Apelin receptor agonists for treatment of muscle disorders

Abstract

The apelin receptor modulator can improve the physical performance of a human patient, slow the progression of age-related weakness, and can reduce age-related muscle weakness. The present disclosure provides methods of treating muscle disorders using specific classes of apelin receptor modulators (e.g., agonists). The muscle condition may be an age-related muscle condition. Also provided is a method of maintaining and / or increasing muscle mass, muscle function, and / or muscle strength in an elderly subject by administering an apelin receptor modulator. In some embodiments, an apelin receptor modulator (e.g., an agonist) is BGE-105 or a pharmaceutically acceptable salt thereof.

Description

[0001] 1. Cross - reference to related applications

[0002] This application claims the benefit of the following U.S. Provisional Applications: No. 63 / 410,576, filed September 27, 2022; No. 63 / 413,436, filed October 5, 2022; No. 63 / 428,405, filed November 28, 2022; No. 63 / 429,946, filed December 2, 2022; No. 63 / 478,291, filed January 3, 2023; No. 63 / 478,302, filed January 3, 2023; No. 63 / 478,474, filed January 4, 2023; No. 63 / 489,935, filed March 13, 2023; No. 63 / 493,987, filed April 3, 2023; No. 63 / 512,888, filed July 10, 2023; No. 63 / 517,584, filed August 3, 2023; No. 63 / 520,333, filed August 17, 2023, each of which is hereby incorporated by reference in its entirety. 2. Background of the Invention

[0003] As a person ages, the human body accumulates physiological and pathophysiological changes; these cumulative age - related changes make a person vulnerable to various external and internal stress factors that can lead to death. Frailty is highly prevalent in the elderly population and is considered synonymous with disability, disease, and other high - risk characteristics that lead to falls, disability, nursing home admission, hospitalization, and death. Frailty is regarded as a clinical syndrome that can be characterized by frailty indices, which are composite measures of such age - related changes. With the increasing median age of the population, there is a growing need for drugs that can reduce or counteract the accumulation of age - related deficits (including frailty) in elderly individuals. 3. Summary of the Invention

[0004] The present disclosure provides methods of treating muscle disorders, particularly a variety of age - related muscle disorders, using specific classes of apelin receptor modulators. In some embodiments, the apelin receptor modulator is an apelin receptor agonist.

[0005] We applied bioinformatics and machine - learning methods to analyze human data using a survival prediction model and discovered an association between apelin protein levels and future aging outcomes. We found that higher circulating levels of apelin were associated with a reduced all - cause mortality, i.e., a longer lifespan (p = 0.0002). In addition, our analysis showed that higher apelin levels were associated with better future physical performance and frailty indices.

[0006] Based on this discovery, we tested the effect of the apelin receptor modulator BGE-105 on aged mice in a frailty model. The structure of BGE-105 is shown below:

[0007]

[0008] BGE-105 (also known as AMG-986) is known to activate the apelin receptor and induce cardiovascular responses in rats (Ason et al., JCI Insight. 5(8):1-16(2020)). Clinical trials have been conducted on AMG-986 to study its safety, tolerability, and pharmacokinetics in healthy subjects and subjects with heart failure (NCT03276728) who have impaired renal function (NCT03318809). However, the effect of this compound on muscle loss and function in the elderly is unclear.

[0009] In the first set of experiments, we demonstrated that aged mice (24 months old) treated with BGE-105 showed a statistically significant increase in spontaneous motor activity (p = 0.00228) and a statistically significant improvement in grip strength (p = 0.04) compared to age-matched controls, indicating improved physical health and enhanced muscle strength.

[0010] In addition, aged mice (18 months old) were first injected with cardiotoxin and then treated with BGE-105, showing a significant increase in the levels of multiple mRNA transcripts that are markers of muscle regeneration.

[0011] Third, immortalized muscle progenitor cells from human patients showed dose-dependent cell growth and differentiation in relation to BGE-105 concentration.

[0012] Furthermore, orally administered BGE-105 significantly reduced muscle atrophy in aged, activity-restricted mice (20 months old) compared to activity-restricted mice injected with the vehicle.

[0013] A Phase I clinical study was conducted, showing that BGE-105 can prevent or reduce muscle atrophy in bedridden human patients. A wide range of muscle dynamics, patient serum biomarkers, and proteomic analyses were extensively evaluated, and the results were consistent with the efficacy demonstrated in the clinical study.

[0014] These analyses further support the use of apelin receptor modulators (such as BGE-105) to treat or prevent various key or chronic muscle disorders, including but not limited to diaphragmatic atrophy, critical care myopathy, frailty, COPD-related muscle dysfunction, sarcopenia, or other muscle disorders as described herein.

[0015] Thus, apelin receptor modulators (such as BGE-105) can improve physical performance, counteract age-related frailty, and can alleviate age-related muscle weakness.

[0016] Accordingly, a first aspect of the present disclosure provides a method of treating or preventing a muscle disorder in a subject, the method comprising administering to a subject in need thereof an effective dose of an apelin receptor modulator. In some aspects of the invention, the modulator is an apelin receptor agonist, such as an apelin receptor agonist of formula (I) or (II) as described herein. In some embodiments, the muscle disorder is an age-related muscle disorder. In some embodiments, the apelin receptor agonist is BGE-105 or a pharmaceutically acceptable salt thereof.

[0017] Another aspect of the present disclosure provides a method of maintaining and / or increasing muscle mass, muscle function, and / or muscle strength in a subject. In some embodiments, the subject is an elderly human. The method may comprise administering to a subject in need thereof an effective dose of an apelin receptor agonist, such as an apelin receptor agonist of formula (I) or (II) as described herein. In some embodiments, the apelin receptor agonist is BGE-105 or a pharmaceutically acceptable salt thereof.

[0018] In some embodiments of the methods of the present disclosure, the subject is a human and has or is determined to have one or more of the following: low muscle strength, low muscle force, low muscle mass, low muscle volume. In some embodiments, the muscle is skeletal muscle. In some embodiments, the muscle is the diaphragm, tibialis anterior, tibialis posterior, gastrocnemius, sartorius, vastus intermedius, vastus lateralis, vastus medialis, soleus, rectus femoris, or extensor digitorum longus.

[0019] In some embodiments of the methods of the present disclosure, the subject is a human and has or is determined to have one or more of the following diseases: diabetes, insulin insensitivity or resistance, cardiovascular disease, neurological disease, and chronic obstructive pulmonary disease (COPD).

[0020] In some embodiments of the methods of the present disclosure, the subject is a human and has low muscle strength, low muscle force, low muscle mass, and / or low muscle volume due to disuse atrophy after restricted activity.

[0021] In some embodiments of the methods of the present disclosure, the subject is a human and has diaphragmatic dysfunction and / or diaphragmatic atrophy. In some embodiments of the methods of the present disclosure, the subject is a human and has critical illness myopathy. 4. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] These and other features, aspects, and advantages of the present invention will be better understood from the following description and the accompanying drawings, wherein:

[0023] Figure 1 Shows the structure of BGE-105.

[0024] FIG. 2A to FIG. 2D Graphically represents the results of a bioinformatics survival model that studied the relationship between a given protein serum level and the risk of future all-cause mortality (i.e., lifespan) or the ability to maintain full activity in a cohort of healthy aging humans, using non-public clinical outcome data and proteomic data generated from archived samples. Figure 2A Shows the Kaplan-Meier curves of the survival probabilities of the top 20% (upper 20%) and bottom 20% (lower 20%) groups of human apelin protein levels, indicating that higher circulating levels of apelin in humans are associated with a reduced risk of all-cause mortality (p = 0.0002). Figure 2B Shows a model of similar protein levels and full activity, where higher circulating levels of apelin are associated with an increased retention of full activity (p = 0.0082). The hazard ratio of apelin is Figure 2A 0.88 in Figure 2B and 0.89 in Figure 2A and Figure 2B In both cases, the hazard ratios given are based on a continuous Cox proportional hazards analysis that fits the entire distribution of apelin measurements. Figure 2C Shows the serum abundance of the apelin protein module in the Honolulu Heart Study (HHS) cohort (highlighted by the oval). Each node represents a protein, and the edges between the nodes represent significant correlations. Figure 2D Shows the relationship between the first principal component of the apelin protein module and mortality. The relative mortality (logarithmic; y-axis) is obtained from a multivariate Cox regression model of the first principal component (PC1) after adjusting for age, pack-years of smoking, and alcohol status. The reference value used is the median of PC1.

[0025] FIG. 3A to FIG. 3I Shows the effects of BGE-105 on the activity and muscle strength of 24-month-old C57BL / 6 mice. Figure 3A and Figure 3D Shows the results of repeated experiments on C57BL / 6 mice on an activity wheel in the cage, with the readings in km / day. In Figure 3A and Figure 3DAmong them, solid dots and related connecting lines represent mice treated with BGE-105, while hollow dots and related connecting lines represent mice not treated with BGE-105. Separately, the Kendall rank correlation coefficient tau p = 0.00228 and 1.14e-04. In Figure 3B and Figure 3C , hollow dots represent mice treated with BGE-105, and solid dots represent mice treated with only the vehicle. The results show that on average, the fall latency (an indicator of increased muscle strength) of mice treated with BGE-105 is increased compared to mice treated with the vehicle in the grid hang test. At the end of the study, the wet tissue weight of the tibialis anterior muscle (TA) of mice in the BGE-105 treatment group was significantly higher ( Figure 3F ), and the wet tissue weights of the gastrocnemius and quadriceps muscles showed an upward trend ( Figure 3G and Figure 3H ), with no difference in the heart ( Fig. 3I ). The body weight of mice in the BGE-105 treatment group was also higher ( Figure 3E ), but the increase was not significant, only at the p-value cutoff.

[0026] FIG. 4A to FIG. 4D Depicts that the levels of pAMPK ( FIG. 4A to FIG. 4B ) and pAkt ( FIG. 4C to FIG. 4D ) are elevated in BGE-105-treated mice compared to vehicle-treated mice.

[0027] FIG. 4E to FIG. 4F Depicts that the soleus muscle contains approximately half as many APLNR receptors per unit mass as the heart, which potentially explains the stronger response in heart tissue.

[0028] FIG. 5A to FIG. 5B Depicts the levels of apelin receptor protein found in rat tissues. FIG. 5C to FIG. 5D Depicts that oral administration of BGE-105 to rats for 5 consecutive days induces phosphorylation of Akt in the TA in a dose-dependent manner, with the strongest response elicited by 50 mg / kg BID. FIG. 5E to FIG. 5F Depicts the same result for Erk. Figure 5G to Figure 5I Depicts the effect of chronic administration of BGE-105 on the level of apelin receptor protein in the TA.

[0029] FIG. 6A to FIG. 6B Shows that in cells stably expressing the human apelin receptor, the potency of BGE-105 is 10-fold higher than that of Pyr 1 -apelin-13; in cells stably expressing the mouse apelin receptor, the potency of BGE-105 is 30-fold higher than that of Pyr 1 -apelin-13.

[0030] 7A to 7FShows the effect of administering PBS, Pyr 1 -Apelin-13 (apelin) (0.5 μmol / kg / day), BA1 (BGE-105 50 mg / kg / day), or BA2 (BGE-105 200 mg / kg / day) on the transcript levels in the tibialis anterior muscle of aged (18-month-old) mice at 3 or 7 days after injection of cardiotoxin.

[0031] Figure 7G to Figure 7L Shows the effect of administering PBS, Pyr 1 -Apelin-13 (apelin) (0.5 μmol / kg / day), BA1 (BGE-105 50 mg / kg / day), or BA2 (BGE-105 200 mg / kg / day) on the transcript levels in the gastrocnemius muscle of aged (18-month-old) mice at 3 or 7 days after injection of cardiotoxin.

[0032] Figure 7M to Figure 7R Shows the effect of administering PBS, Pyr 1 -Apelin-13 (apelin) (0.5 μmol / kg / day), BA1 (BGE-105 50 mg / kg / day), or BA2 (BGE-105 200 mg / kg / day) on the transcript levels in the tibialis muscle of young (3-month-old) mice at 3 or 7 days after injection of cardiotoxin.

[0033] Figures 7S to 7X Shows the effect of administering PBS, Pyr 1 -Apelin-13 (apelin) (0.5 μmol / kg / day), BA1 (BGE-105 50 mg / kg / day), or BA2 (BGE-105 200 mg / kg / day) on the transcript levels in the gastrocnemius muscle of young (3-month-old) mice at 3 or 7 days after injection of cardiotoxin.

[0034] Figure 7Y to Figure 7Z Shows the cross-sectional area of the tibialis muscle after receiving PBS, Pyr 1 -Apelin-13 (apelin), BA1 (BGE-105 50 mg / kg / d), and BA2 (BGE-105 200 mg / kg / d) treatment at 3 and 7 days after injection of cardiotoxin.

[0035] Figures 7AA to 7BB Shows for PBS, Pyr 1- The amount of centrally nucleated fibers (CNM) as part of the regeneration process after cardiotoxin injection in treatments with apelin-13 (apelin), BA1 (BGE-105 50 mg / kg / day), and BA2 (BGE-105 200 mg / kg / day). The mice were 18 months old. Figure 7AA Representative distribution showing DAPI-stained nuclei and eMHC-positive stained fibers. Figure 7BB Quantification showing the amount of centrally nucleated fibers (CNM).

[0036] FIG. 8A to FIG. 8C Showing the ability of BGE-105 to increase the proliferation of immortalized human muscle cells from young (25-year-old) and old (79-year-old) subjects.

[0037] Figures 8D to 8K Showing the expression levels of PAX7, MYf5, MyoD, and MyoG in immortalized muscle cells from old (79-year-old) and young (25-year-old) subjects after incubation with DMSO (0.1%), Pyr 1 - Apelin-13 (apelin / Ape) (1 nM) or BGE-105 (BA) at 0.05, 0.5, 5, or 50 nM. Fig.8D Showing the level of PAX7 in young cells. Fig. 8E Showing the level of MYf5 in young cells after treatment. Figure 8F Showing the expression level of MyoD in young cells after treatment. Figure 8G Showing the expression level of MyoG in young cells after treatment. Figure 8H Showing the level of PAX7 in old cells after treatment. Figure 8I Showing the expression level of Myf5 in cells from old donors after treatment. Figure 8J Showing the level of MyoD in cells from old donors after treatment. Figure 8K Showing the level of MyoG in old cells after treatment.

[0038] FIG. 9A to FIG. 9M Showing the role of BGE-105 in preventing disuse-induced muscle atrophy in old mice.

[0039] Fig.10 Providing an overview of the administration of BGE-105 in a Phase I clinical study in Example 9, including single ascending dose (SAD) cohorts 1-3 or multiple dose (MD) cohorts 1A-1C.

[0040] Fig.11Provide an overview of the screening and pre-treatment of the SAD cohorts in Part A of Example 9. Abbreviations: AE = adverse event; ECG = electrocardiogram; HbsAg = hepatitis B surface antigen; HCV = hepatitis C virus; HIV = human immunodeficiency virus; PD = pharmacodynamics; SAD = single ascending dose; TBD = to be determined.

[0041] Fig.12 Provide an overview of the treatment and follow-up of the SAD cohorts in Part A of Example 9. Abbreviations: AE = adverse event; ECG = electrocardiogram; EOS = end of study; EOT = end of treatment; FU = follow-up; IP = investigational product; IV = intravenous; LD = loading dose; MD = maintenance dose; PD = pharmacodynamics; PK = pharmacokinetics; SAD = single ascending dose; TBD = to be determined.

[0042] Fig.13 Provide an overview of the screening and pre-treatment of the MD cohorts in Part B of Example 9. Abbreviations: AP = before-after; BMR = basal metabolic rate; D3-Cr = deuterated creatine; ECG = electrocardiogram; FSR = fractional synthesis rate; HbsAg = hepatitis B surface antigen; HCV = hepatitis C virus; HIV = human immunodeficiency virus; PD = pharmacodynamics; TBD = to be determined.

[0043] Fig.14 Provide an overview of the treatment and follow-up of the MD cohorts in Part B of Example 9. Abbreviations: AP = before-after; BMR = basal metabolic rate; D3-Cr = deuterated creatine; ECG = electrocardiogram; FSR = fractional synthesis rate; HbsAg = hepatitis B surface antigen; HCV = hepatitis C virus; HIV = human immunodeficiency virus; PD = pharmacodynamics; TBD = to be determined.

[0044] Fig.15 Show preliminary PK data from 3 SAD cohorts indicating dose proportionality. Cmax remained within the expected range, and for the highest dose (240 mg / 1440 mg), AUC last was 1062 μg*hr / mL.

[0045] Fig.16 Show the mean percent change in HOMA-IR relative to baseline.

[0046] Fig.17Show the expression levels of the murine apelin receptor (APJ / APLNR) in the heart, diaphragm, tibialis anterior muscle (TA), and brain tissues of adult mice. As shown, APLNR is highly expressed in the diaphragm, consistent with typical apelin target tissues (skeletal and cardiac muscle). Tissues were taken from 7-month-old mice and homogenized. Protein blotting was performed using 10 μg of protein, and APJ (Invitrogen #5H5L9) and loading control GAPDH (Abcam #EPR1689) were probed. Bands were quantified using a BioRad ChemiDoc XRS+ molecular imager. The APJ signal was normalized to GAPDH.

[0047] FIG. 18A to FIG. 18B Show the effects of BGE-105 on the reduction of rest-induced thigh circumference, vastus lateralis muscle diameter % (thickness), vastus lateralis muscle cross-sectional area (CSA) %, muscle degradation, and cumulative protein synthesis rate % (measured by biopsy). Fig.18B Show the thigh circumference of patients during (middle subfigure) or after (left subfigure) 10 days of bed rest who received placebo (cohort 1A of the MD study of Example 9) and BGE-105 treatment (cohort 1B of the MD study of Example 9), expressed as a percentage change relative to baseline. p = 0.0004.

[0048] Fig.19 Show the effects of BGE-105 treatment on the muscle diameter and cross-sectional area of the vastus lateralis muscle measured by ultrasound in patients after 10 days of bed rest (left subfigure). The vastus lateralis muscle diameter (middle subfigure) or vastus lateralis muscle cross-sectional area (left subfigure) of patients treated with BGE-105 or placebo is expressed as the mean percentage change relative to baseline. p = 0.0297.

[0049] FIG. 20A to FIG. 20C Show the fatty degeneration of the vastus lateralis muscle via echo density measured using an ultrasound muscle mass grading scale (subfigures A - B). Fig. 20A Schematic diagram of grade 1 normal muscle (1, open box), Fig. 20B Schematic diagram of grade 2 muscle containing some fat streaks (2, striped box). The figure shows the large muscle mass observed at baseline and after 10 days of bed rest in each of the placebo group and the BGE-105 group ( Fig. 20C )(p = 0.0019).

[0050] Fig.21 Show the muscle protein synthesis rate in the vastus lateralis muscle measured by micro-biopsy in individual subjects of the BGE-105 treatment group and the placebo group. Muscle protein synthesis was measured after 10 days of bed rest, and the fractional synthesis rate was presented standardized by subject. p = 0.0043.

[0051] FIG. 22A to FIG. 22B Illustration of the timeline of the Phase 2 POC trial for ICU diaphragmatic atrophy or critical illness myopathy. Fig. 22B Illustration of the design of the Phase 2 POC trial for mechanically ventilated patients.

[0052] FIG. 23A to FIG. 23B Shows the characteristics of healthy volunteers with 1b-stage bed rest muscle atrophy in the MD study and the incidence of treatment-emergent adverse events (Part B of Example 9). The placebo group is represented as Cohort 1A of MD in the study of Part B of Example 9, and the BGE-105 treatment group is represented as Cohort 1B of MD in the study of Part B.

[0053] Fig.24 Shows the effect of BGE-105 on the rest-induced reduction in thigh circumference of subjects participating in the 1b-stage MD, Part B clinical trial of Example 9. Thigh circumference was measured in patients receiving placebo or BGE-105 treatment and expressed as the percentage change relative to baseline. Subjects receiving BGE-105 maintained or increased their thigh circumference during the 10-day bed rest period. Measurements were taken 15 cm above the midpoint of the patella. P = 0.0004.

[0054] FIG. 25A to FIG. 25B Shows the effect of BGE-105 on the rest-induced reduction in calf circumference of subjects participating in the 1b-stage MD, Part B clinical trial of Example 9. Fig.25A Shows a graph measuring the calf circumference of patients receiving placebo and BGE-105 treatment during or after the 10-day bed rest period and expressed as the percentage change relative to baseline. Calf circumference is expressed as the percentage change from baseline to day 10 (right subgraph). p = 0.0024. Measurements were taken 15 cm above the midpoint of the patella. Fig.25B Shows the percentage change in calf circumference exhibited by different subjects from baseline to day 10.

[0055] Fig.26 Shows the effect of BGE-105 on the gastrocnemius diameter and gastrocnemius cross-sectional area measured by ultrasound after 10 days of bed rest. The gastrocnemius diameter (middle subgraph, p = 0.836) or gastrocnemius cross-sectional area (left subgraph, p = 0.278) of patients treated with BGE-105 or placebo is expressed as the mean percentage change relative to baseline. As shown, compared with the gastrocnemius diameter (decrease of 2.942%) and cross-sectional area (decrease of 7.758%) in the BGE-105 treatment group, the decrease in the gastrocnemius diameter (decrease of 10.982%) and cross-sectional area (decrease of 15.72%) in the placebo group was greater.

[0056] FIG. 27A to FIG. 27BShow the effects of BGE-105 on the measurements of thigh, calf circumference, cross-sectional area of the vastus lateralis muscle, and cross-sectional area of the gastrocnemius muscle in female subjects. Measure the thigh circumference (p = 0.0033), calf circumference (p = 0.0146), cross-sectional area of the vastus lateralis muscle (p = 0.066), and cross-sectional area of the gastrocnemius muscle (p = 0.4381) of subjects receiving placebo or BGE-105, and express them as percentage changes relative to baseline. As shown in the figure, compared with the thigh circumference (decrease of 1.674%) and calf circumference (decrease of 1.166%) in the BGE-105 treatment group, the thigh circumference (decrease of 6.06%) and calf circumference (decrease of 5.486%) in the placebo group decreased to a greater extent ( Fig.27A ). As shown in the figure, compared with the cross-sectional area of the vastus lateralis muscle (decrease of 9.676%) and cross-sectional area of the gastrocnemius muscle (decrease of 5.558%) in the BGE-105 treatment group, the cross-sectional area of the vastus lateralis muscle (decrease of 23.67%) and cross-sectional area of the gastrocnemius muscle (decrease of 15.32%) in the placebo group decreased to a greater extent ( Fig.27B ).

[0057] Fig.28 Show the rate of muscle myofibrillar protein synthesis in the vastus lateralis muscle of patients treated with BGE-105 and patients treated with placebo. Measure the average rate of myofibrillar synthesis in the vastus lateralis muscle and express it as a change relative to baseline (left subfigure). The average myofibrillar FSR / day is expressed as a percentage change relative to baseline (right subfigure). As shown in the figure, the rate of muscle myofibrillar protein synthesis decreased to a greater extent in the placebo group (decrease of 58%) compared with the BGE-105 treatment group (decrease of 36%).

[0058] Fig.29 Illustrate the measurements taken during the 1b-phase bed rest atrophy study to evaluate the various muscle dynamics shown in Example 9.

[0059] Fig.30 Illustrate the description of the enabling assessment techniques used to evaluate muscle dynamics in the 1b-phase bed rest atrophy study of Example 9.

[0060] Fig.31 Illustrate the location and method of ultrasonic measurement of leg muscles. Ultrasonic measurement of the skeletal muscle circumference, cross-sectional area, color flow analysis, anterior-posterior diameter, and echo density of the vastus lateralis muscle and the gastrocnemius muscle.

[0061] Fig.32 Illustrate the incorporation of deuterated water into muscle proteins for calculating the fractional synthesis rate. Deuterium can be measured invasively (via tissue biopsy) and non-invasively (via blood and urine - virtual biopsy).

[0062] Fig.33Microbiopsies of the vastus lateralis muscle were performed at multiple time points (baseline on day 0 of bed rest, day 5 of bed rest, and at the end of the 10-day bed rest period) to measure the fractional synthesis rate of hundreds of muscle proteins. Biopsies were collected at three time points using a microneedle: at baseline, in the middle of the entire treatment period, and at the end of the treatment period.

[0063] Fig.34 Flowchart depicting the assessment of total muscle mass using a D3-creatine tracer during the Phase 1b study of Example 9. Validation data for human MRI muscle volume (r = 0.87, p < 0.01).

[0064] Fig.35 Shows the correlation between skeletal muscle atrophy and reduced muscle protein synthesis in elderly patients (>65 years old). Muscle atrophy is represented as the change in lean leg mass (g) (left subfigure). Muscle fractional synthesis rate was measured before and after the 10-day bed rest period in patients treated with BGE-105 or placebo and is represented as a percentage change (right subfigure).

[0065] Fig.36A Shows the effect of BGE-105 on muscle protein synthesis in vastus lateralis muscle microbiopsies of patients treated with BGE-105 or placebo. Shown is the ratio of protein FSR in the BGE-105 group compared to the placebo group after 10 days of bed rest (middle subfigure). Shown is the comparison of cumulative protein synthesis in patients treated with placebo and BGE-105 after 5 days of bed rest and after 10 days of bed rest (right subfigure). Fig.36B Shows fractional synthesis in BGE-105-treated patients and placebo-treated patients on day 11 of bed rest. As shown, BGE-105 resulted in higher muscle protein synthesis in the vastus lateralis muscle compared to placebo-treated patients, as measured by microbiopsy.

[0066] Fig.37 Shows the step counts ratio of patients wearing a wearable activity device during the period from day 10 to day 60 (after the bed rest period) in the Phase 1b MD B part clinical trial of Example 9.

[0067] FIG. 38A to FIG. 38D Shows the proteomic profiling of serum collected from subjects in the Phase 1b clinical trial of Example 9. Serum levels of 11 treated subjects and 11 placebo subjects were collected on day 1 (baseline), day 5, and day 11 and subjected to proteomic analysis. Fig.38A Shows the changes in the number of proteins related to frailty (functional), walking speed, instrumental activities of daily living (IADL) (functional tools), and grip strength in BGE-105-treated patients in the Phase 1b clinical trial of Example 9. Fig.38BBGE-105 shifts the serum proteome towards a healthier state, demonstrating the benefits of naturally high levels of apelin in subjects treated with BGE-105. Fig.38C Changes in baseline energy expenditure in subjects treated with BGE-105 and placebo-treated subjects are shown using SomaSignal tests on proteomics data. Fig.38D Changes in maximal cardiorespiratory fitness (VO2) and basal metabolic rate in subjects treated with BGE-105 and placebo-treated subjects are shown using SomaSignal tests on proteomics data.

[0068] Fig.39 Provide a clinical overview of a Phase 2 clinical study in patients with ICU diaphragmatic atrophy.

[0069] Figures 40 to 62 Show details and results of snRNAseq analysis of human muscle tissue samples from a Phase 1b clinical study of BGE-105 for the treatment of muscle atrophy.

[0070] Fig.40 Illustrate the workflow of a nucleic acid isolation kit used in BGE105 clinical trial streamlined sample preparation. 10x Genomics single-cell library technology is used to evaluate tissue samples.

[0071] Fig.41 Show that 11 cell types have been identified, consistent with published muscle atlases. Two methods and two annotations are used. The top 20 variable genes in each cluster are used as markers for cell type annotation.

[0072] Fig.42 Show consistency between the top 20 cell type-specific expressed genes and known cell type markers. The chart shows the log2(fold change) in expression in specific cell types of the top 20 cell type-specific expressed genes compared to the remaining genes. All samples are used for analysis.

[0073] Fig.43 Show differentially expressed genes associated with BGE105 identified for each cell type.

[0074] Fig.44 Show that signaling pathways controlling muscle loss and promoting muscle growth are enriched in genes associated with BGE-105 treatment in fast skeletal muscle. Genes with significant Significance (padj < 0.1) are labeled.

[0075] Fig.45The therapeutic relevance of the most significant genes (padj < 0.1) in muscle growth / loss-related signaling pathways was as expected. Shown are the detections of genes related to fast skeletal muscle treatment (padj < 0.001) and slow skeletal muscle (type I) treatment.

[0076] Fig.46 On day 11, the overall expression levels of VEGFA, PPRGC1A, and COL1A1 in the treatment group were higher than those in the placebo group.

[0077] Fig.47 On day 11, the overall expression levels of TNNC1 and MYH7 in the treatment group were higher than those in the placebo group. Shown are the overall expression levels: the average value of the gene in all cells of each patient. Cell types were ignored. Both TNNC1 and MYH7 are slow skeletal muscle cell markers. Fig.48 For fast skeletal muscle, 10 groups of 5+ enriched pathways were identified.

[0078] Fig.49 For slow skeletal muscle, 13 groups of 5+ enriched pathways were identified.

[0079] Fig.50 Shown is the cell type-specific pattern of differential gene expression related to BGE-105 treatment identified in muscle biopsies.

[0080] Fig.51 The differential regulatory pathways indicate that BGE-105 has beneficial effects on key muscle and adipocyte processes.

[0081] Fig.52 BGE-105 prevents the downregulation of contractile proteins in fast and slow skeletal muscles induced by bed rest. The p-value reflects the change after 10 days of bed rest compared to the baseline.

[0082] Fig.53 BGE-105 prevents the downregulation of the mitochondrial biogenesis regulator PGC-1α and all respiratory complexes induced by bed rest. Shown are representative genes.

[0083] Fig.54 BGE-105 prevents the harmful expression levels of genes involved in muscle metabolic processes. Shown are the differential gene expressions in the insulin signaling pathway (p = 2.61E-03), AMPK signaling pathway (p = 5.04E-03), and glucagon signaling pathway (p = 2.61E-03).

[0084] Fig.55 BGE-105 prevents the bed rest-induced upregulation of genes involved in triglyceride storage and fatty acid metabolism, which is a potential mechanism for promoting fat reduction.

[0085] Fig.56 It was shown that more endothelial cells expressed APLNR in the treatment group. A small number of cells expressed APLNR (272 cells). 46% of these cells were located within endothelial cells. T-test of APLNR expression levels in the cells detected between the treatment group and the placebo group: at day 6, t.stat = 2.02, p.val = 0.04; at day 11, t.stat = 2.33, p.val = 0.02.

[0086] Fig.57 For cell differentiation trajectory and pseudotime inference.

[0087] Fig.58 Graph showing that the degree of cell differentiation of fast / slow skeletal muscle, macrophages, T / NK cells, and muscle stem cells decreased after treatment. Baseline muscle stem cells (treatment group and placebo group) were set as the root.

[0088] Figure 59 to Figure 61 Diagram showing the results of the secondary analysis. Evaluating signals from protein synthesis rate analysis. Investigating aging and muscle characteristics in published studies.

[0089] Fig.59 It was shown that BGE-105 led to relatively higher muscle protein synthesis in the vastus lateralis (p < 0.005) as measured by microdialysis. Shown is the muscle protein synthesis rate after 10 days of bed rest. Asterisks indicate 10 significant genes: TPM2, PYGM, MYH2, TNNI2, TNNC2, TNNC1, ENO3, ALDOA, ATP5F1B, MDH2.

[0090] Fig.60 Showing the validation of muscle protein synthesis assay results in snRNA-seq analysis. Compared with the placebo group, 15 out of 18 muscle proteins were highly expressed statistically in the fast skeletal muscle of the treatment group. In the FSR analysis, there were 13 myofibrillar proteins (5 proteins were significant in the FSR analysis: TPM2, TNNC1, TNNC2, TNNI2, MYH2) and 5 significant non-myofibrillar proteins (5 proteins: ENO3, PYGM, ALDOA, MDH2, ATP5F1B). All 38 proteins in heavy water were also measured in the snRNA-seq data.

[0091] Fig.61 It was shown that BGE-105 treatment shifted the fast / slow skeletal muscle transcriptome towards a state associated with young muscle. Transcripts negatively correlated with BGE-105 treatment showed enrichment for muscle aging characteristics. See Perez et al. (2022), Aging.

[0092] Fig.62 Show a graph indicating the percentage of cells in the sample with mitochondrial readings exceeding 5%. The Y-axis represents the percentage of cells with MT readings > 5% in the sample compared to the baseline (62 samples = 21 patients * 3 time points). P-values for the t-test: at day 6, p = 0.08; at day 11, p = 0.04. Low-quality cells (cells with less than 200 genes or less than 500 readings were removed) were removed before calculating the proportion of cells with more than 5% mitochondrial readings for each patient. The total number of remaining MT cells (> 5%) was then considered.

[0093] Fig.63 Show that BGE-105 maintains the rate of structural protein synthesis to preserve muscle mass. Determine the fractional synthesis rate (FSR) of skeletal muscle proteins detected in the vastus lateralis muscle tissue (collected by micro-needle biopsy). Considering the different baseline fractional synthesis rates, the FSR was adjusted proportionally across subjects within each protein. The two-sample t-test comparison value between groups was statistically significant at p < 0.005.

[0094] Fig.64 Show that BGE-105 prevents the downregulation of the mitochondrial biogenesis regulator PGC-1α and mitochondrial respiratory complexes induced by bed rest. Representative genes for each respiratory complex are shown.

[0095] Subfigures A - B of Figure 65 show that treatment with BGE-105 shifts the proteome towards an estimated higher basal metabolic rate and VO2 max as tested by SomaSignal. Subfigure A: Validation of the resting energy expenditure test by comparing plasma proteomics using SomaLogic with resting energy expenditure measured by indirect calorimetry and the Abbreviated Weir method. Plasma samples were from adults aged 29 - 64 years in the UK (N = 9022). Subfigure B: Validation of the VO2 max test by comparing plasma proteomics using SomaLogic with VO2 max derived from oxygen consumption at maximal exercise (in ml.kg / min) during a standard graded cardiopulmonary exercise test (CPET). Plasma samples were from adults aged 15 - 65 years in North America (N = 743).

[0096] Fig.66Shown is that BGE-105 improves recovery after bed rest via a wearable accelerometer. Days 1 to 10: treatment and bed rest period. Normal activities resumed on day 11. Both groups showed a rapid recovery of steps after bed rest, but the recovery was more pronounced in the azelaprag group, resulting in a separation of the curves between approximately days 15 and 36, after which the curves became indistinguishable. The Spearman correlation coefficient was used to test for an increase in the separation between the curves; the p-values at the end of the first, second, and third weeks after bed rest were 0.028, 0.00092, and 0.00002, respectively.

[0097] Fig.67 The figure shows the flow chart of the BGE-105 clinical trial, a single-center, double-blind, phase 2A randomized controlled trial (RCT), investigating the effects on muscle mass (primary outcome), muscle strength, and frailty outcomes.

[0098] Fig.68 Shown is that BGE-105 prevents the downregulation of contractile proteins in fast and slow skeletal muscles induced by bed rest. Shown are single-nucleus transcriptomic data of the percentage change of sarcoplasmic / endoplasmic reticulum calcium ATPase type 2 (SERCA2) and myosin light chain 3 (MYL3) relative to baseline. The p-value reflects the change compared to baseline after 10 days of bed rest.

[0099] FIG. 69A to FIG. 69B Shown is that BGE-105 prevents the downregulation of the mitochondrial biogenesis regulator PGC-1α and all respiratory complexes induced by bed rest. Fig.69A Shown is a summary of the downregulation of respiratory complexes. Fig.69B Shown are representative genes. The p-value reflects the change compared to baseline after 10 days of bed rest.

[0100] FIG. 70A to FIG. 70B Shown is that BGE-105 preserves the gene expression involved in glucose metabolism. The p-value reflects the change compared to baseline after 10 days of bed rest. Fig.70A Shown is a summary of the preserved gene expression in the glucagon, insulin, and AMPK pathways. Fig.70B Shown are the differential expressions in the insulin signaling pathway (p = 2.61E-03) and the AMPK signaling pathway (p = 5.04E-03). 5. Specific implementation

[0101] 5.1. Apelin receptor modulators and frailty

[0102] 5.1.1. Survival prediction model

[0103] The present disclosure describes a bioinformatics model, generally relating to constructing a survival prediction model that outputs a survival metric. Such survival metrics may be related to observables associated with survival, such as life expectancy and / or risk of death. The survival prediction model can be constructed by selecting observables related to the survival period ("aging metrics"). Such aging metrics may include variables related to all-cause mortality, such as certain clinical factors. The survival prediction model can utilize one or more survival biomarkers in combination with one or more aging metrics to generate a survival metric.

[0104] In some embodiments, the survival prediction model of the present disclosure is based on survival modeling, using clinical outcome data proprietary to the Human Health Aging Group and proteomic data generated from archived samples to examine the relationship between the serum level of apelin and the future risk of all-cause mortality in the Human Health Aging Group. Additionally, the Cox proportional hazards model is used to examine the relationship between apelin and events of decline in mobility (such as decline in walking, stair climbing, or transfer mobility, as indicated by difficulty self-reporting these activities), generating a hazard ratio and associated p-value for apelin.

[0105] We applied such bioinformatics and machine learning methods, using the survival prediction model to analyze human data, and found an association between apelin receptor levels and future aging outcomes. We found that higher circulating levels of apelin were associated with a reduced all-cause mortality (p = 0.0002) - i.e., a longer lifespan. See, for example Figure 2A . Additionally, our analysis confirmed that in healthy elderly human subjects, higher circulating levels of apelin were associated with better future physical performance. Figure 2B Showing a similar model, indicating that higher circulating levels of apelin were associated with an increased preservation of full mobility (p = 0.0082).

[0106] 5.1.2. Expression of Apelin Receptor in Elderly Subjects

[0107] The relationship between mouse or human age and apelin receptor expression was also confirmed. The expression of apelin receptor in skeletal muscle decreases with age. Samples from frail elderly patients showed a more significant decrease in apelin receptor levels. More details are provided in the experimental section, see, for example, Example 1 and Figures 2A-2D .

[0108] 5.1.3. Studies in Elderly Mice

[0109] We confirmed that elderly mice (24 months old) treated with BGE-105 showed a statistically significant increase in spontaneous activity (p = 0.002) and improvement in grip strength (p = 0.04) compared to age-matched control groups, indicating improved physical health and enhanced muscle strength ( Figures 3A-3I)。In addition, aged mice (18 months old) that were first injected with cardiotoxin and then treated with BGE-105 showed significantly higher levels of multiple transcripts that indicate muscle regeneration ( Figures 7A-7X )。Cells from these mice were cryopreserved and tissue sections were stained. The sections showed a dose-dependent increase in central nucleated fibers (indicating muscle growth) ( Figure 7AA-7BB )。More details are provided in the experimental section, see for example Examples 2-5 and Figures 3-7.

