Apelin receptor modulators for treatment of disorders or diseases associated with BBB permeability

Abstract

The present disclosure provides methods of using particular classes of apelin receptor modulators to reduce blood brain barrier (BBB) permeability in a subject in need thereof, and in particular methods of treating various disorders, conditions, and diseases associated with and associated with increased BBB permeability. The present disclosure also provides methods of treating a neurodegenerative disease, delirium, and / or dementia in a subject in need thereof using specific classes of apelin receptor modulators. The disclosure also provides methods of using specific classes of apelin receptor modulators to reduce neuroinflammation in a subject in need thereof. In some embodiments, the apelin receptor modulator (e.g., agonist) is BGE-105 or a pharmaceutically acceptable salt thereof.

Description

[0001] 1. Cross-reference to related applications

[0002] This application claims priority to and the benefit of the following U.S. provisional applications: 63 / 347,073 filed May 31, 2022; 63 / 413,430 filed October 5, 2022; 63 / 478,330 filed January 3, 2023; and 63 / 478,336 filed January 3, 2023; each of which is incorporated herein by reference in its entirety. 2. Background technology

[0003] Neurodegenerative diseases occur when nerve cells in the brain or peripheral nervous system lose function over time and eventually die. The likelihood of developing a neurodegenerative disease increases dramatically with age. Common neurodegenerative diseases include Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), multiple system atrophy (MSA), prion diseases, delirium, dementia, and postoperative cognitive dysfunction.

[0004] The blood-brain barrier (BBB) ​​separates the systemic circulation from the brain, regulates the transport of most molecules and protects the brain microenvironment. Central to the BBB are the unique features of brain endothelial cells, which are abutted and sealed by specialized junctions (tight junctions, TJs) and exhibit minimal vesicular transport (transcytosis), thereby preventing the transport of hydrophilic molecules from the blood to the brain and from the brain to the blood.

[0005] Many diseases and physiological stressors that affect the CNS also alter the functional integrity of the BBB. Changes in various physiological properties of the BBB have been associated with BBB breakdown or disruption associated with normal aging, cognitive impairment, and various neurodegenerative disorders and diseases including dementia. These changes can affect the ability of the BBB to selectively restrict the transport of substances from the blood to the brain. BBB disruption can lead to BBB leakage and vascular cognitive impairment.

[0006] AD is a common form of dementia. AD brain pathology begins before the onset of clinical symptoms. An early pathological feature of AD associated with cognitive decline is BBB dysfunction characterized by barrier leakage.

[0007] Additionally, there is a growing recognition of the role of astrocytes in disorders including AD, PD, HD, and ALS. Increasing evidence suggests that neuroinflammation plays an important role in the pathogenesis of ALS. Although certain treatments (such as anti-inflammatory or immunosuppressive therapies) may help alleviate some of the physical or psychiatric symptoms associated with neurodegenerative diseases, there is still a need to slow their progression or cure the disease.

[0008] As the median age of the population increases, there is an increasing need for drugs that reduce or eliminate age-related deficits that lead to cognitive impairment. Therefore, there remains a need for effective treatments that can treat disorders and diseases associated with BBB permeability and / or neuroinflammation. In addition, there remains a need for effective treatments that can treat neurodegenerative diseases. 3. Summary of the invention

[0009] One aspect of the disclosure includes methods of using a particular class of apelin receptor modulators to reduce blood-brain barrier (BBB) ​​permeability in a subject in need thereof, and in particular methods of treating various disorders associated with increased or abnormal BBB permeability. In some embodiments, the apelin receptor modulator is an apelin receptor agonist.

[0010] Another aspect of the disclosure includes a method of reducing blood-brain barrier (BBB) ​​permeability in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an apelin receptor agonist to reduce BBB permeability.

[0011] Another aspect of the disclosure includes a method of treating a disorder associated with increased BBB permeability in a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of an apelin receptor agonist.

[0012] Another aspect of the disclosure includes a method of treating blood-brain barrier dysfunction in a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of an apelin receptor agonist.

[0013] Another aspect of the disclosure includes a method of treating a neurodegenerative or neurodegenerative disease in a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of an apelin receptor agonist.

[0014] Another aspect of the disclosure includes a method of reducing neuroinflammation in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of an apelin receptor agonist.

[0015] Another aspect of the disclosure includes a method of treating a neurodegenerative disease in a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of an apelin receptor agonist.

[0016] The inventors used a survival prediction model, applied bioinformatics methods and machine learning methods to analyze human data, and found an association between apelin protein levels and age-related cognitive decline. Based on the cognitive abilities screening instrument (CASI) score, we found that higher circulating levels of apelin were significantly associated with reduced cognitive decline.

[0017] The inventors tested the effect of apelin receptor modulator BGE-105 on normal adult or aged mice in the BBB permeability model. BGE-105 has the following structure:

[0018]

[0019] BGE-105 (also known as AMG-986) is known to activate apelin receptors and induce cardiovascular responses in rats (Ason et al., JCI Insight. 5(8): 1-16 (2020)). Clinical trials with AMG-986 are underway to investigate its safety, tolerability, and pharmacokinetics in healthy subjects, subjects with heart failure (NCT03276728), and subjects with impaired renal function (NCT03318809). However, the effects of this compound on BBB permeability function in elderly individuals are unclear.

[0020] Apelin is a peptide hormone that is widely expressed throughout the body. i / o The protein-coupled receptor APJ performs signaling to exert multiple beneficial effects on cellular function. In the central nervous system, APJ is primarily expressed in astrocytes, which play an important role in age-related neuroinflammation and neurodegeneration. The inventors found that in preclinical models of neurodegeneration, direct brain administration of apelin had a disease-modifying effect through its effects on apoptosis, inflammation, and autophagy. The inventors combined multi-omics analysis and computational analysis of a proprietary longitudinal human aging cohort to identify a new link between higher circulating levels of apelin and retention of cognitive function. The inventors also observed that apelin pathway activity decreased with age. Based on the connection between apelin and cognitive aging, the relationship between apelin, inflammation, and neurodegeneration, and the expression of APJ on endothelial cells, the inventors hypothesized that apelin pathway activation could reduce inflammatory signaling in astrocytes and improve blood-brain barrier integrity in aged mice.

[0021] In a first set of experiments, the inventors demonstrated that normal mice (12 months old) treated with BGE-105 exhibited a significant reversal of LPS-induced BBB permeability, demonstrating in vivo activity in an acute cognitive impairment model.

[0022] In a second set of experiments, BGE-105 reversed BBB permeability in aged mice (26 months of age), demonstrating in vivo activity in a model of age-related cognitive impairment.

[0023] In a third set of experiments, BGE-105 improved the cellular function of astrocytes and protected neurons from cell death induced by reactive astrocyte mixture (RAC). The data suggest that BG5-105 can effectively reduce the neurotoxicity of degenerated or aged astrocytes and that BGE-105 can be used to treat neurodegenerative diseases and / or related neurodegenerative conditions.

[0024] Thus, exemplary apelin receptor modulators reduce BBB permeability, improve astrocyte cellular function, and thereby protect neurons from immune activation or inflammatory reactive astrocyte mixture (RAC)-induced cell death in vitro and in vivo in models of cognitive impairment, including acute or age-related cognitive impairment. These results suggest that apelin receptor modulators (such as BGE-105) will effectively reduce BBB permeability and restore astrocyte cellular function, thereby treating acute delirium in patients or neurodegenerative diseases or dementia in elderly patients.

[0025] Thus, a first aspect of the present disclosure provides a method for reducing blood-brain barrier (BBB) ​​permeability in a subject in need thereof, the method comprising administering to the subject a therapeutically 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 apelin receptor agonist is BGE-105 or a pharmaceutically acceptable salt thereof.

[0026] In another aspect, the present disclosure provides a method for treating a disorder associated with increased or abnormal BBB permeability in a subject, the method comprising administering to a subject in need thereof a therapeutically 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.

[0027] In some embodiments of the method of the present disclosure, the subject is a person and suffers from or is identified as suffering from or showing cognitive impairment. In certain embodiments, the obstacle associated with increased or abnormal BBB permeability is acute cognitive impairment, such as postoperative cognitive dysfunction (POCD) or intensive care unit delirium. In certain embodiments, the obstacle associated with increased or abnormal BBB permeability is a neurodegenerative disease, such as dementia (e.g., Alzheimer's disease) or vascular dementia. In various embodiments, the neurodegenerative disease is selected from Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), stroke, Huntington's disease (HD), multiple sclerosis (MS), traumatic brain injury (TBI), dementia and / or inflammation (e.g., neuroinflammation, peripheral inflammation, etc.).

[0028] In another aspect, the present disclosure provides a method for reducing neuroinflammation in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount 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.

[0029] In another aspect, the present disclosure provides a method for alleviating and / or treating a neurodegenerative disease in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount 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.

[0030] In some embodiments of the methods of the present disclosure, the subject is a human and suffers from or is identified as suffering from or exhibiting cognitive impairment. In some embodiments, the subject has increased neurotoxicity and / or neuroinflammation detected in astrocytes, increased astrocyte cytokine or chemokine release, activated or increased NF- K B signal transduction transcriptional response, and / or insufficient glutamate clearance. In some embodiments, the expression of one or more immune biomarkers (such as cytokines or chemokines (e.g., CCL2, IL-10, CCL1 1, CCL5, CXCL1, IL-6, CXCL11)) of the subject changes (increases or decreases). In various embodiments, the subject is old. In various embodiments, the subject has increased BBB permeability (e.g., increased BBB leakage) and / or astrocyte neurotoxicity (e.g., neuroinflammation). In various embodiments, the subject suffers from one or more neurodegenerative diseases selected from the following: Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), stroke, Huntington's disease (HD), multiple sclerosis (MS), traumatic brain injury (TBI), dementia and / or inflammation (e.g., neuroinflammation, peripheral inflammation, etc.).

[0031] In another aspect, the disclosure provides methods of using a particular class of apelin receptor modulators to treat a neurodegenerative disease in a subject in need thereof. In some embodiments, the apelin receptor modulator is an apelin receptor agonist.

[0032] In various embodiments, the subject suffers from one or more neurodegenerative diseases selected from the group consisting of Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), stroke, Huntington's disease (HD), multiple sclerosis (MS).

[0033] In another aspect, the disclosure provides methods of using a particular class of apelin receptor modulators to treat dementia or delirium in a subject in need thereof. In some embodiments, the apelin receptor modulator is an apelin receptor agonist.

[0034] In another aspect, the disclosure provides methods of using a particular class of apelin receptor modulators to treat traumatic brain injury (TBI) in a subject in need thereof. In some embodiments, the apelin receptor modulator is an apelin receptor agonist.

[0035] In another aspect, the disclosure provides methods of using a particular class of apelin receptor modulators to reduce neuroinflammation in a subject in need thereof. In some embodiments, the apelin receptor modulator is an apelin receptor agonist. 4. Description of the drawings

[0036] These and other features, aspects and advantages of the present invention will be better understood with reference to the following description and accompanying drawings, in which:

[0037] Figure 1 The structure of BGE-105 is shown.

[0038] Figure 2A - Figure 2C shows the Kaplan-Meier curve of bioinformatics analysis, which illustrates the relationship between apelin peptide and cognitive decline. The Y-axis represents the probability of CASI decline. The X-axis represents the number of years (10 years). The purple line represents the group participants with APLN levels in the lower 10% of the group, the pink line represents the participants with the highest APLN levels (upper 10%), and the gray line is between 10%-90%.

