one of the following:(a) the level of tryptophan;(b) the level of KynA;(c) the ratio of Kyn to tryptophan;(d) the ratio of KynA to Kyn;(e) the ratio of KynA to tryptophan;(f) the ratio of Quinolinic acid to KynA; and / or(g) the ratio of Quinolinic acid to Kyn.

8. The method of any one of claims 5-7, wherein the sample comprises a plasma sample.

9. The method of any one of claims 5-8, wherein before the treatment, the subject has at least one of the following:(i) the plasma level of tryptophan is above 70 pmol / L;(ii) the molar ratio of KynA to Kyn in plasma is below 0.3;(iii) the molar ratio of KynA to tryptophan in plasma is below 0.01;(iv) the molar ratio of Quinolinic acid to KynA in plasma is above 5; and / or(v) the molar ratio of Quinolinic acid to Kyn in plasma below 0.1.

10. The method of claim 6, wherein the positive change in the aforementioned biomarkers, and ratios thereof, comprises at least one of the following:(i) the plasma level of tryptophan after the administration of the treatment is decreased by at least 5 pmol / mL compared to the baseline before the treatment;(ii) the increase in plasma level of Kyn A after the treatment compared to the baseline before the treatment is at least 0.01 pmol / mL;(iii) the plasma level of KynA after the treatment is at least doubled compared to the baseline before the treatment;(iv) the molar ratio of Kyn to tryptophan in plasma is increased;(v) the increase in the molar ratio of KynA to tryptophan in plasma after the treatment compared to the baseline before the treatment is at least 0.01;(vi) the molar ratio of KynA to tryptophan in plasma is increased;(vii) the plasma level of KynA is increased and the plasma level of Tryptophan is decreased;(viii) the decrease in the molar ratio of Quinolinic acid (QA) to KynA in plasma after the treatment compared to the baseline before the treatment is at least 0.5; and / or(ix) the increase in the molar ratio of Quinolinic acid (QA) to Kyn in plasma after the treatment compared to the baseline before the treatment is at least 0.01.

11. The method of any one of claims 5-10, wherein the efficacy of or responsiveness to the dose or dosing regimen of 4-chloro-N-[2-[(4-chlorophenyl)methyl]-3-oxo-l,2,4-thiadiazol-5- yl]benzamide, or a salt thereof, or a prodrug thereof, is determined when the plasma level of 4- chloro-N-[2-[(4-chlorophenyl)methyl]-3-oxo-l,2,4-thiadiazol-5- yl]benzamide reaches a steady state.

12. The method of any one of claims 5-11, wherein the efficacy of or responsiveness to the dose or dosing regimen of 4-chloro-N-[2-[(4-chlorophenyl)methyl]-3-oxo-l,2,4-thiadiazol-5- yl]benzamide, or a salt thereof, or a prodrug thereof, is determined at least two weeks after the treatment.

13. A method of treating a disease, disorder, or condition ameliorated by the activation of AMPK in a subject in need thereof, comprising:(a) administering to the subject 4-chloro-N-[2-[(4-chlorophenyl)methyl]-3-oxo-l,2,4- thiadiazol-5- yl]benzamide, or a salt thereof, or a prodrug thereof;(b) monitoring the subject’s plasma levels of tryptophan after step (a); and(c) administering to the subject a tryptophan supplement if the subject’s plasma tryptophan levels are below 25 pmol / L.

14. The method of claim 13, wherein the tryptophan supplement comprises an NAD+ precursor.

15. The method of claim 13, wherein the tryptophan supplement comprises nicotinamide riboside.

16. The method of any one of the preceding claims, wherein the 4-chloro-N-[2-[(4- chlorophenyl)methyl]-3-oxo-l,2,4-thiadiazol-5- yl]benzamide, or a salt thereof, or a prodrug thereof, is a sodium salt of 4-chloro-N-[2-[(4-chlorophenyl)methyl]-3-oxo-l,2,4-thiadiazol-5- yl]benzamide.

17. The method of any one of claims 5-16, wherein the disease, disorder, or condition is associated with and / or affected by reduced AMPK and Pgcla signaling.

18. A method of treating depression in a subject in need thereof, comprising administering to the subject 4-chloro-N-[2-[(4-chlorophenyl)methyl]-3-oxo-l,2,4-thiadiazol-5- yl]benzamide, or a salt thereof, or a prodrug thereof.

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