[0110] 5.1.4. BGE-105 Activates the Apelin Pathway In Vitro

[0111] We demonstrated that immortalized human muscle from young and old patients showed increased proliferation after treatment with increasing doses of BGE-105 ( Figures 8B-8C )。Young cells showed a significant increase in cell proliferation at 50 nM, and old cells had a significant increase in cell proliferation at 5 nM. More details are provided in the experimental section, see for example Example 6 and Figure 8.

[0112] 5.1.5. BGE-105 Prevents Muscle Atrophy in Activity-Restricted Mice

[0113] We demonstrated that 20-month-old mice that were activity-restricted and treated with BGE-105 showed a significant improvement in maintaining the muscle weight of the tibialis anterior muscle compared to the vehicle-treated control group. ( Fig.9D and 9E )。Compared to the vehicle, there was a nearly significant improvement in the muscle weight of the extensor digitorum longus muscle ( Fig.9F and 9G ), and there was also a significant improvement in the soleus muscle compared to the vehicle-treated control ( Figure 9H and 9I ).

[0114] The percentage of atrophy of the tibialis anterior muscle decreased significantly, the percentage of atrophy of the extensor digitorum longus muscle decreased nearly significantly, the percentage of atrophy of the soleus muscle improved slightly, and there was no improvement in the gastrocnemius muscle ( Fig.9A )。More details are provided in the experimental section, see for example Example 7 and Figure 9.

[0115] 5.2. Methods for Treating Muscle Disorders

[0116] The clinical studies described herein show that BGE-105 prevents or alleviates muscle atrophy in human patients, including bedridden patients. Extensive evaluations of several muscle dynamics, patient serum biomarkers, and proteomic analyses were also performed, and these evaluations were consistent with our demonstration of clinical efficacy in humans.

[0117] Accordingly, in a first aspect, the present disclosure provides a method of treating a muscle disorder in a subject, such as an age-related muscle disorder, using an apelin receptor modulator. The method comprises administering to the subject a therapeutically effective amount of an apelin receptor modulator of formula (I) or (II) (e.g., as described herein). In some embodiments, the subject is an elderly subject. In some embodiments, the subject is human but not elderly.

[0118] "Age-related muscle disorder" (which may be interchangeably referred to herein as "aging-related muscle disorder") refers to a degenerative disease or disorder or injury related to muscle in a mammalian subject. In some embodiments, the muscle is skeletal muscle. Skeletal muscle is considered an organ of the muscular system. Skeletal muscle may include muscle tissue responsible for bone movement. For example, skeletal muscle may include muscle that is under conscious or voluntary control, such as striated muscle.

[0119] In some embodiments, other parts of the mammal are also affected by age-related muscle disorders, such as blood vessels (e.g., arteries), nerves, bones, or skin. In some embodiments, age-related muscle disorders are associated with inflammation and / or impairment of mitochondrial function.

[0120] Examples of muscle disorders that can be targeted for treatment according to the methods of the present disclosure include, but are not limited to: sarcopenia, frailty, muscle weakness due to hip fracture, reduced risk of hip fracture, ICU-related muscle weakness, muscle atrophy, diaphragmatic muscle weakness, reduced risk of hip fracture, ICU-related muscle weakness, muscle atrophy, diaphragmatic dysfunction, diaphragmatic atrophy, ventilator-induced diaphragmatic dysfunction (VIDD), muscle weakness related to limited mobility, muscle weakness related to immobility, recovery from muscle injury, muscle wasting, and critical illness myopathy. In certain embodiments, the muscle disorder is acute muscle atrophy (e.g., in a bedridden patient). In certain embodiments, the muscle disorder is chronic muscle atrophy. In certain embodiments, the muscle disorder is ICU diaphragmatic atrophy. In certain embodiments, the muscle disorder is critical illness myopathy.

[0121] In some embodiments, the muscle disorder is sarcopenia. Sarcopenia is a disorder characterized by loss of skeletal muscle mass and function. When the disorder is associated with aging, it may also be referred to as age-related sarcopenia. The diagnosis of sarcopenia can be achieved by assessing low muscle mass plus the presence of low muscle function (low muscle strength / muscle weakness or low physical performance) (see, for example, Cruz-Jentoft et al., (2010) Sarcopenia: European consensus on definition and diagnosis Report of the European Working Group on Sarcopenia in Older People. Age and Ageing; 39:412-423; Muscaritoli et al., (2010) Consensus definition of sarcopenia, cachexia and pre-cachexia: joint document elaborated by Special Interest Groups (SIG) "cachexia-anorexia in chronic wasting diseases" and "nutrition in geriatrics". Clin Nutr. Apr, 29(2):154-9; Fielding et al. (2011) Sarcopenia: An Undiagnosed Condition in Older Adults. Current Consensus Definition: Prevalence, Etiology, and Consequences. International Working Group on Sarcopenia. J Am Med Dir Assoc, 12:249-256; and Studenski et al. (2014) The FNIH Sarcopenia Project: Rationale, study description, conference recommendations and final estimates. J Gerontol A Biol Sci Med Sci 69(5):547-558).

[0122] Frailty is an age-related condition characterized by increased vulnerability to external stress. It is strongly associated with adverse outcomes, including mortality, nursing home admission, and falls. In some embodiments, a muscle condition is a condition associated with one or more characteristic measurements of frailty. In some embodiments, a subject is classified as frail. In some embodiments, a subject is classified as pre-frail and at high risk of developing frailty. Frailty can be diagnosed and / or characterized according to various frailty indices, which are composite measures of age-related changes in frailty, such as methods based on Fried’s frailty scale (see, e.g., Fried, et al., Frailty in older adults: evidence for a phenotype. J Gerontol A Biol Sci Med Sci. 2001, 56: M146-M156) and / or Mitnitski’s Frailty Index (see, e.g., Mitnitski et al., Frailty, fitness and late-life mortality in relation to chronological and biological age. BMC Geriatr. 2002, 2: 1-10).

[0123] In some embodiments, the muscle condition is muscle atrophy. Muscle atrophy refers to the wasting or loss of any muscle tissue due to lack of use. Muscle atrophy leads to muscle weakness and disability. In some embodiments, the muscle condition is muscle weakness associated with restricted mobility, which refers to the atrophy or loss of any muscle tissue due to restricted mobility, for example, for medical reasons.

[0124] In some embodiments, the muscle condition is muscle weakness, also known as muscle fatigue, which is a condition characterized by the subject's inability to generate force with skeletal muscle. Muscle weakness typically occurs after muscle atrophy.

[0125] Various endpoints can be used to measure muscle atrophy, such as the fractional synthesis rate (FSR) of skeletal muscle proteins in a liquid biopsy. Other measurements of muscle atrophy include diaphragm thickness, echo density (e.g., vastus lateralis), muscle circumference (e.g., muscle of the thigh / vastus lateralis), muscle cross-sectional area, etc. Muscle circumference can be measured using ultrasound. Ultrasound can be used to evaluate diaphragm dysfunction, predict extubation success or failure, quantify respiratory effort, and detect atrophy in, for example, mechanically ventilated subjects.

[0126] In some embodiments, the muscle disorder is a skeletal muscle disorder. In some embodiments, the muscle disorder is not a cardiovascular disorder. In some embodiments, the subject does not have or is not determined to have a cardiovascular disease or disorder. In some embodiments, the subject does not have heart failure or is not at risk of heart failure.

[0127] In some embodiments, the age-related muscle disorder is associated with a loss of function of skeletal muscle, a decline in regenerative capacity, or impaired healing following injury. In some embodiments, the age-related muscle disorder is associated with a loss of function of muscle stem cells.

[0128] In some embodiments, the muscle disorder is caused by insulin insensitivity associated with muscle atrophy. Type 2 diabetes may be associated with accelerated muscle loss, decreased muscle function, and increased disability during aging.

[0129] 5.2.1. Patient Age

[0130] In some embodiments of the method of treating a muscle disorder in a subject, the subject has or is suspected of having an age-related muscle disorder.

[0131] In some embodiments, the subject is human. The subject can be a human patient who has or is at risk of an age-related muscle disorder. In some embodiments, the patient is at least 40 years old. In some embodiments, the patient is at least 50 years old. In some embodiments, the patient is at least 60 years old. In some embodiments, the patient is at least 65 years old. In some embodiments, the patient is at least 70 years old. In some embodiments, the patient is at least 75 years old. In some embodiments, the patient is at least 80 years old. In some embodiments, the patient is at least 85 years old. In some embodiments, the patient is at least 90 years old. In certain embodiments, the patient is 40 - 50 years old, 50 - 60 years old, 60 - 70 years old, 70 - 80 years old, or 80 - 90 years old.

[0132] 5.2.2. Patient Assessment

[0133] A variety of different assessment methods can be used to identify a subject for treatment according to the methods of the present disclosure.

[0134] The diagnosis of sarcopenia can be determined or confirmed by the presence of low muscle quantity or quality. Sarcopenia is considered severe when low muscle strength or force, low muscle quantity / quality, and low physical performance are all detected. In some embodiments, the patient has a lower muscle quantity or quality compared to a standard representing healthy human subjects (e.g., subjects of the same or younger age).

[0135] Appendicular lean body mass (ALBM) can be used to assess low muscle mass. In some embodiments, low muscle mass is represented by ALBM adjusted for body mass index (BMI), < 0.789 kg for males or < 0.512 kg for females, where ALBM can be measured by dual-energy X-ray absorptiometry (DXA).

[0136] Low muscle mass can be assessed by appendicular skeletal muscle index (ASMI). In some embodiments, low muscle mass is represented by an appendicular skeletal muscle index (ASMI) of less than 7.26 kg / m 2 for males or less than 5.5 kg / m 2 for females, where the ASMI is defined as appendicular skeletal muscle mass divided by the square of height and the ASMI is measured by dual-energy X-ray absorptiometry (DXA).

[0137] Low muscle strength can include low grip strength and can be determined using a handgrip strength test. In some embodiments, low grip strength is assessed by measuring the amount of static force that the hand squeezes around a hand dynamometer. For example, in a handgrip strength test, the value is less than 30 kg, such as less than 26 kg, for males or less than 20 kg, such as less than 16 kg, for females.

[0138] In some embodiments, a human subject has or is determined to have low muscle strength. In some embodiments, a human subject has or is determined to have low muscular force.

[0139] In some embodiments, a human subject has or is determined to have low lower limb muscle mass. In some embodiments, a human subject has or is determined to have low upper limb muscle mass. In some embodiments, a human subject has or is determined to have decreased muscle function. In some embodiments, a human subject has or is determined to have decreased muscle strength.

[0140] In some embodiments, a human subject has or is determined to have low muscle volume. In some embodiments, muscle volume is skeletal muscle volume. In some embodiments, the muscle is skeletal muscle. In some embodiments, the muscle is the diaphragm, tibialis anterior, tibialis posterior, gastrocnemius, sartorius, vastus intermedius, vastus lateralis, vastus medialis, soleus, rectus femoris, or extensor digitorum longus.

[0141] In some embodiments, the muscle volume is selected from the muscle volumes of one or more of the following upper limb muscles: abductor of shoulder, adductor of shoulder, flexor of elbow, extensor of elbow, flexor of wrist, and extensor of wrist.

[0142] In some embodiments, muscle mass is evaluated after administration. In some embodiments, muscle mass is evaluated at least one day after administration. In some embodiments, muscle mass is evaluated at least one week after administration. In some embodiments, muscle mass is evaluated at least one month after administration.

[0143] In some embodiments, the muscle disorder is a skeletal muscle disorder. In some embodiments, skeletal muscle expresses the apelin receptor, and administration of an apelin receptor modulator activates the apelin / APJ system (APLNR gene) in the muscle tissue of a subject. The muscle of interest expresses the apelin receptor, and in some embodiments, the expression level of the apelin receptor can be evaluated or determined in the muscle tissue of a subject before and / or after treatment. In some embodiments, the subject has or is determined to have low circulating apelin levels. The circulating apelin levels can be evaluated in a biological sample obtained from the subject, for example, using a quantitative assay (such as an ELISA assay or LC / MS) for determining the amount of apelin peptide in the sample.

[0144] In some embodiments, the muscle disorder is a diaphragmatic disorder. In some embodiments, the diaphragmatic disorder is diaphragmatic atrophy. In some embodiments, the diaphragmatic disorder is diaphragmatic dysfunction. Diaphragmatic dysfunction ranges from partial loss of the ability to generate pressure (weakness) to complete loss of diaphragmatic function (paralysis). Patients with bilateral diaphragmatic paralysis or severe diaphragmatic weakness are prone to dyspnea or recurrent respiratory failure. They may experience severe dyspnea at rest, lying flat, with exertion, or when immersed in water above the waist. In addition, patients with bilateral diaphragmatic paralysis are at higher risk of sleep fragmentation and hypoventilation during sleep.

[0145] In some embodiments of the methods of the present disclosure, the subject is human and has or is determined to have one or more of diabetes, insulin insensitivity, cardiovascular disease, and neurological disease.

[0146] In some embodiments, the subject is human and has or is determined to have diaphragmatic atrophy. In certain embodiments, the subject is a human undergoing mechanical ventilation (e.g., mechanical ventilation at the time of diagnosis). In certain embodiments, the subject is human and has or is determined to have diaphragmatic atrophy caused by mechanical ventilation. In some embodiments, the subject is human and uses a ventilator (such as mechanical ventilation). In some embodiments, the subject is human and has a loss of muscle mass during periods of restricted activity and bed rest. In some embodiments, the subject is human and has acute myopathy. In some embodiments, the subject is human, hospitalized, and has acute myopathy. In some embodiments, the subject has a chronic medical condition associated with muscle aging.

[0147] In some embodiments, the patient is at bed rest. In some embodiments, the subject has chronic obstructive pulmonary disease (COPD). In some embodiments, the subject with COPD is at bed rest and has bed rest-induced muscle atrophy / muscle loss. In some embodiments, the subject with COPD is at bed rest and has bed rest-induced muscle degeneration. In some embodiments, the subject with COPD is at bed rest and has muscle loss associated with myopathy. In some embodiments, the subject with COPD has severe or acute muscle loss.

[0148] In some embodiments of the methods of the present disclosure, the subject is human and has or is determined to have hypoxic respiratory failure. Hypoxic respiratory failure can be measured by stratifying diaphragmatic thickness.

[0149] Various endpoints can be used, such as the fractional synthesis rate (FSR) of skeletal muscle proteins in a liquid biopsy, to measure muscle atrophy. Other measurements of muscle atrophy include diaphragmatic thickness, echo density (e.g., vastus lateralis), muscle circumference (e.g., muscle of the thigh / vastus lateralis), muscle cross-sectional area, etc. Muscle circumference can be measured using ultrasound. Ultrasound can be used to evaluate diaphragmatic dysfunction, predict extubation success or failure, quantify respiratory effort, and detect atrophy in, for example, mechanically ventilated subjects.

[0150] Diaphragmatic atrophy can be measured by changes in diaphragmatic thickness. For example, diaphragmatic thickness can be measured in subjects undergoing mechanical ventilation before ventilation, during ventilation, days after using a ventilator, after treatment, etc. (see, e.g., Schepens et al., (2015) Crit Care; 19:422).

[0151] 5.3. Methods for Maintaining Muscle Mass or Muscle Strength

[0152] Aspects of the present disclosure include a method for maintaining and / or increasing muscle mass and / or muscle strength in a subject in need thereof. In some embodiments, the subject is elderly. In various embodiments, an apelin receptor modulator (e.g., as described herein) is administered to the elderly subject to maintain or increase the muscle mass and / or muscle strength of the subject's skeletal muscle. In some embodiments, the apelin receptor modulator is an apelin receptor agonist.

[0153] In some embodiments, the elderly subject is human and at least 60 years old. In some embodiments, the patient is at least 65 years old. In some embodiments, the patient is at least 70 years old. In some embodiments, the patient is at least 75 years old. In some embodiments, the patient is at least 80 years old. In some embodiments, the patient is at least 85 years old. In some embodiments, the patient is at least 90 years old. In certain embodiments, the patient is 60 - 70 years old, 70 - 80 years old, or 80 - 90 years old.

[0154] The muscle mass and / or muscle strength of the subject can be monitored during treatment and compared to a baseline assessment conducted prior to using an apelin receptor modulator. In some embodiments, the apelin receptor modulator is an apelin receptor agonist. In some embodiments, the muscle mass or muscle strength of the subject is maintained at least at the baseline level during treatment. In some embodiments, the subject is one in whom muscle mass and / or muscle strength declines over time, and administration of an apelin receptor modulator according to the methods of the present disclosure can reverse and / or improve such decline. In some embodiments, the apelin receptor modulator is an apelin receptor agonist.

[0155] Appendicular lean body mass (ALBM) can be used to assess low muscle mass. In some embodiments, low muscle mass is represented by ALBM adjusted for body mass index (BMI), <0.789 kg for males or <0.512 kg for females, where ALBM can be measured by dual - energy X - ray absorptiometry (DXA).

[0156] Low muscle mass can be assessed by appendicular skeletal muscle index (ASMI). In some embodiments, low muscle mass is represented as an appendicular skeletal muscle index (ASMI) of less than 7.26 kg / m 2 for males or less than 5.5 kg / m 2 for females, where the ASMI is defined as appendicular skeletal muscle mass divided by the square of height, and the ASMI is measured by dual - energy X - ray absorptiometry (DXA).

[0157] Low muscle strength can be determined using a hand - grip strength test. In some embodiments, in the hand - grip strength test, low muscle strength is indicated by a value of less than 30 kg, such as less than 26 kg, for males or less than 20 kg, such as less than 16 kg, for females.

[0158] In some embodiments, muscle mass is evaluated before and after administration of an apelin receptor agonist. In some embodiments, muscle mass is evaluated at least one day after administration. In some embodiments, muscle mass is evaluated at least one week after administration. In some embodiments, muscle mass is evaluated at least one month after administration.

[0159] In some embodiments, muscle strength is evaluated before and after administration of an apelin receptor agonist. In some embodiments, muscle strength is evaluated at least one day after administration. In some embodiments, muscle strength is evaluated at least one week after administration. In some embodiments, muscle strength is evaluated at least one month after administration.

[0160] In some embodiments, the subject has or is determined to have low circulating apelin levels. Circulating apelin levels can be evaluated in a biological sample obtained from the subject.

[0161] In some embodiments, the subject has or is determined to have altered serum protein levels that are associated with frailty, sarcopenia, muscle atrophy, or muscle weakness in the longitudinal aging cohort data of BioAge.

[0162] 5.4. Apelin Receptor Modulators

[0163] Apelin is an endogenous ligand of the apelin receptor (also known as APJ or APLNR). The apelin receptor is a member of the rhodopsin-like G protein-coupled receptor (GPCR) family. The apelin / APJ system is distributed in different peripheral organ tissues and can play various roles in the physiology and pathophysiology of many organs. The apelin / APJ system is involved in multiple cellular activities such as proliferation, migration, apoptosis, or inflammation. Apelin receptor modulators can directly or indirectly, competitively or non-competitively activate the APJ system. Accordingly, the apelin receptor modulators of the present disclosure can be referred to as apelin receptor agonists.

[0164] As further described below, in some embodiments of the methods of the present disclosure, the apelin receptor modulator (e.g., an apelin receptor agonist) is a compound described in U.S. Patent No. 9,573,936 or 9,868,721, the disclosures of which are incorporated herein by reference in their entirety.

[0165] As is known to those skilled in the art, certain compounds of the present disclosure can exist in one or more tautomeric forms. Since a single chemical structure can only represent one tautomeric form, it should be understood that, for convenience, reference to a compound of a given structural formula also includes the tautomers of the structure represented by that structural formula.

[0166] In some embodiments, the apelin receptor modulator is a compound of formula (I) or (II):

[0167]

[0168] or a pharmaceutically acceptable salt thereof, a tautomer thereof, a pharmaceutically acceptable salt of the tautomer, a stereoisomer of any of the foregoing, or a mixture thereof, wherein:

[0169] R 1 is an unsubstituted pyridyl, pyridone, or pyridine N-oxide, or a pyridyl, pyridone, or pyridine N-oxide substituted with 1, 2, 3, or 4 R 1a substituents;

[0170] R 1a is independently selected, in each occurrence, from -F, -Cl, -Br, -I, -CN, -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 perhaloalkyl, -OH, -O-(C1-C6 alkyl), -O-(C1-C6 haloalkyl), -O-(C1-C6 perhaloalkyl), -C2-C6 alkenyl, -O-(C1-C6 alkyl)-OH, -O-(C1-C6 alkyl)-O-(C1-C6 alkyl), -O-(C1-C6 haloalkyl)-OH, -O-(C1-C6 haloalkyl)-O-(C1-C6 alkyl), -O-(C1-C6 perhaloalkyl)-OH, -O-(C1-C6 perhaloalkyl)-O-(C1-C6 alkyl), -NH2, -NH(C1-C6 alkyl), -N(C1-C6 alkyl)2, -C(═O)-(C1-C6 alkyl), -C(═O)OH, -(C═O)-O-(C1-C6 alkyl), -C(═O)NH2, -C(═O)NH(C1-C6 alkyl), -C(═O)N(C1-C6 alkyl)2, phenyl, -C(═O)-(heterocyclic group) or a heterocyclic group, wherein the heterocyclic group of the -C(═O)-(heterocyclic group) or heterocyclic group is a 3- to 7-membered ring containing 1, 2, or 3 heteroatoms selected from N, O, and S;

[0171] R 2 is selected from -H and C1-C4 alkyl, or is absent in the compound of formula II;

[0172] R 3 is selected from unsubstituted C1-C 10 alkyl, C1-C 1a alkyl substituted with 1, 2, or 3 R 10 substituents, a group of formula -(CR 3b R 3c )-Q, a group of formula -NH-(CR 3b R 3c )-Q, a group of formula -(CR 3b R 3c )-C(═O)-Q, a group of formula -(CR 3d R 3e )-(CR3f R 3g )-Q groups, the group of formula -(CR 3b ═CR 3c )-Q groups and the group of formula -(heterocyclic group)-Q, wherein the heterocyclic group of said -(heterocyclic group)-Q has 5 to 7 ring members with 1, 2 or 3 of them being heteroatoms selected from N, O or S, and is unsubstituted or substituted by 1, 2 or 3 R 3h substituents;

[0173] R 1a is independently selected from -F, -Cl, -CN, -OH, -O-(C1-C6 alkyl), -O-(C1-C6 haloalkyl), -O-(C1-C6 perhaloalkyl), -O-(C1-C6 alkyl)-OH, -O-(C1-C6 alkyl)-O-(C1-C6 alkyl), C2-C6 alkenyl, C2-C6 alkynyl, -NH2, -NH(C1-C6 alkyl) and -N(C1-C6 alkyl)2 in each case;

[0174] R 3b and R 3c are independently selected from -H, -F, -Cl, -CN, -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 perhaloalkyl, -OH, -O-(C1-C6 alkyl), -O-(C1-C6 haloalkyl), -O-(C1-C6 perhaloalkyl), -O-(C1-C6 alkyl)-OH, -O-(C1-C6 alkyl)-O-(C1-C6 alkyl), -NH2, -NH(C1-C6 alkyl) and -N(C1-C6 alkyl)2;

[0175] R 3d and R 3e are independently selected from -H, -F, -Cl, -CN, -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 perhaloalkyl, -OH, -O-(C1-C6 alkyl), -O-(C1-C6 haloalkyl), -O-(C1-C6 perhaloalkyl), -O-(C1-C6 alkyl)-OH, -O-(C1-C6 alkyl)-O-(C1-C6 alkyl), -NH2, -NH(C1-C6 alkyl) and -N(C1-C6 alkyl)2;

[0176] R 3f and R 3gindependently selected from -H, -F, -Cl, -CN, -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 perhaloalkyl, -OH, -O-(C1-C6 alkyl), -O-(C1-C6 haloalkyl), -O-(C1-C6 perhaloalkyl), -O-(C1-C6 alkyl)-OH, -O-(C1-C6 alkyl)-O-(C1-C6 alkyl), -NH2, -NH(C1-C6 alkyl), and -N(C1-C6 alkyl)2;

[0177] R 3h in each case independently selected from -F, -Cl, -CN, -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 perhaloalkyl, -OH, -O-(C1-C6 alkyl), -O-(C1-C6 haloalkyl), -O-(C1-C6 perhaloalkyl), -O-(C1-C6 alkyl)-OH, -O-(C1-C6 alkyl)-O-(C1-C6 alkyl), -NH2, -NH(C1-C6 alkyl), -N(C1-C6 alkyl)2, and oxo;

[0178] Q is a monocyclic or bicyclic C6-C 10 aryl group, a monocyclic or bicyclic heteroaryl group having 5 to 10 ring members containing 1, 2, or 3 heteroatoms selected from N, O, or S, a C3-C8 cycloalkyl group, or a 3- to 7-membered heterocyclic group containing 1, 2, or 3 heteroatoms selected from N, O, or S, wherein the C6-C 10 aryl group, the heteroaryl group, the cycloalkyl group, and the heterocyclic group are unsubstituted or substituted by 1, 2, 3, or 4 R Q substituents;

[0179] R Q in each case independently selected from -F, -Cl, -Br, -I, -CN, -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 perhaloalkyl, -C2-C6 alkenyl, -C2-C6 alkynyl, -OH, -O-(C1-C6 alkyl), -O-(C1-C6 haloalkyl), -O-(C1-C6 perhaloalkyl), -NH2, -NH(C1-C6 alkyl), -N(C1-C6 alkyl)2, -C(═O)-(C1-C6 alkyl), -C(═O)OH, -C(═O)-O-(C1-C6 alkyl), -C(═O)NH2, -C(═O)NH(C1-C6 alkyl), -C(═O)N(C1-C6 alkyl)2, -S(═O)2-(C1-C6 alkyl), phenyl, and heteroaryl, and the Q heterocyclic group may be substituted by 1 oxo R Q substituent;

[0180] R 4Selected from monocyclic or bicyclic C6-C 10 aryl groups, monocyclic or bicyclic heteroaryl groups having 5 to 10 ring members containing 1, 2 or 3 heteroatoms independently selected from N, O and S, and monocyclic or bicyclic heterocyclic groups having 5 to 10 ring members containing 1, 2, 3 or 4 heteroatoms independently selected from N, O and S, wherein the C6-C 10 aryl group, the heteroaryl group or the heterocyclic group is unsubstituted or substituted by 1, 2 or 3 R 4a substituents;

[0181] R 4a is independently selected in each case from -F, -Cl, -Br, -I, -CN, -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 perhaloalkyl, -OH, -O-(C1-C6 alkyl), -O-(C1-C6 haloalkyl), -O-(C1-C6 perhaloalkyl), -NH2, -NH(C1-C6 alkyl), -N(C1-C6 alkyl)2, -C(═O)-(C1-C6 alkyl), -C(═O)OH, -C(═O)-O-(C1-C6 alkyl), -C(═O)NH2, -C(═O)NH(C1-C6 alkyl) and -C(═O)N(C1-C6 alkyl)2, and the heterocyclic R 4 group may be further substituted by 1 oxo substituent; and

[0182] Further, wherein:

[0183] If R 4 is an unsubstituted or substituted benzene ring and R 3 is a group of formula -(CR 3b ═CR 3c )-Q, then at least one of the following is true:

[0184] a) R 4 is substituted by at least one -O-(C1-C6 alkyl) group;

[0185] b) Q is not oxadiazole;

[0186] c) R 3b is not -H;

[0187] d) R 3c is not -H;

[0188] e) R 1 is not 2-pyridyl; or

[0189] f) R 4 is substituted by two or more -O-(C1-C6 alkyl) groups.

[0190] In some embodiments, the apelin receptor modulator is a compound of formula (I) or (II):

[0191]

[0192] or a pharmaceutically acceptable salt thereof, a tautomer thereof, a pharmaceutically acceptable salt of the tautomer, a stereoisomer of any of the foregoing, or a mixture thereof, wherein:

[0193] R 1 is an unsubstituted pyridyl, pyridone, or pyridine N-oxide, or is a pyridyl, pyridone, or pyridine N-oxide substituted with 1, 2, 3, or 4 R 1a substituents;

[0194] R 1a is independently selected, in each occurrence, from -F, -Cl, -Br, -I, -CN, -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 perhaloalkyl, -OH, -O-(C1-C6 alkyl), -O-(C1-C6 haloalkyl), -O-(C1-C6 perhaloalkyl), -C2-C6 alkenyl, -O-(C1-C6 alkyl)-OH, -O-(C1-C6 alkyl)-O-(C1-C6 alkyl), -O-(C1-C6 haloalkyl)-OH, -O-(C1-C6 haloalkyl)-O-(C1-C6 alkyl), -O-(C1-C6 perhaloalkyl)-OH, -O-(C1-C6 perhaloalkyl)-O-(C1-C6 alkyl), -NH2, -NH(C1-C6 alkyl), -N(C1-C6 alkyl)2, -C(═O)-(C1-C6 alkyl), -C(═O)OH, -(C═O)-O-(C1-C6 alkyl), -C(═O)NH2, -C(═O)NH(C1-C6 alkyl), -C(═O)N(C1-C6 alkyl)2, phenyl, -C(═O)-(heterocyclic group), or a heterocyclic group, wherein the heterocyclic group of -C(═O)-(heterocyclic group) or the heterocyclic group is a 3- to 7-membered ring containing 1, 2, or 3 heteroatoms selected from N, O, and S;

[0195] R 2 is selected from -H or C1-C4 alkyl, or is absent in the compound of formula II;

[0196] R 3 is a group of formula -(CR 3d R 3e )-(CR 3f R 3g )-Q;

[0197] R 3d and R 3eindependently selected from -H, -F, -Cl, -CN, -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 perhaloalkyl, -OH, -O-(C1-C6 alkyl), -O-(C1-C6 haloalkyl), -O-(C1-C6 perhaloalkyl), -O-(C1-C6 alkyl)-OH, -O-(C1-C6 alkyl)-O-(C1-C6 alkyl), -NH2, -NH(C1-C6 alkyl) or -N(C1-C6 alkyl)2;

[0198] R 3f and R 3g are independently selected from -H, -F, -Cl, -CN, -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 perhaloalkyl, -OH, -O-(C1-C6 alkyl), -O-(C1-C6 haloalkyl), -O-(C1-C6 perhaloalkyl), -O-(C1-C6 alkyl)-OH, -O-(C1-C6 alkyl)-O-(C1-C6 alkyl), -NH2, -NH(C1-C6 alkyl) or -N(C1-C6 alkyl)2;

[0199] Q is a monocyclic or bicyclic C6-C 10 aryl group, a 5- to 10-membered monocyclic or bicyclic heteroaryl group containing 1, 2 or 3 heteroatoms selected from N, O or S, or a 3- to 7-membered heterocyclic group containing 1, 2 or 3 heteroatoms selected from N, O or S, wherein the C6-C 10 aryl group, the heteroaryl group, the cycloalkyl group and the heterocyclic group are unsubstituted or substituted by 1, 2, 3 or 4 R Q substituents;

[0200] R Q is independently selected from -F, -Cl, -Br, -I, -CN, -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 perhaloalkyl, -C2-C6 alkenyl, -C2-C6 alkynyl, -OH, -O-(C1-C6 alkyl), -O-(C1-C6 haloalkyl), -O-(C1-C6 perhaloalkyl), -NH2, -NH(C1-C6 alkyl), -N(C1-C6 alkyl)2, -C(═O)-(C1-C6 alkyl), -C(═O)OH, -C(═O)-O-(C1-C6 alkyl), -C(═O)NH2, -C(═O)NH(C1-C6 alkyl), -C(═O)N(C1-C6 alkyl)2, -S(═O)2-(C1-C6 alkyl), phenyl or heteroaryl group, and the Q heterocyclic group can be substituted by 1 oxo R Q substituent;

[0201] R 4 selected from monocyclic or bicyclic C6-C10 An aryl group, a 5- to 10-membered monocyclic or bicyclic heteroaryl group containing 1, 2 or 3 heteroatoms independently selected from N, O and S, or a 5- to 10-membered monocyclic or bicyclic heterocyclic group containing 1, 2, 3 or 4 heteroatoms independently selected from N, O and S, wherein the C6-C 10 aryl group, the heteroaryl group or the heterocyclic group is unsubstituted or substituted with 1, 2 or 3 R 4a substituents;

[0202] R 4a is independently selected in each case from -F, -Cl, -Br, -I, -CN, -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 perhaloalkyl, -OH, -O-(C1-C6 alkyl), -O-(C1-C6 haloalkyl), -O-(C1-C6 perhaloalkyl), -NH2, -NH(C1-C6 alkyl), -N(C1-C6 alkyl)2, -C(═O)-(C1-C6 alkyl), -C(═O)OH, -C(═O)-O-(C1-C6 alkyl), -C(═O)NH2, -C(═O)NH(C1-C6 alkyl) or -C(═O)N(C1-C6 alkyl)2, and the heterocyclic R 4 group can be further substituted with 1 oxo substituent.

[0203] As described above, the eparineptide receptor agonist compounds of the present disclosure can exist in a variety of tautomeric forms. This is especially true for the compounds of Formula I, wherein R 2 is H. These forms are shown below as tautomer A and tautomer B:

[0204]

[0205] The eparineptide receptor agonist compounds of the present disclosure are structurally described and are generally named as compounds in the "tautomer A" form. However, it is specifically considered and known that the compounds exist in the "tautomer B" form, and therefore the compounds in the "tautomer B" form are expressly considered to be part of the present disclosure. Accordingly, the compounds of Formula I and Formula II are recited in the claims. Depending on the compound, some compounds may exist predominantly in one form rather than the other. In addition, depending on the compound and the energy required to convert one tautomer to another, some compounds may exist as a mixture at room temperature, while other compounds may be isolated in one tautomeric form or the other.

[0206] In some embodiments of Formula (I) and (II), R 1 is an unsubstituted pyridyl group or a pyridyl group substituted with 1 or 2 R 1a substituents.

[0207] In some embodiments of formulas (I) and (II), R 1a is independently selected in each case from: -CH3, -CH2CH3, -F, -Cl, -Br, -CN, -CF3, -CH═CH2, -C(═O)NH2, -C(═O)NH(CH3), -C(═O)N(CH3)2, -C(═O)NH(CH2CH3), -OH, -OCH3, -OCHF2, -OCH2CH3, -OCH2CF3, -OCH2CH2OH, -OCH2C(CH3)2OH, -OCH2C(CF3)2OH, -OCH2CH2OCH3, -NH2, -NHCH3, -N(CH3)2, phenyl, and a group of the following formula:

[0208] wherein the symbol when drawn across a bond, represents a point of attachment to the remainder of the molecule.

[0209] In some embodiments of formulas (I) and (II), R 1 is selected from:

[0210]

[0211]

[0212] wherein the symbol when drawn across a bond, represents a point of attachment to the remainder of the molecule.

[0213] In some embodiments of formulas (I) and (II), R 1 is selected from:

[0214]

[0215] wherein the symbol when drawn across a bond, represents a point of attachment to the remainder of the molecule.

[0216] In some embodiments of formulas (I) and (II), R 2 is -H.

[0217] In some embodiments of formulas (I) and (II), R 4 is phenyl, pyridyl, pyrimidinyl, isoxazolyl, indolyl, naphthyl, or pyridinyl, any of which may be unsubstituted or substituted with 1, 2, or 3 R 4a substituents. In some embodiments of formulas (I) and (II), R 4 is substituted with 1 or 2 R 4aSubstituted phenyl. In some embodiments of Formulas (I) and (II), one or two R 4a substituents are -O-(C1-C2 alkyl) groups.

[0218] In some embodiments of Formulas (I) and (II), R 4a is independently selected from -CH3, -F, -Cl, -Br, -CN, -CF3, -OCH3, -OCHF2, -OCH2CH3, -C(═O)OCH3, -C(═O)CH3 or -N(CH3)2 in each case.

[0219] In some embodiments of Formulas (I) and (II), R 4 is selected from:

[0220]

[0221]

[0222] wherein the symbol , when drawn across a bond, represents a point of attachment to the remainder of the molecule.

[0223] In some embodiments of Formulas (I) and (II), R 3 is selected from groups of the formula -(CR 3b R 3c )-Q, groups of the formula -NH-(CR 3b R 3c )-Q, groups of the formula -(CR 3b R 3c )-C(═O)-Q, groups of the formula -(CR 3d R 3e )-(CR 3f R 3g )-Q, groups of the formula -(CR 3b ═CR 3c )-Q or groups of the formula -(heterocyclic group)-Q, wherein the heterocyclic group of the -(heterocyclic group)-Q contains a 5- to 7-membered ring and is unsubstituted or substituted with 1, 2 or 3 R 3h substituents, and 1, 2 or 3 ring members of the 5- to 7-membered ring are heteroatoms selected from N, O or S.

[0224] In some embodiments of Formulas (I) and (II), Q is selected from pyrimidinyl, pyridinyl, isoxazolyl, thiazolyl, imidazolyl, phenyl, tetrahydropyrimidinone, cyclopropyl, cyclobutyl, cyclohexyl, morpholinyl, pyrrolidinyl, pyrazinyl, imidazo[1,2-a]pyridinyl, pyrazolyl or oxetanyl, any of which is substituted with 1, 2 or 3 R Q substituents.

[0225] In some embodiments of formulas (I) and (II), Q is a 5- or 6-membered monocyclic heteroaryl having 1 or 2 heteroatoms selected from N, O, or S, and Q is unsubstituted or substituted with 1 or 2 R Q substituents.

[0226] In some embodiments of formulas (I) and (II), Q is selected from:

[0227]

[0228]

[0229] where the symbol , when drawn across a bond, represents a point of attachment to the remainder of the molecule.

[0230] In some embodiments of formulas (I) and (II), R 3 is a group of formula -(heterocyclyl)-Q, wherein the heterocyclyl of -(heterocyclyl)-Q has 5 to 7 ring members and is unsubstituted or substituted with 1, 2, or 3 R 3h substituents, wherein 1, 2, or 3 of the 5 to 7 ring members are heteroatoms selected from N, O, or S.

[0231] In some embodiments of formulas (I) and (II), R 3 is a group of formula -(CR 3d R 3e )-(CR 3f R 3g )-Q.

[0232] In some embodiments of formulas (I) and (II), R 3 has the formula:

[0233]

[0234]

[0235] where the symbol , when drawn across a bond, represents a point of attachment to the remainder of the molecule.