[0039] Figure 3A-3B BBB permeability was shown to be reduced after administration of BGE-105 in an LPS-induced BBB mouse model. The BBB permeability model was established by intraperitoneal injection of 3 mg / kg LPS. Figure 3A, mice were divided into three groups: Group 1: mice with PO administration of vehicle "control", Group 2: mice with PO administration of vehicle for 1 week, followed by LPS on the 8th day, and Group 3: mice with BGE-105 for 1 week, followed by LPS on the 8th day. Group 3 mice were administered BGE-105 at 50mg / kg (BID) for one week (7 days), followed by LPS stimulation on the 8th day. The healthy control group mice received PO vehicle (BID) for one week (7 days), followed by an intraperitoneal injection of saline. The PO vehicle or BGE-105 was retained until the endpoint Evans blue (Evans blue) was determined to measure BBB permeability. 23 hours after LPS or saline injection, mice were intravenously injected with Evans blue (EB) (2%, 75ul / 30g BW). The mice were euthanized, and the whole brain was then removed to microdissect specific brain regions (olfactory bulb, hippocampus) from each hemisphere. Figure 3B It was shown that mice treated with BGE-105 exhibited a significant reversal of LPS-induced BBB permeability in the olfactory bulb (OB) and hippocampus (HPF) compared to vehicle-treated mice (LPS vs. LPS+BGE105).

[0040] Figure 4A-4C showed that BBB permeability was reduced after treatment with BGE-105 in aged mice. Figure 4A The differences in blood-brain barrier permeability in 3-month-old mice (young), 13-month-old mice (adult), and 22-month-old mice (old) are shown. The BBB permeability is increased in both the olfactory bulb and hippocampus regions of the brain as shown in naturally aged mice compared to young and adult mice. Figure 4B Schematic diagram of the evaluation of the activity of BGE-105 on BBB permeability in aged mice that exhibit age-related increase in BBB permeability. Figure 4C It was shown that pretreatment with BGE-105 significantly reversed the age-induced increase in BBB permeability in OBs compared with HPFs (-28%; p<0.01, Mann-Whitney U test).

[0041] Figure 5 showed that BGE-105 reduced circulating levels of the peripheral inflammatory marker CXCL1 in aged mice.

[0042] Figure 6 showed that BGE-105 reduced circulating levels of the peripheral inflammatory marker CXCL13 in aged mice.

[0043] Figure 7 BGE-105 was shown to increase the concentration of total brain-derived neurotrophic factor (BDNF) expression in the hippocampus of aged mice.

[0044] Figure 8 Show that BGE-105 weakens the CXCL1, CD3 and IL-6 gene expression induced by RAC. When compared with the control (CON), the CXCL1 expression in the reactive astrocytes (RAC) induced by RAC is significantly upregulated by about 1200-1600 times (p < 0.0001) (left small figure). When compared with the RAC group, the expression of biomarker CXCL1 is significantly reduced by BGE-105 treatment (RAC+105) (p = 0.0204). When compared with the control (CON) group, the C3 expression in the reactive astrocytes (RAC) induced by RAC is significantly upregulated by about 20-30 times (p < 0.0001) (middle small figure). Compared with the RAC group (RAC), the expression of biomarker C3 is significantly reduced by BGE-105 treatment (RAC+105) by 10-20 times (p = 0.0015). Figure 8 The right panel shows that IL-6 was upregulated by about 10-15 fold in RAC-induced reactive astrocytes (RAC) (p<0.0001). Treatment with BGE-105 (RAC+105) significantly reduced the expression of IL-6 by about 5-10 fold (p<0.0001).

[0045] Fig. 9 BGE-105 was shown to reduce CXCL1 and IL-6 release from astrocytes. Fig. 9 The left panel shows that BGE-105 treatment (RAC+105, Group 4) greatly reduces the release of CXCL1 from astrocytes, and thus reduces the concentration of CXCL1 (p=0.0032). When compared with control astrocytes (CON, Group 1), reactive astrocytes (RAC, Group 3) increase the release of CXCL1 by about 30000-40000 times (p<0.0001). Similar results were observed in Group 6 (RAC+BAY) by treating with NF-κB inhibitors (RAC+BAY, p<0.0001). Fig. 9 The right panel shows that the reactive astrocytes (RAC) (RAC+BGE-105, the 4th group) treated with BGE-105 reduce the IL-6 release of astrocytes, and thus reduce the concentration of IL-6 (p=0.0008). When compared with control astrocytes (CON), reactive astrocytes (RAC) increase the release of IL-6 by about 1600-2400 times (p<0.0001). Similar results of the 6th group (RAC+BAY) were observed with NF-κB inhibitor treatment (RAC+BAY, p<0.0001).

[0046] Figures 10A-10DBGE-105 is shown to activate apelin receptor (APJ) signaling in astrocytes. Protein expression of APJ, p-AKT, t-AKT, and β-actin in control astrocytes, cells treated with reactive astrocyte cocktail (RAC), and RAC cells treated with BGE-105 (RAC+BGE-105 [50 nM]) is shown. Fig. 10A The expression of APJ was shown to be comparable in all three experimental groups (control (CON), RAC, RAC + BGE-105). When normalized to β-actin expression, no significant fold change in expression was detected ( Fig. 10B ). Fig. 10C The expression of p-AKT in the RAC+BGE-105 group was shown to be increased, while the expression of t-AKT in all three experimental groups remained comparable. Compared with t-AKT, and normalized with β-actin expression, the expression level of p-AKT increased significantly by about 2-fold in response to RAC+BGE-105 treatment ( Fig. 10D ). p-AKT: phosphorylated AKT.

[0047] Figures 11A-11D BGE-105 was shown to reduce cytokine release in a dose-dependent manner. Fig.11A Relative fold changes in expression of various cytokines are shown. The legend on the right ranges from 0-fold change (blue) to 1-fold change (or no change, marked in white) to 4+-fold change (dark red). Fig.11A Shown is a panel of cytokines and chemokines release detected in astrocytes treated with reactive astrocyte mixture (RAC) and different doses of BGE-105 (RAC+BGE-105 [10 nM, 50 nM, 250 nM]) compared to RAC. Fig. 11B Transcription factor enrichment showing the following biomarkers: IKBKB, IRF1, STAT6, NF- K B1 and RELA. Fig. 11C Fold changes in cytokine and chemokine release detected from different treatments (RAC, RAC+105(10 nM), RAC+105(50 nM), RAC+105(250 nM)) are shown. Fig. 11C It was shown that the number of cytokines that were downregulated (or <1-fold change) increased with increasing doses of BGE-105. Treatment with BGE-105 (RAC + BGE-105 [50 nM and 250 nM]) significantly reduced the release of cytokines and chemokines. Fig.11D Protein bands of cytokines and chemokines quantified by Western blotting and presented in dot blots are shown.

[0048] Fig.12 showed that treatment of reactive astrocytes with BGE-105 reduced CXCL1 and IL-6 release from astrocytes. Fig.12 The left panel shows that the concentration / release of CXCL1 in astrocytes treated with reactive astrocyte mixture (RAC) increased by about 40,000-60,000 pg / ml compared to the control (CON) group. BGE-105 (RAC+BGE-105[50nM, 250nM]) significantly reduced the concentration / release of CXCL1 to about 40,000 pg / ml (50nM) or about 20,000-40,000 pg / ml (250nM). It should be noted that no significant difference in CXCL1 concentration / release was detected between the BGE-105 (RAC+BGE-105[50nM]) treated group and the apelin peptide (APL) (RAC+APL[50nM]) treated group. Fig.12 The right panel shows that treatment with RAC increased the concentration / release of IL-6 by about 10,000 pg / ml compared to the control (CON) group. BGE-105 (RAC+BGE-105[50 nM, 250 nM]) significantly reduced the concentration / release of IL-6 to about 5,000-10,000 pg / ml (50 nM) or about 10,000 pg / ml (250 nM). It should be noted that no significant difference in IL-6 concentration / release was detected between the BGE-105 (RAC+BGE-105[50 nM]) treated group and the Apelin (APL) (RAC+APL[50 nM]) treated group.

[0049] Figure 13A-13C BGE-105 was shown to inhibit NF- K B activation and I K Bα phosphorylation. Fig.13A The expression of NF-κB in control astrocytes, astrocytes treated with reactive astrocyte cocktail (RAC), and astrocytes treated with BGE-105 (RAC+BGE-105 [50 nM]) was shown. K B. pI K Bα、tI K Protein expression of Bα and β-actin. Fig. 13B NF-κB normalized to β-actin is shown. K B p65 expression. Compared with the control (CON), RAC increased NF- K However, treatment with BGE-105 (RAC+BGE-105 (50 nM)) significantly reduced the expression of NF- K Expression of Bp65. Fig. 13C Shown is the pI normalized to β-actinK Bα / tI K The ratio of Bα expression. RAC has increased pI K Bα / tI K The ratio of Bα expression was significantly reduced by BGE-105 treatment (RAC+BGE105). K Bα / tI K The ratio of Bα expression.

[0050] Figure 14A-14B showed that reactive astrocytes treated with BGE-105 were responsive to NF- K B signal transduction transcriptional response has a dose-dependent effect. Fig.14A is a heat map showing the expression of NF- K B. Expression of biomarkers related to the signal transduction pathway and in response to the treatment of BGE-105 (RAC+BGE-105 [50 nM, 250 nM]). The legend shows the fold change of expression compared to the control group (not shown), ranging from -10-fold change (blue) to 0-fold change (or no change, marked in white), up to 30-fold change (dark red). Fig. 14B Shows relative to the control, from Fig.14A Fold changes in the expression of an exemplary subset of biomarkers (Rela, Il2, Cxcl3, and Csf3) of RACs were shown. Two pro-inflammatory genes were downregulated (Rela [decreased by 182%], Cxcl3 [decreased by 55%]) and two anti-inflammatory / neurotrophic genes were upregulated (Il2 increased by 184%], Csf3 [increased by 16%]) in response to treatment of reactive astrocytes (RAC+BGE-105 [250nM]) with a higher dose of BGE-105 (250nM).

[0051] Fig.15 BGE-105 was shown to improve insufficient glutamate clearance in RAC astrocytes. Glutamate clearance in RAC astrocytes (RAC) was significantly reduced, which may contribute to glutamate excitotoxicity in neurodegeneration / aging. BGE-105 (RAC+BGE-105 [10nM, 50nM, 250nM]) and apelin (APL) (RAC+APL [50nM]) significantly and statistically increased the percentage of glutamate uptake.

[0052] Figure 16A-16BReactive astrocytes treated with BGE-105 showed increased cell viability after challenge with RAC conditioned medium. The percentage change in viability of astrocytes relative to RAC in control (RAC) and BGE-105 (RAC+BGE105 [50 nM, 250 nM]) and apelin (APL) (RAC+APL [50 nM]) treated groups is shown. Fig.16A It was shown that BGE-105 (RAC+BGE105 [250 nM]) significantly and statistically increased cell viability by about 30% compared to RAC. Fig. 16B It was shown that exogenous BGE-105 (RAC+ex105 [50 nM]), where BGE-105 was added directly to neurons along with toxic RAC medium, did not provide a statistical change in cell viability relative to RAC. Exogenous BGE-105 did not directly protect neurons from RAC-induced neurotoxicity. 5. Specific implementation methods

[0053] 5.1 Methods of reducing BBB permeability using apelin receptor modulators

[0054] As outlined above, one aspect of the present disclosure provides methods for reducing blood-brain barrier (BBB) ​​permeability in a subject in need thereof using an apelin receptor modulator (e.g., using an apelin receptor agonist of a specific structural class). In some embodiments, the methods of the present disclosure provide for reducing BBB permeability in a subject, providing for restoring BBB permeability levels to normal or healthy levels, reducing BBB permeability, reducing BBB leakage, maintaining BBB integrity, or any combination thereof.