[0236] In some embodiments of formulas (I) and (II), R 3 has the formula:

[0237]

[0238] where the symbol , when drawn across a bond, represents a point of attachment to the remainder of the molecule.

[0239] In specific embodiments of formulas (I) and (II), the epratuzumab receptor agonist is:

[0240] (1R,2S)-N-(4-(2,6-Dimethoxyphenyl)-5-(6-methoxy-2-pyridyl)-4H-1,2,4-triazol-3-yl)-1-methoxy-1-(5-methyl-2-pyrimidinyl)-2-propanesulfonamide;

[0241] (2S,3R)-N-(4-(2,6-Dimethoxyphenyl)-5-(5-methyl-3-pyridyl)-4H-1,2,4-triazol-3-yl)-3-(5-methyl-2-pyrimidinyl)-2-butanesulfonamide;

[0242] (1R,2S)-N-(4-(2,6-Dimethoxyphenyl)-5-(5-methyl-3-pyridyl)-4H-1,2,4-triazol-3-yl)-1-methoxy-1-(5-methyl-2-pyrimidinyl)-2-propanesulfonamide;

[0243] (1R,2S)-N-(4-(2,6-Dimethoxyphenyl)-5-(5-methyl-3-pyridyl)-4H-1,2,4-triazol-3-yl)-1-hydroxy-1-(5-methyl-2-pyrimidinyl)-2-propanesulfonamide;

[0244] (1S,2R)-1-(5-Chloro-2-pyrimidinyl)-N-(4-(2,6-dimethoxyphenyl)-5-(5-methyl-3-pyridyl)-4H-1,2,4-triazol-3-yl)-1-methoxy-2-propanesulfonamide;

[0245] (1S,2R)-N-(4-(2,6-Dimethoxyphenyl)-5-(5-methyl-3-pyridyl)-4H-1,2,4-triazol-3-yl)-1-methoxy-1-(5-methyl-2-pyrazinyl)-2-propanesulfonamide;

[0246] (1R,2S)-N-(4-(2,6-Dimethoxyphenyl)-5-(5-methyl-3-pyridyl)-4H-1,2,4-triazol-3-yl)-1-hydroxy-1-(5-methyl-2-pyrazinyl)-2-propanesulfonamide;

[0247] (1R,2S)-N-(4-(2,6-Dimethoxyphenyl)-5-(3-pyridyl)-4H-1,2,4-triazol-3-yl)-1-methoxy-1-(5-methyl-2-pyrimidinyl)-2-propanesulfonamide;

[0248] (2S,3R)-N-(4-(2,6-Dimethoxyphenyl)-5-(3-pyridyl)-4H-1,2,4-triazol-3-yl)-3-(5-methyl-2-pyrimidinyl)-2-butanesulfonamide;

[0249] (1R,2S)-1-(5-chloro-2-pyrimidinyl)-N-(4-(2,6-dimethoxyphenyl)-5-(3-pyridinyl)-4H-1,2,4-triazol-3-yl)-1-ethoxy-2-propanesulfonamide;

[0250] (1R,2S)-N-(4-(2,6-dimethoxyphenyl)-5-(5-methyl-3-pyridinyl)-4H-1,2,4-triazol-3-yl)-1-ethoxy-1-(5-methyl-2-pyrimidinyl)-2-propanesulfonamide;

[0251] (1S,2R)-N-(4-(2,6-dimethoxyphenyl)-5-(3-pyridinyl)-4H-1,2,4-triazol-3-yl)-1-methoxy-1-(5-methyl-2-pyrazinyl)-2-propanesulfonamide;

[0252] (1R,2S)-N-(4-(2,6-dimethoxyphenyl)-5-(6-methyl-2-pyridinyl)-4H-1,2,4-triazol-3-yl)-1-hydroxy-1-(5-methyl-2-pyrimidinyl)-2-propanesulfonamide;

[0253] (1R,2S)-N-(4-(2,6-dimethoxyphenyl)-5-(3-pyridinyl)-4H-1,2,4-triazol-3-yl)-1-ethoxy-1-(5-methyl-2-pyrimidinyl)-2-propanesulfonamide;

[0254] (1R,2S)-N-(4-(2,6-dimethoxyphenyl)-5-(5-methyl-3-pyridinyl)-4H-1,2,4-triazol-3-yl)-1-(5-fluoro-2-pyrimidinyl)-1-methoxy-2-propanesulfonamide;

[0255] (2S,3R)-N-(4-(2,6-dimethoxyphenyl)-5-(5-methyl-3-pyridinyl)-4H-1,2,4-triazol-3-yl)-3-(5-methyl-2-pyrazinyl)-2-butanesulfonamide;

[0256] (1R,2S)-N-(4-(2,6-dimethoxyphenyl)-5-(3-pyridinyl)-4H-1,2,4-triazol-3-yl)-1-ethoxy-1-(5-fluoro-2-pyrimidinyl)-2-propanesulfonamide;

[0257] (1S,2S)-N-(4-(2,6-dimethoxyphenyl)-5-(5-methyl-3-pyridinyl)-4H-1,2,4-triazol-3-yl)-1-(1-methylethoxy)-1-(5-methyl-2-pyrimidinyl)-2-propanesulfonamide;

[0258] (1R,2S)-N-(4-(2,6-dimethoxyphenyl)-5-(5-methyl-3-pyridyl)-4H-1,2,4-triazol-3-yl)-1-(1-methylethoxy)-1-(5-methyl-2-pyrimidinyl)-2-propanesulfonamide;

[0259] (1S,2R)-1-(5-chloro-2-pyrimidinyl)-N-(4-(2,6-dimethoxyphenyl)-5-(3-pyridyl)-4H-1,2,4-triazol-3-yl)-1-methoxy-2-propanesulfonamide;

[0260] (1R,2S)-N-(4-(2,6-dimethoxyphenyl)-5-(5-methyl-3-pyridyl)-4H-1,2,4-triazol-3-yl)-1-methoxy-1-(5-methoxy-2-pyrazinyl)-2-propanesulfonamide;

[0261] (2S,3R)-N-(4-(2,6-dimethoxyphenyl)-5-(3-pyridyl)-4H-1,2,4-triazol-3-yl)-3-(5-methyl-2-pyrazinyl)-2-butanesulfonamide;

[0262] (1R,2S)-N-(4-(2,6-dimethoxyphenyl)-5-(5-methyl-3-pyridyl)-4H-1,2,4-triazol-3-yl)-1-ethoxy-1-(5-fluoro-2-pyrimidinyl)-2-propanesulfonamide;

[0263] (1R,2S)-N-(4-(4,6-dimethoxy-5-pyrimidinyl)-5-(6-methoxy-2-pyridyl)-4H-1,2,4-triazol-3-yl)-1-methoxy-1-(5-methyl-2-pyrimidinyl)-2-propanesulfonamide;

[0264] (1R,2R)-1-(5-chloro-2-pyrimidinyl)-N-(4-(2,6-dimethoxyphenyl)-5-(3-pyridyl)-4H-1,2,4-triazol-3-yl)-1-ethoxy-2-propanesulfonamide; or

[0265] (1S,2S)-N-(4-(2,6-dimethoxyphenyl)-5-(5-methyl-3-pyridyl)-4H-1,2,4-triazol-3-yl)-1-ethoxy-1-(5-methyl-2-pyrimidinyl)-2-propanesulfonamide.

[0266] In a specific embodiment of formulas (I) and (II), the eparineptide receptor agonist is (1R,2S)-N-(4-(2,6-dimethoxyphenyl)-5-(6-methoxy-2-pyridinyl)-4H-1,2,4-triazol-3-yl)-1-methoxy-1-(5-methyl-2-pyrimidinyl)-2-propanesulfonamide or a pharmaceutically acceptable salt thereof.

[0267] In a specific embodiment of formulas (I) and (II), the eparineptide receptor agonist is (1R,2S)-N-(4-(2,6-dimethoxyphenyl)-5-(5-methyl-3-pyridinyl)-4H-1,2,4-triazol-3-yl)-1-methoxy-1-(5-methyl-2-pyrimidinyl)-2-propanesulfonamide or a pharmaceutically acceptable salt thereof.

[0268] In a specific embodiment of formulas (I) and (II), the eparineptide receptor agonist is (1R,2S)-N-(4-(2,6-dimethoxyphenyl)-5-(5-methyl-3-pyridinyl)-4H-1,2,4-triazol-3-yl)-1-hydroxy-1-(5-methyl-2-pyrimidinyl)-2-propanesulfonamide or a pharmaceutically acceptable salt thereof.

[0269] In a specific embodiment of formulas (I) and (II), the eparineptide receptor agonist is (1S,2R)-1-(5-chloro-2-pyrimidinyl)-N-(4-(2,6-dimethoxyphenyl)-5-(5-methyl-3-pyridinyl)-4H-1,2,4-triazol-3-yl)-1-methoxy-2-propanesulfonamide or a pharmaceutically acceptable salt thereof.

[0270] In a specific embodiment of formulas (I) and (II), the eparineptide receptor agonist is (1S,2R)-N-(4-(2,6-dimethoxyphenyl)-5-(5-methyl-3-pyridinyl)-4H-1,2,4-triazol-3-yl)-1-methoxy-1-(5-methyl-2-pyrazinyl)-2-propanesulfonamide or a pharmaceutically acceptable salt thereof.

[0271] In a specific embodiment of formulas (I) and (II), the eparineptide receptor agonist is (1R,2S)-N-(4-(2,6-dimethoxyphenyl)-5-(5-methyl-3-pyridinyl)-4H-1,2,4-triazol-3-yl)-1-hydroxy-1-(5-methyl-2-pyrazinyl)-2-propanesulfonamide or a pharmaceutically acceptable salt thereof.

[0272] In one specific embodiment of formulas (I) and (II), the eparrinone receptor agonist is (1R,2S)-N-(4-(2,6-dimethoxyphenyl)-5-(3-pyridyl)-4H-1,2,4-triazol-3-yl)-1-methoxy-1-(5-methyl-2-pyrimidinyl)-2-propanesulfonamide or a pharmaceutically acceptable salt thereof.

[0273] In one specific embodiment of formulas (I) and (II), the eparrinone receptor agonist is (2S,3R)-N-(4-(2,6-dimethoxyphenyl)-5-(3-pyridyl)-4H-1,2,4-triazol-3-yl)-3-(5-methyl-2-pyrimidinyl)-2-butanesulfonamide or a pharmaceutically acceptable salt thereof.

[0274] In one specific embodiment of formulas (I) and (II), the eparrinone receptor agonist is (1R,2S)-1-(5-chloro-2-pyrimidinyl)-N-(4-(2,6-dimethoxyphenyl)-5-(3-pyridyl)-4H-1,2,4-triazol-3-yl)-1-ethoxy-2-propanesulfonamide or a pharmaceutically acceptable salt thereof.

[0275] In one specific embodiment of formulas (I) and (II), the eparrinone receptor agonist is (1R,2S)-N-(4-(2,6-dimethoxyphenyl)-5-(5-methyl-3-pyridyl)-4H-1,2,4-triazol-3-yl)-1-ethoxy-1-(5-methyl-2-pyrimidinyl)-2-propanesulfonamide or a pharmaceutically acceptable salt thereof.

[0276] In one specific embodiment of formulas (I) and (II), the eparrinone receptor agonist is (1S,2R)-N-(4-(2,6-dimethoxyphenyl)-5-(3-pyridyl)-4H-1,2,4-triazol-3-yl)-1-methoxy-1-(5-methyl-2-pyrazinyl)-2-propanesulfonamide or a pharmaceutically acceptable salt thereof.

[0277] In one specific embodiment of formulas (I) and (II), the eparrinone receptor agonist is (1R,2S)-N-(4-(2,6-dimethoxyphenyl)-5-(6-methyl-2-pyridyl)-4H-1,2,4-triazol-3-yl)-1-hydroxy-1-(5-methyl-2-pyrimidinyl)-2-propanesulfonamide or a pharmaceutically acceptable salt thereof.

[0278] In a specific embodiment of formulas (I) and (II), the eparineptide receptor agonist is (1R,2S)-N-(4-(2,6-dimethoxyphenyl)-5-(3-pyridyl)-4H-1,2,4-triazol-3-yl)-1-ethoxy-1-(5-methyl-2-pyrimidinyl)-2-propanesulfonamide or a pharmaceutically acceptable salt thereof.

[0279] In a specific embodiment of formulas (I) and (II), the eparineptide receptor agonist is (1R,2S)-N-(4-(2,6-dimethoxyphenyl)-5-(5-methyl-3-pyridyl)-4H-1,2,4-triazol-3-yl)-1-(5-fluoro-2-pyrimidinyl)-1-methoxy-2-propanesulfonamide or a pharmaceutically acceptable salt thereof.

[0280] In a specific embodiment of formulas (I) and (II), the eparineptide receptor agonist is (2S,3R)-N-(4-(2,6-dimethoxyphenyl)-5-(5-methyl-3-pyridyl)-4H-1,2,4-triazol-3-yl)-3-(5-methyl-2-pyrazinyl)-2-butanesulfonamide or a pharmaceutically acceptable salt thereof.

[0281] In a specific embodiment of formulas (I) and (II), the eparineptide receptor agonist is (1R,2S)-N-(4-(2,6-dimethoxyphenyl)-5-(3-pyridyl)-4H-1,2,4-triazol-3-yl)-1-ethoxy-1-(5-fluoro-2-pyrimidinyl)-2-propanesulfonamide or a pharmaceutically acceptable salt thereof.

[0282] In a specific embodiment of formulas (I) and (II), the eparineptide receptor agonist is (1S,2S)-N-(4-(2,6-dimethoxyphenyl)-5-(5-methyl-3-pyridyl)-4H-1,2,4-triazol-3-yl)-1-(1-methylethoxy)-1-(5-methyl-2-pyrimidinyl)-2-propanesulfonamide or a pharmaceutically acceptable salt thereof.

[0283] In a specific embodiment of formulas (I) and (II), the eparineptide receptor agonist is (1R,2S)-N-(4-(2,6-dimethoxyphenyl)-5-(5-methyl-3-pyridyl)-4H-1,2,4-triazol-3-yl)-1-(1-methylethoxy)-1-(5-methyl-2-pyrimidinyl)-2-propanesulfonamide or a pharmaceutically acceptable salt thereof.

[0284] In a specific embodiment of formulas (I) and (II), the eparineptide receptor agonist is (1S,2R)-1-(5-chloro-2-pyrimidinyl)-N-(4-(2,6-dimethoxyphenyl)-5-(3-pyridinyl)-4H-1,2,4-triazol-3-yl)-1-methoxy-2-propanesulfonamide or a pharmaceutically acceptable salt thereof.

[0285] In a specific embodiment of formulas (I) and (II), the eparineptide receptor agonist is (1R,2S)-N-(4-(2,6-dimethoxyphenyl)-5-(5-methyl-3-pyridinyl)-4H-1,2,4-triazol-3-yl)-1-methoxy-1-(5-methoxy-2-pyrazinyl)-2-propanesulfonamide or a pharmaceutically acceptable salt thereof.

[0286] In a specific embodiment of formulas (I) and (II), the eparineptide receptor agonist is (2S,3R)-N-(4-(2,6-dimethoxyphenyl)-5-(3-pyridinyl)-4H-1,2,4-triazol-3-yl)-3-(5-methyl-2-pyrazinyl)-2-butanesulfonamide or a pharmaceutically acceptable salt thereof.

[0287] In a specific embodiment of formulas (I) and (II), the eparineptide receptor agonist is (1R,2S)-N-(4-(2,6-dimethoxyphenyl)-5-(5-methyl-3-pyridinyl)-4H-1,2,4-triazol-3-yl)-1-ethoxy-1-(5-fluoro-2-pyrimidinyl)-2-propanesulfonamide or a pharmaceutically acceptable salt thereof.

[0288] In a specific embodiment of formulas (I) and (II), the eparineptide receptor agonist is (1R,2S)-N-(4-(4,6-dimethoxy-5-pyrimidinyl)-5-(6-methoxy-2-pyridinyl)-4H-1,2,4-triazol-3-yl)-1-methoxy-1-(5-methyl-2-pyrimidinyl)-2-propanesulfonamide or a pharmaceutically acceptable salt thereof.

[0289] In a specific embodiment of formulas (I) and (II), the eparineptide receptor agonist is (1R,2R)-1-(5-chloro-2-pyrimidinyl)-N-(4-(2,6-dimethoxyphenyl)-5-(3-pyridinyl)-4H-1,2,4-triazol-3-yl)-1-ethoxy-2-propanesulfonamide or a pharmaceutically acceptable salt thereof.

[0290] In one specific embodiment of formulas (I) and (II), the eparineptide receptor agonist is (1S,2S)-N-(4-(2,6-dimethoxyphenyl)-5-(5-methyl-3-pyridyl)-4H-1,2,4-triazol-3-yl)-1-ethoxy-1-(5-methyl-2-pyrimidinyl)-2-propanesulfonamide or a pharmaceutically acceptable salt thereof.

[0291] In one specific embodiment of formulas (I) and (II), the eparineptide receptor agonist is (1R,2S)-N-(4-(2,6-dimethoxyphenyl)-5-(6-methoxy-2-pyridyl)-4H-1,2,4-triazol-3-yl)-1-methoxy-1-(5-methyl-2-pyrimidinyl)-2-propanesulfonamide, or a pharmaceutically acceptable salt thereof, its tautomer, a pharmaceutically acceptable salt of the tautomer, a stereoisomer of any of the foregoing, or a mixture thereof.

[0292] In one specific embodiment of formulas (I) and (II), the eparineptide receptor agonist is (1R,2S)-N-(4-(2,6-dimethoxyphenyl)-5-(5-methyl-3-pyridyl)-4H-1,2,4-triazol-3-yl)-1-methoxy-1-(5-methyl-2-pyrimidinyl)-2-propanesulfonamide, or a pharmaceutically acceptable salt thereof, its tautomer, a pharmaceutically acceptable salt of the tautomer, a stereoisomer of any of the foregoing, or a mixture thereof.

[0293] In one specific embodiment of formulas (I) and (II), the eparineptide receptor agonist is (1R,2S)-N-(4-(2,6-dimethoxyphenyl)-5-(5-methyl-3-pyridyl)-4H-1,2,4-triazol-3-yl)-1-hydroxy-1-(5-methyl-2-pyrimidinyl)-2-propanesulfonamide, or a pharmaceutically acceptable salt thereof, its tautomer, a pharmaceutically acceptable salt of the tautomer, a stereoisomer of any of the foregoing, or a mixture thereof.

[0294] In one specific embodiment of formulas (I) and (II), the eparineptide receptor agonist is (1S,2R)-1-(5-chloro-2-pyrimidinyl)-N-(4-(2,6-dimethoxyphenyl)-5-(5-methyl-3-pyridyl)-4H-1,2,4-triazol-3-yl)-1-methoxy-2-propanesulfonamide, or a pharmaceutically acceptable salt thereof, its tautomer, a pharmaceutically acceptable salt of the tautomer, a stereoisomer of any of the foregoing, or a mixture thereof.

[0295] In one specific embodiment of formulas (I) and (II), the eparineptide receptor agonist is (1S,2R)-N-(4-(2,6-dimethoxyphenyl)-5-(5-methyl-3-pyridyl)-4H-1,2,4-triazol-3-yl)-1-methoxy-1-(5-methyl-2-pyrazinyl)-2-propanesulfonamide, or a pharmaceutically acceptable salt thereof, a tautomer thereof, a pharmaceutically acceptable salt of the tautomer, a stereoisomer of any of the foregoing, or a mixture thereof.

[0296] In one specific embodiment of formulas (I) and (II), the eparineptide receptor agonist is (1R,2S)-N-(4-(2,6-dimethoxyphenyl)-5-(5-methyl-3-pyridyl)-4H-1,2,4-triazol-3-yl)-1-hydroxy-1-(5-methyl-2-pyrazinyl)-2-propanesulfonamide, or a pharmaceutically acceptable salt thereof, a tautomer thereof, a pharmaceutically acceptable salt of the tautomer, a stereoisomer of any of the foregoing, or a mixture thereof.

[0297] In one specific embodiment of formulas (I) and (II), the eparineptide receptor agonist is (1R,2S)-N-(4-(2,6-dimethoxyphenyl)-5-(3-pyridyl)-4H-1,2,4-triazol-3-yl)-1-methoxy-1-(5-methyl-2-pyrimidinyl)-2-propanesulfonamide, or a pharmaceutically acceptable salt thereof, a tautomer thereof, a pharmaceutically acceptable salt of the tautomer, a stereoisomer of any of the foregoing, or a mixture thereof.

[0298] In one specific embodiment of formulas (I) and (II), the eparineptide receptor agonist is (2S,3R)-N-(4-(2,6-dimethoxyphenyl)-5-(3-pyridyl)-4H-1,2,4-triazol-3-yl)-3-(5-methyl-2-pyrimidinyl)-2-butanesulfonamide, or a pharmaceutically acceptable salt thereof, a tautomer thereof, a pharmaceutically acceptable salt of the tautomer, a stereoisomer of any of the foregoing, or a mixture thereof.

[0299] In one specific embodiment of formulas (I) and (II), the eparineptide receptor agonist is (1R,2S)-1-(5-chloro-2-pyrimidinyl)-N-(4-(2,6-dimethoxyphenyl)-5-(3-pyridyl)-4H-1,2,4-triazol-3-yl)-1-ethoxy-2-propanesulfonamide, or a pharmaceutically acceptable salt thereof, a tautomer thereof, a pharmaceutically acceptable salt of the tautomer, a stereoisomer of any of the foregoing, or a mixture thereof.

[0300] In a specific embodiment of formulas (I) and (II), the epalrestat receptor agonist is (1R,2S)-N-(4-(2,6-dimethoxyphenyl)-5-(5-methyl-3-pyridinyl)-4H-1,2,4-triazol-3-yl)-1-ethoxy-1-(5-methyl-2-pyrimidinyl)-2-propanesulfonamide, or a pharmaceutically acceptable salt thereof, a tautomer thereof, a pharmaceutically acceptable salt of the tautomer, a stereoisomer of any of the foregoing, or a mixture thereof.

[0301] In a specific embodiment of formulas (I) and (II), the epalrestat receptor agonist is (1S,2R)-N-(4-(2,6-dimethoxyphenyl)-5-(3-pyridinyl)-4H-1,2,4-triazol-3-yl)-1-methoxy-1-(5-methyl-2-pyrazinyl)-2-propanesulfonamide, or a pharmaceutically acceptable salt thereof, a tautomer thereof, a pharmaceutically acceptable salt of the tautomer, a stereoisomer of any of the foregoing, or a mixture thereof.

[0302] In a specific embodiment of formulas (I) and (II), the epalrestat receptor agonist is (1R,2S)-N-(4-(2,6-dimethoxyphenyl)-5-(6-methyl-2-pyridinyl)-4H-1,2,4-triazol-3-yl)-1-hydroxy-1-(5-methyl-2-pyrimidinyl)-2-propanesulfonamide, or a pharmaceutically acceptable salt thereof, a tautomer thereof, a pharmaceutically acceptable salt of the tautomer, a stereoisomer of any of the foregoing, or a mixture thereof.

[0303] In a specific embodiment of formulas (I) and (II), the epalrestat receptor agonist is (1R,2S)-N-(4-(2,6-dimethoxyphenyl)-5-(3-pyridinyl)-4H-1,2,4-triazol-3-yl)-1-ethoxy-1-(5-methyl-2-pyrimidinyl)-2-propanesulfonamide, or a pharmaceutically acceptable salt thereof, a tautomer thereof, a pharmaceutically acceptable salt of the tautomer, a stereoisomer of any of the foregoing, or a mixture thereof.

[0304] In a specific embodiment of formulas (I) and (II), the epalrestat receptor agonist is (1R,2S)-N-(4-(2,6-dimethoxyphenyl)-5-(5-methyl-3-pyridinyl)-4H-1,2,4-triazol-3-yl)-1-(5-fluoro-2-pyrimidinyl)-1-methoxy-2-propanesulfonamide, or a pharmaceutically acceptable salt thereof, a tautomer thereof, a pharmaceutically acceptable salt of the tautomer, a stereoisomer of any of the foregoing substances, or a mixture thereof.

[0305] In a specific embodiment of formulas (I) and (II), the eparineptide receptor agonist is (2S,3R)-N-(4-(2,6-dimethoxyphenyl)-5-(5-methyl-3-pyridinyl)-4H-1,2,4-triazol-3-yl)-3-(5-methyl-2-pyrazinyl)-2-butenesulfonamide, or a pharmaceutically acceptable salt thereof, a tautomer thereof, a pharmaceutically acceptable salt of the tautomer, a stereoisomer of any of the foregoing, or a mixture thereof.

[0306] In a specific embodiment of formulas (I) and (II), the eparineptide receptor agonist is (1R,2S)-N-(4-(2,6-dimethoxyphenyl)-5-(3-pyridinyl)-4H-1,2,4-triazol-3-yl)-1-ethoxy-1-(5-fluoro-2-pyrimidinyl)-2-propanesulfonamide, or a pharmaceutically acceptable salt thereof, a tautomer thereof, a pharmaceutically acceptable salt of the tautomer, a stereoisomer of any of the foregoing, or a mixture thereof.

[0307] In a specific embodiment of formulas (I) and (II), the eparineptide receptor agonist is (1S,2S)-N-(4-(2,6-dimethoxyphenyl)-5-(5-methyl-3-pyridinyl)-4H-1,2,4-triazol-3-yl)-1-(1-methylethoxy)-1-(5-methyl-2-pyrimidinyl)-2-propanesulfonamide, or a pharmaceutically acceptable salt thereof, a tautomer thereof, a pharmaceutically acceptable salt of the tautomer, a stereoisomer of any of the foregoing, or a mixture thereof.

[0308] In a specific embodiment of formulas (I) and (II), the eparineptide receptor agonist is (1R,2S)-N-(4-(2,6-dimethoxyphenyl)-5-(5-methyl-3-pyridinyl)-4H-1,2,4-triazol-3-yl)-1-(1-methylethoxy)-1-(5-methyl-2-pyrimidinyl)-2-propanesulfonamide, or a pharmaceutically acceptable salt thereof, a tautomer thereof, a pharmaceutically acceptable salt of the tautomer, a stereoisomer of any of the foregoing, or a mixture thereof.

[0309] In a specific embodiment of formulas (I) and (II), the eparineptide receptor agonist is (1S,2R)-1-(5-chloro-2-pyrimidinyl)-N-(4-(2,6-dimethoxyphenyl)-5-(3-pyridinyl)-4H-1,2,4-triazol-3-yl)-1-methoxy-2-propanesulfonamide, or a pharmaceutically acceptable salt thereof, a tautomer thereof, a pharmaceutically acceptable salt of the tautomer, a stereoisomer of any of the foregoing, or a mixture thereof.

[0310] In one specific embodiment of formulas (I) and (II), the apelin receptor agonist is (1R,2S)-N-(4-(2,6-dimethoxyphenyl)-5-(5-methyl-3-pyridyl)-4H-1,2,4-triazol-3-yl)-1-methoxy-1-(5-methoxy-2-pyrazinyl)-2-propanesulfonamide, or a pharmaceutically acceptable salt thereof, a tautomer thereof, a pharmaceutically acceptable salt of the tautomer, a stereoisomer of any of the foregoing, or a mixture thereof.

[0311] In one specific embodiment of formulas (I) and (II), the apelin receptor agonist is (2S,3R)-N-(4-(2,6-dimethoxyphenyl)-5-(3-pyridyl)-4H-1,2,4-triazol-3-yl)-3-(5-methyl-2-pyrazinyl)-2-butanesulfonamide, or a pharmaceutically acceptable salt thereof, a tautomer thereof, a pharmaceutically acceptable salt of the tautomer, a stereoisomer of any of the foregoing, or a mixture thereof.

[0312] In one specific embodiment of formulas (I) and (II), the apelin receptor agonist is (1R,2S)-N-(4-(2,6-dimethoxyphenyl)-5-(5-methyl-3-pyridyl)-4H-1,2,4-triazol-3-yl)-1-ethoxy-1-(5-fluoro-2-pyrimidinyl)-2-propanesulfonamide, or a pharmaceutically acceptable salt thereof, a tautomer thereof, a pharmaceutically acceptable salt of the tautomer, a stereoisomer of any of the foregoing, or a mixture thereof.

[0313] In one specific embodiment of formulas (I) and (II), the apelin receptor agonist is (1R,2S)-N-(4-(4,6-dimethoxy-5-pyrimidinyl)-5-(6-methoxy-2-pyridyl)-4H-1,2,4-triazol-3-yl)-1-methoxy-1-(5-methyl-2-pyrimidinyl)-2-propanesulfonamide, or a pharmaceutically acceptable salt thereof, a tautomer thereof, a pharmaceutically acceptable salt of the tautomer, a stereoisomer of any of the foregoing, or a mixture thereof.

[0314] In one specific embodiment of formulas (I) and (II), the apelin receptor agonist is (1R,2R)-1-(5-chloro-2-pyrimidinyl)-N-(4-(2,6-dimethoxyphenyl)-5-(3-pyridyl)-4H-1,2,4-triazol-3-yl)-1-ethoxy-2-propanesulfonamide, or a pharmaceutically acceptable salt thereof, a tautomer thereof, a pharmaceutically acceptable salt of the tautomer, a stereoisomer of any of the foregoing, or a mixture thereof.

[0315] In a specific embodiment of formulas (I) and (II), the epalrestat receptor agonist is (1S,2S)-N-(4-(2,6-dimethoxyphenyl)-5-(5-methyl-3-pyridyl)-4H-1,2,4-triazol-3-yl)-1-ethoxy-1-(5-methyl-2-pyrimidinyl)-2-propanesulfonamide, or a pharmaceutically acceptable salt thereof, a tautomer thereof, a pharmaceutically acceptable salt of the tautomer, a stereoisomer of any of the foregoing, or a mixture thereof.

[0316] In a specific embodiment of formulas (I) and (II), the epalrestat receptor agonist is (1R,2S)-N-(4-(2,6-difluorophenyl)-5-(6-methoxy-2-pyridyl)-4H-1,2,4-triazol-3-yl)-1-methoxy-1-(5-methyl-2-pyrimidinyl)-2-propanesulfonamide, or a pharmaceutically acceptable salt thereof, a tautomer thereof, a pharmaceutically acceptable salt of the tautomer, a stereoisomer of any of the foregoing, or a mixture thereof.

[0317] In a specific embodiment of formulas (I) and (II), the epalrestat receptor agonist is (1R,2S)-N-(4-(4,6-dimethoxy-5-pyrimidinyl)-5-(2-pyridyl)-4H-1,2,4-triazol-3-yl)-1-methoxy-1-(5-methyl-2-pyrimidinyl)-2-propanesulfonamide, or a pharmaceutically acceptable salt thereof, a tautomer thereof, a pharmaceutically acceptable salt of the tautomer, a stereoisomer of any of the foregoing, or a mixture thereof.

[0318] In a specific embodiment of formulas (I) and (II), the epalrestat receptor agonist is N-(4-(2,6-dimethoxyphenyl)-5-(5-methyl-3-pyridyl)-4H-1,2,4-triazol-3-yl)-1-isopropoxy-1-(5-methyl-2-pyrimidinyl)-2-propanesulfonamide, or a pharmaceutically acceptable salt thereof, a tautomer thereof, a pharmaceutically acceptable salt of the tautomer, a stereoisomer of any of the foregoing, or a mixture thereof.

[0319] In a specific embodiment of formulas (I) and (II), the epalrestat receptor agonist is (1S,2S)-N-(4-(2,6-dimethoxyphenyl)-5-(5-methyl-3-pyridyl)-4H-1,2,4-triazol-3-yl)-1-isopropoxy-1-(5-methyl-2-pyrimidinyl)-2-propanesulfonamide, or a pharmaceutically acceptable salt thereof, a tautomer thereof, a pharmaceutically acceptable salt of the tautomer, a stereoisomer of any of the foregoing, or a mixture thereof.

[0320] In one specific embodiment of formulas (I) and (II), the eparineptide receptor agonist is N-(4-(2,6-dimethoxyphenyl)-5-(5-methyl-3-pyridyl)-4H-1,2,4-triazol-3-yl)-3-(5-methyl-2-pyrimidinyl)-2-butenesulfonamide, or a pharmaceutically acceptable salt thereof, a tautomer thereof, a pharmaceutically acceptable salt of the tautomer, a stereoisomer of any of the foregoing, or a mixture thereof.

[0321] In one particular embodiment of formulas (I) and (II), the eparineptide receptor agonist is (2S,3R)-N-(4-(2,6-dimethoxyphenyl)-5-(5-methyl-3-pyridyl)-4H-1,2,4-triazol-3-yl)-3-(5-methyl-2-pyrimidinyl)-2-butenesulfonamide (BGE-105) or a pharmaceutically acceptable salt thereof.

[0322] In one specific embodiment of formulas (I) and (II), the eparineptide receptor agonist is

[0323]

[0324] (BGE-105) or a pharmaceutically acceptable salt thereof.

[0325] U.S. Patent Nos. 9,573,936, 9,868,721, 9,745,286, 9,656,997, 9,751,864, 9,656,998, 9,845,310, 10,058,550, 10,221,162 and 10,344,016 (the disclosures of which are incorporated herein by reference in their entireties) describe eparineptide receptor agonists of formula (I) or (II), and methods for synthesizing such triazole agonists of eparineptide receptors, including BGE-105. See, for example, Example 263.0 of U.S. Patent No. 9,573,936.

[0326] If any variable occurs more than once in a chemical formula, its definition at each occurrence is independent of its definition at all other occurrences. If a chemical structure and a chemical name conflict, the chemical structure determines the identity of the compound. The compounds of the present disclosure may contain one or more chiral centers and / or double bonds and thus may exist as stereoisomers, such as double bond isomers (i.e., geometric isomers), enantiomers, or diastereomers. Accordingly, any chemical structure described in the specification in relative configuration, whether in whole or in part, encompasses all possible enantiomers and stereoisomers of the depicted compound, including stereoisomerically pure forms (e.g., geometrically pure, enantiomerically pure, or diastereomerically pure) and mixtures of enantiomers and stereoisomers. Mixtures of enantiomers and stereoisomers can be resolved into the component enantiomers or stereoisomers using separation techniques or chiral synthesis techniques well known to those skilled in the art.

[0327] Certain compounds of the present disclosure may have asymmetric carbon atoms (optical centers) or double bonds; racemates, enantiomers, diastereomers, geometric isomers, and individual isomers are all intended to be encompassed within the scope of the present invention. In addition, atropisomers and mixtures thereof, such as those resulting from restricted rotation about two aromatic or heteroaromatic rings bonded to each other, are also intended to be encompassed within the scope of the present invention. For example, when R 4 is phenyl and is substituted by groups bonded to two C atoms adjacent to the point of attachment of the N atom of the triazole, rotation of the phenyl group may be restricted. In some cases, the rotational barrier is high enough that different atropisomers can be separated and isolated.

[0328] Unless otherwise indicated, the term "stereoisomer" or "stereoisomerically pure" refers to a stereoisomer of a compound that is substantially free of other stereoisomers of that compound. For example, a stereoisomerically pure compound having one chiral center will be substantially free of the opposite enantiomer of that compound. A stereoisomerically pure compound having two chiral centers is substantially free of other diastereomers of that compound. A typical stereoisomerically pure compound contains greater than about 80% by weight of one stereoisomer of the compound and less than about 20% by weight of other stereoisomers of that compound, more preferably greater than about 90% by weight of one stereoisomer of the compound and less than about 10% by weight of other stereoisomers of that compound, even more preferably greater than about 95% by weight of one stereoisomer of the compound and less than about 5% by weight of other stereoisomers of that compound, and most preferably greater than about 97% by weight of one stereoisomer of the compound and less than about 3% by weight of other stereoisomers of that compound. If the stereochemistry of a structure or a portion of a structure is not indicated, for example, by bold or dashed lines, then the structure or the portion of the structure is to be interpreted as encompassing all of its stereoisomers. A bond drawn with a wavy line represents a mixture of two stereoisomers.

[0329] The various compounds of the present disclosure contain one or more chiral centers and may exist as racemic mixtures of enantiomers, mixtures of diastereomers, or enantiomers or optically pure compounds. The present invention encompasses the use of stereoisomerically pure forms of such compounds, as well as the use of mixtures of these forms. For example, mixtures containing equal or unequal amounts of the enantiomers of a particular compound of the present invention can be used in the methods and compositions of the present invention. These isomers can be synthesized asymmetrically or resolved using standard techniques (e.g., chiral columns or chiral resolving agents).

[0330] The compounds of the present disclosure include, but are not limited to, compounds of Formula I and all pharmaceutically acceptable forms thereof. Pharmaceutically acceptable forms of the compounds described herein include pharmaceutically acceptable salts, solvates, crystal forms (including polymorphs and clathrates), chelates, non-covalent complexes, prodrugs, and mixtures thereof. In certain embodiments, the compounds described herein are in the form of a pharmaceutically acceptable salt. The term "compound" includes not only the compound itself, but also its pharmaceutically acceptable salts, its solvates, its chelates, its non-covalent complexes, its prodrugs, and any mixtures of the foregoing. In some embodiments, the term "compound" encompasses the compound itself, its pharmaceutically acceptable salt, a tautomer of the compound, a pharmaceutically acceptable salt of the tautomer, and an ester prodrug such as a (C1-C4) alkyl ester. In other embodiments, the term "compound" encompasses the compound itself, its pharmaceutically acceptable salt, a tautomer of the compound, and a pharmaceutically acceptable salt of the tautomer.

[0331] The term "solvate" refers to a compound formed by the interaction of a solvent with a compound. Suitable solvates are pharmaceutically acceptable solvates, such as hydrates, including monohydrates and hemihydrates.

[0332] The compounds of the present disclosure may also contain unnatural proportions of atomic isotopes on one or more atoms constituting such compounds. For example, the compounds may be radiolabeled with a radioactive isotope, such as, for example, tritium ( 3 H), iodine-125 ( 125 I), or carbon-14 ( 14 C). The radiolabeled compounds can be used as therapeutic or prophylactic agents, research reagents (such as assay reagents), and diagnostic agents (such as in vivo imaging agents). All isotopic variants of the compounds of the present invention, whether radioactive or not, are intended to be encompassed within the scope of the present invention. For example, if a variable is said to be or shown as H, this means that the variable may also be deuterium (D) or tritium (T).

[0333] The term "pharmaceutically acceptable salt" refers to a salt that is acceptable for administration to a subject. Examples of pharmaceutically acceptable salts include, but are not limited to: mineral acid salts, such as hydrochloride, hydrobromide, hydroiodide, phosphate, sulfate, and nitrate; sulfonate salts, such as methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and trifluoromethanesulfonate; organic acid salts, such as oxalate, tartrate, citrate, maleate, succinate, acetate, trifluoroacetate, benzoate, mandelate, ascorbate, lactate, gluconate, and malate; amino acid salts, such as glycinate, lysinate, arginate, ornithinate, glutamate, and aspartate; inorganic salts, such as lithium salt, sodium salt, potassium salt, calcium salt, and magnesium salt; and salts with organic bases, such as ammonium salt, triethylamine salt, diisopropylamine salt, and cyclohexylamine salt. The term "salt" as used herein encompasses hydrated salts.

[0334] Other examples of pharmaceutically acceptable salts include anions of the compounds of the present disclosure complexed with suitable cations. For therapeutic use, the salts of the compounds of the present disclosure may be pharmaceutically acceptable. However, salts of non-pharmaceutically acceptable acids and bases may also be used, for example, in the preparation or purification of pharmaceutically acceptable compounds.

[0335] The essentially basic compounds contained in the disclosed compositions and methods are capable of forming a variety of salts with various inorganic and organic acids. The acids useful for preparing pharmaceutically acceptable acid addition salts of such basic compounds are those that form non-toxic acid addition salts, i.e., salts containing pharmacologically acceptable anions, including but not limited to: malates, oxalates, chloride salts, bromide salts, iodide salts, nitrates, sulfates, bisulfates, phosphates, acid phosphates, isonicotinate salts, acetates, lactates, salicylates, citrates, tartrates, oleates, tannates, pantothenates, bitartrates, ascorbates, succinates, maleates, gentisates, fumarates, gluconates, glucuronates, galacturonates, formates, benzoates, glutamates, mesylates, esylates, benzenesulfonates, p-toluenesulfonates, and pamoates (i.e., 1,1'-methylene-bis-(2-hydroxy-3-naphthoates)).

[0336] The acidic delivery compounds contained in the disclosed compositions and methods are capable of forming basic salts with various pharmacologically acceptable cations. Examples of such salts include alkali metal or alkaline earth metal salts, especially calcium, magnesium, sodium, lithium, zinc, potassium, and iron salts.

[0337] Additionally, if the compounds or their salts of the present invention form hydrates or solvates, these are also included within the scope of the compounds or their salts of the present invention.

[0338] Compounds containing basic or acidic moieties contained in the compositions and methods of the present invention can also form pharmaceutically acceptable salts with various amino acids. The disclosed compounds can contain both acidic and basic groups; for example, an amino group and a carboxylic acid group. In such cases, the compounds can exist in the form of acid addition salts, zwitterions, or base salts.

[0339] 5.4.1. Pharmaceutical Compositions

[0340] The apelin receptor agonist compounds used in the methods described herein can be formulated in any suitable pharmaceutical composition for administration by any suitable route of administration. The pharmaceutical composition can include a compound or its pharmaceutically acceptable salt, its tautomer, a pharmaceutically acceptable salt of the tautomer, a stereoisomer of any of the foregoing, or a mixture thereof according to any embodiment described herein, and at least one pharmaceutically acceptable excipient, carrier, or diluent. In some such embodiments, the compound or its pharmaceutically acceptable salt, its tautomer, a pharmaceutically acceptable salt of the tautomer, a stereoisomer of any of the foregoing, or a mixture thereof is present in an amount effective to treat a muscle disorder (e.g., as described herein) to activate the APJ receptor.

[0341] Suitable routes of administration include, but are not limited to, oral, topical, and intravenous routes of administration. Suitable routes also include pulmonary administration, including by oral inhalation. The most suitable route may depend on the condition and state of the recipient. The formulation may conveniently be presented in unit dosage form and may be prepared by any method known in the pharmaceutical art.

[0342] In some embodiments, the pharmaceutical composition is formulated for oral delivery, while in other embodiments, the pharmaceutical composition is formulated for intravenous delivery. In some embodiments, the pharmaceutical composition is formulated for once-daily or QD oral administration and, in some such formulations, is a tablet, wherein the effective amount of the active ingredient ranges from 5 mg to 60 mg, 6 mg to 58 mg, 10 mg to 40 mg, 15 mg to 30 mg, 16 mg to 25 mg, or 17 mg to 20 mg. In some such compositions, the amount of the active ingredient is 17 mg.

[0343] All methods include the step of combining an eparineptide agonist or a salt thereof with a carrier consisting of one or more excipients. Generally, the formulation is prepared by uniformly and intimately mixing the active ingredient with a liquid carrier or a finely divided solid carrier or both, and then, if necessary, shaping the product into the desired formulation.

[0344] In certain embodiments, the route of administration for the methods described herein is parenteral administration. In certain embodiments, the route of administration for the methods described herein is intravenous administration (e.g., intravenous infusion). In certain embodiments, the route of administration for the methods described herein is oral administration. In certain embodiments, the route of administration for the methods described herein is continuous intravenous infusion.

[0345] Formulations of the methods of the invention suitable for oral administration may: exist as discrete units, such as capsules, cachets, or tablets, each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or suspension in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. The active ingredient may also be presented as a bolus, electuary, or paste.

[0346] Formulations for parenteral administration include aqueous and non-aqueous sterile injection solutions, which may contain antioxidants, buffers, bacteriostatic agents, and solutes that render the formulation isotonic with the blood of the intended recipient. Formulations for parenteral administration also include aqueous and non-aqueous sterile suspensions, which may include suspending agents and thickening agents. The formulations may be presented in unit-dose multi-dose containers, such as sealed ampoules and vials, and may be stored under lyophilized (freeze-dried) conditions, requiring only the immediate addition of a sterile liquid carrier, such as saline, phosphate-buffered saline (PBS), etc., prior to use. Temporary injection solutions and suspensions may be prepared from the sterile powders, granules, and tablets described above.

[0347] The pharmaceutical composition may comprise one or more pharmaceutical excipients. Any suitable pharmaceutical excipients can be used, and those of ordinary skill in the art are capable of selecting suitable pharmaceutical excipients. Pharmaceutical excipients include, for example, those described in the Handbook of Pharmaceutical Excipients, 8th Edition, Revised (2017).

[0348] 5.4.2. Administration Regimen

[0349] In various embodiments, an apelin receptor agonist (e.g., as described herein) is administered in a dose (e.g., as described herein) sufficient to treat age-related muscle disorders.

[0350] In various embodiments, an apelin receptor agonist (e.g., as described herein) is administered in a method for maintaining and / or increasing muscle mass and / or muscle strength in elderly subjects. In some embodiments, the elderly subject is human and is at least 50 years old, at least 55 years old, at least 60 years old, or at least 65 years old.

[0351] In various embodiments, the dose of the apelin receptor agonist is at least 0.01 mg / kg, such as at least 0.5 mg / kg or at least 1 mg / kg. In certain embodiments, the dose is 25 mg / kg to 1000 mg / kg per day. In certain embodiments, the dose is 25 mg / kg to 1500 mg / kg per day.

[0352] In some embodiments, the apelin receptor agonist is administered in a dose (a uniform dose) independent of the patient's body weight or surface area.

[0353] In various embodiments, the dosage is 1 - 5000 mg. In various embodiments, the dosage is 25 - 2000 mg. In some embodiments, the dosage is at least 60 mg, at least 100 mg, at least 120 mg, at least 140 mg, at least 160 mg, at least 180 mg, at least 200 mg, at least 220 mg, at least 240 mg, at least 260 mg, at least 280 mg, at least 300 mg, at least 320 mg, at least 340 mg, at least 360 mg, at least 380 mg, at least 400 mg, at least 420 mg, at least 440 mg, at least 460 mg, at least 480 mg, at least 500 mg, at least 520 mg, at least 550 mg, at least 580 mg, at least 600 mg, at least 650 mg, at least 700 mg, at least 750 mg, at least 800 mg, at least 850 mg, at least 900 mg, at least 950 mg, at least 1000 mg, at least 1100 mg, at least 1200 mg, at least 1300 mg, at least 1400 mg, at least 1440 mg or at least 100 mg. In various embodiments, the dosage is 25 - 2000 mg. In some embodiments, the dosage is at least 200 mg. In some embodiments, the dosage is at least 240 mg. In some embodiments, the dosage is at least 60 mg. In some embodiments, the dosage is at least 360 mg. In some embodiments, the dosage is at least 120 mg. In some embodiments, the dosage is at least 720 mg.

[0354] The eparifermin receptor agonist can be administered in a single dose or multiple doses.

[0355] In some embodiments, the dose is administered daily. In some embodiments, the dose is a single ascending dose (SAD). In some embodiments, the single ascending dose includes a first dose of at least 60 mg and a second dose of at least 360 mg. In some embodiments, the dose is a single ascending dose (SAD). In some embodiments, the single ascending dose includes a first dose of at least 120 mg and a second dose of at least 720 mg. In some embodiments, the dose is a single ascending dose (SAD). In some embodiments, the single ascending dose includes a first dose of at least 240 mg and a second dose of at least 1440 mg. In some embodiments, the dose is a single ascending dose (SAD). In some embodiments, the single ascending dose includes a first dose of at least 20 mg, at least 30 mg, at least 40 mg, at least 50 mg, at least 60 mg, at least 70 mg, at least 80 mg, at least 90 mg, at least 100 mg, at least 110 mg, at least 120 mg, at least 130 mg, at least 140 mg, at least 150 mg, at least 160 mg, at least 170 mg, at least 180 mg, at least 190 mg, at least 200 mg, at least 210 mg, at least 220 mg, at least 230 mg, at least 240 mg, at least 250 mg, at least 260 mg, at least 270 mg, or at least 280 mg, and a second dose of at least 20 mg, at least 30 mg, at least 40 mg, at least 50 mg, at least 60 mg, at least 70 mg, at least 80 mg, at least 90 mg, at least 100 mg, at least 110 mg, at least 120 mg, at least 130 mg, at least 140 mg, at least 150 mg, at least 160 mg, at least 170 mg, at least 180 mg, at least 190 mg, at least 200 mg, at least 210 mg, at least 220 mg, at least 230 mg, at least 240 mg, at least 250 mg, at least 260 mg, at least 270 mg, at least 280 mg, at least 290 mg, at least 300 mg, at least 310 mg, at least 320 mg, at least 330 mg, at least 340 mg, at least 350 mg, at least 360 mg, at least 400 mg, at least 450 mg, at least 500 mg, at least 550 mg, at least 600 mg, at least 700 mg, at least 800 mg, at least 900 mg, at least 1000 mg, at least 1100 mg, at least 1200 mg, at least 1300 mg, at least 1400 mg, at least 1500 mg, at least 1600 mg, at least 1700 mg, at least 1800 mg, at least 1900 mg, or at least 2000 mg.

[0356] In some embodiments, the dose is administered as multiple sub-doses that are equal or unequal. In some embodiments, the dose is administered as multiple doses. In some embodiments, the dose is administered intravenously for 1 hour per day for at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, or at least 10 days.

[0357] In certain embodiments, the dose is administered continuously (e.g., by intravenous infusion) for a period of time. In certain embodiments, the dose is administered as a loading intravenous infusion dose for a period of time (e.g., 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 1 hour, 2 hours, 3 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, or 10 hours). In certain embodiments, after the loading dose, the dose is administered as a maintenance intravenous infusion dose for a period of time (e.g., 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 1 hour, 2 hours, 3 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, or 48 hours). In certain embodiments, after a loading dose and a washout period of 24 hours or 48 hours, the dose is administered as a maintenance intravenous infusion dose for a period of time (e.g., 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 1 hour, 2 hours, 3 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, or 48 hours). In certain embodiments, after a first loading dose and a washout period of 24 hours or 48 hours, the dose is administered as a loading intravenous infusion dose for a period of time (e.g., 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 1 hour, 2 hours, 3 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, or 10 hours), and then as a maintenance dose for a period of time (e.g., 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, or 48 hours).

[0358] In certain embodiments, the apalutamide receptor agonist is administered as follows: by intravenous infusion at a loading dose for 1 hour; a washout period of 48 hours; and by intravenous infusion at a loading dose for 1 hour, followed by a maintenance dose by intravenous infusion for 22 hours. In certain embodiments, the apalutamide receptor agonist is administered as follows: by intravenous infusion at a loading dose for 1 hour, then a maintenance dose is administered by intravenous infusion for 119 hours.

[0359] In some embodiments, the apalutamide receptor agonist is administered orally, intravenously, intranasally, or intramuscularly. In some embodiments, the apalutamide receptor agonist is administered orally.

[0360] In some embodiments, the apalutamide receptor agonist is administered once monthly, twice monthly, three times monthly, every other week (qow), once weekly (qw), twice weekly (biw), three times weekly (tiw), four times weekly, five times weekly, six times weekly, every other day (qod), once daily (qd), twice daily (qid), or three times daily (tid) for a period of about one day to about one week, about two weeks to about four weeks, about one month to about two months, about two months to about four months, about four months to about six months, about six months to about eight months, about eight months to about one year, about one year to about two years, or about two years to about four years or longer. In some embodiments, the apalutamide receptor agonist is administered continuously for at least 10 minutes, at least 20 minutes, at least 30 minutes, at least 40 minutes, at least 50 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, at least 10 hours, at least 11 hours, at least 12 hours, at least 13 hours, at least 14 hours, at least 15 hours, at least 16 hours, at least 17 hours, at least 18 hours, at least 19 hours, at least 20 hours, at least 21 hours, at least 22 hours, at least 23 hours, at least 24 hours, at least 48 hours, at least 72 hours, at least 100 hours, at least 110 hours, at least 115 hours, at least 120 hours, or at least 125 hours.

[0361] 5.4.3. Dosage Form

[0362] In some embodiments, the eparineptide receptor modulator or a salt thereof is administered as a suspension. In other embodiments, the eparineptide receptor modulator or a salt thereof is administered as a solution. In some embodiments, the eparineptide receptor modulator or a salt thereof is administered in a solid dosage form. In certain embodiments, the solid dosage form is a capsule. In certain embodiments, the solid dosage form is a tablet. In specific embodiments, the eparineptide receptor modulator is in crystalline or amorphous form. In certain embodiments, the eparineptide receptor modulator is in amorphous form. In some embodiments, the eparineptide receptor modulator is an eparineptide receptor agonist.

[0363] In one of the methods, the eparineptide receptor modulator or a pharmaceutical composition comprising the same is administered intravenously, topically, orally, by inhalation, by infusion, by injection, intraperitoneally, intramuscularly, subcutaneously, intratympanically, by intra-articular administration, by intra-mammary administration, by topical administration or by absorption through the epithelial or mucocutaneous lining. In certain embodiments, the eparineptide receptor modulator or a pharmaceutical composition comprising the same is administered by intravenous infusion, in capsule form or as a tablet.

[0364] 5.5. Definitions

[0365] Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention belongs.

[0366] The terms "individual", "host" and "subject" are used interchangeably and refer to an animal to be treated, including but not limited to: humans and non-human primates; rodents, including rats and mice; bovines; equines; sheep; felines; and canines. "Mammal" refers to one or more members of any mammalian species. Non-human animal models, namely mammals, non-human primates, murine, rabbits, etc., can be used for experimental studies. The term "patient" refers to a human subject.

[0367] The term "modulator" refers to a compound or composition that modulates the level or activity or function of a target, which can be but is not limited to the eparineptide receptor. In some embodiments, the modulator compound can agonize or activate the target, such as the eparineptide receptor. An agonist or activator of a target can increase the level of activity or signal transduction associated with that target.

[0368] The terms "treatment", "treating" and their grammatical variants are used in the broadest sense in the clinical field. Thus, these terms do not require a cure or complete remission of the disease, and these terms encompass obtaining any clinically desired pharmacological and / or physiological effect, including improvement in physiological measures associated with "normal", non-pathological aging. Unless otherwise stated, "treatment" and "treating" do not cover prevention.

[0369] The phrase "therapeutically effective amount" means the amount of a compound that, when administered to a mammal or other subject for treating or preventing a disease, disorder, or condition, is sufficient to effect treatment of the disease, disorder, or condition. A "therapeutically effective amount" may vary depending on the compound, the disease and its severity, and the age, weight, etc. of the subject to be treated.

[0370] Ranges: Throughout this disclosure, various aspects of the invention are presented in range format. Ranges include the recited endpoints. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as a rigid limitation on the scope of the invention. Thus, the description of a range should be considered to specifically disclose all possible sub-ranges as well as the individual numerical values within that range. For example, a description of a range such as 1 to 6 should be considered to specifically disclose sub-ranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, and the individual numbers within that range, such as 1, 2, 3, 4, 5, 5, 3, and 6. This applies regardless of how broad the range is.

[0371] Unless specifically stated otherwise or apparent from the context, the term "or" as used herein should be understood to be inclusive.

[0372] Unless specifically stated otherwise or apparent from the context, the terms "a", "an", and "the" as used herein should be understood to be singular or plural. That is, the articles "a" and "an" are used herein to refer to one or more (i.e., at least one) grammatical objects of the article. For example, "an element" means one element or more than one element.

[0373] Unless specifically stated otherwise or apparent from the context, the term "about" as used herein should be understood to be within the normal tolerances in the art, e.g., within 2 standard deviations of the mean, and is intended to encompass variations of ±20% or ±10% of the recited value, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1%.

[0374] Unless otherwise stated or understood from the context, when percentages are provided regarding the amounts of components or substances in a composition, the percentages should be understood to be weight percentages.

[0375] It should be understood that the order of steps or the order of performing certain acts is not important so long as the present disclosure remains operable. In addition, two or more steps or acts may be performed simultaneously.

[0376] The terms "pharmaceutically acceptable excipient", "pharmaceutically acceptable diluent", "pharmaceutically acceptable carrier" and "pharmaceutically acceptable adjuvant" are used interchangeably and refer to an excipient, diluent, carrier or adjuvant used in the preparation of a pharmaceutical composition which is generally safe, non-toxic and neither biologically nor otherwise undesirable, and includes excipients, diluents, carriers and adjuvants acceptable for veterinary and human pharmaceutical use. The phrase "pharmaceutically acceptable excipient" includes one or more such excipients, diluents, carriers and / or adjuvants.

[0377] "Alkyl" refers to a saturated branched or straight-chain monovalent hydrocarbon group obtained by removing a hydrogen atom from a single carbon atom of a parent alkane. Typical alkyl groups include, but are not limited to: methyl, ethyl, propyl (e.g., propan-1-yl and propan-2-yl), butyl (e.g., butan-1-yl, butan-2-yl, 2-methylpropan-1-yl, 2-methylpropan-2-yl), tert-butyl, etc. In certain embodiments, the alkyl group contains 1 to 20 carbon atoms. In some embodiments, the alkyl group contains 1 to 10 carbon atoms or 1 to 6 carbon atoms, while in other embodiments, the alkyl group contains 1 to 4 carbon atoms. In still other embodiments, the alkyl group contains 1 or 2 carbon atoms. Branched alkyl groups contain at least 3 carbon atoms and typically contain 3 to 7 carbon atoms, or in some embodiments contain 3 to 6 carbon atoms. An alkyl group having 1 to 6 carbon atoms may be referred to as a (C1-C6) alkyl group, and an alkyl group having 1 to 4 carbon atoms may be referred to as a (C1-C4) alkyl group. This nomenclature may also be applied to alkyl groups having different numbers of carbon atoms. When the alkyl group is a further substituted substituent, the term "alkyl" may also be used, in which case the bond between the second hydrogen atom and the carbon atom of the alkyl substituent is replaced by a bond to another atom, such other atom being, for example but not limited to, a halogen, O, N or S atom. For example, the group -O-(C1-C6 alkyl)-OH would be considered such a group in which the -O atom is bonded to the C1-C6 alkyl group and one of the H atoms bonded to the C atom of the C1-C6 alkyl group is replaced by a bond to the O atom of the -OH group. Another example is that the group -O-(C1-C6 alkyl)-O-(C1-C6 alkyl) would be considered such a group in which the -O atom is bonded to the first C1-C6 alkyl group, and one of the H atoms bonded to the C atom of the first C1-C6 alkyl group is replaced by a bond to the second O atom, which second O atom is bonded to the second C1-C6 alkyl group.

[0378] "Alkenyl" refers to an unsaturated branched or straight-chain hydrocarbon group having at least one carbon-carbon double bond obtained by removing a hydrogen atom from a single carbon atom of a parent alkene. The group can be in the Z or E (cis or trans) form with respect to the double bond. Typical alkenyl groups include, but are not limited to: vinyl; propenyl, such as prop-1-en-1-yl, prop-1-en-2-yl, prop-2-en-1-yl (allyl), and prop-2-en-2-yl; butenyl, such as but-1-en-1-yl, but-1-en-2-yl, 2-methyl-prop-1-en-1-yl, but-2-en-1-yl, but-2-en-2-yl, but-1,3-dien-1-yl, and but-1,3-dien-2-yl; and the like. In certain embodiments, the alkenyl group has 2 to 20 carbon atoms, and in other embodiments, 2 to 6 carbon atoms. An alkenyl group having 2 to 6 carbon atoms can be referred to as a (C2-C6) alkenyl group.

[0379] "Alkynyl" refers to an unsaturated branched or straight-chain hydrocarbon having at least one carbon-carbon triple bond obtained by removing a hydrogen atom from a single carbon atom of a parent alkyne. Typical alkynyl groups include, but are not limited to: ethynyl, propynyl, butynyl, 2-pentynyl, 3-pentynyl, 2-hexynyl, 3-hexynyl, and the like. In certain embodiments, the alkynyl group has 2 to 20 carbon atoms, and in other embodiments, 2 to 6 carbon atoms. An alkynyl group having 2 to 6 carbon atoms can be referred to as a -(C2-C6) alkynyl group.

[0380] "Alkoxy" refers to the group -OR, where R represents an alkyl group as defined herein. Representative examples include, but are not limited to: methoxy, ethoxy, propoxy, butoxy, cyclohexyloxy, and the like. Typical alkoxy groups contain 1 to 10 carbon atoms, 1 to 6 carbon atoms, or 1 to 4 carbon atoms in the R group. An alkoxy group containing 1 to 6 carbon atoms can be designated as an -O-(C1-C6) alkyl or -O-(C1-C6 alkyl) group. In some embodiments, the alkoxy group can contain 1 to 4 carbon atoms and can be designated as an -O-(C1-C4) alkyl or -O-(C1-C4 alkyl) group.

[0381] "Aryl" refers to a monovalent aromatic hydrocarbon group obtained by removing a hydrogen atom from a single carbon atom of a parent aromatic ring system. Aryl encompasses monocyclic carbocyclic aromatic rings, such as benzene. Aryl also encompasses bicyclic carbocyclic aromatic ring systems, where each ring is aromatic, such as naphthalene. Thus, aryl can include fused ring systems, where each ring is a carbocyclic aromatic ring. In certain embodiments, the aryl group contains 6 to 10 carbon atoms. Such groups can be referred to as C6-C 10An aryl group. However, an aryl does not cover a heteroaryl in any way or overlap with a heteroaryl defined separately below. Thus, if one or more carbocyclic aromatic rings are fused to an aromatic ring containing at least one heteroatom, the resulting ring system is a heteroaryl, not an aryl as defined herein.

[0382] "Carbonyl" means the group -C(O) or -C(=O) group.

[0383] "Carboxyl" means the group -C(O)OH.

[0384] "Cyano" means the group -CN.

[0385] "Cycloalkyl" means a saturated cyclic alkyl group obtained by removing a hydrogen atom from a single carbon atom of a parent cycloalkane. Typical cycloalkyls include, but are not limited to, groups derived from cyclopropane, cyclobutane, cyclopentane, cyclohexane, cycloheptane, cyclooctane, etc. A cycloalkyl can be described by the number of carbon atoms in the ring. For example, a cycloalkyl having 3 to 7 ring members can be referred to as a (C3-C7) cycloalkyl, and a cycloalkyl having 4 to 7 ring members can be referred to as a (C4-C7) cycloalkyl. In certain embodiments, the cycloalkyl can be a (C3-C 10 ) cycloalkyl, a (C3-C8) cycloalkyl, a (C3-C7) cycloalkyl, a (C3-C6) cycloalkyl or a (C4-C7) cycloalkyl, and these can be referred to in alternative language as C3-C 10 cycloalkyl, C3-C8 cycloalkyl, C3-C 10 cycloalkyl, C3-C6 cycloalkyl or C4-C7 cycloalkyl.

[0386] "Heterocyclic group" means a cyclic group containing at least one saturated or unsaturated but non-aromatic cyclic ring. The heterocyclic group contains at least one heteroatom as a ring member. Typical heteroatoms include O, S and N, and are independently selected. Heterocyclic groups include monocyclic ring systems and bicyclic ring systems. Bicyclic heterocyclic groups include at least one non-aromatic ring having at least one heteroatom ring member, which can be fused to a cycloalkyl ring or can be fused to an aromatic ring, where the aromatic ring can be a carbocyclic ring or can contain one or more heteroatoms. The point of attachment of a bicyclic heterocyclic group can be on the non-aromatic cyclic ring containing at least one heteroatom, or on another ring of the heterocyclic group. For example, a heterocyclic group obtained by removing a hydrogen atom from one of the 9-membered heterocyclic compounds shown below can be attached to a 5-membered or 6-membered ring of the rest of the molecule.

[0387]

[0388] In some embodiments, the heterocyclic group contains 5 to 10 ring members, 1, 2, 3, or 4 of which, or 1, 2, or 3 of which are independently selected from the heteroatoms O, S, or N. In other embodiments, the heterocyclic group contains 3 to 7 ring members, 1, 2, or 3 of which are heteroatoms independently selected from O, S, or N. In such 3- to 7-membered heterocyclic groups, when the ring contains only 3 members, only 1 ring member is a heteroatom, and when the ring contains 4 members, it contains 1 or 2 heteroatoms. In some embodiments, the heterocyclic group contains 3 or 4 ring members, 1 of which is a heteroatom selected from O, S, or N. In other embodiments, the heterocyclic group contains 5 to 7 ring members, 1, 2, or 3 of which are independently selected from the heteroatoms O, S, or N. Typical heterocyclic groups include, but are not limited to, groups derived from epoxides, aziridines, azetidines, imidazolidines, morpholines, piperazines, piperidines, hexahydropyrimidines, 1,4,5,6-tetrahydropyrimidines, pyrazolidines, pyrrolidines, quinuclidines, tetrahydrofurans, tetrahydropyrans, benzimidazolone, pyridone, etc. Substituted heterocyclic groups also include ring systems substituted with one or more oxo(=O) or oxide (-O - ) substituents, such as piperidinyl N-oxide, morpholinyl-N-oxide, 1-oxo-1-thiomorpholinyl, pyridonyl, benzimidazolonyl, benz[d]oxazol-2(3H)-one, 3,4-dihydroisoquinolin-1(2H)-one, indolin-1-one, 1H-imidazo[4,5-c]pyridin-2(3H)-one, 7H-purin-8(9H)-one, imidazolidin-2-one, 1H-imidazol-2(3H)-one, 1,1-dioxo-1-thiomorpholinyl, etc.

[0389] "Halogenated" or "halogen" refers to a fluorine, chlorine, bromine, or iodine group.

[0390] "Haloalkyl" refers to an alkyl group in which at least one hydrogen is replaced by a halogen. Thus, the term "haloalkyl" includes monohaloalkyl (alkyl substituted by one halogen atom) and polyhaloalkyl (alkyl substituted by two or more halogen atoms). Representative "haloalkyls" include difluoromethyl, 2,2,2-trifluoroethyl, 2,2,2-trichloroethyl, etc. Unless otherwise specified, the term "perhaloalkyl" refers to an alkyl group in which each hydrogen atom is replaced by a halogen atom. For example, the term "perhaloalkyl" includes, but is not limited to, trifluoromethyl, pentachloroethyl, 1,1,1-trifluoro-2-bromo-2-chloroethyl, etc.

[0391] "Heteroaryl" refers to a monovalent heteroaromatic group obtained by removing a hydrogen atom from a single atom of a parent heteroaromatic ring system. Heteroaryl generally includes 5- to 14-membered, but more typically 5- to 10-membered, aromatic monocyclic, bicyclic, and tricyclic rings containing one or more (e.g., 1, 2, 3, or 4, or in certain embodiments, 1, 2, or 3) heteroatoms selected from O, S, or N, with the remaining ring members being carbon. In monocyclic heteroaryl, the monocyclic ring is aromatic and contains at least one heteroatom. In some embodiments, the monocyclic heteroaryl can include 5 or 6 ring members and can contain 1, 2, 3, or 4 heteroatoms, 1, 2, or 3 heteroatoms, 1 or 2 heteroatoms, or 1 heteroatom, where the heteroatoms are independently selected from O, S, or N. In bicyclic aromatic rings, both rings are aromatic. In bicyclic heteroaryl groups, at least one ring must contain a heteroatom, but it is not necessary for both rings to contain heteroatoms, although it is permitted for both rings to contain heteroatoms. For example, the term "heteroaryl" includes 5- to 7-membered heteroaromatic rings fused to a carbocyclic aromatic ring or to another heteroaromatic ring. In tricyclic aromatic rings, all three rings are aromatic and at least one ring contains at least one heteroatom. For fused bicyclic and tricyclic heteroaromatic ring systems where only one ring contains one or more heteroatoms, the point of attachment can be on the ring containing at least one heteroatom or on a carbocyclic ring. When the total number of S and O atoms in the heteroaryl exceeds 1, these heteroatoms are not adjacent to each other. In certain embodiments, the total number of S and O atoms in the heteroaryl does not exceed 2. In certain embodiments, the total number of S and O atoms in the aromatic heterocycle does not exceed 1. Heteroaryl does not encompass aryl as defined above or overlap with these aryl groups. Examples of heteroaryl include, but are not limited to, groups derived from acridine, carbazole, cinnoline, furan, imidazole, indazole, indole, indazine, isobenzofuran, isochromanone, isoindole, isoquinoline, isothiazole, 2H-benzo[d][1,2,3]triazole, isoxazole, naphthyridine, oxadiazole, oxazole, phthalazine, phenanthridine, phenanthroline, phenazine, phthalazine, pteridine, purine, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine, pyrrole, pyrrolizine, quinazoline, quinoline, quinazoline, quinoxaline, tetrazole, thiadiazole, thiazole, thiophene, triazole, etc. In certain embodiments, the heteroaryl can be a 5- to 20-membered heteroaryl, such as, for example, a 5- to 14-membered or 5- to 10-membered heteroaryl. In certain embodiments, the heteroaryl can be a heteroaryl derived from thiophene, pyrrole, benzothiophene, 2H-benzo[d][1,2,3]triazole, benzofuran, indole, pyridine, quinoline, imidazole, benzimidazole, oxazole, tetrazole, and pyrazine.

[0392] As described herein, the text relates to various embodiments of the compounds, compositions, and methods of the present invention. The various embodiments described are intended to provide various illustrative examples and should not be construed as an alternative to the description of the species. On the contrary, it should be noted that the descriptions of the various embodiments provided herein may have overlapping scopes. The embodiments discussed herein are merely illustrative and are not intended to limit the scope of the technology.

[0393] 6. Other Embodiments

[0394] Aspects of the present disclosure are further described in the numbered clauses below.

[0395] 1. A method of treating a muscle disorder in a subject, the method comprising administering to the subject in need thereof an effective dose of an apelin receptor agonist.

[0396] 2. The method according to clause 1, wherein the muscle disorder is an age-related muscle disorder.

[0397] 3. The method according to any one of clauses 1 to 2, wherein the subject is human and at least 40 years old.

[0398] 4. The method according to clause 3, wherein the subject is at least 50 years old.

[0399] 5. The method according to clause 4, wherein the subject is at least 60 years old.

[0400] 6. The method according to clause 5, wherein the subject is at least 65 years old.

[0401] 7. The method according to clause 6, wherein the subject is at least 70 years old.

[0402] 8. The method according to clause 7, wherein the subject is at least 75 years old.

[0403] 9. The method according to clause 8, wherein the subject is at least 80 years old.

[0404] 10. The method according to any one of clauses 1 to 9, wherein the muscle disorder is a skeletal muscle disorder.

[0405] 11. The method according to any one of clauses 1 to 10, wherein the muscle expresses the apelin receptor, and administering the apelin receptor agonist activates the apelin / APJ (APLNR) system in the muscle tissue of the subject.

[0406] 12. The method according to any one of clauses 1 to 11, wherein the muscle disorder is not a cardiovascular disorder.

[0407] 13. The method according to any one of clauses 1 to 11, wherein the subject does not have heart failure or is not at risk of heart failure.

[0408] 14. The method according to any one of clauses 1 to 13, wherein the muscle disorder is associated with inflammation and / or mitochondrial function impairment.

[0409] 15. The method according to any one of clauses 1 to 14, wherein the muscle disorder is associated with loss of muscle function, decreased muscle regeneration ability, or decreased healing ability after muscle injury.

[0410] 16. The method according to any one of clauses 1 to 15, wherein the muscle disorder is associated with loss of function of muscle stem cells.

[0411] 17. The method according to any one of clauses 1 to 16, wherein the muscle disorder is selected from sarcopenia, frailty, hip fracture, ICU-related myasthenia, mechanical ventilation-related myasthenia, immobility-related myasthenia, recovery from muscle injury, muscle atrophy, diaphragmatic atrophy, critical illness myopathy, and muscle wasting.

[0412] 18. The method according to any one of clauses 1 to 17, wherein the muscle disorder is associated with insulin insensitivity or resistance, or type 2 diabetes.

[0413] 19. The method according to any one of clauses 1 to 18, wherein the subject has or is determined to have low muscle strength or low muscle power.

[0414] 20. The method according to any one of clauses 17 to 19, wherein the subject has or is determined to have chronic obstructive pulmonary disease (COPD).

[0415] 21. The method according to any one of clauses 1 to 20, wherein the subject has or is determined to have low lower limb muscle mass.

[0416] 22. The method according to any one of clauses 1 to 21, wherein the subject has or is determined to have low upper limb muscle mass.

[0417] 23. The method according to any one of clauses 1 to 22, wherein the subject has or is determined to have low muscle volume.

[0418] 24. The method according to clause 23, wherein the muscle volume is skeletal muscle volume.

[0419] 25. The method according to clause 24, wherein the muscle is the tibialis anterior, tibialis posterior, gastrocnemius, sartorius, vastus intermedius, vastus lateralis, vastus medialis, soleus, rectus femoris, extensor digitorum longus, or diaphragm.

[0420] 26. The method according to any one of clauses 1 to 25, wherein the eparineptide receptor agonist is administered orally, intravenously, intranasally, or intramuscularly.

[0421] 27. The method according to any one of clauses 1 to 26, wherein the dose is administered daily.

[0422] 28. The method according to any one of clauses 1 to 27, wherein the dose is administered in multiple equal or unequal sub-doses.

[0423] 29. The method according to any one of clauses 1 to 28, wherein the dose is administered at varying dosing intervals.

[0424] 30. The method according to any one of clauses 1 to 29, wherein the dose is 200 mg.

[0425] 31. The method according to any one of clauses 1 to 30, further comprising assessing muscle mass after administration.

[0426] 32. The method according to clause 31, wherein the muscle mass is assessed at least one day after administration.

[0427] 33. The method according to clause 32, wherein the muscle mass is assessed at least one week or at least two weeks after administration.

[0428] 34. The method according to clause 33, wherein the muscle mass is assessed at least one month after administration.

[0429] 35. The method according to any one of clauses 1 to 34, wherein the subject has low circulating levels of eparineptide.

[0430] 36. A method of maintaining and / or increasing muscle mass and / or muscle strength in a human subject, the method comprising administering an effective dose of an eparineptide receptor agonist to a subject in need thereof.

[0431] 37. The method according to clause 36, wherein the subject is at least 60 years of age.

[0432] 38. The method according to clause 37, wherein the subject is at least 65 years of age.

[0433] 39. The method according to clause 38, wherein the subject is at least 70 years of age.

[0434] 40. The method according to clause 39, wherein the subject is at least 75 years of age.

[0435] 41. The method according to clause 40, wherein the subject is at least 80 years of age.

[0436] 42. The method according to any one of clauses 36 to 41, wherein the human subject has or is determined to have low muscle strength.

[0437] 43. The method according to any one of clauses 36 to 42, wherein the human subject has or is determined to have low muscle power.

[0438] 44. The method according to any one of clauses 36 to 43, wherein the human subject has or is determined to have low lower limb muscle mass.

[0439] 45. The method according to any one of clauses 36 to 44, wherein the human subject has or is determined to have low upper limb muscle mass.

[0440] 46. The method according to any one of clauses 36 to 45, wherein the human subject has or is determined to have low muscle volume.

[0441] 47. The method according to clause 46, wherein the muscle volume is skeletal muscle volume.

[0442] 48. The method according to clause 47, wherein the muscle is the diaphragm, tibialis anterior, tibialis posterior, gastrocnemius, sartorius, vastus intermedius, vastus lateralis, vastus medialis, soleus, rectus femoris or extensor digitorum longus.

[0443] 49. The method according to any one of clauses 47 to 48, wherein the muscle is skeletal muscle.

[0444] 50. The method according to any one of clauses 36 to 49, wherein the human subject is receiving mechanical ventilation.

[0445] 51. The method according to any one of clauses 36 to 50, wherein the human subject has or is determined to have a reduced diaphragm thickness compared to a human subject not receiving mechanical ventilation.

[0446] 52. The method according to any one of clauses 36 to 51, wherein the human subject has or is determined to have diaphragm atrophy.

[0447] 53. The method according to any one of clauses 36 to 52, wherein the human subject has or is determined to have ventilator-induced diaphragmatic dysfunction (VIDD).

[0448] 54. The method according to any one of clauses 36 to 53, wherein the human subject has or is determined to have hypoxic respiratory failure.

[0449] 55. The method according to any one of clauses 48 to 54, wherein the muscle expresses the apelin receptor.

[0450] 56. A method as described in any one of clauses 36 to 55, wherein the human subject has low circulating levels of apelin.

[0451] 57. A method as described in any one of clauses 36 to 56, wherein the apelin receptor agonist is administered orally, intravenously, intranasally, or intramuscularly.

[0452] 58. A method as described in any one of clauses 36 to 57, wherein the dose is administered daily.

[0453] 59. A method as described in any one of clauses 36 to 58, wherein the dose is administered in multiple equal or unequal sub - doses.