[0055] During aging, various mechanisms lead to BBB breakdown and increase BBB permeability. Increased BBB permeability is associated with dementia and various neurodegenerative diseases. See, for example, Hussain et al. "Blood-Brain Barrier Breakdown: An Emerging Biomarker of Cognitive Impairment in Normal Aging and Dementia", Front. Neurosci., August 19, 2021 (doi.org / 10.3389 / fnins.2021.688090). Postoperative delirium is associated with the breakdown of the BBB. This increased permeability is dynamic and associated with neuroinflammatory responses and lactate responses. See, for example, Taylor, Jennifer et al., (“Postoperative delirium and changes in the blood-brain barrier, neuroinflammation, and cerebrospinal fluid lactate: a prospective cohort study” Neuroscience And Neuroanaesthesia, Vol. 129, No. 2, pp. 219-230, August 2022).

[0056] The inventors have shown that BGE-105 reverses BBB permeability in aged mice (26 months old), demonstrating in vivo activity in a model of age-related cognitive impairment.

[0057] Systemic inflammation can also cause BBB permeability to increase and cause cognitive impairment, such as acute cognitive impairment. In certain embodiments, acute cognitive impairment is dementia, delirium or postoperative cognitive dysfunction. The acute onset of such delirium can also induce damage and facilitate long-term cognitive decline. In addition, it is known that peripheral inflammation can promote BBB leakage and cognitive impairment. Preliminary studies using BGE-105 to aged mice show that BGE-105 reduces the circulating levels of two cytokines (CXCL1 / 13) associated with mortality, neutrophil recruitment and inflammatory propagation.

[0058] The inventors further demonstrated that normal mice (12 months old) treated with BGE-105 exhibited a significant reversal of LPS-induced BBB permeability, demonstrating in vivo activity in an acute cognitive impairment model.

[0059] Thus, exemplary apelin receptor modulators reduce BBB permeability in vivo in models of cognitive impairment, including acute or age-related cognitive impairment. These results suggest that apelin receptor modulators (such as BGE-105) will be effective in treating disorders associated with BBB permeability. In some embodiments, the method of reducing BBB permeability treats acute delirium in a patient. In some embodiments, the method of reducing BBB permeability treats a neurodegenerative disease or dementia in an elderly patient. In some embodiments, reducing BBB permeability treats delirium caused by trauma. In some embodiments, reducing BBB permeability treats delirium caused by trauma from hip fracture or cardiovascular surgery, delirium caused by trauma, or delirium caused by surgery. In some embodiments, reducing BBB permeability treats inflammation, such as neuroinflammation or peripheral inflammation.

[0060] As described herein, BBB permeability can be assessed or determined during treatment via various direct and / or indirect methods. In some embodiments, the reduction in BBB permeability is determined by comparing with normal or healthy levels of BBB permeability, and its changes are assessed over time. In some embodiments, the reduction in BBB permeability is determined by comparing with the baseline increase level of BBB permeability assessed in the subject before treatment. In some embodiments, BBB permeability can be indirectly assessed via assessing one or more symptoms of cognitive impairment (e.g., as described herein).

[0061] 5.1.1 Acute cognitive impairment model

[0062] The present disclosure describes the in vivo evaluation of apelin receptor agonists in a mouse model commonly associated with inflammation-induced acute cognitive impairment.

[0063] In the first set of experiments (see Figure 3A ), the activity of exemplary apelin receptor agonists was evaluated in 12-month-old mice that were challenged with LPS to induce increased BBB permeability in the olfactory bulb (OB) and hippocampus (HPF). Figure 3B (Control vs. LPS). Further details are provided in Example 2 of the Experimental section. The inventors demonstrated that mice treated with BGE-105 exhibited a significant reversal of LPS-induced BBB permeability compared to vehicle-treated mice. Figure 3B (LPS compared to LPS+BGE105).

[0064] 5.1.2 Models of cognitive impairment in elderly subjects

[0065] In a second set of experiments, the activity of exemplary apelin receptor agonists was assessed against age-related increases in BBB permeability in an aged mouse model. Figure 4Ashowed that BBB permeability was increased in aged mice in this model.

[0066] The inventors demonstrated that pretreatment of mice with BGE-105 significantly reversed the age-induced increase in BBB permeability in aged mice. See e.g. Figure 4C , aging vs aging+BGE-105 in the olfactory bulb (OB) compared to HPF (-28%) or cortical floor plate (CTX) (data not shown). Further details are provided in Example 3 of the Experimental section, demonstrating in vivo activity in a model of age-related cognitive impairment.

[0067] 5.2 Methods of treating diseases or disorders associated with BBB permeability

[0068] Thus, in one aspect, the present disclosure provides methods of using apelin receptor modulators to treat disorders associated with BBB permeability.

[0069] The method comprises administering to a subject in need thereof a therapeutically effective amount of an apelin receptor modulator of Formula (I) or (II) (eg, as described herein), such as BGE-105.

[0070] "BBB permeability-related disorders" (interchangeably referred to herein as "BBB permeability-related disorders" and "BBB permeability-related disorders") refer to disorders or conditions that cause or are susceptible to increased BBB permeability and / or abnormal BBB permeability in a mammalian subject. In other words, disorders associated with BBB permeability can include disorders or conditions that have an effect on BBB breakdown or BBB permeability. For example, such disorders can include one or more neurodegenerative diseases, including disorders that cause neurodegeneration, neuroinflammation, and / or cognitive impairment. In some embodiments, increased BBB permeability or abnormal permeability leads to acute or chronic cognitive impairment.

[0071] In some embodiments, the disorder associated with BBB permeability is dementia. Dementia is not a specific disease, but covers various conditions characterized by impaired cognitive ability and problems with memory, language, thinking or judgment that interfere with daily activities. In some embodiments, the disorder is vascular dementia (VaD). Vascular dementia is a neurodegenerative disease characterized by the loss of cognitive function caused by ischemic, ischemic-hypoxic or hemorrhagic brain damage caused by cardiovascular disease and cardiovascular pathological changes. After AD, VaD is also considered to be the second most common type of dementia. The symptoms of VaD include cognitive loss, headache, insomnia and memory loss.

[0072] In some embodiments, the disorder associated with BBB permeability is characterized by cognitive impairment. In some embodiments, cognitive impairment occurs in a subject having or at risk of having a neurodegenerative disease or a related or associated disorder.

[0073] In some embodiments, the subject is showing one or more symptoms of age-related cognitive impairment. Mild cognitive impairment (MCI) is a stage between the expected cognitive decline of normal aging and the more severe decline of dementia. The symptoms of MCI can remain stable for several years, or in some cases progress to dementia. Acute cognitive impairment may be associated with acute inflammation (such as inflammation associated with surgery or other injuries). In certain embodiments, acute cognitive impairment is referred to as or characterized as delirium. Delirium is a disease that affects the brain and can suddenly appear within a few hours or days of injury or other causes. Some characteristics of delirium include difficulty concentrating (inattention), sudden changes in behavior and confusion. For most people, delirium is short-lived, usually only a few days.

[0074] In some embodiments, the disorder associated with BBB permeability is delirium. In some embodiments, the disorder associated with BBB permeability is postoperative delirium. In certain embodiments, the disorder associated with BBB permeability is intensive care unit (ICU) delirium. Patients in the intensive care unit (ICU) may be at risk for ICU delirium. In some cases, about two-thirds of ICU patients will experience delirium, with those on ventilators often being at greatest risk. ICU delirium should be diagnosed and treated as soon as possible because patients with ICU delirium, if not treated, may have poor outcomes, leading to long-term problems such as depression and anxiety.

[0075] In certain embodiments, the disorder associated with BBB permeability is postoperative cognitive dysfunction (POCD). POCD is a state in which a patient's memory and learning ability decline after surgery. Although patients over 60 are more often affected by POCD, patients of all age groups are at risk. It is reported that symptoms of POCD include: difficulty remembering and recalling; inability to complete tasks that were not previously difficult; problems with intellectual performance; difficulty handling multiple tasks; reduced psychomotor skills; difficulty understanding language; and problems with social integration.

[0076] In some embodiments, the disorder associated with BBB permeability is inflammation. In some embodiments, the disorder associated with BBB permeability is neuroinflammation. In some embodiments, the disorder associated with BBB permeability is a disorder associated with the central nervous system (CNS). In some embodiments, the disorder associated with BBB permeability is peripheral inflammation.

[0077] In some embodiments, the disorder associated with BBB permeability is a neurodegenerative disease or a related or associated condition.Any neurodegenerative disease associated with altered BBB permeability and function can be targeted for treatment according to the methods of the present disclosure.

[0078] In certain embodiments, the neurodegenerative disease is Alzheimer's disease (AD). AD can be referred to as a neurodegenerative disease and is also a type of dementia. In AD patients, the BBB exhibits leakage of cerebral blood vessels, perivascular accumulation of fibrinogen, albumin, thrombin, and immunoglobulin (IgG), loss of TJs, and degeneration of ECs and pericytes.

[0079] In certain embodiments, the neurodegenerative disease is Parkinson's disease (PD).

[0080] In certain embodiments, the neurodegenerative disease is amyotrophic lateral sclerosis (ALS).

[0081] In certain embodiments, the neurodegenerative disease is multiple sclerosis (MS).

[0082] In some embodiments, the obstacle or disease is brain injury. Brain injury (such as ischemic, hemorrhagic or traumatic) can lead to dysfunction of BBB. In certain embodiments, the obstacle related to BBB permeability is traumatic brain injury (TBI). The method of the present disclosure can provide the treatment of post-traumatic dysfunction of BBB.

[0083] In some embodiments, the disorder associated with BBB permeability is stroke. In some embodiments, the disorder associated with BBB permeability is ischemic stroke. In some embodiments, the disorder associated with BBB permeability is hemorrhage. In some embodiments, the disorder associated with BBB permeability is amyloid-β induced memory loss.

[0084] In certain embodiments, the disorder associated with BBB permeability is stroke. In certain embodiments, the disorder associated with BBB permeability is ischemic stroke. The methods disclosed herein can provide treatment for post-stroke dysfunction of the BBB.

[0085] In certain embodiments, the disorder associated with BBB permeability is neuroinflammation. In some embodiments, the disorder associated with BBB permeability is peripheral inflammation. The methods disclosed herein can provide treatment for neuroinflammation (such as peripheral inflammation).

[0086] 5.3 Methods for treating dementia

[0087] Thus, in one aspect, the present disclosure provides methods of treating dementia in a subject using an apelin receptor modulator.

[0088] In some embodiments, the method comprises administering to a subject in need thereof a therapeutically effective amount of an apelin receptor modulator of Formula (I) or (II) (eg, as described herein), such as BGE-105.

[0089] In certain embodiments, the dementia is acute dementia. In certain embodiments, the dementia is chronic or progressive dementia. In certain embodiments, the subject suffers from vascular dementia (VaD).