[0454] 60. A method as described in any one of clauses 36 to 59, wherein the dose is administered intravenously.

[0455] 61. A method as described in clause 60, wherein the dose is administered intravenously as a loading dose of at least 60 mg, followed by a maintenance dose of at least 360 mg.

[0456] 62. A method as described in clause 60, wherein the dose is administered intravenously as a loading dose of at least 120 mg, followed by a maintenance dose of at least 720 mg.

[0457] 63. A method as described in clause 60, wherein the dose is administered intravenously as a loading dose of at least 240 mg, followed by a maintenance dose of at least 1440 mg.

[0458] 64. A method as described in any one of clauses 61 to 63, wherein the loading dose is administered for at least 1 hour.

[0459] 65. A method as described in any one of clauses 61 to 64, wherein the maintenance dose is administered for at least 20 hours.

[0460] 66. A method as described in any one of clauses 61 to 64, wherein the maintenance dose is administered for at least 22 hours.

[0461] 67. A method as described in any one of clauses 61 to 64, wherein the maintenance dose is administered for at least 100 hours.

[0462] 68. A method as described in any one of clauses 36 to 60, wherein the dose is at least 60 mg.

[0463] 69. A method as described in any one of clauses 36 to 60, wherein the dose is at least 75 mg.

[0464] 70. The method according to any one of clauses 36 to 60, wherein the dose is at least 120 mg.

[0465] 71. The method according to any one of clauses 36 to 60, wherein the dose is at least 240 mg.

[0466] 72. The method according to any one of clauses 36 to 60, wherein the dose is at least 150 mg.

[0467] 73. The method according to any one of clauses 36 to 60, wherein the dose is at least 300 mg.

[0468] 74. The method according to any one of clauses 36 to 60, wherein the dose is at least 375 mg.

[0469] 75. The method according to any one of clauses 36 to 60, wherein the dose is 75 - 150 mg.

[0470] 76. The method according to any one of clauses 36 to 60, wherein the dose is 150 - 300 mg.

[0471] 77. The method according to any one of clauses 36 to 60, wherein the dose is 240 - 1440 mg.

[0472] 78. The method according to any one of clauses 36 to 60, wherein the dose is 75 mg.

[0473] 79. The method according to any one of clauses 36 to 60, wherein the dose is 150 mg.

[0474] 80. The method according to any one of clauses 36 to 60, wherein the dose is 240 mg.

[0475] 81. The method according to any one of clauses 36 to 60, wherein the dose is 300 mg.

[0476] 82. The method according to any one of clauses 36 to 60, wherein the dose is 375 mg.

[0477] 83. The method according to any one of clauses 36 to 60, wherein the dose is 450 mg.

[0478] 84. The method according to any one of clauses 36 to 60, wherein the dose is a single escalating dose of 60 mg / 360 mg.

[0479] 85. The method according to any one of clauses 36 to 60, wherein the dose is a single escalating dose of 120 mg / 720 mg.

[0480] 86. The method according to any one of clauses 36 to 60, wherein the dose is a single ascending dose of 240 mg / 1440 mg.

[0481] 87. The method according to any one of clauses 36 to 86, further comprising assessing muscle mass or muscle thickness after administration.

[0482] 88. The method according to clause 87, wherein the muscle mass is assessed at least one day after administration.

[0483] 89. The method according to clause 87, wherein the muscle mass is assessed at least one week after administration.

[0484] 90. The method according to clause 88, wherein the muscle mass is assessed at least one month after administration.

[0485] 91. The method according to any one of clauses 1 to 90, wherein the apelin receptor agonist is of formula (I) or (II):

[0486]

[0487] or a pharmaceutically acceptable salt thereof, a tautomer thereof, a pharmaceutically acceptable salt of the tautomer, a stereoisomer of any of the foregoing, or a mixture thereof,

[0488] wherein:

[0489] R 1 is an unsubstituted pyridyl, pyridone or pyridine N-oxide, or a pyridyl, pyridone or pyridine N-oxide substituted with 1, 2, 3 or 4 R 1a substituents;

[0490] R 1aindependently selected from -F, -Cl, -Br, -I, -CN, -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 perhaloalkyl, -OH, -O-(C1-C6 alkyl), -O-(C1-C6 haloalkyl), -O-(C1-C6 perhaloalkyl), -C2-C6 alkenyl, -O-(C1-C6 alkyl)-OH, -O-(C1-C6 alkyl)-O-(C1-C6 alkyl), -O-(C1-C6 haloalkyl)-OH, -O-(C1-C6 haloalkyl)-O-(C1-C6 alkyl), -O-(C1-C6 perhaloalkyl)-OH, -O-(C1-C6 perhaloalkyl)-O-(C1-C6 alkyl), -NH2, -NH(C1-C6 alkyl), -N(C1-C6 alkyl)2, -C(═O)-(C1-C6 alkyl), -C(═O)OH, -(C═O)-O-(C1-C6 alkyl), -C(═O)NH2, -C(═O)NH(C1-C6 alkyl), -C(═O)N(C1-C6 alkyl)2, phenyl, -C(═O)-(heterocyclic group) or a heterocyclic group, wherein the heterocyclic group of the -C(═O)-(heterocyclic group) or heterocyclic group is a 3- to 7-membered ring containing 1, 2 or 3 heteroatoms selected from N, O and S;

[0491] R 2 selected from -H and C1-C4 alkyl, or absent in the compound of formula II;

[0492] R 3 selected from unsubstituted C1-C 10 alkyl, C1-C 1a alkyl substituted with 1, 2 or 3 R 10 substituents, a group of formula -(CR 3b R 3c )-Q, a group of formula -NH-(CR 3b R 3c )-Q, a group of formula -(CR 3b R 3c )-C(═O)-Q, a group of formula -(CR 3d R 3e )-(CR 3f R 3g )-Q, a group of formula -(CR 3b ═CR 3c )-Q and a group of formula -(heterocyclic group)-Q, wherein the heterocyclic group of the -(heterocyclic group)-Q has 5 to 7 ring members in which 1, 2 or 3 are heteroatoms selected from N, O or S, and is unsubstituted or substituted with 1, 2 or 3 R 3h substituents;

[0493] R 1aindependently selected from -F, -Cl, -CN, -OH, -O-(C1-C6 alkyl), -O-(C1-C6 haloalkyl), -O-(C1-C6 perhaloalkyl), -O-(C1-C6 alkyl)-OH, -O-(C1-C6 alkyl)-O-(C1-C6 alkyl), C2-C6 alkenyl, C2-C6 alkynyl, -NH2, -NH(C1-C6 alkyl), and -N(C1-C6 alkyl)2 in each case independently;

[0494] R 3b and R 3c independently selected from -H, -F, -Cl, -CN, -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 perhaloalkyl, -OH, -O-(C1-C6 alkyl), -O-(C1-C6 haloalkyl), -O-(C1-C6 perhaloalkyl), -O-(C1-C6 alkyl)-OH, -O-(C1-C6 alkyl)-O-(C1-C6 alkyl), -NH2, -NH(C1-C6 alkyl), and -N(C1-C6 alkyl)2;

[0495] R 3d and R 3e independently selected from -H, -F, -Cl, -CN, -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 perhaloalkyl, -OH, -O-(C1-C6 alkyl), -O-(C1-C6 haloalkyl), -O-(C1-C6 perhaloalkyl), -O-(C1-C6 alkyl)-OH, -O-(C1-C6 alkyl)-O-(C1-C6 alkyl), -NH2, -NH(C1-C6 alkyl), and -N(C1-C6 alkyl)2;

[0496] R 3f and R 3g independently selected from -H, -F, -Cl, -CN, -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 perhaloalkyl, -OH, -O-(C1-C6 alkyl), -O-(C1-C6 haloalkyl), -O-(C1-C6 perhaloalkyl), -O-(C1-C6 alkyl)-OH, -O-(C1-C6 alkyl)-O-(C1-C6 alkyl), -NH2, -NH(C1-C6 alkyl), and -N(C1-C6 alkyl)2;

[0497] R 3hindependently selected from -F, -Cl, -CN, -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 perhaloalkyl, -OH, -O-(C1-C6 alkyl), -O-(C1-C6 haloalkyl), -O-(C1-C6 perhaloalkyl), -O-(C1-C6 alkyl)-OH, -O-(C1-C6 alkyl)-O-(C1-C6 alkyl), -NH2, -NH(C1-C6 alkyl), -N(C1-C6 alkyl)2 and oxo in each case;

[0498] Q is a monocyclic or bicyclic C6-C 10 aryl group, a monocyclic or bicyclic heteroaryl group having 5 to 10 ring members containing 1, 2 or 3 heteroatoms independently selected from N, O or S, a C3-C8 cycloalkyl group or a 3- to 7-membered heterocyclic group containing 1, 2 or 3 heteroatoms independently selected from N, O or S, wherein the C6-C 10 aryl group, the heteroaryl group, the cycloalkyl group and the heterocyclic group are unsubstituted or substituted by 1, 2, 3 or 4 R Q substituents;

[0499] R Q is independently selected from -F, -Cl, -Br, -I, -CN, -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 perhaloalkyl, -C2-C6 alkenyl, -C2-C6 alkynyl, -OH, -O-(C1-C6 alkyl), -O-(C1-C6 haloalkyl), -O-(C1-C6 perhaloalkyl), -NH2, -NH(C1-C6 alkyl), -N(C1-C6 alkyl)2, -C(═O)-(C1-C6 alkyl), -C(═O)OH, -C(═O)-O-(C1-C6 alkyl), -C(═O)NH2, -C(═O)NH(C1-C6 alkyl), -C(═O)N(C1-C6 alkyl)2, -S(═O)2-(C1-C6 alkyl), phenyl and heteroaryl in each case, and the Q heterocyclic group may be substituted by 1 oxo R Q substituent;

[0500] R 4 is selected from a monocyclic or bicyclic C6-C 10 aryl group, a monocyclic or bicyclic heteroaryl group having 5 to 10 ring members containing 1, 2 or 3 heteroatoms independently selected from N, O and S, and a monocyclic or bicyclic heterocyclic group having 5 to 10 ring members containing 1, 2, 3 or 4 heteroatoms independently selected from N, O and S, wherein the C6-C 10 aryl group, the heteroaryl group or the heterocyclic group is unsubstituted or substituted by 1, 2 or 3 R 4a substituents;

[0501] R 4a Independently selected from -F, -Cl, -Br, -I, -CN, -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 perhaloalkyl, -OH, -O-(C1-C6 alkyl), -O-(C1-C6 haloalkyl), -O-(C1-C6 perhaloalkyl), -NH2, -NH(C1-C6 alkyl), -N(C1-C6 alkyl)2, -C(═O)-(C1-C6 alkyl), -C(═O)OH, -C(═O)-O-(C1-C6 alkyl), -C(═O)NH2, -C(═O)NH(C1-C6 alkyl), and -C(═O)N(C1-C6 alkyl)2 in each case, and the heterocyclic group R 4 group may be further substituted by 1 oxo substituent; and

[0502] Further, wherein:

[0503] If R 4 is an unsubstituted or substituted benzene ring and R 3 is a group of formula -(CR 3b ═CR 3c )-Q, then at least one of the following is true:

[0504] a) R 4 is substituted by at least one -O-(C1-C6 alkyl) group;

[0505] b) Q is not oxadiazole;

[0506] c) R 3b is not -H;

[0507] d) R 3c is not -H;

[0508] e) R 1 is not 2-pyridyl; or

[0509] f) R 4 is substituted by two or more -O-(C1-C6 alkyl) groups.

[0510] 92. The method according to clause 91, wherein R 1 is an unsubstituted pyridyl or a pyridyl substituted by 1 or 2 R 1a substituents.

[0511] 93. The method according to any one of clauses 91 to 92, wherein R 1aIndependently selected in each case from: -CH3, -CH2CH3, -F, -Cl, -Br, -CN, -CF3, -CH═CH2, -C(═O)NH2, -C(═O)NH(CH3), -C(═O)N(CH3)2, -C(═O)NH(CH2CH3), -OH, -OCH3, -OCHF2, -OCH2CH3, -OCH2CF3, -OCH2CH2OH, -OCH2C(CH3)2OH, -OCH2C(CF3)2OH, -OCH2CH2OCH3, -NH2, -NHCH3, -N(CH3)2, phenyl, and a group of the following formula:

[0512] wherein the symbol when drawn across a bond, represents the point of attachment to the remainder of the molecule.

[0513] 94. The method according to any one of clauses 91 to 93, wherein R 1 is selected from:

[0514]

[0515] wherein the symbol when drawn across a bond, represents the point of attachment to the remainder of the molecule.

[0516] 95. The method according to any one of clauses 91 to 93, wherein R 2 is -H.

[0517] 96. The method according to any one of clauses 91 to 94, wherein R 4 is phenyl, azaphenyl, pyrimidinyl, isoxazolyl, indolyl, naphthyl, or pyridyl, any one of which may be unsubstituted or substituted with 1, 2, or 3 R 4a substituents.

[0518] 97. The method according to clause 96, wherein R 4 is phenyl substituted with 1 or 2 R 4a substituents.

[0519] 98. The method according to clause 97, wherein the 1 or 2 R 4a substituents are -O-(C1-C2 alkyl) groups.

[0520] 99. The method according to any one of clauses 1 to 97, wherein R 4a is independently selected in each case from -CH3, -F, -Cl, -Br, -CN, -CF3, -OCH3, -OCHF2, -OCH2CH3, -C(═O)OCH3, -C(═O)CH3, or -N(CH3)2.

[0521] 100. The method according to any one of clauses 91 to 99, wherein R 3 is selected from the group of formula -(CR 3b R 3c )-Q, the group of formula -NH-(CR 3b R 3c )-Q, the group of formula -(CR 3b R 3c )-C(═O)-Q, the group of formula -(CR 3d R 3e )-(CR 3f R 3g )-Q, the group of formula -(CR 3b ═CR 3c )-Q or the group of formula -(heterocyclic group)-Q, wherein the heterocyclic group of -(heterocyclic group)-Q has 5 to 7 ring members with 1, 2 or 3 of them being heteroatoms selected from N, O or S, and is unsubstituted or substituted by 1, 2 or 3 R 3h substituents.

[0522] 101. The method according to any one of clauses 91 to 100, wherein Q is selected from pyrimidinyl, pyridinyl, isoxazolyl, thiazolyl, imidazolyl, phenyl, tetrahydropyrimidinone, cyclopropyl, cyclobutyl, cyclohexyl, morpholinyl, pyrrolidinyl, pyrazinyl, imidazo[1,2-a]pyridinyl, pyrazolyl or oxetanyl, any of which is unsubstituted or substituted by 1, 2 or 3 R Q substituents.

[0523] 102. The method according to any one of clauses 91 to 101, wherein Q is a monocyclic heteroaryl group having 5 or 6 ring members with 1 or 2 heteroatoms selected from N, O or S, and Q is unsubstituted or substituted by 1 or 2 R Q substituents.

[0524] 103. The method according to any one of clauses 91 to 102, wherein R 3 is a group of formula -(CR 3d R 3e )-(CR 3f R 3g )-Q.

[0525] 104. The method according to any one of clauses 91 to 102, wherein R 3 has the following formula:

[0526]

[0527]

[0528] wherein the symbol When drawn through the bond, represents the point of attachment to the rest of the molecule.

[0529] 105. The method according to any one of clauses 1 to 104, wherein the apelin receptor agonist is (2S,3R)-N-(4-(2,6-dimethoxyphenyl)-5-(5-methyl-3-pyridyl)-4H-1,2,4-triazol-3-yl)-3-(5-methyl-2-pyrimidinyl)-2-butenesulfonamide, or a pharmaceutically acceptable salt thereof, a tautomer thereof, a pharmaceutically acceptable salt of the tautomer, a stereoisomer of any of the foregoing, or a mixture thereof.

[0530] 106. The method according to clause 105, wherein the apelin receptor agonist is (2S,3R)-N-(4-(2,6-dimethoxyphenyl)-5-(5-methyl-3-pyridyl)-4H-1,2,4-triazol-3-yl)-3-(5-methyl-2-pyrimidinyl)-2-butenesulfonamide or a pharmaceutically acceptable salt thereof.

[0531] 7. Examples

[0532] The following are examples for implementing the specific embodiments of the present invention. These examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention in any way. We have made every effort to ensure the accuracy of the numbers used (such as quantities, temperatures, etc.), but of course some experimental errors and deviations should be allowed.

[0533] Unless otherwise specified, the practice of the present invention will employ conventional methods of protein chemistry, biochemistry, recombinant DNA technology, and pharmacology within the skill of the art. Such techniques are explained in detail in the literature.

[0534] 7.1. Example 1: Bioinformatics analysis determines the relationship between apelin and all-cause mortality and decline in mobility events in a human healthy aging cohort

[0535] Based on the establishment of a survival model, using unpublished clinical outcome data and proteomic data generated from archived samples, a survival prediction model was used to examine the relationship between serum levels of apelin and the future risk of all-cause death in a human healthy aging cohort. By combining proteomic data with the phenotypes and clinical data of the participants, a "muscle aging score" for all measured proteins was calculated, which reflects a comprehensive risk ratio related to longitudinal grip strength decline and mortality. This analysis found that apelin (APLN), a myokine secreted by skeletal muscle in response to exercise, is a target for muscle aging. In addition, in this cohort, apelin protein levels were associated with an increased probability of longevity (living to ≥90 years) and grip strength maintenance (living to ≥90 years and no decline in grip strength from baseline by ≥10 kg) ( Figure 1 b).

[0536] In addition, the relationship between the levels of apelin and events of decreased mobility (e.g., reduction in walking, stair climbing, or transfer activities, as indicated by the difficulty of these activities self-reported) was examined. Using the Cox proportional hazards model, the hazard ratio and associated p-value of apelin were obtained.

[0537] As Figure 2A shown, Kaplan-Meier curves of survival probability were generated for humans with apelin protein levels in the top 20% (blue) and bottom 20% (red). In humans, we found that higher circulating levels of apelin were associated with a reduced all-cause mortality (p = 0.0002). Figure 2B A similar model is shown, in which we found that higher circulating levels of apelin were associated with increased mobility (p = 0.0082). The hazard ratio of apelin was generated using the Cox proportional hazards model ( Figure 2A 0.88 in Figure 2B , 0.89 in Figure 2A and Figure 2B ). In both cases, the hazard ratios given were for continuous Cox proportional hazards analyses that fit the entire distribution of apelin measurements.

[0538] Next, rank-based inverse normalization of the protein levels was performed, and pairwise Spearman correlation coefficients between the normalized levels of all 4,575 proteins were calculated. Among the 590 proteins significantly correlated with apelin (Benjamini-Hochberg FDR < 0.05) (referred to as the apelin protein module; Figure 2C ), hypergeometric tests showed significant enrichment of proteins associated with all-cause mortality (p = 1.04E-10).

[0539] After adjustment for age, pack-years of smoking, and monthly alcohol consumption, the association between the first principal component (PC1) of the apelin protein module and mortality was then tested using a multivariate Cox regression model. The contribution of PC1 to relative mortality in this model (i.e., termplot) was in the range of 1.43 to 0.77 ( Figure 2D ), with the median of PC1 as the reference.

[0540] Figure 2C The serum abundances of the apelin protein module in the HHS cohort are shown (highlighted by the green oval). Each node represents a protein, and the edges between the nodes represent significant correlations. Figure 2DDisplay of the first principal component of the apelin protein module and mortality. The relative mortality (logarithm; y-axis) was derived from a multivariate Cox regression model of PC1 after adjustment for age, pack-years of smoking, and alcohol status. The reference value used was the median of PC1.

[0541] 7.2. Example 2: BGE-105 improves the activity level of aged mice (frailty study)

[0542] Based on the findings of the association between baseline apelin and apelin receptor protein levels and future aging outcomes in otherwise healthy aged humans described in Example 1, an apelin receptor agonist was administered to aged mice to evaluate the effect of the agonist on spontaneous physical activity compared to age-matched control groups.

[0543] BGE-105 has the structure shown below ( Figure 1 ):

[0544]

[0545] It is known that BGE-105 activates the apelin receptor and induces cardiovascular responses in rats (Ason et al., JCI Insight. 5(8):1-16 (2020)). Clinical trials have also been conducted with BGE-105 to study the safety, tolerability, and pharmacokinetics in healthy subjects and subjects with impaired renal function (NCT03318809) or heart failure (NCT03276728).

[0546] In the current study, aged mice (24 months old) were treated daily with BGE-105 (ad libitum in water) for 2 months. These animals were housed with access to a spontaneous running wheel that wirelessly transmitted running data to a computer for analysis. The spontaneous running wheel activity level was measured daily, and body weight was measured every two weeks. The effect of BGE-105 on preventing frailty in mice was examined.

[0547] The formal test consisted of calculating the Spearman correlation coefficient between these daily differences and the days (e.g., days 1, 2, 3, etc. of the experiment) and testing the null hypothesis that this correlation coefficient was equal to 0.

[0548] The first day of the study (Study Day 1) started with animal acclimation, and then BGE-105 treatment began on Study Day 19 (Phase Day 1). The study ended on Study Day 83. Activity wheel monitoring started on Study Day 1 and ended on Study Day 83 (Phase Day 64). The analyzed data were collected at the end of the study. The total duration of activity monitoring after the start of BGE-105 treatment was 64 days. For the frailty part of the study, mice were evaluated using an activity monitoring wheel that was passively monitored by a computer monitoring system.

[0549] As shown in Table 1, the study included C57BL / 6 strain mice at 23 to 24 months of age. Mice at 18 to 24 months of age are known to be associated with humans aged 56 to 69 years, while mice older than 24 months are associated with humans over 69 years of age (Flurkey, Currer, and Harrison, 2007. “The mouse in biomedical research” in James G. Fox (ed.), American College of Laboratory Animal Medicine series, Elsevier, AP: Amsterdam; Boston). This age range meets the definition of “old age,” which is defined as the appearance of senescent changes in biomarkers in the animal body.

[0550]

[0551] The mice were treated with BGE-105 at a dose concentration of 275 μg / mL. BGE-105 was dissolved in deionized water at 275 μg / mL. BGE-105 was administered by free access to drinking water. The compound is weakly acidic when dissolved, producing a solution with a pH of 4.5. The deionized water was adjusted to pH 8.5 by adding 1N NaOH. The vehicle control group drank water of the same pH without the drug (free access).

[0552] Table 2 provides the study parameters for Groups 1-2. The study parameters for the mice in Groups 1-2 included animal acclimation, animal welfare, such as checking the animal body weight, clinical examination, administration of treatment, activity monitoring, and blood collection on specific study days and / or stage days.

[0553]

[0554]

[0555]

[0556] Activity monitoring wheel test

[0557] The activity monitoring wheel is a rotating running wheel for monitoring. The running wheel can monitor the voluntary running wheel that operates 24 hours a day. A computer monitoring system is used to passively and wirelessly monitor the activity. The activity level of the running wheel is monitored daily. The running wheel data is reported as the median number of rotations per day for each group (BGE-105 treatment and control). The activity level of the mice is measured as the number of times each mouse turns the wheel per day and is converted into kilometers run per day using the running wheel diameter. In each experimental group, the daily median of the activity is calculated. For each experiment, the baseline period before the start of the experiment is used to calculate the median baseline activity level of each mouse. These baselines are subtracted from the future measurements of the same mouse. The daily corrected median obtained during the experiment is plotted for each day of the experiment, and a smooth curve is plotted using local regression (LOESS). The daily differences between the running distances of each group are calculated, and the Kendall rank correlation tau test is used to test for an increasing trend.

[0558] Study Results

[0559] As Figure 3A shown, the BGE-105-treated mice were significantly more active than the control group (Kendall rank correlation tau p-value = 0.00228). The study was conducted twice, and each replication produced similar results ( Figure 3D , Kendall rank correlation tau p-value = 1.14e-04).

[0560] During the course of these experiments, the activity levels of both groups of old mice decreased ( Figure 3A and Figure 3D ), but the decrease in the treatment group was significantly smaller, resulting in a gradual divergence of the activity curves between the drug-treated group and the vehicle-treated group. In both experiments, by the end of the experimental period, the BGE-105-treated mice ran at least 1 km more per day on average than the mice treated with the vehicle.

[0561] Grid suspension test

[0562] A 3×3-foot metal grid is suspended using four 20-gallon plastic buckets, which is suspended approximately 3 feet above the ground. The ground directly below the grid is covered with soft material. The metal grid is placed on its side so that it is perpendicular to the surface of the bucket. The mouse is placed on the grid and carefully dropped so that the mouse starts to hang. Once the grid is completely parallel to the horizontal plane (i.e., the floor), the timer is started. When the mouse falls onto the soft padded floor, the timer stops, the fall time is recorded, and a graph is plotted.

[0563] Study Results

[0564] To determine whether the increase in wheel activity was accompanied by an increase in muscle strength, we performed a grid hanging test on mice, which measures forelimb grip strength, near the end of the frailty study. Mice were tested at 24 months and again at 26 months, 64 days after treatment with BGE-105 or vehicle. The mean fall latency increased in mice treated with BGE-105 (p = 0.04, Mann-Whitney U test)( Figure 3B and Figure 3C ). Thus, the increase in running activity observed in BGE-105-treated mice was accompanied by an improvement in forelimb grip strength. At the end of the study, the wet weight of TA tissue was significantly increased in BGE-105-treated mice( Figure 3F ), and there was a trend towards an increase in the wet weight of gastrocnemius and quadriceps muscles( Figure 3G and Figure 3H ), while there was no difference in the heart( Fig. 3I ). The body weight of BGE-105-treated mice was also higher( Figure 3E ), but the increase was not significant, only at the p-value cut-off (0.0850).

[0565] 7.3. Example 3: BGE-105 activates the apelin receptor signaling pathway.

[0566] Administration of an apelin receptor agonist induces phosphorylation and activation of AMPK in cardiac tissue. Tissue samples were lysed on an Omni Bead Ruptor 12 homogenizer using T-PER tissue protein extraction reagent (Thermo Fisher Scientific #78510) containing EDTA and protease / phosphatase inhibitors. Total protein was extracted and then quantified using the PierceTM BCA Protein Assay Kit. Equal amounts of total protein were loaded per lane on a 4-12% SDS-PAGE gel and then transferred to a PVDF membrane. The membrane was blocked and blotted with anti-phospho-AMPKα-Thr172 (Cell Signaling Technology, CST #2535), total AMPKα (CST #2532), anti-phospho-Akt-Ser473 (CST #4060), total Akt (CST #4685), anti-phospho-ERK-1 / 2-Thr202 / Tyr204 for Erk1 and Thr185 / Tyr187 for Erk2 (CST #4370), total ERK-1 / 2 (CST #9107) or anti-APLNR receptor (abcam, ab214369) antibodies. Band intensities were normalized to the loading control anti-β-actin (CST #3700) or anti-GAPDH (abcam, ab181602) antibodies. Immunoreactive proteins were detected using SuperSignal TMDetection was performed using the West Femto substrate (Thermo Fisher Scientific #34095) and quantification was carried out using Image LabTM software (Bio-Rad Laboratories, Inc.).

[0567] After oral administration of 45 mg / kg BGE-105 or vehicle to mice, the level of pAMPK in the heart of the BGE-105 treatment group was significantly higher than that of the vehicle control group, FIG. 4A to FIG. 4B . We also evaluated the effect of BGE-105 on skeletal muscle. Apelin induced phosphorylation of Akt in soleus muscle and improved glucose homeostasis. In the BGE-105 treatment group, we observed a slightly significant increase in the level of pAkt in soleus muscle (p = 0.0516), FIG. 4C to FIG. 4D . β-actin was used as an internal control. The number of apelin receptors per unit mass of soleus muscle was approximately half that of the heart, FIG. 4E to FIG. 4F , which potentially explains the stronger response in heart tissue.

[0568] Differences among rodent tissues were relatively conserved: similar to mice, the level of apelin receptors in the heart of rat tissues was 2-fold higher than that in soleus muscle, FIG. 5A to FIG. 5B . The level of apelin receptors in the tibialis anterior (TA) of rats was similar to that in soleus muscle, while the levels of apelin receptors in quadriceps and gastrocnemius muscles were below the detection limit of our Western blot analysis. Oral administration of BGE-105 to rats for 5 consecutive days induced phosphorylation of Akt in TA in a dose-dependent manner, with 50 mg / kg BID eliciting the strongest response, FIG. 5C to FIG. 5D . A similar trend was also observed for phosphorylation of Erk, FIG. 5E to FIG. 5F . Given that pErk is downstream of pAkt, this observation is consistent with the known signaling pathway. Next, we evaluated the effect of chronic administration of BGE-105 on the level of apelin receptors. After oral administration of BGE-105 at the lowest and highest doses (50 mg / kg QD and 200 mg / kg BID, respectively) for 5 consecutive days, BGE-105 caused a moderate but not significant decrease in the level of apelin receptors in TA, Figure 5G to Figure 5I .

[0569] 7.4. Example 4: BGE-105 activates the apelin receptor in a manner similar to apelin.

[0570] We compared the ability of BGE-105 and Pyr 1 -apelin-13 to activate the APLNR receptor and recruit β-arrestin using the PathHunter β-arrestin assay.

[0571] Comparison of BGE-105 with Pyr 1 -apelin-13 in terms of EC50 for β-arrestin recruitment in mouse or human APLNR. APLNR activation was determined by β-arrestin recruitment measured by the ProLink β-gal complementation technology (93-0001, DiscoveRx). Briefly, CHO cells stably expressing APLNR were seeded and incubated overnight at 37 °C. Compounds were tested in duplicate and serially diluted 3-fold (<1% DMSO) to obtain a 10-point curve. The compounds and cells were incubated at 37 °C for 3 hours. After the incubation period, the detection reagent was added and the plate chemiluminescence signal was measured 30 minutes later at room temperature.

[0572] In cells stably expressing human APLNR, BGE-105 was 10-fold more potent than Pyr 1 -apelin-13: BGE-105, EC50 = 0.1 nM; Pyr 1 -apelin-13, EC50 = 1.2 nM, Fig. 6A . In cells stably expressing mouse APLNR, BGE-105 was 30-fold more potent than Pyr 1 -apelin-13: BGE-105, EC50 = 0.8 nM; Pyr 1 -apelin-13 (EC50 = 25 nM), Fig. 6A .

[0573] Although the potency increase was comparable for human and mouse APLNR, the maximum effect (Emax) was different: human APLNR, Emax = 114%; mouse APLNR, Emax = 65%. Repeating the mouse APLNR β-arrestin assay with freshly prepared BGE-105 gave similar data: Emax = 60%, Figure 6B . Thus, the potency of BGE-105 as an APLNR agonist may be higher in humans than in mice.

[0574] 7.5. Example 5: BGE-105 accelerates the regeneration of CTX-induced muscle injury in aged mice.

[0575] Muscle regeneration impairment can lead to age-related muscle weakness. This is especially true for the elderly engaged in physical activities. Exercise-induced muscle hypertrophy is related to the activation and regenerative-promoting ability of muscle stem cells. We evaluated the effect of oral treatment with BGE-105 during muscle regeneration ( 7A to 7BB)。For this purpose, we injected cardiotoxin (CTX) into the left tibialis anterior and gastrocnemius muscles of 18-month-old male mice (and 3-month-old male mice), and then orally administered BGE-105 (50 or 200 mg / kg / day) for 3 or 7 consecutive days.

[0576] Thirty minutes before injury and 1 day after injury, buprenorphine (Centravet, 0.1 mg / kg) was intraperitoneally injected into the mice. On the day of injury, the mice were anesthetized by inhaled isoflurane, and the hair on the hind limbs was shaved off. Then, a 22-gauge needle (Hamilton) was used to inject 10 μM cardiotoxin (CTX, Latoxan, #L8102), with two injections of 25 μl into the left tibialis anterior muscle and two injections of 50 μl into the left gastrocnemius muscle. The mice were euthanized by cervical dislocation on the 3rd and 7th days after injury, and the muscles (injected with PBS and CTX) were cut into two parts. One part was quickly frozen in liquid nitrogen for total RNA extraction, and the other part was embedded in OCT and frozen in isopentane cooled with liquid nitrogen for histological analysis.

[0577] The mouse muscle samples were dissected and cryopreserved in OCT in isopentane cooled with liquid nitrogen. Then the samples were cut into 10-μm-thick sections on a cryostat and fixed with 4% paraformaldehyde (PFA) at room temperature for 15 minutes. The muscle cryosections (10 μm) were stained with hematoxylin / eosin or immunolabeled with laminin (Abcam) and embryonic myosin. Briefly, the sections were blocked in PBS plus 4% BSA, 2% goat serum, 0.01% Triton X-100 for 1 hour. Then the sections were incubated with the primary antibody overnight. After washing with PBS, the sections were incubated with the secondary antibody anti-Ig2b AF 488 (Life Technology) for 1 hour. Finally, the slides were mounted in ProLong Gold antifade reagent containing DAPI (Molecular Probes of Life Technology). Images were taken using a digital camera (Nanozoomer, Hamamatsu) connected to an electric fluorescence microscope or using an Olympus VS120 virtual microscope slide scanning system. The areas covered by eMHC-positive fibers and degenerated regions in the whole section were manually determined using the measurement tool of VS-ASW FL software. The size of myofibers with central nuclei was calculated by laminin / DAPI staining of all fibers in the section using an automated image processing algorithm developed in-house with MetaXpress software (Molecular Devices), and the area measurement of the whole section was performed.

[0578] Study Results

[0579] 7A to 7FShows the effects of administering PBS, Pyr 1 -apelin-13 (apelin) (0.5 μmol / kg / day), BA1 (BGE-105 50 mg / kg / day), or BA2 (BGE-105 200 mg / kg / day) on the transcript levels of the tibialis anterior muscle of aged (18-month-old) mice. Fig. 7A Shows that after 7 days of administering apelin (injected) and two doses of BGE-105 (P.O.), the Pax7 level increased significantly. Figure 7B Shows that after 3 days and 7 days of injecting apelin and two doses of BGE-105, the MyoD level increased significantly. Figure 7C Shows that after 7 days of administering apelin and two doses of BGE-105, the MyoG level increased significantly. Fig.7D Shows that after 7 days of injecting apelin and two doses of BGE-105 (P.O.), the MyHC3 level increased significantly. Fig. 7E Shows that after 7 days of injecting apelin and two doses of BGE-105, the MyHC8 level changed significantly. Figure 7F Shows that after 7 days of injecting apelin and two doses of BGE-105, the Myf5 level changed significantly.

[0580] Figure 7G to Figure 7L Shows that after 3 days or 7 days of injecting cardiotoxin, administering PBS, Pyr 1 -apelin-13 (apelin) (0.5 μmol / kg / day), BA1 (BGE-105 50 mg / kg / day), or BA2 (BGE-105 200 mg / kg / day) on the transcript levels of the gastrocnemius muscle of aged (18-month-old) mice. Figure 7G Shows that the Pax7 level did not change significantly. Figure 7H Shows that after 3 days and 7 days of injecting apelin and two doses of BGE-105, the MyoD level increased significantly in both cases. Fig.7I Shows that the MyoG level did not change significantly. Figure 7J Shows that for the larger BGE-105 injection, the MyHC3 level increased significantly only 7 days after injection. Figure 7K No change in the MyHC8 level was shown for any injection at any time point. Figure 7L Shows that the Myf5 level did not change significantly.

[0581] Figure 7M to Figure 7R Shows that after 3 days or 7 days of injecting cardiotoxin, administering PBS, Pyr 1- Effects of apelin-13 (apelin) (0.5 μmol / kg / day), BA1 (BGE-105 50 mg / kg / day), or BA2 (BGE-105 200 mg / kg / day) on the transcript levels in the tibialis muscle of young (3-month-old) mice. Figure 7M It shows that the Pax7 level has no change. Figure 7N It shows that the MyoD level has no significant increase. Fig.7O It shows that the MyoG level has no difference. Figure 7P It shows that the MyHC3 level has no significant change. Figure 7Q It shows that the MyHC8 level has no significant change. Figure 7R It shows that the Myf5 level has no difference.

[0582] Figures 7S to 7X It shows that at 3 or 7 days after injection of cardiotoxin, PBS, Pyr 1 - Effects of apelin-13 (apelin) (0.5 μmol / kg / day), BA1 (BGE-105 50 mg / kg / day), or BA2 (BGE-105 200 mg / kg / day) on the transcript levels in the gastrocnemius muscle of young (3-month-old) mice. Figure 7S It shows that the Pax7 level has no change. Figure 7T It shows that the MyoD level has no change. Figure 7U It shows that the MyoG level has no change. Figure 7V It shows that for the lower BGE-105 dose, the MyHC3 level increases only at 3 days after injection. Figure 7W It shows that there is no significant increase in the MyHC8 level at 3 days after injection of apelin and both BGE-105 doses. Figure 7X It shows that the Myf5 level has no change.

[0583] Figure 7Y to Figure 7Z It shows that at days 3 and 7 after injection of cardiotoxin, those receiving PBS, Pyr 1 - Cross-sectional areas of the tibialis muscle after treatment with apelin-13 (apelin), BA1 (BGE-105 50 mg / kg / d), and BA2 (BGE-105 200 mg / kg / d). Figure 7Y It shows representative histological cross-sectional slices of the tibialis muscle at 3 and 7 days after injection with PBS, apelin, BA1, or BA2. Figure 7Z It shows quantification of cross-sectional histological slides, which shows a significant increase in the cross-sectional areas of apelin, BA1, and BA2 at 3 and 7 days after injection.

[0584] Figures 7AA to 7BB It shows that for PBS, Pyr 1- The amount of central nucleated myofibers (CNM) as part of the regeneration process after cardiotoxin injection in treatments with apelin-13 (apelin), BA1 (BGE-105 50 mg / kg / day), and BA2 (BGE-105 200 mg / kg / day). The mice were 18 months old. Figure 7AA Representative distribution showing DAPI-stained nuclei and eMHC-positive stained myofibers. Figure 7BB Quantification showing the amount of central nucleated myofibers (CNM). The amount of CNM increased significantly after treatment with the higher of apelin treatment and the two BGE-105 treatments.