[0090] 5.4 Methods for treating cognitive impairment or cognitive dysfunction

[0091] Thus, in one aspect, the present disclosure provides methods of using apelin receptor modulators to treat cognitive impairment or cognitive dysfunction in a subject.

[0092] In some embodiments, the method comprises administering to a subject in need thereof a therapeutically effective amount of an apelin receptor modulator of Formula (I) or (II) (eg, as described herein), such as BGE-105.

[0093] In certain embodiments, the subject suffers from acute, mild or progressive cognitive impairment. In certain embodiments, the subject suffers from postoperative cognitive dysfunction (POCD).

[0094] 5.5 Methods of treating delirium

[0095] Thus, in one aspect, the present disclosure provides methods of treating delirium in a subject using an apelin receptor modulator.

[0096] In some embodiments, the method comprises administering to a subject in need thereof a therapeutically effective amount of an apelin receptor modulator of Formula (I) or (II) (eg, as described herein), such as BGE-105.

[0097] In some embodiments, the delirium is postoperative delirium. In some embodiments, delirium is diagnosed in patients after surgery (e.g., cardiovascular surgery). In some embodiments, delirium is diagnosed after a fracture (e.g., a hip fracture). In some embodiments, the delirium is ICU-induced delirium. In some embodiments, delirium is diagnosed due to trauma.

[0098] 5.6 Methods to reduce neuroinflammation

[0099] Thus, in one aspect, the present disclosure provides methods of using apelin receptor modulators to reduce neuroinflammation in a subject.

[0100] In some embodiments, the method comprises administering to a subject in need thereof a therapeutically effective amount of an apelin receptor modulator of Formula (I) or (II) (eg, as described herein), such as BGE-105.

[0101] 5.7 Methods of using apelin receptor modulators to reduce neurotoxicity

[0102] As summarized above, the present disclosure provides methods of using apelin receptor modulators (eg, using an apelin receptor modulator) to reduce neurotoxicity or neurodegeneration in a subject in need thereof.

[0103] In some embodiments, the method comprises administering to a subject in need thereof a therapeutically effective amount of an apelin receptor modulator of Formula (I) or (II) (eg, as described herein), such as BGE-105.

[0104] In some embodiments, the methods disclosed herein provide for reducing neurotoxicity or neurodegeneration in a subject, providing methods for reducing neurotoxicity or neurodegeneration of astrocytes, reducing astrocyte cytokine or chemokine release, inhibiting or reducing NF- K B signaling transcriptional response, and / or restoring deficient glutamate clearance to normal or healthy levels, or any combination thereof.

[0105] Astrocytes are primary cells that express the apelin receptor (Aplnr). BGE-105 is an agonist of the apelin receptor. The inventors show that the apelin receptor is expressed in large quantities in astrocytes and activated by BGE-105, and BGE-105 attenuates the astrocyte inflammatory response after exposure to RAC in mouse astrocytes. The inventors show that BGE-105 also limits RAC-induced astrocyte inflammation by modifying NF-κB signaling. BGE-105 improves the cellular function of astrocytes and protects neurons from RAC-induced cell death, which indicates that BGE-105 can effectively reduce the neurotoxicity of degenerated or aged astrocytes and can therefore be used to treat neurodegenerative diseases.

[0106] 5.8 Methods of treating neurodegenerative diseases or disorders

[0107] Thus, in one aspect, the present disclosure provides methods of using apelin receptor modulators to treat a neurodegenerative disease or disorder in a subject.

[0108] In some embodiments, the method comprises administering to a subject in need thereof a therapeutically effective amount of an apelin receptor modulator of Formula (I) or (II) (eg, as described herein), such as BGE-105.

[0109] In some embodiments, the neurodegenerative disease is selected from Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), stroke, Huntington's disease (HD) and multiple sclerosis (MS). In some embodiments, the disorder is traumatic brain injury (TBI).

[0110] 5.9 Patient age

[0111] In some embodiments of the methods of the disclosure, the subject is elderly.

[0112] In some embodiments, the subject is a human. When the subject is a human, the subject may be referred to as a patient. 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 some 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.

[0113] 5.10 Patient Assessment

[0114] A variety of direct and / or indirect assessment methods can be used to determine the subject in need of treatment according to the method disclosed herein. The reduction in BBB permeability can be determined by comparing with a baseline level, which is determined, for example, by a suitable assessment method before treatment, directly (e.g., via an imaging method) or indirectly (e.g., via a level determined by assessing relevant biomarkers in a subject's sample). In some embodiments, the reduction in BBB permeability achieved by practicing the method disclosed herein is 10% or more reduction compared to baseline BBB permeability, such as 20% or more, 30% or more, 40% or more, or 50% or more reduction, as determined by a suitable direct or indirect assessment method.

[0115] A reduction in neurotoxicity associated with a neurodegenerative disease can be determined by comparison with a baseline level, such as a level determined by a suitable assessment method prior to treatment, directly (e.g., via an imaging method) or indirectly (e.g., via assessment of relevant biomarkers in a sample from a subject). In some embodiments, a reduction in neurotoxicity associated with a neurodegenerative disease achieved by practicing the methods of the present disclosure is a 10% or greater reduction, such as a 20% or greater, 30% or greater, 40% or greater, or a 50% or greater reduction compared to baseline neurotoxicity, as determined by a suitable direct or indirect assessment method.

[0116] BBB permeability and dysfunction or neurodegenerative diseases (including related conditions) can be assessed using a variety of methods, such as by imaging techniques or electroencephalography. See, for example, WO 2021053684. Various other methods can be used to identify biomarkers associated with BBB breakdown and increased BBB permeability in a subject. See, for example, Hussain et al. 2021. Blood-Brain Barrier Breakdown: An Emerging Biomarker of Cognitive Impairment in Normal Aging and Dementia. Front Neurosci. 2021 Aug 19; 15: 688090. In addition to imaging, biochemical and genomic analysis, other methods for identifying and assessing neurodegenerative diseases include a combination of more detailed clinical assessments (covering specific cognitive and neurophysiological tests). See, for example, Henley et al., 2005. Biomarkers for neurodegenerative diseases. Current Opinion in Neurology 18(6): pp. 698-705, December 2005.

[0117] In some embodiments, the subject's BBB permeability, neurotoxicity, neurodegeneration, and / or cognitive impairment are assessed after administration. In some embodiments, the assessment is performed at least one day after administration. In some embodiments, the assessment is performed at least one week after administration. In some embodiments, the assessment is performed at least one month after administration.

[0118] In certain embodiments, the subject is a human and is undergoing mechanical ventilation (eg, undergoing mechanical ventilation at the time of diagnosis). In certain embodiments, the subject is a human and is using a ventilator.

[0119] In some embodiments, the patient is on bed rest. In some embodiments, the patient is on a ventilator.

[0120] In some embodiments, the human subject suffers from or is identified as suffering from cognitive impairment (e.g., acute, mild, severe cognitive impairment). In some embodiments, the human subject suffers from or is identified as suffering from postoperative cognitive dysfunction. In some embodiments, the human subject suffers from or is identified as suffering from dementia. In some embodiments, the human subject suffers from or is identified as suffering from delirium (e.g., postoperative delirium, ICU-induced delirium). Cognitive impairment can be assessed using a variety of diagnostic methods.

[0121] In some embodiments, the patient has or is identified as having increased neurotoxicity. In some embodiments, the patient suffers from or is identified as suffering from neuroinflammation. In certain embodiments, the neuroinflammation is peripheral inflammation. In some embodiments, the human subject suffers from or is at risk of suffering from neurodegeneration.

[0122] In some embodiments, the patient suffers from or is identified as suffering from a neurodegenerative disease or disorder selected from: Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), stroke, Huntington's disease (HD), multiple sclerosis (MS), and traumatic brain injury (TBI).

[0123] In certain embodiments, the human subject has or is at risk of developing Alzheimer's disease (AD).

[0124] In certain embodiments, the human subject has or is at risk of developing Parkinson's disease (PD).

[0125] In certain embodiments, the human subject has or is at risk of developing amyotrophic lateral sclerosis (ALS).

[0126] In certain embodiments, the human subject has or is at risk of developing multiple sclerosis (MS).

[0127] In certain embodiments, the human subject has or is at risk of developing Huntington's disease (HD).

[0128] In certain embodiments, the human subject has or is at risk of developing neuroinflammation. In certain embodiments, the human subject has or is at risk of developing peripheral inflammation.

[0129] In some embodiments, the human subject suffers from or is at risk of having a stroke. In some embodiments, the human subject suffers from or is at risk of having a hemorrhage.

[0130] In some embodiments, the human subject has hyperplasticity (e.g., hyperplasticity associated with a neurodegenerative disease) or is at risk of having it. In some embodiments, the human subject has innate immune activation (e.g., associated with a neurodegenerative disease) or is at risk of developing it. In some embodiments, the human subject has blood-brain barrier dysfunction or is at risk of developing it.

[0131] 5.11 Apelin receptor modulators

[0132] 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 different roles in the physiology and pathophysiology of many organs. The apelin / APJ system is involved in various cellular activities, such as proliferation, migration, apoptosis or inflammation. Apelin receptor modulators can directly or indirectly, competitively or non-competitively activate the APJ system.

[0133] The suitability of apelin receptor modulators for use in the methods of the present disclosure for treating disorders, neurodegenerative diseases or conditions associated with increased or abnormal BBB permeability can be assessed in any of a number of animal models of neurodegenerative disease and / or BBB permeability, for example, animal models for Huntington's disease (see, e.g., Mangiarini et al., 1996, Cell 87:493-506; Lin et al., 2001, Hum. Mol. Genet. 10:137-144), Alzheimer's disease (Hsiao, 1998, Exp. Gerontol, 33:883-889; Hsiao et al., 1996, Science 274:99-102), Parkinson's disease (Kim et al., 2002, Nature 418:50-56), and amyotrophic lateral sclerosis (Zhu et al., 2002, Nature 417:74-78).

[0134] 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. Pat. Nos. 9,573,936 or 9,868,721, the disclosures of which are incorporated herein by reference in their entireties.

[0135] As known to those skilled in the art, certain compounds of the present disclosure may exist in one or more tautomeric forms. Since a chemical structure can only be used to represent one tautomeric form, for convenience, it is understood that reference to a compound of a given structural formula includes tautomers of the structure represented by the structural formula.

[0136] In some embodiments, the apelin receptor modulator is a compound of formula (I) or (II):

[0137]

[0138] 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:

[0139] R 1 is unsubstituted pyridyl, pyridone or pyridine N-oxide, or is substituted by 1, 2, 3 or 4 R 1a a pyridyl, pyridonyl or pyridine N-oxide substituted with a substituent;

[0140] R 1a is independently selected from -F, -Cl, -Br, -I, -CN, -C 1 -C 6 Alkyl, -C 1 -C 6 Haloalkyl, -C 1 -C 6 Perhaloalkyl, -OH, -O-(C 1 -C 6 Alkyl), -O-(C 1 -C 6 Haloalkyl), -O-(C 1 -C 6 Perhaloalkyl), -C 2 -C 6 Alkenyl, -O-(C 1 -C 6 Alkyl)-OH, -O-(C 1 -C 6 alkyl)-O-(C 1 -C 6 Alkyl), -O-(C 1 -C 6 Haloalkyl)-OH, -O-(C 1 -C 6 (C 1 -C 6 Alkyl), -O-(C 1 -C 6 Perhaloalkyl)-OH, -O-(C 1 -C 6 Perhaloalkyl)-O-(C 1 -C 6 Alkyl), -NH 2 、-NH(C 1 -C 6 Alkyl), -N(C 1 -C 6 alkyl) 2 、-C(=O)-(C 1 -C 6 alkyl), -C(=O)OH, -(C=O)-O-(C 1 -C 6 alkyl), -C(=O)NH 2 、-C(=O)NH(C 1-C 6 alkyl), -C(=O)N(C 1 -C 6 alkyl) 2 , phenyl, -C(=O)-(heterocyclyl) or heterocyclyl, wherein the heterocyclyl of the -C(=O)-(heterocyclyl) or the heterocyclyl is a 3- to 7-membered ring containing 1, 2 or 3 heteroatoms selected from N, O and S;

[0141] R 2 Selected from -H and C 1 -C 4 Alkyl, or, in compounds of formula II, is absent;

[0142] R 3 Selected from unsubstituted C 1 -C 10 Alkyl, 1, 2 or 3 R 1a Substituent-substituted C 1 -C 10 Alkyl, formula -(CR 3b R 3c )-Q, a group of formula -NH-(CR 3b R 3c )-Q group, the formula -(CR 3b R 3c )-C(=O)-Q, a group of the formula -(CR 3d R 3e )-(CR 3f R 3g )-Q group, the formula -(CR 3b =CR 3c )-Q and -(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 are substituted, wherein 1, 2 or 3 ring members are heteroatoms selected from N, O and S;

[0143] R 1a is independently selected from -F, -Cl, -CN, -OH, -O-(C 1 -C 6 Alkyl), -O-(C 1 -C 6 Haloalkyl), -O-(C 1 -C 6 Perhaloalkyl), -O-(C 1 -C 6 Alkyl)-OH, -O-(C 1 -C 6 alkyl)-O-(C 1 -C 6Alkyl), C 2 -C 6 Alkenyl, C 2 -C 6 Alkynyl, -NH 2 、-NH(C 1 -C 6 Alkyl) and -N(C 1 -C 6 alkyl) 2 ;

[0144] R 3b and R 3c Independently selected from -H, -F, -Cl, -CN, -C 1 -C 6 Alkyl, -C 1 -C 6 Haloalkyl, -C 1 -C 6 Perhaloalkyl, -OH, -O-(C 1 -C 6 Alkyl), -O-(C 1 -C 6 Haloalkyl), -O-(C 1 -C 6 Perhaloalkyl), -O-(C 1 -C 6 Alkyl)-OH, -O-(C 1 -C 6 alkyl)-O-(C 1 -C 6 Alkyl), -NH 2 、-NH(C 1 -C 6 Alkyl) and -N(C 1 -C 6 alkyl) 2 ;

[0145] R 3d and R 3e Independently selected from -H, -F, -Cl, -CN, -C 1 -C 6 Alkyl, -C 1 -C 6 Haloalkyl, -C 1 -C 6 Perhaloalkyl, -OH, -O-(C 1 -C 6 Alkyl), -O-(C 1 -C 6 Haloalkyl), -O-(C 1 -C 6 Perhaloalkyl), -O-(C 1-C 6 Alkyl)-OH, -O-(C 1 -C 6 alkyl)-O-(C 1 -C 6 Alkyl), -NH 2 、-NH(C 1 -C 6 Alkyl) and -N(C 1 -C 6 alkyl) 2 ;

[0146] R 3f and R 3g Independently selected from -H, -F, -Cl, -CN, -C 1 -C 6 Alkyl, -C 1 -C 6 Haloalkyl, -C 1 -C 6 Perhaloalkyl, -OH, -O-(C 1 -C 6 Alkyl), -O-(C 1 -C 6 Haloalkyl), -O-(C 1 -C 6 Perhaloalkyl), -O-(C 1 -C 6 Alkyl)-OH, -O-(C 1 -C 6 alkyl)-O-(C 1 -C 6 Alkyl), -NH 2 、-NH(C 1 -C 6 Alkyl) and -N(C 1 -C 6 alkyl) 2 ;

[0147] R 3h is independently selected from -F, -Cl, -CN, -C 1 -C 6 Alkyl, -C 1 -C 6 Haloalkyl, -C 1 -C 6 Perhaloalkyl, -OH, -O-(C 1 -C 6 Alkyl), -O-(C 1 -C 6 Haloalkyl), -O-(C 1 -C 6Perhaloalkyl), -O-(C 1 -C 6 Alkyl)-OH, -O-(C 1 -C 6 alkyl)-O-(C 1 -C 6 Alkyl), -NH 2 、-NH(C 1 -C 6 Alkyl), -N(C 1 -C 6 alkyl) 2 and oxo;

[0148] Q is a monocyclic or bicyclic C 6 -C 10 Aryl, monocyclic or bicyclic heteroaryl with 5 to 10 ring members containing 1, 2 or 3 heteroatoms selected from N, O or S, C 3 -C 8 Cycloalkyl, or a 3- to 7-membered heterocyclic group containing 1, 2 or 3 heteroatoms selected from N, O or S, wherein the C 6 -C 10 The aryl, heteroaryl, cycloalkyl and heterocyclyl groups are unsubstituted or substituted with 1, 2, 3 or 4 R Q Substituent substitution;

[0149] R Q is independently selected from -F, -Cl, -Br, -I, -CN, -C 1 -C 6 Alkyl, -C 1 -C 6 Haloalkyl, -C 1 -C 6 Perhaloalkyl, -C 2 -C 6 Alkenyl, -C 2 -C 6 Alkynyl, -OH, -O-(C 1 -C 6 Alkyl), -O-(C 1 -C 6 Haloalkyl), -O-(C 1 -C 6 Perhaloalkyl), -NH 2 、-NH(C 1 -C 6 Alkyl), -N(C 1 -C 6 alkyl) 2 、-C(=O)-(C 1 -C 6 alkyl), -C(=O)OH, -C(=O)-O-(C1 -C 6 alkyl), -C(=O)NH 2 、-C(=O)NH(C 1 -C 6 alkyl), -C(=O)N(C 1 -C 6 alkyl) 2 、-S(=O) 2 -(C 1 -C 6 alkyl), phenyl and heteroaryl, and the Q heterocyclic group may be substituted with 1 oxo R Q Substituent substitution;

[0150] R 4 Selected from monocyclic or bicyclic C 6 -C 10 aryl, monocyclic or bicyclic heteroaryl having 5 to 10 ring members containing 1, 2 or 3 heteroatoms independently selected from N, O and S, and monocyclic or bicyclic heterocyclyl having 5 to 10 ring members and containing 1, 2, 3 or 4 heteroatoms independently selected from N, O and S, wherein the C 6 -C 10 The aryl group, the heteroaryl group or the heterocyclic group is unsubstituted or substituted by 1, 2 or 3 R 4a Substituent substitution;

[0151] R 4a is independently selected from -F, -Cl, -Br, -I, -CN, -C 1 -C 6 Alkyl, -C 1 -C 6 Haloalkyl, -C 1 -C 6 Perhaloalkyl, -OH, -O-(C 1 -C 6 Alkyl), -O-(C 1 -C 6 Haloalkyl), -O-(C 1 -C 6 Perhaloalkyl), -NH 2 、-NH(C 1 -C 6 Alkyl), -N(C 1 -C 6 alkyl) 2 、-C(=O)-(C 1 -C 6 alkyl), -C(=O)OH, -C(=O)-O-(C 1 -C 6 alkyl), -C(=O)NH 2、-C(=O)NH(C 1 -C 6 alkyl) and -C(=O)N(C 1 -C 6 alkyl) 2 , and the heterocyclic group R 4 may be further substituted with 1 oxo substituent; and

[0152] Further wherein:

[0153] If R 4 is an unsubstituted or substituted benzene ring and R 3 It is the formula -(CR 3b =CR 3c )-Q, then at least one of the following is true:

[0154] a) R 4 By at least one -O-(C 1 -C 6 Alkyl) group substitution;

[0155] b) Q is not oxadiazole;

[0156] c) R 3b Not -H;

[0157] d) R 3c Not -H;

[0158] e)R 1 is not 2-pyridyl; or

[0159] f)R 4 By two or more -O-(C 1 -C 6 alkyl) group.

[0160] In some embodiments, the apelin receptor modulator is a compound of formula (I) or (II):

[0161]

[0162] 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:

[0163] R 1 is unsubstituted pyridyl, pyridone or pyridine N-oxide, or is substituted by 1, 2, 3 or 4 R 1a a pyridyl, pyridonyl or pyridine N-oxide substituted with a substituent;

[0164] R 1ais independently selected from -F, -Cl, -Br, -I, -CN, -C 1 -C 6 Alkyl, -C 1 -C 6 Haloalkyl, -C 1 -C 6 Perhaloalkyl, -OH, -O-(C 1 -C 6 Alkyl), -O-(C 1 -C 6 Haloalkyl), -O-(C 1 -C 6 Perhaloalkyl), -C 2 -C 6 Alkenyl, -O-(C 1 -C 6 Alkyl)-OH, -O-(C 1 -C 6 alkyl)-O-(C 1 -C 6 Alkyl), -O-(C 1 -C 6 Haloalkyl)-OH, -O-(C 1 -C 6 (C 1 -C 6 Alkyl), -O-(C 1 -C 6 Perhaloalkyl)-OH, -O-(C 1 -C 6 Perhaloalkyl)-O-(C 1 -C 6 Alkyl), -NH 2 、-NH(C 1 -C 6 Alkyl), -N(C 1 -C 6 alkyl) 2 、-C(=O)-(C 1 -C 6 alkyl), -C(=O)OH, -C(=O)-O-(C 1 -C 6 alkyl), -C(=O)NH 2 、-C(=O)NH(C 1 -C 6 alkyl), -C(=O)N(C 1 -C 6 alkyl) 2, phenyl, -C(=O)-(heterocyclyl) or heterocyclyl, wherein the heterocyclyl of the -C(=O)-(heterocyclyl) or the heterocyclyl is a 3- to 7-membered ring containing 1, 2 or 3 heteroatoms selected from N, O or S;

[0165] R 2 Select from -H or C 1 -C 4 Alkyl, or, in compounds of formula II, is absent;

[0166] R 3 It is the formula -(CR 3d R 3e )-(CR 3f R 3g )-Q group;

[0167] R 3d and R 3e Independently selected from -H, -F, -Cl, -CN, -C 1 -C 6 Alkyl, -C 1 -C 6 Haloalkyl, -C 1 -C 6 Perhaloalkyl, -OH, -O-(C 1 -C 6 Alkyl), -O-(C 1 -C 6 Haloalkyl), -O-(C 1 -C 6 Perhaloalkyl), -O-(C 1 -C 6 Alkyl)-OH, -O-(C 1 -C 6 alkyl)-O-(C 1 -C 6 Alkyl), -NH 2 、-NH(C 1 -C 6 Alkyl) or -N(C 1 -C 6 alkyl) 2 ;

[0168] R 3f and R 3g Independently selected from -H, -F, -Cl, -CN, -C 1 -C 6 Alkyl, -C 1 -C 6 Haloalkyl, -C 1 -C 6 Perhaloalkyl, -OH, -O-(C 1 -C6 Alkyl), -O-(C 1 -C 6 Haloalkyl), -O-(C 1 -C 6 Perhaloalkyl), -O-(C 1 -C 6 Alkyl)-OH, -O-(C 1 -C 6 alkyl)-O-(C 1 -C 6 Alkyl), -NH 2 、-NH(C 1 -C 6 Alkyl) or -N(C 1 -C 6 alkyl) 2 ;