[0585] 7A to 7F The results presented in [ ] show that, as previously demonstrated with apelin-13, BGE-105 treatment enhances the regeneration of the tibialis muscle 7 days after CTX injection. In fact, the expression levels of genes involved in muscle regeneration (Pax7, MyoD, MyoG, Myf5, MyHC3, and MyHC8) in animals treated with BGE-105 were significantly higher than those of littermates treated with PBS. This effect was not obvious in the gastrocnemius muscle ( Figure 7G to Figure 7L ), indicating that BGE-105 is more effective on muscle types with high receptor density (such as the tibialis muscle). In addition, BGE-105 was not as effective in young mice ( Figures 7M to 7X ), suggesting that it is most effective on aging muscles with impaired repair capacity. Muscle size (cross-sectional area) analysis confirmed the positive effect of BGE-105 on the muscle regeneration of the tibialis muscle in old mice 3 and 7 days after CTX injection ( Figure 7Y to Figure 7Z ). Finally, BGE-105 treatment promoted the accumulation of centrally nucleated regenerated myofibers ( Figures 7AA to 7BB ), indicating that this compound can have a beneficial effect on regeneration.

[0586] Overall, this effect was less obvious in the gastrocnemius muscle, indicating that the APJ agonist is most effective in tissues with high APJ receptor density (such as the tibialis anterior muscle). Its effect on young mice was also poor, suggesting that the APJ agonist is most effective on aging muscles with impaired repair capacity.

[0587] 7.6. Example 6: BGE-105 promotes the early proliferation and differentiation of human myoblasts.

[0588] Immortalized human cells from 25-year-old (25-HMC) and 79-year-old (79-HMC) male donors were grown from the proliferation stage until they reached 80% confluence, differentiated, and became myotubes. From day 1 to day 4, the cells were treated with 1 nM of Pyr 1 - apelin-13, 0.05, 0.5, 5, 50 nM of BGE-105, or vehicle (<0.1% DMSO) ( FIG. 8A to FIG. 8K)。The early proliferation markers (Pax7, Myf5, MyoD, and MyoG) were evaluated via RT-PCR ( Figures 8D to 8K )。

[0589] Study Results

[0590] Short-term (from day 0 to day 4 post-seeding) BGE-105 treatment induced a significant increase in cell proliferation of both young and old donor cells ( FIG. 8B to FIG. 8C )。BGE-105 treatment also increased the expression of muscle cell differentiation markers (such as Pax7 and MyoD) in young donor cells ( FIG. 8D to FIG. 8G ) as well as the expression of Pax7, Myf5, MyoD, and MyoG in old donor cells ( Figures 8H to 8K )。Collectively, these results support the use of BGE-105 in human muscle physiology of the elderly population.

[0591] FIG. 8A to FIG. 8C Demonstrates the ability of BGE-105 to increase the proliferation of immortalized human muscle cells from young (25 years old) and old (79 years old) subjects. Fig. 8A Shows the experimental protocol where the first incubation (treatment #1) was performed during in vitro cell proliferation (day 0 to day 4), and the second incubation (treatment #2) was performed during the differentiation into myotubes stage (day 4 to day 18). The treatment groups were DMSO (0.1%), Pyr 1 -apelin-13 (1 nM), or 0.05, 0.5, 5, or 50 nM of BA (BGE-105). Figure 8B Shows the cell proliferation (measured at day 4) from young subjects, which increased with 5 nM of BGE-105 treatment and significantly increased with 50 nM of BGE-105 treatment. Figure 8C Shows the cell proliferation from old subjects, which significantly increased with 5 nM of BGE-105 treatment and increased with 50 nM of BGE-105 treatment.

[0592] Figures 8D to 8K Shows the expression levels of PAX7, MYf5, MyoD, and MyoG in immortalized myocytes from elderly (79 years old) and young (25 years old) subjects after incubation with DMSO (0.1%), Pyr 1 -apelin-13 (apelin / Ape) (1 nM), or 0.05, 0.5, 5, or 50 nM of BGE-105 (BA). Fig.8D Shows the levels of PAX7 in young cells. 5 and 50 nM of BGE-105 reproduced the levels of apelin. Fig. 8EShow the level of MYf5 in young cells after treatment. For any amount of BGE-105, the MYf5 level did not change relative to the control. Figure 8F Show the level of MyoD expression in young cells after treatment. At 5 nM of BGE-105, the MyoD expression increased significantly. Figure 8G Show the level of MyoG expression in young cells after treatment. There was no significant change after treatment. Figure 8H Show the level of PAX7 in senescent cells after treatment. There was a significant increase at all treatment doses compared to the control. Figure 8I Show the expression level of Myf5 in cells from senescent donors after treatment. At 5 and 50 nM, the levels were the same as those with apelin treatment. Figure 8J Show the level of MyoD in cells from senescent donors after treatment. It increased at all treatment levels with increasing dose, approaching the expression level induced by apelin. Figure 8K Show the level of MyoG in senescent cells after treatment. It increased at all treatment levels, approaching the expression level induced by apelin.

[0593] 7.7. Example 7: BGE-105 prevents disuse-induced muscle atrophy in aged mice.

[0594] BGE-105 activates pathways beneficial to skeletal muscle physiology, particularly the pAkt / pErk pathway, which plays a key role in regulating muscle mass. Limitation of limb movement leads to loss of skeletal muscle mass, accompanied by a significant decrease in the transcript level of apelin. Therefore, we tested whether BGE-105 could rescue muscle atrophy caused by long-term movement limitation. Since skeletal muscle atrophy caused by movement limitation is exacerbated during aging, we evaluated the effect of BGE-105 on the maintenance of muscle mass in aged mice with movement limitation of the plantar flexor muscle group (soleus, TA, EDL, gastrocnemius). Animals were orally administered a vehicle or BGE-105 at a dose of 50 mg / kg BID; after one week of treatment, the right hind limb was immobilized with a cast, and the muscle was allowed to atrophy for 21 days.

[0595] Male C57 / Bl6 mice at 20 months of age (n = 10 / group) were administered P.O. vehicle or BGE-105 at 50 mg / kg BID at ZT1 and ZT11.5. One week after treatment, a modified hindlimb cast was applied to one limb of the mice. The mice were anesthetized by inhalation of isoflurane, the hindlimb was wiped with povidone iodine and then ethanol, and loosely wrapped with surgical gauze. A custom-made plastic fixation device was placed on the limb to fully extend the foot, thus minimizing the in vivo load on the plantar flexor muscle group. The device was fixed to the hindlimb using Vetbond, and then the animal was returned to the cage. After 3 weeks of treatment after cast application, the mice were euthanized 1 hour after the last ZT1 dose, tissues were isolated, weighed, and then quickly frozen in liquid nitrogen for subsequent western blot analysis.

[0596] Research Results

[0597] Activity limitation caused significant atrophy of the casted limb in all muscle types in the vehicle treatment group, Figure 9A . Notably, the wet weight of the tibialis anterior muscle (TA) in the placebo group decreased by 25%, but only by 3% in the BGE-105 group, Figures 9D to 9E . Similarly, the extensor digitorum longus muscle (EDL) in the placebo group atrophied by 14%, but there was no significant atrophy in the BGE-105 group, Figures 9F to 9G . A modest alleviation of atrophy was observed in the soleus muscle, but this effect was not significant, Figures 9H to 9I .

[0598] In these animals, the apelin receptor density in the gastrocnemius muscle was significantly lower than that in other muscles, Figures 9J to 9K , which potentially explains why BGE-105 has a poorer effect in this muscle. Figures 9L to 9M The apelin receptor levels after one month of treatment with BGE-105 at 50 mg / kg BID are shown. Again, we observed that chronic activation of the apelin receptor by BGE-105 led to a significant but not significant decrease in apelin levels. Therefore, although BGE-105 did downregulate the apelin receptor after long-term treatment, the effect was not significant at the tested dose.

[0599] Our data indicate that aged mice treated with BGE-105 were protected from some muscle mass loss induced by activity limitation. Therefore, BGE-105 may have clinical benefits in preventing disuse atrophy in humans.

[0600] 7.8. Example 8: BGE-105 prevents disuse atrophy of human muscles

[0601] Two groups of healthy elderly subjects with moderate activity (e.g., N = 10 subjects / group) were bedridden for 10 consecutive days. Except for using the toilet, they consumed an isocaloric diet that provided the recommended dietary allowance of protein (0.8 g / kg protein per day). One group took 200 mg of BGE-105 daily, and the other group took a placebo. Measurements before and after bed rest included muscle function and protein synthesis.

[0602] It was shown that BGE-105 prevented or alleviated atrophy of the immobilized human muscle during disuse.

[0603] 7.9. Example 9: Safety, Tolerability, Pharmacokinetics and Pharmacodynamics of BGE-105 in Healthy Adult Patients (Phase 1b Clinical Study)

[0604] The study described herein characterized the effect of apelin-13 of BGE-105 with both single-dose and multiple-dose. The target indications included the treatment of improving frailty in the elderly and improving muscle function. Two groups (Group A "Part A", single ascending dose (SAD) and Group B "Part B", multiple dose (MD)) of healthy elderly subjects participated in the study for approximately 42 days, including a screening / pre-treatment period of up to 14 days, a treatment period of 5 days for Part A and 7 days for Part B, and a follow-up of 27 days after the first administration of the study drug (BGE-105 or placebo).

[0605] In Part A, 24 subjects were recruited (3 SAD cohorts, 8 subjects in each cohort), with at least 12 subjects aged ≥ 65 years in total (≥ 4 subjects in each cohort). The remaining subjects were aged ≥ 18 years. In each cohort, 6 subjects received BGE-105 and 2 subjects received a placebo, for a total of 18 subjects treated with BGE-105 and 6 subjects treated with placebo, for a total of 24 subjects. In addition to characterizing the PD effects associated with acute BGE-105 exposure, in the SAD cohorts, a 48-hour "drug holiday" ( Figure 10 the washout period in

[0606] In Part B, 30 subjects were recruited, all of whom were ≥65 years of age. The 30 eligible subjects were to be enrolled in cohort 1A (placebo), cohort 1B (240 mg of BGE-105 daily), or group 1C (240 mg / 1440 mg) for treatment. Cohort 1A included 10 subjects receiving placebo normal saline (NS), cohort 1B included 10 subjects each receiving BGE-105, and cohort 1C included up to 10 subjects each receiving BGE-105. Subjects participated in this study for approximately 81 days, including a screening period of up to 16 days; a 5-day (day -5 to day -1) outpatient pretreatment period with heavy water and D3-creatine (D3-Cr); a 10-day treatment period of bed rest with heavy water / D3-Cr and BGE-105 or placebo; and a follow-up period after dosing (days 11 to 60), including 2 follow-ups on days 11, 12, 13, 14, 21, 30, and 60 after the first administration of the study drug (BGE-105 or placebo). In the multiple-dose cohorts (1A, 1B, 1C), PD parameters were measured and evaluated to assess the effects on insulin sensitivity and changes in muscle index during the bed rest period, in order to better inform decisions on dose selection and further development directions.

[0607] Goals and Endpoints

[0608] Primary objective. To evaluate the safety and tolerability of single and multiple escalating doses of BGE-105 in healthy adult subjects (≥18 years of age in Part A, ≥65 years of age in Part B) following intravenous (IV) infusion of BGE-105, with a focus on elderly subjects (≥65 years of age in Part A).

[0609] Safety assessment. The safety assessment is summarized in Table 6.

[0610]

[0611] Additional physical examinations included vital signs. The ECG assessment included continuous telemetry monitoring.

[0612] Secondary Goals and Endpoints

[0613] Secondary objectives. The secondary objectives include: characterizing the pharmacodynamic (PD) effects of BGE-105 following IV infusion in healthy adult subjects; characterizing the pharmacokinetics (PK) of BGE-105 following IV infusion in healthy adult subjects; and characterizing the PK / PD relationships of BGE-105 on predefined biomarkers (including but not limited to glucose, insulin, and insulin sensitivity) and PD variables (such as systolic and diastolic blood pressure, heart rate changes), and in the multiple-dose cohorts (Part B), measuring the muscle protein synthesis rate by administering heavy water and vastus lateralis muscle micro (small needle) biopsies, measuring total skeletal muscle mass of D3-creatine (D3-Cr) from urine samples, and measuring muscle circumference, cross-sectional area, color flow analysis, anteroposterior (AP) diameter, and echo density of the vastus lateralis and gastrocnemius muscles by ultrasound.

[0614] Secondary endpoints. The secondary endpoints are summarized in Table 7.

[0615]

[0616]

[0617]

[0618] This study is a randomized, placebo-controlled, double-blind, single ascending dose (SAD) and single-blind, multiple-dose (MD) study involving up to 72 healthy adult subjects. There are a total of 6 cohorts: 3 cohorts in Part A (8 subjects per cohort) and 3 cohorts in Part B (10 subjects per cohort). The cohorts can be split and dosed 1 day apart to collect data on major procedure days (e.g., the 10 subjects in the MD cohort can be split into 5 groups, each dosed 1 day apart). The study design is as Figure 10 shown.

[0619] In the 3 cohorts of Part A (SAD cohorts), a total of 6 healthy males or females received BGE-105 via intravenous infusion (at least 3 subjects ≥65 years old and the remaining subjects ≥18 years old) and 2 healthy males or females received placebo (at least 1 subject ≥65 years old and the remaining subjects ≥18 years old).

[0620] Following a 24-hour baseline period in the outpatient setting, all subjects received an LD, 1-hour infusion on Day 1. After a 48-hour washout period (during which PK and PK / PD data were collected), the subjects received a 23-hour infusion (1-hour LD followed by 22 hours of maintenance dose). See Figure 10 the different dose levels in each SAD cohort. For example, see Table 3 below. Part A is double-blind. The cohorts in Part A proceed sequentially starting from SAD cohort 1.

[0621] In each of the three cohorts in Part B, 30 subjects were recruited, and they received treatment in Cohort 1A, Cohort 1B, or Cohort 1C, respectively. Cohort 1A included 10 subjects who all received a 1-hour infusion of placebo NS from Day 1 to Day 10 for 10 days. Cohort 1B included 10 subjects, and each subject received a 1-hour infusion of BGE-105 at a dose of 240 mg from Day 1 to Day 10 for up to 10 days. Cohort 1C included up to 10 subjects, and each subject received no more than 1400 mg of BGE-105 within 24 hours for up to 10 days. The patients remained in bed throughout the 10-day treatment period.

[0622] The dose of BGE-105 in Cohort 1C was no more than 1440 mg within 24 hours, which was the highest dose given in the SAD and was well tolerated. The dosing regimen within 10 days was guided by the HOMA-IR data required for Cohort 1B, and the dose, dosing regimen, or both could be changed.

[0623] The pretreatment period for all three cohorts started on Day -5 and continued until Day -1. All subjects in each cohort were admitted to the ward on Day -2. All subjects remained in bed from Day 1 until Day 10.

[0624] According to Figures 13 to 14 , all subjects in the multiple-dose (Part B) cohorts started receiving heavy water and D3-Cr on Day -5 and continued until Day 10. Figure 13 Provide the screening and pretreatment summary for the MD cohort. Figure 14 Provide the treatment and follow-up summary for the MD cohort.

[0625] According to Figures 13 to 14 , the subjects started receiving heavy water on Day -5 and D3-Cr on Day -3 and continued until Day 10.

[0626] After completing multiple-dose Cohort 1A, the sponsor will conduct a pharmacodynamic evaluation of the results of samples collected from skeletal muscle biopsies, blood, urine, and saliva to measure the effect on skeletal muscle. The HOMA-IR is evaluated to assess insulin resistance. These data are non-blind to the sponsor and are used to confirm the number of days of bed rest (e.g., 10 days or less) in Cohorts 1B and 1C. In addition, between Cohorts 1B and 1C, the sponsor evaluates non-blind HOMA-IR to confirm the dose and dosing regimen of Cohort 1C. The sponsor reviews the data from other measurements (such as ultrasound measurements, pharmacokinetic data, and muscle mass and muscle protein synthesis rate) in a non-blind manner after the end of each cohort.

[0627] The subjects also need to receive additional clinic and home evaluations during the pretreatment period and the follow-up period extended to Day 60. SeeFigures 13 to 14 。

[0628] For the multiple-dose cohorts (Part B), Cohort 1A was conducted to confirm the effects of bed rest on skeletal muscle and on HOMA-IR. Data from Cohort 1A were used to confirm that 10 days of bed rest was sufficient to characterize the effects on the muscle protein synthesis rate after administration of heavy water and via vastus lateralis muscle micro-needle (fine needle) skeletal muscle biopsy, total skeletal muscle mass determination of D3-Cr in fasting urine samples, and muscle perimeter, cross-sectional area, color flow analysis, anteroposterior diameter (AP), and echo density measured by ultrasound of the vastus lateralis and gastrocnemius muscles. If there was evidence that a shorter time could have a measurable effect on skeletal muscle, Cohort 1B and Cohort 1C were subjected to 10 days or less of bed rest.

[0629] The cohorts in Part A and the cohorts in Part B were conducted sequentially starting from SAD Cohort 1. This process was repeated for each cohort in both Part A and Part B of the study.

[0630] Dose rationale. Based on the preliminary results of the SAD cohort (BGE-105-101), it was recommended to increase the IV dose of BGE-105 to 4 times the maximum dose studied in the previously completed Phase 1 trial (BGE-105-101).

[0631] Given that both toxicants had good tolerability, the lack of safety study results in humans and the lack of dose-limiting toxicity (the NOAEL was the maximum dose tested in rats and dogs), this proposed dose increase was reasonable as long as the predicted human exposure did not exceed the exposure in the toxicant species. Considering the highest maximum exposures in rats and dogs, the updated exposure margin was calculated as follows: The maximum exposure in rats (male) occurred in a 14-day intravenous (IV) study at a dose of 300 mg / kg, with an AUC last of 1070 μg*hr / mL; the maximum exposure in dogs occurred in a 9-month oral toxicity study at a dose of 300 mg / kg, with an AUC last of 1310 μg·hr / mL.

[0632] Therefore, based on the PK of a single dose of 240 mg loading dose followed by a 22-hour 1440 mg IV infusion and multiple dosing at this dose (the highest recommended dose used in this study), the predicted human exposure margin was: human:rat = 1036 / 1070 = 0.97; human:dog = 1036 / 1310 = 0.79.

[0633] Therefore, all available safety data continued to support the use of doses up to 240 / 1440 mg, and even at the highest recommended dose, human exposure would not exceed the toxicological coverage.

[0634] Table 3: Cohorts, Doses, and Administration Schedules

[0635]

[0636]

[0637] Abbreviations: h = hour; LD = loading dose; MD = maintenance dose; yo = years old; TBD = to be determined

[0638] *The total number of subjects in Part B may be reduced or increased to a maximum of 48 subjects.

[0639] **From Day -5 through Day 10, heavy water was administered 3 times per day. One D3 - Creatine capsule was taken on Days -3, 6, and 11.

[0640] A placebo product for infusion matching BGE - 105 was prepared by using normal saline of the corresponding IV bag size. If a label was added to the normal saline bag used for preparing the active BGE - 105 for infusion, the label on the placebo normal saline bag should also match to maintain blinding.

[0641] Study duration. Subjects in Part A (SAD) participated in this study for approximately 42 days, including a screening / pre - treatment period of up to 14 days, a treatment period of 5 days, and a follow - up period of 27 days after the first administration of the study drug (BGE - 105 or placebo).

[0642] Subjects in Part B (MD) participated in this study for approximately 81 days, including: a screening period of up to 16 days; a 5 - day outpatient pre - treatment period (Days -5 to -1) using heavy water and D3 - Creatine (D3 - Cr); a 10 - day treatment period of bed rest using heavy water / D3 - Cr and BGE - 105 or placebo; and a post - dosing follow - up period (Days 11 to 60), which included follow - up visits on Days 11, 12, 13, 14, 21, 30, and 60 after the first administration of the study drug (BGE - 105 or placebo).

[0643] Part A: Cohort Screening and Pre - treatment

[0644] Subjects in Part A were admitted to the clinic on Day -2. On Day -1, a baseline assessment was conducted, and the subjects were randomly assigned to receive blinded treatment with the study drug (BGE - 105 or placebo). The protocol summary for the Part A cohorts is outlined in Figure 11 and described in Table 8 below.

[0645]

[0646] Part A: SAD Cohort Treatment and Follow - up

[0647] On Day 1 of Part A, the subject received an LD, 1-hour infusion. On Day 3, after a 48-hour washout period, the subject received a 23-hour infusion (1 hour LD, then 22 hours MD). Not earlier than 24 hours after the end of the infusion, the subject was discharged on Day 5 (End of Treatment Period [EOTP]). The treatment protocol for the SAD cohort in Part A is outlined in Figure 12 and described in Table 9 below.

[0648]

[0649] Part B: MD Cohort Screening and Pretreatment

[0650] Subjects in Part B were admitted on the evening of Day -2 and fasted overnight starting at 10:00 PM for baseline procedures on Day -1. The pretreatment protocol for the MD cohort in Part B is outlined in Figure 13 and described in Table 10.

[0651]

[0652]

[0653] Part B: MD Cohort Treatment and Follow-up

[0654] The treatment protocol for the MD cohort in Part B is outlined in Figure 14 and described in Table 11 below.

[0655]

[0656]

[0657]

[0658] Follow-up Period

[0659] For Part A, on Day 28 (approximately 27 days after the administration of the first dose of the study drug), the subject was contacted by phone for a safety follow-up assessment. The date on which the subject completed the follow-up was the subject's End of Study (or EOS). For Part B, after 10 days of bed rest, the subject was required to stay in the clinic until Day 13 for safety follow-up assessments, laboratory sample collection, and physical function rehabilitation. If it was not logically feasible to return to the clinic on Day 14 for protocol assessment, the subject could choose to stay in the clinic for one additional day. The subject had a telemedicine visit on Day 21 and clinic visits on Days 30 and 60 for safety follow-up assessments.

[0660] Pharmacokinetics and Pharmacodynamics Assessments

[0661] Ultrasound. For the subjects in part B only, ultrasound was performed at the previously described time points to measure the muscle circumference, cross-sectional area, color flow analysis, AP diameter, and echo density of the vastus lateralis and gastrocnemius muscles of one of the legs. The leg to be measured must be consistent.

[0662] Ultrasound images were collected during the following study visits: Day - 1 (baseline), Day 6 (predose), and Day 11 (end of treatment). Ultrasound was performed on both the vastus lateralis and gastrocnemius muscles to measure the cross-sectional area, color flow Doppler, anteroposterior (AP) diameter, and echo density. The muscle circumference was also measured. Throughout the study, all subjects' ultrasound imaging was performed by the same operator. Throughout the study, all subjects' ultrasound readings were performed by the same reader. The ultrasound operator and reader were blinded to the study treatment, regardless of whether the subject received the active study drug BGE - 105 or placebo. For all ultrasounds, the leg to be measured (right leg vs. left leg) and leg position (medial vs. lateral) were consistent.

[0663] The muscle circumference was measured in centimeters (cm) using a tape measure and recorded on the ultrasound worksheet at the time of imaging. The target circumference measurements were obtained at the following markings: the vastus lateralis was measured 15 cm above the mid-patella, and the gastrocnemius was measured 3 inches below the popliteal vessels.

[0664] Ultrasound procedure

[0665] Ultrasound images were collected before performing skeletal muscle microbiopsies. Before the test, subjects were asked to wear shorts on the day of the test to avoid compression of the thigh muscles and expose the upper thigh. The subject needed to lie supine on the examination table with both legs fully extended for at least 5 minutes to allow fluid shift to occur. Each subject was instructed to lie on the non-dominant side to obtain skeletal muscle ultrasound images of the vastus lateralis and gastrocnemius muscles of the dominant leg. The subject's legs were stacked on top of each other with the knees slightly bent. The ultrasound image of the vastus lateralis muscle was captured at the 50% point of the linear distance between the greater trochanter and the lateral epicondyle of the femur.

[0666] To ensure correct probe placement and consistent image capture locations, a dotted line was drawn transversely and longitudinally along the skin surface from the above-mentioned positions. All muscle morphology measurements were obtained using a B-mode, 12 MHz linear probe US (General Electric vivid E9) to provide acoustic contact without compressing the dermal layer of the skin. Longitudinal B-mode and transverse field of view (FOV) images were acquired and analyzed during each examination. For each subject's examination, the ultrasound settings were kept fixed: the image gain was set to 50 decibels (dB), the dynamic range was set to 72, and the image depth was set to 5 cm. Three panoramic transverse images (PTI) were captured in the transverse plane perpendicular to the long axis of the muscle. These images utilized extended field of view ultrasound examination to include the entire area of the vastus lateralis muscle in a single panoramic image.

[0667] In addition, three single longitudinal images (SLI) were captured in the longitudinal plane parallel to the long axis of the muscle. The selection of single static longitudinal images (SLI) included: (i) applying a constant pressure through the probe to minimize muscle compression, (ii) carefully positioning the probe on the thigh using a large amount of ultrasound gel to avoid affecting the measurement values, so that the probe did not directly contact the skin to push the soft tissue; (iii) the superficial fascia (adipose tissue / muscle interface) of the vastus lateralis muscle was as close to horizontal as possible; (iv) the entire length of the image consisted of muscle fibers (without inconsistencies in fascia or probe pressure or position). The same technique was also applied to the ultrasound examination of the gastrocnemius muscle. Either the medial or lateral head of the gastrocnemius muscle could be used for ultrasound assessment, but not both simultaneously. Throughout the study, the same head was used for all gastrocnemius muscle ultrasound examinations.

[0668] Heavy water ingestion and assessment. Only for the subjects in Part B, heavy water, deuterated H2O (D2O), was provided to the subjects at the previously described time points before breakfast, in the afternoon, and after fasting overnight during pre-treatment and treatment.

[0669] Blood samples (10 mL) and urine samples were collected at the previously described time points to evaluate the fractional synthesis rate (FSR) of skeletal muscle proteins.

[0670] Plasma insulin and glucose. Blood samples (4 mL) were collected at the previously described time points in the morning, before breakfast, and after fasting overnight for plasma insulin and blood glucose monitoring.

[0671] Proteomics. Only for the subjects in Part B, blood samples (4 mL) for proteomic analysis were collected at the previously described time points.

[0672] Bioenergetics. Only for the subjects in Part B, blood samples (8 mL) for bioenergetic assessment were collected at the previously described time points.

[0673] Statistical Analysis

[0674] The Statistical Analysis Plan (SAP) provides the statistical methods and definitions for analyzing safety, PK, PD, and non-target metabolomic and proteomic data, and describes the methods to be taken for summarizing other study information (such as subject distribution, demographics and baseline characteristics, study product exposure, and previous and concomitant medications). The SAP also includes a description of how to handle missing, unused, and spurious data. Descriptive statistics are provided according to the nature of the criteria:

[0675] Quantitative variables: sample size, arithmetic mean, standard deviation (SD), standard error of the mean (SEM), minimum, median, and maximum, and quartiles (with geometric mean, arithmetic and geometric coefficient of variation (CV), and quartiles of pharmacokinetic (PK) parameters) when necessary.

[0676] Qualitative variables include: sample size, absolute and relative frequencies for each category. All listings are presented by group and treatment. Details of the statistical analysis are described in the SAP, which is completed before database lock.

[0677] Unless otherwise specified, all calculations are performed using SAS statistical software version 9.3 or higher and / or Phoenix WinNonlin. All safety, PK, PD, and PK / PD data for each treatment group are presented separately in tables and listings. The TLFs are provided in a collated electronic MS Word.rtf file format (i.e., table columns and rows are presented in MS Word table format). The statistical analysis of this study is conducted in a GCP environment (ICH E6).

[0678] Sample size:

[0679] No formal sample size calculation has been performed. The cohort and overall study sizes are based on practical considerations. This study is planned to enroll up to 72 volunteers who receive at least one dose of BGE-105 or placebo.

[0680] In SAD (Part A), 18 subjects received BGE-105 and 6 subjects received placebo.

[0681] For MD (Part B), 30 subjects received BGE-105 or placebo. All Part B subjects were aged ≥ 65 years. The Sponsor may, at its discretion, replace subjects on a case-by-case basis. Replacement subjects will receive the same treatment as the subjects being replaced.

[0682] Analysis population:

[0683] The safety analysis set includes all subjects who received ≥1 administrations of the study drug (BGE-105 or placebo). The safety analysis set is used for safety analysis. Subjects are analyzed based on the actual treatment received.

[0684] The pharmacokinetics (PK) set includes subjects who have received ≥1 administrations of the study drug without any events and / or major protocol deviations that affect PK assessment and with a complete PK profile. Prior to locking of the PK concentration data set, agreement will be reached between the sponsor and the CRO regarding the inclusion / exclusion of subjects with incomplete PK profiles in this set.

[0685] The pharmacodynamics (PD) set includes all enrolled subjects who have completed the study without any protocol deviations that affect PD assessment and with baseline samples and ≥1 post-baseline samples for PD assessment. Prior to locking of the PD concentration data set, agreement will be reached between the sponsor and the CRO regarding the inclusion / exclusion of subjects with incomplete PD profiles in this set.

[0686] The pharmacokinetics / pharmacodynamics (PK / PD) set includes all subjects who are simultaneously in the PD set and the PK set.

[0687] Safety Analysis

[0688] Summarize safety data, using descriptive statistics (number of subjects, mean, median, standard deviation, minimum, and maximum) to summarize continuous variables and frequencies and percentages to summarize discrete variables.

[0689] Adverse events are coded using the Medical Dictionary for Regulatory Activities. For each cohort and treatment group, the number of events, incidence, and percentage of TEAEs are calculated by system organ class, preferred term, and overall for the treatment group. The number and percentage of subjects with TEAEs are further summarized according to severity and relationship to the study drug. AEs related to the study drug, AEs leading to withdrawal, SAsE, and deaths are also summarized and / or listed in a similar manner.

[0690] Summarize clinical laboratory tests, vital signs, and ECG results by treatment group and study visit. If applicable, calculate descriptive statistics for quantitative safety data and differences from baseline. Frequency counts are compiled to categorize qualitative safety data. The baseline for safety data is defined as the last value before the first dose of IP administration. Potential important clinical findings are also summarized and / or listed.

[0691] Pharmacokinetic Analysis:

[0692] List the individual BGE-105 plasma concentrations and provide descriptive statistics including mean, geometric mean, median, range, standard deviation, and coefficient of variation. The corresponding concentration-time profiles (individual and mean) are shown in graphs.

[0693] Derive the relevant plasma PK parameters of BGE-105 by standard non-compartmental methods and tabulate them together with the descriptive statistics and graphs.

[0694] Use an exponential regression model (“power model”) to investigate the linear dose proportionality of Cmax, AUC0-t, and AUC0-inf (if applicable) of BGE-105.

[0695] Pharmacodynamic Analysis:

[0696] Analyze the data on changes relative to baseline within each dose group and treatment group. Treatment comparisons are based on samples taken at matching times during the 24-hour period on Day -1, comparing all treatment groups to all placebo groups, and each individual dose group to all placebo groups. The reported P-values are not adjusted for multiplicity.

[0697] For Part B, first analyze the changes in thigh circumference, cross-sectional area, color flow analysis, AP diameter, and echo density of the vastus lateralis and gastrocnemius muscles measured by ultrasound as linear contrasts comparing all treatment groups to placebo, and then as individual doses to placebo. The reported P-values are not adjusted for multiplicity.

[0698] Compare the skeletal muscle protein synthesis rate (% / time) of individual muscles, first as a linear contrast including all doses to placebo, and then as individual doses to placebo.

[0699] The Benjamini-Hochberg procedure is used to control the false discovery rate for the evaluated protein diversity.

[0700] Analyze the total muscle mass of D3-creatine as changes relative to baseline in each group and treatment group. Treatment comparisons are based on samples taken at matching times, making linear contrasts of all treatment groups to placebo groups and each individual dose group to all placebo groups.

[0701] Microneedle biopsies are analyzed by three different metrics, including target biomarkers, proteomics, and protein changes (exploratory).

[0702] Target biomarkers: Compare the means of the target biomarkers (creatine kinase - muscle [CK-M], etc.) for each MD cohort using analysis of variance (ANOVA).

[0703] Proteomics: Compare the ratio of proteins with high FSR to those with low FSR to the baseline value.

[0704] Protein changes (exploratory): After multiple comparison corrections, individual proteins showing significant changes over time were detected.

[0705] The sit-to-stand test and SPPB were analyzed as within-subject changes and applied to each dose cohort and treatment group. The POMA assessment was an exploratory measure. Any changes relative to baseline within subjects were summarized and applied to each dose cohort and treatment.

[0706] Pharmacokinetics / Pharmacodynamics Analysis:

[0707] List the plasma concentrations of individual biomarkers and provide descriptive statistics including mean, geometric mean, median, range, standard deviation, and coefficient of variation. The corresponding concentration-time profiles (individual and mean) are shown in graphs.

[0708] The relevant PD parameters are listed and tabulated together with descriptive statistics and graphs.

[0709] Use graphical exploratory and simple modeling methods to study the relationship between BGE-105 and plasma and other PD parameters.

[0710] Non-blind data review. After each cohort is completed, the sponsor conducts a non-blind data review. A non-blind data review is conducted after completion of Part A to confirm the study design and dose for Part B. After completion of each MD cohort, a sponsor non-blind data review will also be conducted.

[0711] Results

[0712] The results indicate that BGE-105, using a single dose or multiple doses, prevents frailty and improves muscle function in the elderly.

[0713] After administering BGE-105 by continuous intravenous (IV) infusion at single ascending doses and multiple ascending doses to healthy adult subjects (≥18 years of age in Part A, ≥65 years of age in Part B), with a focus on elderly subjects (≥65 years of age in both Part A and Part B), the BGE-105 administered was shown to be safe and well-tolerated.

[0714] Results of 3 SAD Cohorts in Part A - BGE-105-101 Study

[0715] Pharmacokinetic (PK) data from 3 SAD cohorts demonstrated Figure 15 the dose proportionality shown in C max was maintained within the expected range, and for the highest dose (240 mg / 1440 mg), AUC lastis 1062 μg*hr / mL (Table 4 below):

[0716] For the SAD cohorts in Part A, all doses were well tolerated, including the highest doses of 240 mg / 1440 mg. No new safety issues or trends were observed, nor were there any serious adverse events. The maximum tolerated dose from the 3 SAD cohorts has not been determined. Table 4 provides an overview of the pharmacokinetic parameters for Part A (single ascending dose) of the Phase 1 study BGE-105-101.

[0717]

[0718] Figure 15 Dose proportionality was demonstrated across the 3 SAD cohorts. The dose slope and 95% confidence intervals for Cmax(A) and AUC(B) included 1, indicating linearity of BGE-105 PK across the dose range. The low point estimates and wide confidence intervals may be due to differences between subjects.

[0719] Figure 16 Preliminary results are shown for SAD cohorts 1 - 3 (Part A of the study), where for the placebo group in the SAD study (Part A) of Example 9, HOMA-IR increased by 17% from baseline at the pre-dose visit on Day 3 and by 12% from baseline at the visit on Day 4. In patients treated with BGE-105 (cohorts 1, 2, and 3) in the SAD study (Part A) of Example 9, the percentage change in HOMA-IR relative to baseline decreased at both visits, with the greatest decrease in the highest dose group (cohort 3), indicating a positive effect on insulin sensitivity and an improvement in insulin resistance.

[0720] Results of 3 MD Cohorts in Part B - BGE-105 Study

[0721] In a double-blind placebo-controlled study, 21 healthy volunteers aged ≥65 years were strictly bedridden for 10 days while receiving a daily IV infusion of placebo (n = 10) or a fixed dose of BGE-105 (n = 11). At one day before the start of bed rest (baseline, D-1), Day 5 (D5), and Day 10 (D10) of bed rest, key muscle atrophy endpoints were measured: thigh circumference; cross-sectional area (CSA) and A-P diameter of the vastus lateralis muscle (ultrasound); ultrasound muscle mass grade, which is an index quantifying muscle fat degeneration (ultrasound echo density); and muscle protein synthesis rate (biopsy). Parameters measured during the 10-day bed rest period included thigh circumference, muscle size, muscle mass (e.g., fat degeneration), and muscle protein synthesis rate. Table 5 summarizes the results.

[0722]

[0723] *Effect size was based on Cramer’s V. All percentages were relative to baseline values measured 1 day before the start of dosing.

[0724] Measurement of thigh circumference and vastus lateralis muscle is one of the gold standard markers of skeletal muscle atrophy. The vastus lateralis muscle is the largest muscle in the quadriceps femoris and one of the most frequently studied muscles because (a) it has an important function in terms of activity and disability, (b) the location and structure of the muscle, which facilitates ultrasound measurement and biopsy, and (c) atrophy in elderly patients is more severe than in calf muscles. Endpoint measures were taken at baseline, after 5 days of bed rest, and after 10 days of bed rest, including endpoints such as limb circumference, muscle area measured by ultrasound, muscle mass measured by ultrasound (measuring normal muscle and fat), fractional synthesis rate, etc. Many endpoint measures are biochemically related to muscle size and muscle function, such as thigh circumference, muscle size measured by ultrasound, to calculate the diameter, thickness, and cross-sectional area of the muscle (calculated after ultrasound).

[0725] Figures 18A to 18B The effects of BGE-105 on the reduction of rest-induced thigh circumference, vastus lateralis muscle diameter % (thickness), vastus lateralis muscle cross-sectional area (CSA) %, muscle degeneration, and cumulative protein synthesis rate % (measured by biopsy) are shown, as in Table 5. Measurements were taken 15 cm above the midpoint of the patella. In healthy volunteers aged ≥65 years, BGE-105 significantly reduced muscle atrophy at multiple key endpoints. Compared with the placebo group, the muscle protein synthesis rate was higher in patients treated with BGE-105, providing a potential mechanistic basis for the protective effect of BGE-105 on muscle size. The results of the Phase 1b trial support the investigation of BGE-105 as a treatment for multiple age-related syndromes driven by muscle loss. These conditions include acute myopathy in hospitalized patients on mechanical ventilation and chronic medical conditions. BGE-105 significantly prevented muscle atrophy at multiple endpoints: (circumference (p<0.001), diameter (p<0.01), cross-sectional area (p<0.05), muscle grade (scale) (p<0.005), and cumulative protein synthesis (p<0.005).

[0726] Figure 18A It was shown that thigh circumference, muscle size, muscle mass (such as fatty degeneration), and muscle protein synthesis rate were increased in patients treated with BGE-105 (cohort 1B) compared with the placebo group (cohort 1A).

[0727] Figure 18BShows the thigh circumference of patients during (middle sub - figure) or after (left - hand sub - figure) 10 - day bed rest for those receiving placebo (cohort 1A of the MD study in Example 9) and BGE - 105 treatment (cohort 1B of the MD study in Example 9), expressed as percentage change relative to baseline. p = 0.0004.