[0169] Q is a monocyclic or bicyclic C 6 -C 10 Aryl, monocyclic or bicyclic heteroaryl with 5 to 10 ring members containing 1, 2 or 3 heteroatoms selected from N, O or S, C 3 -C 8 Cycloalkyl, or a 3- to 7-membered heterocyclic group containing 1, 2 or 3 heteroatoms selected from N, O or S, wherein the C 6 -C 10 The aryl, heteroaryl, cycloalkyl and heterocyclyl groups are unsubstituted or substituted with 1, 2, 3 or 4 R Q Substituent substitution;

[0170] R Q is independently selected from -F, -Cl, -Br, -I, -CN, -C 1 -C 6 Alkyl, -C 1 -C 6 Haloalkyl, -C 1 -C 6 Perhaloalkyl, -C 2 -C 6 Alkenyl, -C 2 -C 6 Alkynyl, -OH, -O-(C 1 -C 6 Alkyl), -O-(C 1 -C 6 Haloalkyl), -O-(C 1 -C 6 Perhaloalkyl), -NH 2 、-NH(C 1 -C 6 Alkyl), -N(C 1 -C6 alkyl) 2 、-C(=O)-(C 1 -C 6 alkyl), -C(=O)OH, -C(=O)-O-(C 1 -C 6 alkyl), -C(=O)NH 2 、-C(=O)NH(C 1 -C 6 alkyl), -C(=O)N(C 1 -C 6 alkyl) 2 、-S(=O) 2 -(C 1 -C 6 alkyl), phenyl or heteroaryl, and the Q heterocyclyl may be substituted with 1 oxo substituent;

[0171] R 4 Selected from monocyclic or bicyclic C 6 -C 10 aryl, monocyclic or bicyclic heteroaryl having 5 to 10 ring members containing 1, 2 or 3 heteroatoms independently selected from N, O or S, or monocyclic or bicyclic heterocyclyl having 5 to 10 ring members and containing 1, 2, 3 or 4 heteroatoms independently selected from N, O or S, wherein the C 6 -C 10 The aryl group, the heteroaryl group or the heterocyclic group is unsubstituted or substituted by 1, 2 or 3 R 4a Substituents are substituted; and

[0172] R 4a is independently selected from -F, -Cl, -Br, -I, -CN, -C 1 -C 6 Alkyl, -C 1 -C 6 Haloalkyl, -C 1 -C 6 Perhaloalkyl, -OH, -O-(C 1 -C 6 Alkyl), -O-(C 1 -C 6 Haloalkyl), -O-(C 1 -C 6 Perhaloalkyl), -NH 2 、-NH(C 1 -C 6 Alkyl), -N(C 1 -C 6 alkyl) 2 、-C(=O)-(C 1 -C 6alkyl), -C(=O)OH, -C(=O)-O-(C 1 -C 6 alkyl), -C(=O)NH 2 、-C(=O)NH(C 1 -C 6 alkyl) or -C(=O)N(C 1 -C 6 alkyl) 2 , and the heterocyclic group R 4 The group may be further substituted with 1 oxo substituent.

[0173] As noted above, the apelin receptor agonist compounds disclosed herein may exist in multiple tautomeric forms. 2 This is particularly true for compounds of formula I where is H. These forms are shown below as tautomer A and tautomer B:

[0174]

[0175] The apelin receptor agonist compounds disclosed herein are structurally described and generally designated as compounds in the "tautomer A" form. However, compounds are specifically contemplated and known to exist in the "tautomer B" form, and therefore compounds in the "tautomer B" form are expressly considered to be part of the present disclosure. For this reason, the claims are directed to compounds of Formula I and Formula II. Depending on the compound, some compounds may exist predominantly in one form and not 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.

[0176] In some embodiments of formula (I) and (II), R 1 is unsubstituted pyridyl or substituted with 1 or 2 R 1a The substituent is substituted pyridinyl.

[0177] In some embodiments of formula (I) and (II), R 1a is independently selected from -CH 3 、-CH 2 CH 3 、-F、-Cl、-Br、-CN、-CF 3 、-CH=CH 2 、-C(=O)NH 2 、-C(=O)NH(CH 3 )、-C(=O)N(CH 3 ) 2 、-C(=O)NH(CH 2CH 3 ), -OH, -OCH 3 、-OCHF 2 、-OCH 2 CH 3 、-OCH 2 CF 3 、-OCH 2 CH 2 OH, -OCH 2 C(CH 3 ) 2 OH, -OCH 2 C(CF 3 ) 2 OH, -OCH 2 CH 2 OCH 3 、-NH 2 、-NHCH 3 、-N(CH 3 ) 2 , phenyl and a group of the formula

[0178]

[0179] When drawn across a bond, the symbol Indicates the point of attachment to the rest of the molecule.

[0180] In some embodiments of formula (I) and (II), R 1 Selected from

[0181]

[0182]

[0183] When drawn across a bond, the symbol Indicates the point of attachment to the rest of the molecule.

[0184] In some embodiments of formula (I) and (II), R 1 Selected from

[0185]

[0186]

[0187] When drawn across a bond, the symbol Indicates the point of attachment to the rest of the molecule.

[0188] In some embodiments of formula (I) and (II), R 2 Yes -H.

[0189] In some embodiments of formula (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 In some embodiments of formula (I) and (II), R 4 Is 1 or 2 R 4a In some embodiments of formula (I) and (II), 1 or 2 R 4a The substituent is -O-(C 1 -C 2 alkyl) group.

[0190] In some embodiments of formula (I) and (II), R 4a is independently selected from -CH 3 、-F、-Cl、-Br、-CN、-CF 3 、-OCH 3 、-OCHF 2 、-OCH 2 CH 3 、-C(=O)OCH 3 、-C(=O)CH 3 or -N(CH 3 ) 2 .

[0191] In some embodiments of formula (I) and (II), R 4 Selected from:

[0192]

[0193]

[0194]

[0195] When drawn across a bond, the symbol Indicates the point of attachment to the rest of the molecule.

[0196] In some embodiments of formula (I) and (II), R 3 Select from the formula -(CR 3b R 3c )-Q, a group of formula -NH-(CR 3b R 3c )-Q group, the formula -(CR 3b R 3c )-C(=O)-Q, a group of the formula -(CR 3d R 3e )-(CR3f R 3g )-Q group, the formula -(CR 3b =CR 3c )-Q or a group of the 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 are substituted, 1, 2 or 3 of the ring members are heteroatoms selected from N, O or S.

[0197] In some embodiments of formula (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 may be unsubstituted or substituted with 1, 2 or 3 R Q Substituent substitution.

[0198] In some embodiments of formula (I) and (II), Q is a monocyclic heteroaryl group having 5 or 6 ring members containing 1 or 2 heteroatoms selected from N, O or S, and Q is unsubstituted or substituted with 1 or 2 R Q Substituent substitution.

[0199] In some embodiments of formula (I) and (II), Q is selected from

[0200]

[0201]

[0202]

[0203] When drawn across a bond, the symbol Indicates the point of attachment to the rest of the molecule.

[0204] In some embodiments of formula (I) and (II), R 3 is a radical of the formula -(heterocyclyl)-Q, wherein the heterocyclyl radical of -(heterocyclyl)-Q has 5 or 7 ring members and is unsubstituted or substituted with 1, 2 or 3 R 3h Substituents are substituted, 1, 2 or 3 of the ring members are heteroatoms selected from N, O or S.

[0205] In some embodiments of formula (I) and (II), R 3 It is the formula -(CR 3d R 3e )-(CR 3f R 3g )-Q group.

[0206] In some embodiments of formula (I) and (II), R 3 With the following formula

[0207]

[0208]

[0209] When drawn across a bond, the symbol Indicates the point of attachment to the rest of the molecule.

[0210] In some embodiments of formula (I) and (II), R 3 With the following formula

[0211]

[0212] When drawn across a bond, the symbol Indicates the point of attachment to the rest of the molecule.

[0213] In certain embodiments of formula (I) and (II), the apelin receptor agonist is

[0214] (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;

[0215] (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;

[0216] (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;

[0217] (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;

[0218] (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;

[0219] (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;

[0220] (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;

[0221] (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;

[0222] (2S,3R)-N-(4-(2,6-dimethoxyphenyl)-5-(3-pyridyl)-4H-1,2,4-triazol-3-yl)-3-(5-methyl-2-pyrimidinyl)-2-butanesulfonamide;

[0223] (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;

[0224] (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;

[0225] (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;

[0226] (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;

[0227] (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;

[0228] (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;

[0229] (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;

[0230] (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;

[0231] (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;

[0232] (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;

[0233] (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;

[0234] (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;

[0235] (2S,3R)-N-(4-(2,6-dimethoxyphenyl)-5-(3-pyridyl)-4H-1,2,4-triazol-3-yl)-3-(5-methyl-2-pyrazinyl)-2-butanesulfonamide;

[0236] (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;

[0237] (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;

[0238] (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

[0239] (1S,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.

[0240] In specific embodiments of formula (I) and (II), the apelin 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.

[0241] In specific embodiments of formula (I) and (II), the apelin 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.

[0242] In specific embodiments of formula (I) and (II), the apelin 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.

[0243] In specific embodiments of formula (I) and (II), the apelin 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.

[0244] In specific embodiments of formula (I) and (II), the apelin 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.

[0245] In specific embodiments of formula (I) and (II), the apelin 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.

[0246] In specific embodiments of formula (I) and (II), the apelin receptor agonist is (1R, 2S)-N-(4-(2,6-dimethoxyphenyl)-5-(3-pyridinyl)-4H-1,2,4-triazol-3-yl)-1-methoxy-1-(5-methyl-2-pyrimidinyl)-2-propanesulfonamide or a pharmaceutically acceptable salt thereof.

[0247] In specific embodiments of formula (I) and (II), the apelin receptor agonist is (2S,3R)-N-(4-(2,6-dimethoxyphenyl)-5-(3-pyridinyl)-4H-1,2,4-triazol-3-yl)-3-(5-methyl-2-pyrimidinyl)-2-butanesulfonamide or a pharmaceutically acceptable salt thereof.

[0248] In specific embodiments of formula (I) and (II), the apelin receptor agonist is (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 or a pharmaceutically acceptable salt thereof.

[0249] In specific embodiments of formula (I) and (II), the apelin 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.

[0250] In specific embodiments of formula (I) and (II), the apelin 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.

[0251] In specific embodiments of formula (I) and (II), the apelin 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.

[0252] In specific embodiments of formula (I) and (II), the apelin 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.

[0253] In specific embodiments of formula (I) and (II), the apelin 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.

[0254] In specific embodiments of formula (I) and (II), the apelin 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-butanesulfonamide or a pharmaceutically acceptable salt thereof.

[0255] In specific embodiments of formula (I) and (II), the apelin 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.

[0256] In specific embodiments of formula (I) and (II), the apelin 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.

[0257] In specific embodiments of formula (I) and (II), the apelin 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.

[0258] In specific embodiments of formula (I) and (II), the apelin 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.

[0259] In specific embodiments of formula (I) and (II), the apelin 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.

[0260] In specific embodiments of formula (I) and (II), the apelin 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.

[0261] In specific embodiments of formula (I) and (II), the apelin 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.

[0262] In specific embodiments of formula (I) and (II), the apelin 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.

[0263] In specific embodiments of formula (I) and (II), the apelin 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.

[0264] In specific embodiments of formula (I) and (II), the apelin receptor agonist is (1S, 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.

[0265] In specific embodiments of formula (I) and (II), the apelin 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, a tautomer thereof, a pharmaceutically acceptable salt of the tautomer, a stereoisomer of any of the foregoing, or a mixture thereof.

[0266] In specific embodiments of formula (I) and (II), the apelin 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, a tautomer thereof, a pharmaceutically acceptable salt of the tautomer, a stereoisomer of any of the foregoing, or a mixture thereof.

[0267] In specific embodiments of formula (I) and (II), the apelin 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, a tautomer thereof, a pharmaceutically acceptable salt of the tautomer, a stereoisomer of any of the foregoing, or a mixture thereof.

[0268] In specific embodiments of formula (I) and (II), the apelin 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, a tautomer thereof, a pharmaceutically acceptable salt of the tautomer, a stereoisomer of any of the foregoing, or a mixture thereof.

[0269] In specific embodiments of formula (I) and (II), the apelin 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, a tautomer thereof, a pharmaceutically acceptable salt of the tautomer, a stereoisomer of any of the foregoing, or a mixture thereof.

[0270] In specific embodiments of formula (I) and (II), the apelin 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, a tautomer thereof, a pharmaceutically acceptable salt of the tautomer, a stereoisomer of any of the foregoing, or a mixture thereof.

[0271] In specific embodiments of formula (I) and (II), the apelin receptor agonist is (1R, 2S)-N-(4-(2,6-dimethoxyphenyl)-5-(3-pyridinyl)-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.

[0272] In specific embodiments of formula (I) and (II), the apelin receptor agonist is (2S, 3R)-N-(4-(2,6-dimethoxyphenyl)-5-(3-pyridinyl)-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.

[0273] In specific embodiments of formula (I) and (II), the apelin receptor agonist is (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 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.

[0274] In specific embodiments of formula (I) and (II), the apelin 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.

[0275] In specific embodiments of formula (I) and (II), the apelin 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.

[0276] In specific embodiments of formula (I) and (II), the apelin 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.

[0277] In specific embodiments of formula (I) and (II), the apelin 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.

[0278] In specific embodiments of formula (I) and (II), the apelin 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, or a mixture thereof.

[0279] In specific embodiments of formula (I) and (II), the apelin 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-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.

[0280] In specific embodiments of formula (I) and (II), the apelin 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.

[0281] In specific embodiments of formula (I) and (II), the apelin 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.

[0282] In specific embodiments of formula (I) and (II), the apelin 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.

[0283] In specific embodiments of formula (I) and (II), the apelin 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.

[0284] In specific embodiments of formula (I) and (II), the apelin 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, a tautomer thereof, a pharmaceutically acceptable salt of the tautomer, a stereoisomer of any of the foregoing, or a mixture thereof.

[0285] In specific embodiments of formula (I) and (II), the apelin 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, a tautomer thereof, a pharmaceutically acceptable salt of the tautomer, a stereoisomer of any of the foregoing, or a mixture thereof.

[0286] In specific embodiments of formula (I) and (II), the apelin 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, a tautomer thereof, a pharmaceutically acceptable salt of the tautomer, a stereoisomer of any of the foregoing, or a mixture thereof.

[0287] In specific embodiments of formula (I) and (II), the apelin 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, a tautomer thereof, a pharmaceutically acceptable salt of the tautomer, a stereoisomer of any of the foregoing, or a mixture thereof.

[0288] In specific embodiments of formula (I) and (II), the apelin 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, a tautomer thereof, a pharmaceutically acceptable salt of the tautomer, a stereoisomer of any of the foregoing, or a mixture thereof.

[0289] In specific embodiments of formula (I) and (II), the apelin receptor agonist is (1S, 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.

[0290] In specific embodiments of formula (I) and (II), the apelin receptor agonist is (1R, 2S)-N-(4-(2,6-difluorophenyl)-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, a tautomer thereof, a pharmaceutically acceptable salt of the tautomer, a stereoisomer of any of the foregoing, or a mixture thereof.

[0291] In specific embodiments of formula (I) and (II), the apelin receptor agonist is (1R, 2S)-N-(4-(4,6-dimethoxy-5-pyrimidinyl)-5-(2-pyridinyl)-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.

[0292] In specific embodiments of formula (I) and (II), the apelin receptor agonist is N-(4-(2,6-dimethoxyphenyl)-5-(5-methyl-3-pyridinyl)-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.

[0293] In specific embodiments of formula (I) and (II), the apelin receptor agonist is (1S, 2S)-N-(4-(2,6-dimethoxyphenyl)-5-(5-methyl-3-pyridinyl)-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.

[0294] In specific embodiments of formula (I) and (II), the apelin receptor agonist is N-(4-(2,6-dimethoxyphenyl)-5-(5-methyl-3-pyridinyl)-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.

[0295] In certain embodiments of formula (I) and (II), the apelin receptor agonist is compound 2:

[0296]

[0297] or a pharmaceutically acceptable salt thereof.

[0298] In specific embodiments of formula (I) and (II), the apelin 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-pyrimidinyl)-2-butanesulfonamide (BGE-105) or a pharmaceutically acceptable salt thereof.

[0299] In certain embodiments of formula (I) and (II), the apelin receptor agonist is compound 3

[0300]

[0301] (BGE-105) or a pharmaceutically acceptable salt thereof.

[0302] BGE-105 has the above and Figure 1 Structure shown in . BGE-105 is known to activate apelin receptors and induce 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 its safety, tolerability, and pharmacokinetics in healthy subjects and subjects with impaired renal function (NCT03318809) or heart failure (NCT03276728).

[0303] 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 entirety) describe apelin receptor agonists of Formula (I) or Formula (II) and methods of synthesizing such triazole agonists of apelin receptors (including BGE-105). See, e.g., Example 263.0 of U.S. Patent No. 9,573,936.

[0304] If any variable appears more than once in a chemical formula, its definition at each occurrence is independent of its definition at each other occurrence. If the chemical structure and chemical name conflict, the chemical structure determines the identity of the compound. The compounds disclosed herein may contain one or more chiral centers and / or double bonds and may therefore exist as stereoisomers (such as double bond isomers (i.e., geometric isomers), enantiomers, or diastereomers). Therefore, any chemical structure with relative configuration described in whole or in part within the scope of the specification covers all possible enantiomers and stereoisomers of the compounds shown, including stereoisomerically pure forms (e.g., geometrically pure, enantiomerically pure, or diastereomerically pure forms) as well as enantiomers and mixtures of stereoisomers. Enantiomers and mixtures of stereoisomers may be split into component enantiomers or stereoisomers using separation techniques or chiral synthesis techniques well known to technicians.

[0305] 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 intended to be encompassed within the scope of the present invention. In addition, atropisomers and mixtures thereof, such as those resulting from hindered rotation around two aromatic or heteroaromatic rings bonded to each other, are intended to be encompassed within the scope of the present invention. For example, when R 4 When the phenyl group is substituted with two groups bonded to C atoms adjacent to the point of attachment of the triazole N atom, the rotation of the phenyl group may be restricted. In some cases, the barrier to rotation is high enough that the different atropisomers can be separated and isolated.

[0306] Unless otherwise indicated, the term "stereoisomer" or "stereoisomerically pure" means one stereoisomer of a compound that is substantially free of other stereoisomers of the compound. For example, a stereoisomerically pure compound having one chiral center will be substantially free of the opposite enantiomer of the compound. A stereoisomerically pure compound having two chiral centers will be substantially free of other diastereomers of the 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 the 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 the 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 the 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 the compound. If the stereochemistry of a structure or a portion of a structure is not indicated, for example, by bold or dashed lines, the structure or portion of the structure should be interpreted as encompassing all stereoisomers thereof. Bonds drawn with wavy lines indicate that both stereoisomers are encompassed.

[0307] The various compounds disclosed herein contain one or more chiral centers and may exist as a racemic mixture of enantiomers, a mixture of diastereomers, or an enantiomerically pure compound or an optically pure compound. The present invention encompasses the use of stereoisomerically pure forms of such compounds and the use of mixtures of these forms. For example, mixtures of equal or unequal enantiomers comprising a particular compound of the present invention may be used in the methods and compositions of the present invention. These isomers may be asymmetrically synthesized or resolved using standard techniques (such as chiral columns or chiral resolving agents).

[0308] The compounds disclosed herein 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, crystalline forms (including polymorphs and inclusion compounds), chelates, non-covalent complexes, prodrugs, and mixtures thereof. In certain embodiments, the compounds described herein are in the form of pharmaceutically acceptable salts. The term "compound" encompasses not only the compound itself, but also its pharmaceutically acceptable salts, its solvates, its chelates, its non-covalent complexes, its prodrugs, and mixtures of any of the foregoing. In some embodiments, the term "compound" encompasses the compound itself, its pharmaceutically acceptable salts, tautomers of the compound, pharmaceutically acceptable salts of tautomers, and ester prodrugs (such as (C 1 -C 4In other embodiments, the term "compound" encompasses the compound itself, a pharmaceutically acceptable salt thereof, a tautomer of the compound, and a pharmaceutically acceptable salt of a tautomer.

[0309] The term "solvate" refers to a compound formed by the interaction of a solvent and a compound. Suitable solvates are pharmaceutically acceptable solvates, such as hydrates, including monohydrates and hemihydrates.

[0310] The compounds of the present disclosure may also contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute such compounds. For example, radioactive isotopes (e.g., tritium ( 3 H), iodine-125(1 25 I) or carbon-14 (1 4 C)) The compound is radiolabeled. Radiolabeled compounds can be used as therapeutic or prophylactic agents, research reagents (e.g., assay reagents) and diagnostic reagents (e.g., in vivo imaging agents). All isotopic variants of the compounds of the present invention (whether radioactive or not) are intended to be included within the scope of the present invention. For example, if a variable is called or shown as H, this means that this variable can also be deuterium (D) or tritium (T).

[0311] 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, inorganic acid salts such as hydrochlorides, hydrobromides, hydroiodides, phosphates, sulfates, and nitrates; sulfonates such as methanesulfonates, ethanesulfonates, benzenesulfonates, p-toluenesulfonates, and trifluoromethanesulfonates; organic acid salts such as oxalates, tartrates, citrates, maleates, succinates, acetates, trifluoroacetates, benzoates, mandelates, ascorbates, lactates, gluconates, and malates; amino acid salts such as glycinates, lysine salts, arginine salts, ornithine salts, glutamates, and aspartates; inorganic salts such as lithium salts, sodium salts, potassium salts, calcium salts, and magnesium salts; and salts of organic bases such as ammonium salts, triethylamine salts, diisopropylamine salts, and cyclohexylamine salts. As used herein, the term "salt" encompasses hydrate salts.

[0312] Other examples of pharmaceutically acceptable salts include complexes of anions of the disclosed compounds with suitable cations. For therapeutic uses, salts of the disclosed compounds 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.

[0313] The compounds that are basic in nature and that are included in the compositions and methods of the present invention are capable of forming a wide variety of salts with various inorganic and organic acids. Acids that can be used to prepare 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 malate, oxalate, chloride, bromide, iodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, isonicotinate, acetate, lactate, salicylate, citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucuronate, sucrose salt, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate (i.e., 1,1′-methylene-bis-(2-hydroxy-3-naphthoate)).