[0728] Figure 18B Shows that BGE - 105 significantly prevents bed - rest - induced reduction in thigh circumference (middle sub - figure). The results indicate that, as measured by ultrasound, thigh circumference is related to the thickness and cross - sectional area of constitutive muscle. Elderly patients (>65 years old) experience severe muscle atrophy after 10 days of bed rest, especially in the legs. Overall, elderly patients experience a total lean body mass loss of approximately 3.2% to 4.7%, and a leg lean body mass loss of approximately 6% to 7% (cohort 1A, MD1A). After a 10 - day bed - rest period, the thigh circumference of elderly patients in the placebo group decreased by 6.438%, while that of elderly patients in the BGE - 105 treatment group increased by 0.8%, p<0.0001. Measurements were taken 15 cm above the mid - patella (left - hand sub - figure). Solid lines represent patients receiving treatment, and dashed lines represent the placebo group. Figure 18B Shows that patients receiving BGE - 105 treatment had a greater thigh circumference compared to the placebo group (cohort 1A). All percentages are relative to the baseline value measured 1 day before the start of dosing.

[0729] Muscle size measured as a function of ultrasound showed a 21% reduction in the placebo group (cohort 1A) and a 5.664% reduction in the treatment group (cohort 1B), indicating an approximately 75% improvement in muscle size. Figure 19 Shows that, as measured by ultrasound, BGE - 105 significantly prevents bed - rest - induced reduction in the thickness and cross - sectional area of the vastus lateralis muscle (left - hand sub - figure). After 10 days of bed rest, the diameter of the vastus lateralis muscle in patients receiving BGE - 105 treatment decreased by only 5.664%, while that in the placebo group decreased by 21.18% (p = 0.0075) (middle sub - figure). Similar results were seen in the measurement of the cross - sectional area of the vastus lateralis muscle (right - hand sub - figure). BGE - 105 treatment showed an 8.041% reduction in the cross - sectional area of the vastus lateralis muscle, which was significantly different from the 19.47% reduction in the cross - sectional area of the vastus lateralis muscle observed in the placebo group (p = 0.0297). Percentages are shown as mean percentage change relative to baseline.

[0730] Figures 20A to 20CIt was shown that, via echo density measurement, BGE-105 significantly prevented bed rest-induced fatty degeneration of the vastus lateralis muscle. Muscle grading was performed according to Figures A to B. Echo density measurement determines muscle mass. Echo density measurement showed a 6.4% decrease in the placebo group (cohort 1A), while the treatment group (cohort 1B, 240 mg daily) showed an 0.8% increase.

[0731] The echo density measurement results show the number of patients receiving the ultrasound muscle mass grading scale, which uses a numerical grading system of grade 1 or 2, to classify fatty muscle atrophy by measuring the fat infiltration of biopsy muscle fibers from the patients. Figure 20A Showing the amount of fat in normal muscle (1), Figure 20B Showing muscle with some fat streaks (2), Figure 20C Showing the changes in fatty degeneration from baseline to 10 days of bed rest in the placebo group and the BGE-105 group. Open boxes represent normal muscle (grade 1), and striped boxes represent muscle with some fat streaks (grade 2). After 10 days of bed rest, 8 out of 10 patients (8 / 10) in the placebo group progressed to grade 2, while among the 11 patients treated with BGE-105, only one (1) patient (1 / 11) progressed to grade 2 after 10 days of bed rest. (p = 0.0019). The echo density measurement results showed that the ultrasound muscle mass grade of 80% of the patients increased from grade 1 (normal muscle tissue) to grade 2 (muscle tissue with fat streaks), while in the BGE-105 treatment group (cohort 1B), only 1 patient increased from grade 1 to grade 2, showing statistical significance.

[0732] Figure 21 Showing the muscle protein synthesis rate in the vastus lateralis muscle measured via microdialysis in individual subjects in the BGE-105 treatment group and the placebo group. Muscle protein synthesis was measured 10 days after bed rest, and the fractional synthesis rate normalized by the subject was presented. p = 0.0043.

[0733] Figure 21Shown, via microdialysis measurements, BGE-105 resulted in an elevated muscle protein synthesis rate in the vastus lateralis muscle. The figure represents the ratio of the BGE-105 treatment group to the placebo group. The horizontal line above the x-axis indicates that the muscle protein synthesis level was higher than that of the placebo group after 10 days of bed rest. The muscle protein synthesis rate shows the cumulative muscle synthesis rate within 10 days of bed rest. The cumulative muscle protein synthesis rate of the BGE-105 treatment group (cohort 1A) was significantly increased compared to the placebo group (cohort 1A). After 10 days of bed rest, 8 out of 11 patients treated with BGE-105 (approximately 72.7%) experienced an elevated muscle protein synthesis rate, and the average normalized synthesis rate score ranged from 0.18 to 2.2. In contrast, only 3 out of 10 patients treated with placebo (30%) experienced an elevated muscle protein synthesis rate. The average normalized synthesis rate score ranged from 0.04 to 0.33. The results indicate that BGE-105 can effectively induce or maintain muscle protein synthesis in the vastus lateralis muscle (p = 0.0043).

[0734] Figures 23A to 23B Shows the characteristics of healthy volunteers with 1b-stage bed rest atrophy in the MD study and the incidence of treatment-emergent adverse events (Part B of Example 9). The placebo group is represented as cohort 1A of the MD in the study of Part B of Example 9, and the BGE-105 treatment group is represented as cohort 1B of the MD in the study of Part B. Figure 23B Shows the incidence of treatment-emergent adverse events (TEAEs). Nine (9) out of 10 volunteers in the placebo group (90%) had TEAEs, while seven (7) out of 11 volunteers in the BGE-105 group (64%) had TEAEs.

[0735] Figure 24 Shows the effect of BGE-105 on the rest-induced reduction in thigh circumference of subjects participating in the 1b-stage MD, Part B clinical trial of Example 9. Thigh circumference was measured in patients receiving placebo or BGE-105 treatment and is expressed as the percentage change relative to baseline. The thigh circumference of subjects receiving BGE-105 was completely maintained or increased during the 10-day bed rest period. The measurement was taken 15 cm above the midpoint of the patella. P = 0.0004. After 10 days of treatment, seven (7) out of 11 subjects treated with BGE-105 (64%) had their thigh circumference completely maintained or increased within 10 days of bed rest. P = 0.0004. Among the subjects receiving placebo, none (0%) showed an increase in thigh circumference compared to baseline.

[0736] Figure 25AFigures 25C to 25C show the effect of BGE-105 on the rest-induced reduction in calf circumference in subjects participating in the Phase 1b MD, Part B clinical trial of Example 9. The calf circumferences of patients treated with placebo and BGE-105 were measured during (middle subfigure) or after (left subfigure) 10 days of bed rest and expressed as percentage change relative to baseline. The calf circumference is expressed as the percentage change from baseline to Day 10 (right subfigure). p = 0.0024. Measurements were taken 15 cm above the midpoint of the patella. Figure 25C shows the number of subjects showing percentage change in calf circumference from baseline to Day 10.

[0737] Figure 26 Shows the effect of BGE-105 on the gastrocnemius diameter and gastrocnemius cross-sectional area measured by ultrasound after 10 days of bed rest. The gastrocnemius diameter (middle subfigure, p = 0.836) or gastrocnemius cross-sectional area (left subfigure, p = 0.278) of patients treated with BGE-105 or placebo is expressed as the mean percentage change relative to baseline. As shown, the reduction in gastrocnemius diameter (10.982%) and cross-sectional area (15.72%) in the placebo group was greater than that in the BGE-105 treatment group (reduction in gastrocnemius diameter of 2.942% and cross-sectional area of 7.758%).

[0738] Figures 27A to 27B Shows the effect of BGE-105 on measurements of thigh, calf circumference, vastus lateralis cross-sectional area, and gastrocnemius cross-sectional area in female subjects. The thigh circumference (p = 0.0033), calf circumference (p = 0.0146), vastus lateralis cross-sectional area (p = 0.066), and gastrocnemius cross-sectional area (p = 0.4381) of subjects treated with placebo or BGE-105 were measured and expressed as percentage change relative to baseline. As shown, the reduction in thigh circumference (6.06%) and calf circumference (5.486%) in the placebo group was greater than that in the BGE-105 treatment group (reduction in thigh circumference of 1.674% and calf circumference of 1.166%) ( Figure 27A ). As shown, the reduction in vastus lateralis cross-sectional area (23.67%) and gastrocnemius cross-sectional area (15.32%) in the placebo group was greater than that in the BGE-105 treatment group (reduction in vastus lateralis cross-sectional area of 9.676% and gastrocnemius cross-sectional area of 5.558%) ( Figure 27B ).

[0739] Figure 28Shows the rate of muscle myofibrillar protein synthesis in the vastus lateralis muscle of patients treated with BGE-105 and placebo-treated patients. The mean vastus lateralis muscle myofibril synthesis rate was measured and expressed as a change relative to baseline (left subplot). The mean myofibril FSR / day was expressed as a percentage change relative to baseline (right subplot). As shown, the rate of muscle myofibrillar protein synthesis was reduced to a greater extent in the placebo group (58% reduction) compared to the BGE-105 treatment group (36% reduction).

[0740] Figure 29 Figure shows measurements taken during the 1b phase bed rest atrophy study to evaluate the various muscle dynamics shown in Example 9. Key metrics include serum biomarkers, muscle protein synthesis rate, muscle size, and total muscle mass.

[0741] Figure 30 Figure shows a description of the modalities used in the 1b phase bed rest atrophy study of Example 9 to evaluate muscle dynamics. Proteomic analysis involves performing SomaScan assays on approximately 7k serum proteins that can be mapped to established muscle health and frailty characteristics. The skeletal muscle protein synthesis rate is measured by measuring deuterium incorporation into proteins synthesized in skeletal muscle that escape into the bloodstream (plasma and urine analysis) or are captured by micro-biopsies. Micro-biopsies of the vastus lateralis muscle are used to be able to measure the synthesis rate of hundreds of proteins. It will also be able to explore changes in the levels of specific proteins. Ultrasonography measures the circumference, cross-sectional area, color flow analysis, anterior-posterior diameter, and echo density of the vastus lateralis and gastrocnemius muscles. Measuring the size of the total body creatine pool (via urine) provides an assessment of total muscle mass as approximately 98% of the creatine in the body is sequestered in the sarcomeres.

[0742] Figure 31 Figure shows the location and method of ultrasonic measurement of leg muscles. Ultrasonography measures the skeletal muscle circumference, cross-sectional area, color flow analysis, anterior-posterior diameter, and echo density of the vastus lateralis and gastrocnemius muscles.

[0743] Figure 32 Figure shows the process of incorporating deuterated water into muscle proteins for calculating the fractional synthesis rate. Deuterium can be measured both invasively (via tissue biopsy) and non-invasively (via blood and urine - virtual biopsy).

[0744] Figure 33 Shows micro-biopsies of the vastus lateralis muscle taken at multiple time points (baseline on day 0 of bed rest, day 5 of bed rest, and at the end of the 10-day bed rest period) to measure the fractional synthesis rate of hundreds of muscle proteins. Biopsies were collected at three time points using a micro-needle: baseline, mid-way through the entire treatment period, and at the end of the treatment period.

[0745] Figure 34Flowchart depicting the assessment of total muscle mass using the D3-creatine tracer during the Phase 1b study of Example 9. Validation data for human MRI muscle volume (r = 0.87, p < 0.01). Independent of renal function. Briefly, subjects were administered D3-creatine orally. Subjects reached isotopic steady state (approx. 3 days). Fasting morning urine D3-creatinine and creatinine levels were measured. Creatine pool size and total body muscle mass were calculated. Higher creatine pool size and higher D3-creatinine:total creatinine ratio indicate higher muscle mass. Lower creatine pool size and lower D3-creatinine:total creatinine ratio indicate lower muscle mass.

[0746] Figure 35 Showing the correlation between skeletal muscle atrophy and reduced muscle protein synthesis in elderly patients (>65 years). Muscle atrophy is represented by the change in lean leg mass (g) (left subfigure). Muscle fractional synthesis rate was measured before and after a 10-day bed rest period in patients treated with BGE-105 or placebo and is represented as a percentage change (right subfigure). Data indicate that elderly patients (65+) experience rapid and severe muscle atrophy after 10 days of bed rest. A 9% reduction in total lean leg mass was observed in elderly patients after 10 days of bed rest, while in young control subjects, only a 2% reduction in total lean leg mass was observed after 28 days of bed rest. Approximately 40% of elderly patients showed a reduced muscle protein synthesis rate. The net muscle protein synthesis rate in elderly patients also decreased by approximately 30%. P = 0.02.

[0747] Figure 36A Showing the effect of BGE-105 on muscle protein synthesis in vastus lateralis muscle biopsies of patients treated with BGE-105 or placebo. Shown is the protein FSR ratio of the BGE-105 group compared to the placebo group after 10 days of bed rest (middle subfigure). Shown is the comparison of cumulative protein synthesis in patients treated with placebo and BGE-105 after 5 days and 10 days of bed rest (right subfigure).

[0748] Figure 36B Showing fractional synthesis in BGE-105-treated patients and placebo-treated patients on day 11 of bed rest. As shown, BGE-105 resulted in higher muscle protein synthesis in the vastus lateralis muscle compared to placebo-treated patients, as measured by muscle biopsy.

[0749] Figure 37Shows the step ratio of patients wearing a wearable activity device during the period from day 10 to day 60 (after the bed rest period) in the Phase 1b MD B part clinical trial as described above. This ratio is calculated by dividing the patient's subsequent steps by their baseline steps, i.e., the average daily steps before the bed rest period. Compared with patients receiving placebo treatment, physical activity of patients treated with BGE-105 increased as determined by the number of steps taken using the wearable activity device. The activity level in the BGE-105 treatment group started near baseline and then increased continuously for several weeks before the curve converged again.

[0750] Muscle atrophy, i.e., the loss of muscle mass and strength, is a common feature of human aging, which increases the risk of multiple diseases, shortens lifespan, and reduces quality of life. Hospitalization and a period of forced inactivity greatly accelerate this loss in the elderly.

[0751] Analysis of the inventors' unique human aging cohort indicates that the apelin pathway is a powerful predictor of healthy longevity and muscle function and directly translates to the clinical finding of this study that activation of the apelin pathway with BGE-105 improves muscle physiology in the elderly.

[0752] Analysis of a proprietary human biobank indicates that apelin pathway activity, which declines with age, is positively correlated with lifespan, mobility, and cognitive function. Apelin is the natural ligand of APJ, secreted by skeletal muscle in response to exercise, and regulates multiple aspects of muscle metabolism, growth, and repair.

[0753] This double-blind, placebo-controlled clinical trial evaluated the safety and pharmacodynamics of BGE-105. Twenty-one volunteers underwent 10 days of bed rest while receiving an infusion of BGE-105 or placebo.

[0754] Volunteers taking placebo (n = 10) showed muscle atrophy, manifested as statistically significant decreases in thigh circumference, vastus lateralis muscle dimensions (cross-sectional area and thickness), and muscle mass (fatty degeneration) as measured by ultrasound.

[0755] Treatment with BGE-105 (n = 11) significantly improved muscle atrophy relative to placebo:

[0756] Muscle dimensions: Volunteers treated with BGE-105 showed a 100% increase in thigh circumference (p < 0.001) relative to placebo-treated volunteers, and ultrasound measurements showed a 58% increase in the cross-sectional area of the vastus lateralis muscle (p < 0.05) and a 73% increase in the thickness of the vastus lateralis muscle (p < 0.01).

[0757] Muscle mass: Ultrasonographic echo density measurements showed that the muscle mass grading scale (an index quantifying muscle degeneration) deteriorated in 8 out of 10 placebo - receiving volunteers, while it deteriorated in only 1 out of 11 BGE - 105 - receiving volunteers (p < 0.005).

[0758] Muscle protein synthesis: Proteomic analysis of muscle micro - biopsy samples showed that bed rest decreased muscle protein production, while BGE - 105 significantly improved this effect (p < 0.005). The muscle protein synthesis rate in the drug group was higher than that in the placebo group, providing a potential mechanistic basis for the protective effect of BGE - 105 on muscle size.

[0759] Compared with placebo, the eparineptide agonist BGE - 105 showed statistically significant improvements in muscle size, mass, and protein synthesis during 10 days of bed rest in volunteers aged ≥65 years, and there were no serious adverse reactions. Compared with placebo, BGE - 105 treatment statistically significantly prevented muscle atrophy after 10 days of strict bed rest in healthy volunteers aged 65 years or older.

[0760] On day 10, volunteers receiving BGE - 105 showed improvement in bed - rest - induced atrophy compared with placebo - treated volunteers, as reflected in multiple metrics (table). BGE - 105 was well - tolerated in the study, and no serious adverse reactions were reported. The results are shown in Table 5 above.

[0761] In terms of safety, BGE - 105 was well - tolerated in the study. In healthy volunteers aged ≥65 years, BGE - 105 significantly reduced muscle atrophy at multiple key endpoints. The muscle protein synthesis rate was higher in the BGE - 105 treatment group than in the placebo group, providing a mechanistic basis for the protective effect of BGE - 105 on muscle size. The results of the phase 1b clinical trial support the investigation of BGE - 105 as a treatment for multiple age - related syndromes driven by muscle loss. These conditions include acute myopathy in hospitalized patients receiving mechanical ventilation and chronic diseases common in millions of elderly people for which there are no approved therapeutic agents for prevention or treatment, representing a large unmet clinical need.

[0762] Proteomic Features of Physical Function and Mortality

[0763] The sera collected from the Phase 1B clinical trial subjects (treatment group and placebo group) as described above were subjected to proteomic profiling and analysis. The sera levels collected from 11 treatment group subjects and 10 placebo subjects on Day -1 (baseline), Day 5, and Day 11 were subjected to proteomic profiling. A linear regression model of the interaction term between the treatment group and the date was implemented to identify proteins whose differential abundance between the treatment groups affected the average daily change rate of a given protein. The model was fitted to all measured proteins separately, and the resulting coefficients (on the interaction term) for each protein were used to rank all proteins from most positive to most negative.

[0764] To test whether the effects of BGE-105 on the plasma proteome significantly affected the proteomic signatures of physical function and mortality, enrichment analysis was performed using the GSEA method, using (1) the sorted list of proteins affected by BGE-105 described above and (2) various protein sets. For each human aging cohort phenotype (various physical function phenotypes and mortality), the two protein sets were composed of proteins that were positively and negatively correlated (p < 0.05) with that phenotype (to maintain directionality). Figure 48 and Figure 49 The significant phenotypes in the right panel, their GSEA plots, and p-values (when the protein sets of the significant phenotypes substantially overlapped with those of the phenotypes already shown, the significant phenotypes were not shown).

[0765] The proteomic data showed significant enrichment of proteins associated with muscle function in parallel and future decline; that is, the clinical trial data analysis indicated that the proteome of individuals treated with BGE-105 under bed rest conditions had a tendency to shift towards a healthier functional outcome. This observation reinforced the initial observation of the importance of apelin in maintaining grip strength during the aging process, suggesting potential long-term benefits of treatment with BGE-105. Figures 38A to 38D Shows the proteomic profiling of sera collected from the subjects of the Phase 1b clinical trial of Example 9. The sera levels of 11 treated subjects and 11 placebo subjects were collected on Day -1 (baseline), Day 5, and Day 11 and subjected to proteomic profiling. Figure 38ADisplay the proteomic data from patients in a Phase 1B clinical trial, providing the changes in the number of proteins related to frailty (functional), walking speed, instrumental activities of daily living (IADL) (functional tools), and grip strength in patients treated with BGE-105 in the Phase 1b clinical trial as described above. IADL is a specific category of functional activities being measured. For frailty (functional), among the 992 proteins related to frailty in Phase 1b clinical trial patients, 69 changed due to treatment with BGE-105 (p<0.05). For walking speed, among the 526 proteins related to walking speed in Phase 1b clinical trial patients, 35 changed due to treatment with BGE-105. For grip strength, among the 379 proteins related to grip strength, 58 changed due to treatment with BGE-105. Figure 38B Show that BGE-105 shifts the serum proteome towards a healthier state, recapitulating the benefits of natural high amylin levels in subjects treated with BGE-105. The proteome includes groups for vigorous activity, walking speed, lifespan, physical function, grip strength, and walking speed. Vigorous activity refers to difficulty in performing heavy household chores or lifting 10 pounds. Physical function refers to difficulty in performing physical activities such as walking half a mile, lifting, or grasping objects. Multiple testing adjustment of P-values was performed using the Benjamini Hocheberg method.

[0766] Figures 38A to 38B The proteome definitions of the proteomic data shown below:

[0767] Proteome definition

[0768]

[0769]

[0770] Performing SomaSignal Tests Using Proteomic Data

[0771] Next, implement a linear mixed effects model in the proteomic data to capture the differences in the daily average rate of change between the two groups (treatment group and placebo group) in the SomaSignal test.

[0772] When evaluating resting energy expenditure, 122 aptamers were used, and the Somasignal test was used to predict an individual's resting energy expenditure in calories per day (cal / day). Population used: A study of 9,022 individuals (aged 29 - 64 years) based in the UK. Model performance: CCC = 0.66, R2 = 0.46 (CI: 0.42 - 0.49). Figure 38CShows the change in baseline energy expenditure of BGE-105-treated subjects and placebo-treated subjects using SomaSignal tests on proteomics data. Figure 38C Shows the absolute change in REE (calories / day) compared to baseline (at the end of treatment, p = 0.03). As shown, the change in baseline energy expenditure was mainly driven by the change in muscle mass. As Figure 38C shown, proteomics data predicted that BGE-105 treatment "rescued" the resting energy rate.

[0773] When evaluating cardiorespiratory fitness (VO2)max, 52 aptamers were used, and the predicted peak exercise capacity was estimated using SomaSignal tests. A North America-based study included 743 individuals (aged 15 - 65 years), model performance: CCC = 0.85, R2 = 0.75 (CI: 0.68 - 0.81). Figure 38D Shows the change in cardiorespiratory fitness (VO2)max and basal metabolic rate of BGE-105-treated subjects and placebo-treated subjects using SomaSignal tests on proteomics data. As Figure 38D shown, VO2 max is driven by maximal cardiac output and oxygen uptake in skeletal muscle. Through SomaSignal tests, BGE-105 treatment shifted the proteome towards the predicted higher basal metabolic rate and VO2 max (at the end of treatment, p = 0.004).

[0774] BGE-105 significantly altered the serum levels of some of the same proteins in the BioAge longitudinal aging cohort data that are associated with future physical function decline (assessed by walking speed, activities of daily living (functional instrument), and grip strength). These proteins can then be used to attempt to identify patients who respond to BGE-105 treatment and may have reduced muscle atrophy.

[0775] The results also showed that clinical multi-omics provides predictive benefits for muscle strength, metabolism, and aerobic capacity.

[0776] Results of Clinical Studies on Disuse Atrophy

[0777] The study demonstrated that, compared to placebo, BGE-105 significantly reduced muscle loss, as measured by vastus lateralis muscle thickness (75% reduction in loss) and cross-sectional area (>50% reduction in loss). The maintenance of muscle mass was supported by the maintenance of the skeletal muscle fractional protein synthesis rate (measured by the D2O method and muscle biopsy). Importantly, compared to placebo, the synthesis of myofibrillar structural proteins such as myosin and troponin was preserved.

[0778] 7.10. Example 10: Phase 2 Study of BGE-105 for Preventing Diaphragm Atrophy

[0779] This is a Phase 2 clinical study of BGE-105 for preventing physical dysfunction caused by muscle atrophy in elderly patients who are bedridden for a long time due to severe diseases, surgeries or traumas. This example study examines the efficacy of BGE-105 in preventing diaphragmatic atrophy (DA).

[0780] Patients undergoing mechanical ventilation (MV) experience rapid diaphragmatic atrophy (DA) due to muscle disuse. Diaphragmatic atrophy is very common in critical illness situations. It has a great impact on morbidity and mortality, and there is no standardized treatment method. Given the impact of diaphragmatic atrophy on patients' weaning from the ventilator, preventing diaphragmatic atrophy is one of the most unmet needs in critical care medicine. Diaphragmatic atrophy in the intensive care unit (ICU) leads to poor clinical outcomes and a large waste of resources. Approximately 40 - 75% of patients undergoing mechanical ventilation (MV) develop severe diaphragmatic atrophy due to muscle disuse. DA is the main cause of difficulty in weaning from MV, leading to poor clinical outcomes and increased resources. Generally, patients with DA receive MV for 2 times longer (7 days vs. 4 days), stay in the ICU for 2 times longer (12 days vs. 6 days), and / or have a 4-fold higher in-hospital mortality rate (27% vs. 7%). There is no approved therapy for preventing or treating DA.

[0781] This Phase 2 clinical trial evaluated the role of BGE-105 in preventing adverse outcomes in elderly patients undergoing mechanical ventilation in the intensive care unit (ICU). This study evaluated the ability of BGE-105 to prevent diaphragmatic atrophy in the ICU. These conditions, which affect millions of patients each year, are associated with poor clinical outcomes and a substantial increase in mortality. There is currently no effective treatment, meaning that a huge medical need remains unmet.

[0782] Figures 22A to 22B and Figure 39 Illustration of the overview of the Phase 2 point-of-care (POC) trial in the ICU diaphragm. Figure 22A Illustration of the timeline of the BGE-105 clinical trial. Figure 22B Illustration of the design of the Phase 2 POC trial in patients with mechanical ventilation and ICU diaphragmatic atrophy. This study investigated POC in ICU diaphragmatic atrophy to improve recovery: reducing muscle and peripheral atrophy, and / or improving functional outcomes. Other objectives included optimizing the dose and optimizing the endpoints to characterize the safety, efficacy and reproducibility of the treatment.

[0783] The purpose of this trial is to prevent diaphragmatic and bed rest atrophy in elderly mechanical ventilation patients and to determine the power requirements for patient outcomes.

[0784] Adult mechanical ventilation patients aged ≥ 65 years, with acute hypoxemic respiratory failure (P / F < 300) and stratified according to diaphragm thickness were eligible. Treatment started from ventilation and lasted for 10 days. Approximately 100 patients were enrolled: 50 received placebo treatment and 50 received BGE-105 treatment. BGE-105 / placebo was administered via IV.

[0785] Primary endpoint - ICU diaphragm atrophy: To evaluate the progression of diaphragm atrophy, indicated by the change in diaphragm thickness.

[0786] Secondary endpoint - critical illness myopathy: Ultrasonography of the vastus lateralis muscle, indicated by cross-sectional area (CSA), muscle thickness, and Goutallier grading scale.

[0787] Exploratory endpoints included duration of mechanical ventilation, length of ICU stay, time to discharge, quality of life (QOL) and PRO scales, thigh and calf circumferences, and skeletal muscle biopsy.

[0788] 7.11. Example 11: Phase 2 Study of BGE-105 for the Prevention of Critical Illness Myopathy

[0789] This is a Phase 2 clinical study of BGE-105 for the prevention of muscle atrophy that leads to physical dysfunction in elderly patients on prolonged bed rest due to severe illness, surgery, or trauma. This example studies the efficacy of BGE-105 for the prevention of critical illness myopathy (CIM).

[0790] Critical illness myopathy is very common in the critical illness setting. It has a significant impact on morbidity and mortality, and there is no standardized treatment. Through a Phase 1b study of bedridden atrophy, the risk of critical illness myopathy was significantly reduced. Approximately 40%-90% of ICU patients will develop critical illness myopathy, which is a condition of proximal muscle weakness and atrophy. CIM is also associated with poor clinical outcomes and increased resources, similar to DA (Example 11). Approximately 65% of CIM patients will experience disabling and long-term muscle weakness after transfer out of the ICU, including 15% of patients who still have persistent muscle weakness 1 year later. CIM increases ICU-related costs by approximately 30% and increases in-hospital and 5-year mortality by approximately 15%-25%.

[0791] Figure 22A Illustration of the timeline of the Phase 2 point-of-care (POC) trial for critical illness myopathy. Figure 22B Illustration of the design of the Phase 2 POC trial in mechanically ventilated patients with critical illness myopathy. This study investigated POC in critical illness myopathy to improve recovery time and functional outcomes, optimize dosing, and optimize endpoints to characterize the safety, efficacy, and reproducibility of the treatment.

[0792] 7.12. Example 12: Efficacy of BGE-105 in Improving Muscle Function

[0793] Novel apelin receptor agonist. BGE-105 is a potent orally active small molecule agonist of the apelin APJ receptor and is currently in Phase I clinical trials. Safety, PK, and PD data have been obtained in >220 subjects. There are no approved APJ receptor agonists, and its target is very novel. It is involved in regulating multiple cardiac, vascular, metabolic, muscle (e.g., skeletal muscle), and gastrointestinal functions. Organ dysfunctions associated with age- and disease-related dysregulation of apelin signaling are expected to respond to treatment with an APJ receptor agonist.

[0794] In multiple preclinical models testing aged mice, BGE-105 improves muscle function and frailty in aged mice.

[0795] BGE-105 improves frailty (running wheel activity, grid hanging time). Protects mouse muscle from atrophy upon hindlimb immobilization. Improves muscle regeneration after cardiotoxin challenge.

[0796] BioAge's informatics platform reveals a strong link to longevity and healthspan. Elderly individuals with elevated apelin levels live longer and have improved physical and mental function.

[0797] BGE-105 is undergoing a Phase 1b bed rest trial to explore the potential of BGE-105 to reduce the rate of muscle loss.

[0798] Overall healthy men and women aged >65 years. Single-blind, placebo-controlled 10-day bed rest model with an age / sex / weight-composition standardized diet. Assess safety, pharmacokinetics, changes in insulin sensitivity (HOMA-IR), fractional synthetic rate of skeletal muscle proteins evaluated using proteomics after enrichment of body water with deuterated water, total muscle mass evaluated using deuterated creatine supplementation, volume and cross-sectional area of the vastus lateralis and gastrocnemius muscles by ultrasound, and physical function measured by the 30-second sit-to-stand test.

[0799] Frailty can be measured by detecting serum proteins in the BioAge longitudinal aging cohort data that are associated with future physical function decline as assessed by walking speed, activities of daily living (functional instrument), and grip strength. These proteins can then be used to attempt to identify patients at risk of future sarcopenia, muscle atrophy, or loss of muscle strength who would benefit from treatment with BGE-105. BGE-105 significantly altered the serum levels of some of the same proteins in the BioAge longitudinal aging cohort data that are associated with future physical function decline as assessed by walking speed, activities of daily living (functional instrument), and grip strength. These proteins can then be used to attempt to identify patients who respond to BGE-105 treatment and may have reduced muscle atrophy.

[0800] 7.13. Example 13 A Phase 2 clinical study was conducted in elderly patients on mechanical ventilation to prevent ICU diaphragmatic atrophy and critical illness myopathy

[0801] This example is an update of the clinical protocol for the clinical trial of BGE-105 in preventing ICU diaphragmatic atrophy or critical illness myopathy in elderly patients on mechanical ventilation as described in Examples 10 and 11.

[0802] Patients on mechanical ventilation (MV) develop rapid diaphragmatic atrophy (DA) due to muscle diseases. Among patients on MV, 40 - 75% develop clinically significant DA, which is very common. DA usually starts within 24 hours of MV, and the most obvious changes occur within 3 days. DA is the main cause of difficulty in weaning from MV and is associated with poor clinical outcomes and increased resource utilization, including longer MV time, longer ICU stay, and higher mortality. As Figure 39 outlined, this study evaluated the effect of BGE-105 on preventing ICU diaphragmatic atrophy and / or critical illness myopathy in elderly patients on mechanical ventilation to determine the effect of BGE-105 on atrophy of multiple muscle systems in patients on mechanical ventilation.

[0803]

[0804]

[0805] Results: In adults over 65 years old undergoing invasive mechanical ventilation for acute hypoxemic respiratory failure, BGE-105 can prevent diaphragmatic atrophy during the early period of mechanical ventilation. Patients who respond to BGE-105 treatment may have reduced muscle atrophy.

[0806] 7.14. Example 14 snRNAseq analysis of human muscle tissue samples from healthy adult patients (Phase 1b clinical study)

[0807] This example presents updated proteomic analysis data from biological samples collected from healthy adult patients in the Phase 1b clinical study described in Example 9.

[0808] Figures 40 to 62 Details and results of snRNAseq analysis of human muscle tissue samples from the Phase 1b clinical study of BGE-105 for the treatment of muscular atrophy are shown. These results are consistent with the proof of efficacy in the clinical study described herein.

[0809] Figure 40 The figure shows the nuclear isolation of the 10x Genomics single-cell transcriptome gene expression technology used to evaluate tissue samples. Figure 40 The figure shows the workflow of the nucleic acid isolation kit used in the streamlined sample preparation of the BGE-105 clinical trial. Tissue samples are collected from patients and processed in the laboratory. The tissue is dissociated and cleaned. Debris is removed and the sample (e.g., nuclei) is washed and resuspended in multiple cycles according to standard tissue processing procedures. The nuclei are visualized and counted using standard transcriptome gene expression techniques, and the 10x Genomics single-cell library technology is used to evaluate tissue samples.

[0810] Figure 41 Eleven cell types have been identified, consistent with the published muscle atlas (see, e.g., Perez et al., 2022, Single nuclei profiling identifies cell specific markers of skeletal muscle aging, frailty, and senescence. Aging (Albany NY). 2022 Dec 13;14(23):9393-9422. doi:10.18632 / aging.204435. Epub 2022 Dec 13. PMID: 36516485; PMCID: PMC9792217). The cell type clusters include fast skeletal muscle, slow skeletal muscle (type 1), myofibroblasts, tendon cells, fibroblasts, muscle stem cells, pericytes, smooth muscle, endothelial cells, T / NK cells, and adipocytes. Two methods and two annotations are used. The top 20 variable genes within the clusters are used as markers for cell type annotation in the placebo group and the BGE-105 treatment group.

[0811] Figure 42It is shown that there is a consistency between the top 20 cell type-specifically expressed genes and known cell type markers. The figure shows the log2(fold change) expressed in specific cell types for the top 20 cell type-specifically expressed genes compared to the remaining genes. Analysis was performed using all samples. The biomarkers tested included MYH1, MYH2, MYH3, COL1A1, COL1A2, TNT1, MYH7, PDGFRB, MRC1, CD163, PTPRC, PECAM1, PPARG, PAX7, VWF, CD247, ACTA2, and MYH11. For example, the expression of MYH1 and MYH2 genes was mainly detected in fast skeletal muscle, while the expression of TNT1 and MYH7 genes was mainly detected in slow skeletal muscle.

[0812] Figure 43 It is shown that for each cell type including fast and slow skeletal muscle, differentially expressed genes (DEGs) related to BGE105 were identified.

[0813] Figure 44 It is shown that the signaling pathways controlling muscle loss and promoting muscle growth are enriched in genes related to BGE105 treatment in fast skeletal muscle. Genes with significant Significance (padj < 0.1) were annotated.

[0814] Figure 45 It is shown that the treatment relevance of most significant genes (padj < 0.1) in muscle growth / loss-related signaling pathways is as expected. Shown are the detections of genes related to fast skeletal muscle treatment (padj < 0.001) and slow skeletal muscle (type I) treatment.

[0815] Figure 46 It is shown that on day 11, the overall expression levels of VEGFA, PPRGC1A, and COL1A1 in the treatment group were higher than those in the placebo group.

[0816] Figure 47 It is shown that on day 11, the overall expression levels of TNNC1 and MYH7 in the treatment group were higher than those in the placebo group. Shown are the overall expression levels: the average of the genes in all cells of each patient. Cell types were ignored. Both TNNC1 and MYH7 are slow skeletal muscle cell markers.

[0817] Figure 48It is shown that for fast skeletal muscle, 10 groups of 5+ enriched pathways were identified. These include pathways involved in the regulation and transport of calcium and metal ions, calcium- and calcineurin-mediated signal transduction, ERBB2 signal transduction and related events in cancer, muscle system processes and contraction, cardiac conduction and muscle contraction, development and differentiation of the muscular and skeletal systems, organization and assembly of myofilaments and myofibrils, development and morphogenesis of heart chambers and ventricles, development and differentiation of myocardium and heart, and / or assembly and organization of cell junctions and matrix adhesions.

[0818] Figure 49 It is shown that for slow skeletal muscle, 13 groups of 5+ enriched pathways were identified. These include pathways involved in the regulation of transmembrane transport activity, regulation of lipid storage and foam cell differentiation, development and differentiation of muscle and heart cells, regulation of cardiac conduction and action potential, myocardial tissue and chamber morphogenesis, muscle contraction and regulation, cytoplasmic calcium ion transport and regulation, intracellular calcium release and regulation of myocardial contraction bands, sodium ion transport and regulation, muscle adaptation and hypertrophy, myotube adaptation and hypertrophy, cardiac conduction and multicellular organism signal transduction, and / or assembly and development of myofibers and myofibrils.

[0819] Figure 50 Shows cell type-specific patterns of differential gene expression associated with BGE-105 treatment identified in muscle biopsies collected from the vastus lateralis muscle. Shown are differentially expressed genes detected in fast skeletal muscle (3418) and slow skeletal muscle (type 1) (1571).

[0820] Figure 51 Shows that differential regulatory pathways indicate beneficial effects of BGE-105 on key muscle and adipocyte processes.

[0821] Figure 52 Shows that BGE-105 prevents the downregulation of contractile proteins in both fast and slow skeletal muscles induced by bed rest. The p-values reflect changes after 10 days of bed rest compared to baseline. BGE-105 prevents the reduction of troponin C (TNNC1), which binds calcium to activate muscle contraction (p = 0.014); myosin heavy chain β (MYH7), which is involved in ATPase activity to promote muscle contraction (p = 0.009); and tropomyosin β chain (TPM2), which stabilizes actin to regulate contraction (p = 0.014). These findings reinforce the increased synthesis of structural proteins observed after treatment with BG5-105.

[0822] Figure 53BGE-105 was shown to prevent the downregulation of the mitochondrial biogenesis regulator PGC-1α and all respiratory complexes induced by bed rest. Shown are representative genes, PGC-1α (p = 0.03), COMPLEX 1: NDUFA (p = 0.007), COMPLEX II: SDHD (p = 0.036), COMPLEX III: UQCRH (p = 0.048), COMPLEX IV: COX5A (p = 0.047), COMPLEX V: ATP5F1B (p = 0.038).

[0823] Figure 54 BGE-105 was shown to prevent the detrimental expression levels of genes involved in muscle metabolic processes. Shown are differential gene expressions in the insulin signaling pathway (p = 2.61E-03), AMPK signaling pathway (p = 5.04E-03), and glucagon signaling pathway (p = 2.61E-03). Exemplary genes are GLUT4, EIF4EBP1, PCK2 (insulin signaling pathway); MYLD, EEF2K, PPP2R2D (AMPK signaling pathway); and PHKAA1, PPRA, ADCY2 (glucagon signaling pathway).

[0824] Figure 55 BGE-105 was shown to prevent the bed rest-induced upregulation of genes involved in triglyceride storage and fatty acid metabolism, which is a potential mechanism for promoting fat loss. Exemplary genes include: DGAT2, which catalyzes the last step in the triglyceride synthesis process (p = 0.18); GOS2, which inhibits the activity of lipases involved in triglyceride breakdown (p = 0.014); and / or FABP4, which binds and transports intracellular fatty acids so that they can be metabolized (p = 0.026).

[0825] Figure 56 More endothelial cells were shown to express APLNR in the treatment group. A small fraction of cells expressed APLNR (272 cells). 46% of these cells were located within endothelial cells. T-test of APLNR expression levels in the cells tested between the treatment group and the placebo group: day 6 _t.stat = 2.02, day 6 _p.val = 0.04, day 11 _t.stat = 2.33, day 11 _p.val = 0.02.

[0826] Figure 57For cell differentiation trajectory and pseudotime inference. Pseudotime inference of cell differentiation is a computational method used to model the dynamic changes and transitions between different cell states, such as transitions between progenitor cells, precursor cells, immature cells, and mature cell states. It allows the distinction between early and late stages of biological processes. Pseudotime analysis can help reveal the temporal order of gene expression changes during biological processes, enabling us to understand the underlying molecular mechanisms.

[0827] Figure 58 Graph showing reduced cell differentiation of fast / slow skeletal muscle, macrophages, T / NK cells, and muscle stem cells after treatment. Baseline muscle stem cells (treatment and placebo groups) were set as the root. As shown, muscle stem cells, fast skeletal muscle, slow skeletal muscle (type I), and macrophages in the treatment group had lower differentiation, while T / NK cells in the treatment group had higher differentiation.

[0828] Figures 59 to 61 Illustration of the results of secondary analysis. Evaluation of signals from protein synthesis rate analysis. Investigation of aging and muscle characteristics in published studies.

[0829] Figure 59 Showing that via microdialysis measurement, BGE-105 led to relatively higher vastus lateralis muscle protein synthesis (p < 0.005). Shown is the muscle protein synthesis rate after 10 days of bed rest. Asterisks indicate 10 significant genes: TPM2, PYGM, MYH2, TNNI2, TNNC2, TNNC1, ENO3, ALDOA, ATP5F1B, MDH2.

[0830] Figure 60 Showing the validation of muscle protein synthesis assay results in snRNA-seq analysis. Among 18 muscle proteins, 15 were statistically highly expressed in the fast skeletal muscle of the treatment group compared to the placebo group. In the FSR analysis, there were 13 myofibrillar proteins (5 proteins were significant in the FSR analysis: TPM2, TNNC1, TNNC2, TNNI2, MYH2) and 5 significant non-myofibrillar proteins (5 proteins: ENO3, PYGM, ALDOA, MDH2, ATP5F1B). All 38 proteins in heavy water were also measured in the snRNA-seq data.

[0831] Figure 61 Showing that BGE-105 treatment shifted the fast / slow skeletal muscle transcriptome towards a state associated with young muscle. The transcriptome negatively correlated with BGE-105 treatment showed enrichment of muscle aging characteristics. This result is related to reports on gene expression changes in old muscle versus young muscle (see, for example, Perez et al. (2022), Aging).

[0832] Figure 62 Display a graph showing the percentage of cells in the sample with mitochondrial readings exceeding 5%. The Y-axis is the percentage of cells with MT readings > 5% in the sample compared to the baseline (62 samples = 21 patients × 3 time points). p-values from the t-test: at day 6, p = 0.08; at day 11, p = 0.04. Low-quality cells were removed before calculating the proportion of cells with mitochondrial readings > 5% for each patient (cells with less than 200 genes or less than 500 readings were removed, and the total number of remaining MT cells (> 5%)).

[0833] Figure 68 The left graph and the right sub-graph on the left show that BGE-105 prevents the downregulation of contractile proteins in fast and slow skeletal muscles induced by bed rest. Shown are single-nucleus transcriptomic data of the percentage change of sarcoplasmic / endoplasmic reticulum calcium ATPase type 2 (SERCA2) and myosin light chain 3 (MYL3) relative to the baseline. As shown, compared to the placebo, BGE-105 treatment prevents the downregulation of SERCA2 (p = 0.028) or MYL3 (p = 0.012) after 10 days of bed rest. The p-value reflects the change compared to the baseline after 10 days of bed rest.

[0834] Figures 69A to 69B Show that BGE-105 prevents the downregulation of the mitochondrial biogenesis regulator PGC-1α and all respiratory complexes induced by bed rest. Figure 69A Show a summary of the downregulation of respiratory complexes. Exemplary genes include ATP5PB, NDUFA8, ATP5F1A, NDUFB3, ATP5MG, ATP2A2, PPARGC1A, SDHD, ATP13A3, UQCRB, NDUFA4, COX10, NDUFA9, SDHA, ATP8A1, ATP5F1D, and / or NDUFS4. Figure 69B Show the expression of representative genes in subjects treated with BGE-105 and subjects treated with placebo. For example, BGE-105 prevents the downregulation of PGC-1α (p = 0.029), NDUFA8 (p = 0.011), SDHD (p = 0.033), UQCRB (p = 0.046), COX10 (p = 0.049), and / or ATP5PB (p = 0.0088). The p-value reflects the change compared to the baseline after 10 days of bed rest.

[0835] Figures 70A to 70B Show that BGE-105 preserves the gene expression involved in glucose metabolism. The p-value reflects the change compared to the baseline after 10 days of bed rest. Exemplary genes include LDHB, PPARA, EIF4EBP1, HRAS, MLYCD, PHKA1, PCK2, PPARGC1A, EEF2K. Figure 70AShow a summary of the retained gene expression in the glucagon, insulin, and AMPK pathways. Figure 70B Show differential expression in the insulin signaling pathway (p = 2.61E-03) and differential expression in the AMPK signaling pathway (p = 5.04E-03). For example, BGE-105 prevents the downregulation of PGC-1α (p = 0.029), EIF4BP1 (p = 0.009), PHKA1 (p = 0.015), MLYCD (p = 0.015), EEF2K (p = 0.031), and / or CD36 (p = 0.05).

[0836] In summary, patients responsive to BGE-105 treatment may have reduced muscle atrophy.

[0837] 7.15. Example 15 Enhancing the Resilience of Hospitalized Elderly COPD Patients by Preventing Muscle Atrophy

[0838] COPD Patients Are at Risk of Losing Resilience

[0839] Acute exacerbation of chronic obstructive pulmonary disease (AECOPD) is the second most common cause of emergency hospital admission in the UK, with over 100,000 admissions per year. Among these admitted patients, 20% are readmitted within 30 days, making AECOPD a major cause of readmission. A key factor driving these readmissions is COPD-related muscle dysfunction, which increases skeletal muscle atrophy and mortality and prevents recovery to baseline function.

[0840] The important impact of skeletal muscle atrophy associated with recurrent AECOPD is clear: muscle atrophy leads to a deterioration of resilience, and the direct result of immobility due to hospitalization for AECOPD is a higher incidence of muscle atrophy.

[0841] Pulmonary Rehabilitation Is Insufficient for the Recovery of AECOPD

[0842] Despite receiving standard care rehabilitation during hospitalization and after discharge, elderly COPD patients still experience significant muscle loss, which is often associated with longer hospital stays and increased short-term and long-term morbidity and mortality (12% mortality at 90 days after hospitalization, compared to 4.9% for myocardial infarction).

[0843] Although clinical trials have demonstrated that post - exacerbation pulmonary rehabilitation (PEPR) has the potential to improve physical function and reduce the risk of readmission through exercise programs, education, and behavioral interventions, PEPR has poor effectiveness in practice because less than 10% of AECOPD patients complete PEPR in this context. Even when PEPR is completed, the program may not prevent acute loss of resilience because it is initiated after the most acute and pro - inflammatory phases of the disease. Early intervention may be more successful in preventing acute muscle wasting; however, in the largest trial conducted in this population, the intensity of the exercise intervention was not sufficient to reduce readmission rates or improve physical function and health status over the long term. Therefore, the development of acute muscle wasting represents an important unmet need in AECOPD patients and is a potential feature that can be treated with muscle - targeted pharmacotherapy.

[0844] Apelin Pathway Activation in Preclinical and Clinical Studies Can Reduce Muscle Loss and Improve Frailty

[0845] Apelin is an endogenous peptide exercise hormone that primarily targets skeletal muscle tissue. Apelin has been implicated in improving muscle regeneration and stem cell activation while reducing muscle atrophy and inflammation. In BioAge's human aging cohort, activation of the apelin module predicts human lifespan, suggesting that apelin levels are closely related to lifespan and healthspan (e.g., muscle strength). Additionally, apelin levels typically decline with age, further highlighting the potential importance of apelin agonists in the elderly population. BGE - 105 (also known as azelaprag) is a small - molecule apelin receptor agonist approved for the development of muscle aging.

[0846] BGE-105 in Preclinical Models

[0847] Non - clinical studies in rodents have demonstrated that BGE - 105 is able to mitigate muscle and strength loss seen in various muscle loss models.

[0848] BioAge evaluated the therapeutic effect of BGE - 105 in a murine model of muscle disuse atrophy. Muscle mass loss was induced by hindlimb casting: the right hindlimb was cast for 14 days. The left hindlimb remained mobile and served as an intra - individual control. Treatment with BGE - 105 prevented muscle mass loss in the tibialis anterior muscle of the casted limb relative to the non - casted limb compared to vehicle - treated animals (p < 0.0001).

[0849] BioAge evaluated daily BGE - 105 treatment versus vehicle - only treatment in aged mice (23 - 24 months old) over 2 months. The study demonstrated that animals treated with azelaprag had statistically significant improvements in both total daily running distance (exercise wheel) and grid hang time (inverted grip strength measurement).

[0850] BGE-105 in Clinical Studies on Disuse Atrophy

[0851] BioAge completed a double-blind, non-randomized Ph1b trial in healthy elderly volunteers (≥65 years old) who underwent 10 days of continuous bed rest. BGE-105 (240 mg) or placebo was administered intravenously once daily for 10 days and normal activities were resumed on day 11. Muscle atrophy was evaluated on the day before the start of bed rest (D-1) (baseline), 5 days (D5) and 10 days (D10) after bed rest.

[0852] The study demonstrated that BGE-105 significantly reduced muscle loss compared to placebo, measured by vastus lateralis muscle thickness (75% reduction in loss) and cross-sectional area (>50% reduction in loss) ( Figure 19 ). Maintenance of muscle mass was supported by the maintenance of the skeletal muscle fractional protein synthesis rate (measured by D2O method and muscle biopsy). Importantly, synthesis of myofibrillar structural proteins such as myosin and troponin was maintained compared to placebo ( Figure 63 ).

[0853] Transcriptomics (by single-nucleus RNA sequencing) from the muscle tissue of study subjects demonstrated that BGE-105 prevented the downregulation of the mitochondrial bioregulator PGC-1α and all respiratory complexes induced by bed rest ( Figure 64 ). At the proteomic level, by SomaSignal testing, BGE-105 treatment shifted the plasma proteome towards an estimated higher basal metabolic rate and VO2 max (Figure 65). Previous studies of inpatients have documented that overcoming anabolic resistance in this patient population is a challenge, where nutritional supplements fail to maintain muscle protein synthesis and exercise training is an obstacle for frail elderly patients.

[0854] This study trialed wearable activity monitoring; encouraging accelerometer results indicated that the recovery of activity after bed rest (starting on day 11; bed rest and BGE-105 / placebo treatment were conducted from day 1 to day 10) was more pronounced in the BGE-105 group ( Figure 66 ).

[0855] This provides a human model similar to acute hospitalization in COPD and, as a proof of concept, acute muscle loss can be improved by BGE-105 regardless of nutritional and exercise capacity.

[0856] In summary, previous preclinical and clinical studies have shown that in a series of Phase 1a / 1b trials, over 200 subjects demonstrated target binding, efficacy (including improvements in both overall activity and muscle atrophy), favorable pharmacokinetics, and human safety. BGE-105 is a highly selective and potent apelin receptor agonist. In a 10-day bed rest study, BGE-105 was shown to preserve muscle size and mass in elderly volunteers (≥65 years) compared to placebo.

[0857] BGE-105 can mitigate acute muscle loss and promote functional improvement through multiple mechanisms, including preventing a decrease in muscle protein synthesis and improving metabolic function. Since the use of BGE-105 during the pre-hospitalization period preserves muscle mass and strength, enhanced resilience may also impact the long-term readmission risk in this population. Therefore, addressing frailty and muscle loss is crucial for improving outcomes in these patients. The present disclosure provides methods for intervening in and preventing muscle atrophy to reduce the risk of frailty, enhance resilience, and support recovery.

[0858] Research

[0859] To address this cycle of devastating disease and deteriorating resilience, the present disclosure provides a method for preventing acute skeletal muscle atrophy during AECOPD hospitalization by treating a subject with an apelin receptor APJ agonist (BGE-105) starting during hospitalization.

[0860] Target Population

[0861] This randomized, double-blind Phase 2a study will treat 60 elderly subjects (30 each of BGE-105 and placebo) hospitalized for AECOPD with a long-term hospitalization risk (>5 days). Subjects with a long-term hospitalization risk are defined as: ≥60 years old, having been hospitalized for AECOPD and / or a modified Medical Research Council (mMRC) dyspnea scale score ≥2. Figure 67 The figure shows the flow chart of a single-center, double-blind Phase 2a RCT clinical trial.

[0862] Interventions and Controls

[0863] BGE-105 (or placebo) is administered via a one-hour IV infusion daily for 10 days. Subjects are followed up for 90 days for muscle mass, function, readmission, and mortality. The primary objective is to evaluate whether BGE-105 (240 mg, 1440 mg) can preserve muscle mass after hospitalization for COPD exacerbation compared to conventional treatment.

[0864] The first dose (BGE-10 or placebo) is administered on Day 2 (within 36 hours after admission). The patient is discharged on Day 5, and the treatment ends on Day 10. From Day 5 until Day 90 after the start of treatment, the patient needs to come back for follow-up on Day 30 and Day 60 until the end of the study on Day 90.

[0865] Results

[0866] The primary outcome is muscle mass. Other outcomes are muscle strength and / or frailty outcomes. Based on a 50% reduction in muscle loss difference (supported by a Phase 1b bed rest study), each study group requires 30 subjects for a proof-of-concept study.

[0867] This study correlates various measurement indicators to understand the treatment of BGE-105 and determine whether the resilience of subjects hospitalized due to COPD exacerbation is maintained or improved. These measurement indicators include but are not limited to: grip strength before and after treatment, functional / physical fitness tests (e.g., Short Physical Performance Battery [SPPB]), muscle mass measured by D3-creatine, muscle protein synthesis measured by D2O in micro-biopsies, limb muscle composition and changes measured by biopsies, ultrasound, DEXA, and CT, mortality and readmission rates on Day 30, Day 60, and Day 90, and quality of life assessments. Success is achieved by maintaining the strength, mass, and quality of skeletal muscle, which is due to improved protein synthesis reducing the readmission rate and potentially shortening the initial hospital stay.

[0868] Research Objectives

[0869] This proposed study aims to intervene in and prevent muscle atrophy to improve frailty risk, resilience, and recovery.

[0870] COPD patients hospitalized due to exacerbation are a vulnerable group, and more than half (56%) of them meet the criteria for physical frailty (according to the Fried frailty criteria, see, for example, Fried et al., 2001. Biol Sci Med Sci. 2001 Mar;56(3):M146-56. doi:10.1093 / gerona / 56.3.m146. PMID:11253156), and only 5% of the patients are physically robust. These frail individuals have lower muscle mass at admission and greater muscle mass loss during hospitalization, thus exacerbating the impact of this event. Data show that this loss occurs in the early stage of admission, and most patients experience a clinically significant loss of more than 5% of muscle mass (quadriceps).

[0871] In this study, muscle mass loss was used as a precursor to loss of resilience (manifested as prolonged hospital stay, poor functional recovery, readmission, and death), decline in physical function, and increased frailty. Measuring muscle mass has been widely recognized by multiple methods, is related to muscle performance / strength and frailty (e.g., SPPB, grip strength, HR-QoL), and ultimately to the risk of hospitalization and death in COPD patients. SPPB is a widely used clinical assessment tool designed to evaluate the physical function and performance of the elderly and is supported by the European Working Group on Sarcopenia in Older People (EWGSOP) for assessing physical performance, resilience, and frailty in the elderly. SPPB typically assesses mobility, balance, and lower limb strength through balance, gait speed, and chair stand tests. SPPB provides a quantitative measure of physical function performance and can help identify those at risk of functional challenges and decline. In COPD patients, poorer SPPB performance (i.e., a 1-point decrease) is associated with a higher risk of hospitalization for AECOPD and a longer hospital stay. Conversely, an improvement in SPPB performance (i.e., a 1-point increase) is associated with an increased risk of death and readmission in AECOPD patients.

[0872] Muscle mass, muscle strength and endurance, physical function, frailty, and resilience outcomes were evaluated at 5 time points from baseline (within 36 hours of admission) up to 90 days. In a he...

Claims

1. A method of treating a muscle disorder in a subject, the method comprising administering to a subject in need thereof an effective dose of an apelin receptor agonist.

2. The method of claim 1, wherein the muscle disorder is an age-related muscle disorder.

3. The method of any one of claims 1 to 2, wherein the subject is human and at least 40 years old.

4. The method of claim 3, wherein the subject is at least 50 years old.

5. The method of claim 4, wherein the subject is at least 60 years old.

6. The method of claim 5, wherein the subject is at least 65 years old.

7. The method of claim 6, wherein the subject is at least 70 years old.

8. The method of claim 7, wherein the subject is at least 75 years old.

9. The method of claim 8, wherein the subject is at least 80 years old.

10. The method of any one of claims 1 to 9, wherein the muscle disorder is a skeletal muscle disorder.

11. The method of any one of claims 1 to 10, wherein the muscle expresses an apelin receptor, and administering the apelin receptor agonist activates the apelin / APJ (APLNR) system in the muscle tissue of the subject.

12. The method of any one of claims 1 to 11, wherein the muscle disorder is not a cardiovascular disorder.

13. The method of any one of claims 1 to 11, wherein the subject does not have heart failure or is not at risk of heart failure.

14. The method of any one of claims 1 to 13, wherein the muscle disorder is associated with inflammation and / or mitochondrial function impairment.

15. The method of any one of claims 1 to 14, wherein the muscle disorder is associated with loss of muscle function, decreased muscle regenerative capacity, or decreased healing capacity after muscle injury.

16. The method of any one of claims 1 to 15, wherein the muscle disorder is associated with loss of function of muscle stem cells.

17. The method of any one of claims 1 to 16, wherein the muscle disorder is selected from sarcopenia, frailty, hip fracture, ICU-related myasthenia, mechanical ventilation-related myasthenia, immobility-related myasthenia, recovery from muscle injury, muscle atrophy, diaphragmatic atrophy, critical illness myopathy, and muscle wasting.

18. The method of any one of claims 1 to 17, wherein the muscle disorder is associated with insulin insensitivity or resistance, or type 2 diabetes.

19. The method of any one of claims 1 to 18, wherein the subject has or is determined to have low muscle strength or low muscle power.

20. The method of any one of claims 17 to 19, wherein the subject has or is determined to have chronic obstructive pulmonary disease (COPD).

21. The method of any one of claims 1 to 20, wherein the subject has or is determined to have low lower limb muscle mass.

22. The method of any one of claims 1 to 21, wherein the subject has or is determined to have low upper limb muscle mass.

23. The method according to any one of claims 1 to 22, wherein the subject has or is determined to have a low muscle volume.

24. The method according to claim 23, wherein the muscle volume is skeletal muscle volume.

25. The method according to claim 24, wherein the muscle is tibialis anterior, tibialis posterior, gastrocnemius, sartorius, vastus intermedius, vastus lateralis, vastus medialis, soleus, rectus femoris, extensor digitorum longus or diaphragm.

26. The method according to any one of claims 1 to 25, wherein the apelin receptor agonist is administered orally, intravenously, intranasally or intramuscularly.

27. The method according to any one of claims 1 to 26, wherein the dose is administered daily.

28. The method according to any one of claims 1 to 27, wherein the dose is administered in multiple equal or unequal sub-doses.

29. The method according to any one of claims 1 to 28, wherein the dose is administered at varying dosing intervals.

30. The method according to any one of claims 1 to 29, wherein the dose is 200 mg.

31. The method according to any one of claims 1 to 30, further comprising assessing muscle mass after administration.

32. The method according to claim 31, wherein the muscle mass is assessed at least one day after administration.

33. The method according to claim 32, wherein the muscle mass is assessed at least one week or at least two weeks after administration.

34. The method according to claim 33, wherein the muscle mass is assessed at least one month after administration.

35. The method according to any one of claims 1 to 34, wherein the subject has a low apelin circulating level.

36. A method of maintaining and / or increasing muscle mass and / or muscle strength in a human subject, the method comprising administering an effective dose of an apelin receptor agonist to a subject in need thereof.

37. The method according to claim 36, wherein the subject is at least 60 years old.

38. The method according to claim 37, wherein the subject is at least 65 years old.

39. The method according to claim 38, wherein the subject is at least 70 years old.

40. The method according to claim 39, wherein the subject is at least 75 years old.

41. The method according to claim 40, wherein the subject is at least 80 years old.

42. The method according to any one of claims 36 to 41, wherein the human subject has or is determined to have low muscle strength.

43. The method according to any one of claims 36 to 42, wherein the human subject has or is determined to have low muscle power.

44. The method according to any one of claims 36 to 43, wherein the human subject has or is determined to have low lower limb muscle mass.

45. The method according to any one of claims 36 to 44, wherein the human subject has or is determined to have low upper limb muscle mass.

46. The method according to any one of claims 36 to 45, wherein the human subject has or is determined to have a low muscle volume.

47. The method according to claim 46, wherein the muscle volume is the skeletal muscle volume.

48. The method according to claim 47, wherein the muscle is the diaphragm, tibialis anterior, tibialis posterior, gastrocnemius, sartorius, vastus intermedius, vastus lateralis, vastus medialis, soleus, rectus femoris or extensor digitorum longus.

49. The method according to any one of claims 47 to 48, wherein the muscle is skeletal muscle.

50. The method according to any one of claims 36 to 49, wherein the human subject is receiving mechanical ventilation.

51. The method according to any one of claims 36 to 50, wherein the human subject has or is determined to have a reduced diaphragm thickness compared to a human subject not receiving mechanical ventilation.

52. The method according to any one of claims 36 to 51, wherein the human subject has or is determined to have diaphragm atrophy.

53. The method according to any one of claims 36 to 52, wherein the human subject has or is determined to have ventilator-induced diaphragmatic dysfunction (VIDD).

54. The method according to any one of claims 36 to 53, wherein the human subject has or is determined to have hypoxic respiratory failure.

55. The method according to any one of claims 48 to 54, wherein the muscle expresses the apelin receptor.

56. The method according to any one of claims 36 to 55, wherein the human subject has low circulating levels of apelin.

57. The method according to any one of claims 36 to 56, wherein the apelin receptor agonist is administered orally, intravenously, intranasally or intramuscularly.

58. The method according to any one of claims 36 to 57, wherein the dose is administered daily.

59. The method according to any one of claims 36 to 58, wherein the dose is administered in multiple equal or unequal sub-doses.

60. The method according to any one of claims 36 to 59, wherein the dose is administered intravenously.

61. The method according to claim 60, wherein the dose is administered intravenously as a loading dose of at least 60 mg, followed by a maintenance dose of at least 360 mg.

62. The method according to claim 60, wherein the dose is administered intravenously as a loading dose of at least 120 mg, followed by a maintenance dose of at least 720 mg.

63. The method according to claim 60, wherein the dose is administered intravenously as a loading dose of at least 240 mg, followed by a maintenance dose of at least 1440 mg.

64. The method according to any one of claims 61 to 63, wherein the loading dose is administered for at least 1 hour.

65. The method according to any one of claims 61 to 64, wherein the maintenance dose is administered for at least 20 hours.

66. The method according to any one of claims 61 to 64, wherein the maintenance dose is administered for at least 22 hours.

67. The method according to any one of claims 61 to 64, wherein the maintenance dose is administered for at least 100 hours.

68. The method according to any one of claims 36 to 60, wherein the dose is at least 60 mg.

69. The method according to any one of claims 36 to 60, wherein the dose is at least 75 mg.

70. The method according to any one of claims 36 to 60, wherein the dose is at least 120 mg.

71. The method according to any one of claims 36 to 60, wherein the dose is at least 240 mg.

72. The method according to any one of claims 36 to 60, wherein the dose is at least 150 mg.

73. The method according to any one of claims 36 to 60, wherein the dose is at least 300 mg.

74. The method according to any one of claims 36 to 60, wherein the dose is at least 375 mg.

75. The method according to any one of claims 36 to 60, wherein the dose is 75 - 150 mg.

76. The method according to any one of claims 36 to 60, wherein the dose is 150 - 300 mg.

77. The method according to any one of claims 36 to 60, wherein the dose is 240 - 1440 mg.

78. The method according to any one of claims 36 to 60, wherein the dose is 75 mg.

79. The method according to any one of claims 36 to 60, wherein the dose is 150 mg.

80. The method according to any one of claims 36 to 60, wherein the dose is 240 mg.

81. The method according to any one of claims 36 to 60, wherein the dose is 300 mg.

82. The method according to any one of claims 36 to 60, wherein the dose is 375 mg.

83. The method according to any one of claims 36 to 60, wherein the dose is 450 mg.

84. The method according to any one of claims 36 to 60, wherein the dose is a single escalating dose of 60 mg / 360 mg.

85. The method according to any one of claims 36 to 60, wherein the dose is a single escalating dose of 120 mg / 720 mg.

86. The method according to any one of claims 36 to 60, wherein the dose is a single escalating dose of 240 mg / 1440 mg.

87. The method according to any one of claims 36 to 86, further comprising assessing muscle mass or muscle thickness after administration.

88. The method according to claim 87, wherein the muscle mass is assessed at least one day after administration.

89. The method according to claim 87, wherein the muscle mass is assessed at least one week after administration.

90. The method according to claim 88, wherein the muscle mass is assessed at least one month after administration.

91. The method according to any one of claims 1 to 90, wherein the apelin receptor agonist is of formula (I) or (II): or a pharmaceutically acceptable salt thereof, a tautomer thereof, a pharmaceutically acceptable salt of the tautomer, a stereoisomer of any of the foregoing, or a mixture thereof, wherein: R 1 is an unsubstituted pyridyl, pyridone or pyridine N-oxide, or a pyridyl, pyridone or pyridine N-oxide substituted by 1, 2, 3 or 4 R 1a substituents; R 1a Independently selected from -F, -Cl, -Br, -I, -CN, -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 perhaloalkyl, -OH, -O-(C1-C6 alkyl), -O-(C1-C6 haloalkyl), -O-(C1-C6 perhaloalkyl), -C2-C6 alkenyl, -O-(C1-C6 alkyl)-OH, -O-(C1-C6 alkyl)-O-(C1-C6 alkyl), -O-(C1-C6 haloalkyl)-OH, -O-(C1-C6 haloalkyl)-O-(C1-C6 alkyl), -O-(C1-C6 perhaloalkyl)-OH, -O-(C1-C6 perhaloalkyl)-O-(C1-C6 alkyl), -NH2, -NH(C1-C6 alkyl), -N(C1-C6 alkyl)2, -C(═O)-(C1-C6 alkyl), -C(═O)OH, -(C═O)-O-(C1-C6 alkyl), -C(═O)NH2, -C(═O)NH(C1-C6 alkyl), -C(═O)N(C1-C6 alkyl)2, phenyl, -C(═O)-(heterocyclic group) or heterocyclic group in each case independently, wherein the heterocyclic group of the -C(═O)-(heterocyclic group) or heterocyclic group is a 3- to 7-membered ring containing 1, 2 or 3 heteroatoms selected from N, O and S; R 2 selected from -H and C1-C4 alkyl, or absent in the compounds of formula II; R 3 Selected from unsubstituted C1-C 10 alkyl, C1-C 1a alkyl substituted with 1, 2 or 3 R 10 substituents, group of formula -(CR 3b R 3c )-Q, group of formula -NH-(CR 3b R 3c )-Q, group of formula -(CR 3b R 3c )-C(═O)-Q, group of formula -(CR 3d R 3e )-(CR 3f R 3g )-Q, group of formula -(CR 3b ═CR 3c )-Q and group of formula -(heterocyclic group)-Q, wherein the heterocyclic group of said -(heterocyclic group)-Q has 5 to 7 ring members, 1, 2 or 3 of which are heteroatoms selected from N, O or S, and is unsubstituted or substituted with 1, 2 or 3 R 3h substituents; R 1a independently selected from -F, -Cl, -CN, -OH, -O-(C1-C6 alkyl), -O-(C1-C6 haloalkyl), -O-(C1-C6 perhaloalkyl), -O-(C1-C6 alkyl)-OH, -O-(C1-C6 alkyl)-O-(C1-C6 alkyl), C2-C6 alkenyl, C2-C6 alkynyl, -NH2, -NH(C1-C6 alkyl), and -N(C1-C6 alkyl)2 in each case; R 3b and R 3c are independently selected from -H, -F, -Cl, -CN, -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 perhaloalkyl, -OH, -O-(C1-C6 alkyl), -O-(C1-C6 haloalkyl), -O-(C1-C6 perhaloalkyl), -O-(C1-C6 alkyl)-OH, -O-(C1-C6 alkyl)-O-(C1-C6 alkyl), -NH2, -NH(C1-C6 alkyl), and -N(C1-C6 alkyl)2; R 3d and R 3e are independently selected from -H, -F, -Cl, -CN, -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 perhaloalkyl, -OH, -O-(C1-C6 alkyl), -O-(C1-C6 haloalkyl), -O-(C1-C6 perhaloalkyl), -O-(C1-C6 alkyl)-OH, -O-(C1-C6 alkyl)-O-(C1-C6 alkyl), -NH2, -NH(C1-C6 alkyl), and -N(C1-C6 alkyl)2; R 3f and R 3g are independently selected from -H, -F, -Cl, -CN, -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 perhaloalkyl, -OH, -O-(C1-C6 alkyl), -O-(C1-C6 haloalkyl), -O-(C1-C6 perhaloalkyl), -O-(C1-C6 alkyl)-OH, -O-(C1-C6 alkyl)-O-(C1-C6 alkyl), -NH2, -NH(C1-C6 alkyl), and -N(C1-C6 alkyl)2; R 3h independently selected from -F, -Cl, -CN, -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 perhaloalkyl, -OH, -O-(C1-C6 alkyl), -O-(C1-C6 haloalkyl), -O-(C1-C6 perhaloalkyl), -O-(C1-C6 alkyl)-OH, -O-(C1-C6 alkyl)-O-(C1-C6 alkyl), -NH2, -NH(C1-C6 alkyl), -N(C1-C6 alkyl)2 and oxo in each case; Q is a monocyclic or bicyclic C6-C 10 aryl group, a monocyclic or bicyclic heteroaryl group having 5 to 10 ring members containing 1, 2 or 3 heteroatoms selected from N, O or S, a C3-C8 cycloalkyl group or a 3- to 7-membered heterocyclic group containing 1, 2 or 3 heteroatoms selected from N, O or S, wherein the C6-C 10 aryl group, the heteroaryl group, the cycloalkyl group and the heterocyclic group are unsubstituted or substituted by 1, 2, 3 or 4 R Q substituents; R Q independently selected from -F, -Cl, -Br, -I, -CN, -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 perhaloalkyl, -C2-C6 alkenyl, -C2-C6 alkynyl, -OH, -O-(C1-C6 alkyl), -O-(C1-C6 haloalkyl), -O-(C1-C6 perhaloalkyl), -NH2, -NH(C1-C6 alkyl), -N(C1-C6 alkyl)2, -C(═O)-(C1-C6 alkyl), -C(═O)OH, -C(═O)-O-(C1-C6 alkyl), -C(═O)NH2, -C(═O)NH(C1-C6 alkyl), -C(═O)N(C1-C6 alkyl)2, -S(═O)2-(C1-C6 alkyl), phenyl and heteroaryl in each case, and the Q heterocyclic group may be substituted by 1 oxo R Q substituent; R 4 selected from monocyclic or bicyclic C6-C 10 aryl groups, monocyclic or bicyclic heteroaryl groups having 5 to 10 ring members containing 1, 2 or 3 heteroatoms independently selected from N, O and S, and monocyclic or bicyclic heterocyclic groups having 5 to 10 ring members containing 1, 2, 3 or 4 heteroatoms independently selected from N, O and S, wherein the C6-C 10 aryl group, the heteroaryl group or the heterocyclic group is unsubstituted or substituted with 1, 2 or 3 R 4a substituents; R 4a independently selected in each case from -F, -Cl, -Br, -I, -CN, -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 perhaloalkyl, -OH, -O-(C1-C6 alkyl), -O-(C1-C6 haloalkyl), -O-(C1-C6 perhaloalkyl), -NH2, -NH(C1-C6 alkyl), -N(C1-C6 alkyl)2, -C(═O)-(C1-C6 alkyl), -C(═O)OH, -C(═O)-O-(C1-C6 alkyl), -C(═O)NH2, -C(═O)NH(C1-C6 alkyl) and -C(═O)N(C1-C6 alkyl)2, and the heterocyclic group R 4 group may be further substituted by 1 oxo substituent; and Further, wherein: If R 4 is an unsubstituted or substituted benzene ring and R 3 is a group of formula -(CR 3b ═CR 3c )-Q, then at least one of the following is true: a)R 4 substituted by at least one -O-(C1-C6 alkyl) group; b) Q is not oxadiazole; c)R 3b not - H; d)R 3c not - H; e) R 1 not 2-pyridyl; or f)R 4 substituted by two or more -O-(C1-C6 alkyl) groups.

92. The method according to claim 91, wherein R 1 is an unsubstituted pyridyl group or a pyridyl group substituted with 1 or 2 R 1a substituents.

93. The method according to any one of claims 91 to 92, wherein R 1a is independently selected in each case from: -CH3, -CH2CH3, -F, -Cl, -Br, -CN, -CF3, -CH═CH2, -C(═O)NH2, -C(═O)NH(CH3), -C(═O)N(CH3)2, -C(═O)NH(CH2CH3), -OH, -OCH3, -OCHF2, -OCH2CH3, -OCH2CF3, -OCH2CH2OH, -OCH2C(CH3)2OH, -OCH2C(CF3)2OH, -OCH2CH2OCH3, -NH2, -NHCH3, -N(CH3)2, phenyl and a group of the following formula: where the symbol when drawn through the bond, represents the point of attachment to the remainder of the molecule.

94. The method according to any one of claims 91 to 93, wherein R 1 is selected from: where the symbol when drawn through the bond, represents a point of attachment to the remainder of the molecule.

95. The method according to any one of claims 91 to 93, wherein R 2 is -H.

96. The method according to any one of claims 91 to 94, wherein R 4 is phenyl, azaphenyl, pyrimidinyl, isoxazolyl, indolyl, naphthyl or pyridyl, any of which may be unsubstituted or substituted by 1, 2 or 3 R 4a substituents.

97. The method according to claim 96, wherein R 4 is phenyl substituted by 1 or 2 R 4a substituents.

98. The method according to claim 97, wherein the 1 or 2 R 4a substituents are -O-(C1-C2 alkyl) groups.

99. The method according to any one of claims 1 to 97, wherein R 4a is independently selected in each case from -CH3, -F, -Cl, -Br, -CN, -CF3, -OCH3, -OCHF2, -OCH2CH3, -C(═O)OCH3, -C(═O)CH3 or -N(CH3)2.

100. The method according to any one of claims 91 to 99, wherein R 3 is selected from the group consisting of -(CR 3b R 3c )-Q, -NH-(CR 3b R 3c )-Q, -(CR 3b R 3c )-C(═O)-Q, -(CR 3d R 3e )-(CR 3f R 3g )-Q, -(CR 3b ═CR 3c )-Q or -(heterocyclic group)-Q, wherein the heterocyclic group of -(heterocyclic group)-Q has 5 to 7 ring members, 1, 2 or 3 of which are heteroatoms selected from N, O or S, and is unsubstituted or substituted by 1, 2 or 3 R 3h substituents.

101. The method according to any one of claims 91 to 100, wherein Q is selected from pyrimidinyl, pyridinyl, isoxazolyl, thiazolyl, imidazolyl, phenyl, tetrahydropyrimidinone, cyclopropyl, cyclobutyl, cyclohexyl, morpholinyl, pyrrolidinyl, pyrazinyl, imidazo[1,2-a]pyridinyl, pyrazolyl or oxetanyl, any of which is unsubstituted or substituted with 1, 2 or 3 R Q substituents.

102. The method according to any one of claims 91 to 101, wherein Q is a monocyclic heteroaryl group having 5 or 6 ring members with 1 or 2 heteroatoms selected from N, O or S, and Q is unsubstituted or substituted with 1 or 2 R Q substituents.

103. The method according to any one of claims 91 to 102, wherein R 3 is a group of the formula -(CR 3d R 3e )-(CR 3f R 3g )-Q.

104. The method according to any one of claims 91 to 102, wherein R 3 has the following formula: where the symbol when drawn through the bond, represents the point of attachment to the rest of the molecule.

105. The method according to any one of claims 1 to 104, wherein the eparineptide receptor agonist is (2S,3R)-N-(4-(2,6-dimethoxyphenyl)-5-(5-methyl-3-pyridyl)-4H-1,2,4-triazol-3-yl)-3-(5-methyl-2-pyrimidinyl)-2-butenesulfonamide, or a pharmaceutically acceptable salt thereof, a tautomer thereof, a pharmaceutically acceptable salt of the tautomer, a stereoisomer of any of the foregoing, or a mixture thereof.

106. The method according to claim 105, wherein the eparineptide receptor agonist is (2S,3R)-N-(4-(2,6-dimethoxyphenyl)-5-(5-methyl-3-pyridyl)-4H-1,2,4-triazol-3-yl)-3-(5-methyl-2-pyrimidinyl)-2-butenesulfonamide or a pharmaceutically acceptable salt thereof.

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