[0314] The compounds that are acidic in nature and included in the compositions and methods of the present invention are capable of forming base salts with various pharmacologically acceptable cations. Examples of such salts include alkali metal salts or alkaline earth metal salts, and in particular calcium salts, magnesium salts, sodium salts, lithium salts, zinc salts, potassium salts and iron salts.

[0315] Furthermore, if the compound of the present disclosure or a salt thereof forms a hydrate or a solvate, these are also included in the scope of the compound of the present disclosure or a salt thereof.

[0316] Compounds comprising basic or acidic moieties included in the compositions and methods of the present invention may also form pharmaceutically acceptable salts with various amino acids. The compounds disclosed herein may contain both acidic and basic groups; for example, an amino group and a carboxylic acid group. In this case, the compound may exist as an acid addition salt, a zwitterion, or a base salt.

[0317] 5.12 Pharmaceutical Compositions

[0318] The apelin receptor agonist compounds used in the methods described herein can be formulated in any suitable pharmaceutical composition form for administration by any suitable route of administration. The pharmaceutical composition may include a compound according to any embodiment described herein or a pharmaceutically acceptable salt thereof, a tautomer thereof, a pharmaceutically acceptable salt of a tautomer, a stereoisomer of any of the foregoing, or a mixture thereof and at least one pharmaceutically acceptable excipient, carrier or diluent. In some such embodiments, the compound according to any embodiment or a pharmaceutically acceptable salt thereof, a tautomer thereof, a pharmaceutically acceptable salt of a tautomer, a stereoisomer of any of the foregoing, or a mixture thereof is present in an amount effective for treating muscle disorders (e.g., as described herein) and for activating APJ receptors.

[0319] Suitable routes of administration include, but are not limited to, oral, topical, and intravenous routes of administration. Suitable routes of administration also include intrathecal administration, such as via injection into the subject's spinal canal or into the subarachnoid space. Suitable routes also include pulmonary administration, including inhalation through the mouth. The most suitable route may depend on the recipient's condition and disorder. The formulation can be conveniently presented in unit dosage form and can be prepared by any method known in the pharmaceutical field.

[0320] 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 preparations it is a tablet, wherein the effective amount range of the active ingredient is 5mg to 60mg, 6mg to 58mg, 10mg to 40mg, 15mg to 30mg, 16mg to 25mg or 17mg to 20mg. In some such compositions, the amount of the active ingredient is 17mg. In some embodiments, the pharmaceutical composition is formulated for once daily PO administration, wherein the effective amount range of the active ingredient is 5mg to 300mg, 6mg to 58mg, 10mg to 40mg, 15mg to 30mg, 16mg to 25mg or 17mg to 20mg. In some such compositions, the amount of the active ingredient is 17mg.

[0321] All methods include the step of bringing the apelin agonist or salt thereof into association with the carrier which constitutes one or more excipients. In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product into the desired formulation.

[0322] In certain embodiments, the route of administration used in the methods described herein is parenteral administration. In certain embodiments, the route of administration used in the methods described herein is intravenous administration (e.g., intravenous infusion). In certain embodiments, the route of administration used in the methods described herein is oral administration. In certain embodiments, the route of administration used in the methods described herein is continuous intravenous infusion.

[0323] Formulations of the methods of the invention suitable for oral administration may be presented 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.

[0324] Preparations for parenteral administration include aqueous and non-aqueous sterile injection solutions, which may contain antioxidants, buffers, antibacterial agents, and solutes that make the preparations isotonic with the blood of the intended recipient. Preparations for parenteral administration also include aqueous and non-aqueous sterile suspensions, which may include suspending agents and thickening agents. Preparations can be presented in multi-dose containers (e.g., sealed ampoules and vials) of unit doses, and can be stored under freeze drying (lyophilization) conditions, requiring only the addition of sterile liquid carriers (e.g., saline, phosphate buffered saline (PBS) or the like) immediately before use. Ready-to-use injection solutions and suspensions can be prepared by previously described sterile powders, granules, and tablets.

[0325] The pharmaceutical composition may include one or more pharmaceutical excipients. Any suitable pharmaceutical excipient may be used, and one of ordinary skill in the art will be able to select a suitable pharmaceutical excipient. Pharmaceutical excipients include, for example, those described in Handbook of Pharmaceutical Excipients, 8th revised edition (2017).

[0326] 5.13 Dosage Regimen

[0327] In various embodiments, an apelin receptor agonist (e.g., as described herein) is administered at a dose sufficient to reduce BBB permeability and / or treat a disorder associated with increased BBB permeability (e.g., as described herein), and / or treat a neurodegenerative disease (e.g., increased neurotoxicity as described herein).

[0328] In various embodiments, an apelin receptor agonist (e.g., as described herein) is administered to an elderly subject in need thereof. In some embodiments, the elderly subject is a human and is at least 40 years old, at least 50 years old, at least 55 years old, at least 60 years old, at least 65 years old, at least 70 years old, at least 75 years old, or at least 80 years old.

[0329] In various embodiments, the dosage 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 dosage is 25 mg / kg to 1000 mg / kg per day.

[0330] In some embodiments, the apelin receptor agonist is administered at a dose that is independent of the patient's body weight or surface area (flat dose).

[0331] 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, or at least 100 mg. In various embodiments, the dosage is 25-2000 mg. In some embodiments, the dosage is at least 200 mg.

[0332] The apelin receptor agonist may be administered in a single dose or in multiple doses.

[0333] In some embodiments, the dose is administered daily.

[0334] In some embodiments, a dose is administered in multiple equally or unequally divided sub-doses.

[0335] In certain embodiments, the dosage is continuously administered (e.g., IV infusion) for a period of time. In certain embodiments, the dosage is administered 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) with an intravenous infusion dosage. In certain embodiments, after the dosage, the dosage is administered 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) with an intravenous infusion maintenance dosage. In certain embodiments, the dose is administered as an intravenous infusion 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, 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) following the dose and a 24 or 48 hour washout period. In certain embodiments, the dose is administered as an 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) followed by a second 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) after a first dose and a 24-hour or 48-hour washout period.

[0336] In some embodiments, the apelin receptor agonist is administered orally, intravenously, intranasally, or intramuscularly. In some embodiments, the apelin receptor agonist is administered orally. In some embodiments, the apelin receptor agonist is administered intrathecally. In some embodiments, the apelin receptor agonist is administered intravenously. In some embodiments, the subject is a human patient on a ventilator.

[0337] In some embodiments, the apelin receptor agonist is administered once a month, twice a month, three times a month, once every other week (qow), once a week (qw), twice a week (biw), three times a week (tiw), four times a week, five times a week, six times a week, once every other day (qod), once a day (qd), twice a day (qid), or three times a day (tid) for a period of time ranging from 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 1 year, about 1 year to about 2 years, or about 2 years to about 4 years, or longer. In some embodiments, the apelin 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 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.

[0338] 5.14 Dosage Form

[0339] In some embodiments, the apelin receptor modulator or a salt thereof is administered in the form of a suspension. In other embodiments, the apelin receptor modulator or a salt thereof is administered in the form of a solution. In some embodiments, the apelin receptor modulator or a salt thereof is administered in a solid dosage form. In a specific embodiment, the solid dosage form is a capsule. In a specific embodiment, the solid dosage form is a tablet. In a specific embodiment, the apelin receptor modulator is in crystalline or amorphous form. In a specific embodiment, the apelin receptor modulator is in amorphous form. In some embodiments, the apelin receptor modulator is an apelin receptor agonist.

[0340] In one aspect of these methods, an apelin receptor modulator or a pharmaceutical composition comprising the same is administered intravenously, topically, orally, by inhalation, by infusion, by injection, intraperitoneally, intramuscularly, subcutaneously, intraaurally, by intraarticular administration, by intramammary administration, by topical administration, or by absorption through epithelial or mucocutaneous linings. In certain embodiments, an apelin receptor modulator or a pharmaceutical composition comprising the same is administered via intravenous infusion.

[0341] 5.15 Definitions

[0342] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0343] The terms "individual," "host," and "subject" are used interchangeably and refer to animals to be treated, including but not limited to humans and non-human primates, rodents (including rats and mice), cattle, horses, sheep, cats, and dogs. "Mammal" means one or more members of any mammalian species. Non-human animal models (i.e., mammals, non-human primates, mice, rabbits, etc.) can be used for experimental studies. The term "patient" refers to a human subject.

[0344] The term "modulator" refers to a compound or composition that modulates the level or activity or function of a target, which may be, but is not limited to, an apelin receptor. In some embodiments, a modulator compound may agonize or activate a target (such as an apelin receptor). An agonist or activator of a target may increase the level of activity or signaling associated with the target.

[0345] The terms "treating," "treatment," and grammatical variations thereof are used in the broadest sense as understood in the clinical art. Thus, these terms do not require a cure or complete alleviation of a disease, and these terms encompass obtaining any clinically desired pharmacological and / or physiological effect, including improvements in physiological measures associated with "normal" nonpathological aging. Unless otherwise indicated, "treating" and "treatment" do not encompass prevention.

[0346] The phrase "therapeutically effective amount" refers to the amount of a compound that, when administered to a mammal or other subject for treating a disease, condition or disorder, is sufficient to effect treatment of the disease, condition or disorder. The "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.

[0347] Range: Throughout this disclosure, various aspects of the disclosure are presented in range format. Ranges include the endpoints listed. It should be understood that the description in range format is only for convenience and brevity, and should not be interpreted as a rigid limitation on the scope of the disclosure. Therefore, it should be considered that the description of the range specifically discloses all possible sub-ranges and single numerical values ​​within the range. For example, it should be considered that the description of a range such as 1 to 6 specifically discloses sub-ranges (such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc.) and single numbers within the range (e.g., 1, 2, 3, 4, 5, 5.3 and 6). This applies regardless of the width of the range.

[0348] Unless expressly stated or obvious from the context, as used herein, the term "or" should be construed as inclusive.

[0349] Unless expressly stated or apparent from the context, as used herein, the terms "a", "an", and "the" are to be understood as singular or plural. That is, the articles "a" and "an" are used herein to refer to one or more than one (i.e., at least one) grammatical object of the article. For example, "an element" means one element or more than one element.

[0350] Unless otherwise indicated or apparent from the context, as used herein, the term "about" is understood to be within the normal tolerance range in the art, such as within 2 standard deviations of the mean, and is intended to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the stated value. Unless otherwise indicated or apparent from the context, where a percentage is provided with respect to the amount of a component or material in a composition, the percentage is understood to be a percentage based on weight.

[0351] It should be understood that the order of steps or the order in which certain actions are performed is not important, as long as the present disclosure remains operable. In addition, two or more steps or actions may be performed simultaneously.

[0352] 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 that can be used to prepare a pharmaceutical composition that is generally safe, non-toxic and neither biologically nor otherwise undesirable, and include excipients, diluents, carriers and adjuvants that are acceptable for veterinary use as well as human pharmaceutical use. The phrase "pharmaceutically acceptable excipient" includes one and more than one such excipient, diluent, carrier and / or adjuvant.

[0353] "Alkyl" refers to a saturated branched or straight chain monovalent hydrocarbon group derived from a single carbon atom of a parent alkane by removing a hydrogen atom. Typical alkyl groups include, but are not limited to, methyl, ethyl, propyl (such as prop-1-yl and prop-2-yl), butyl (such as but-1-yl, but-2-yl, 2-methyl-prop-1-yl, 2-methyl-prop-2-yl, tert-butyl, etc.). In certain embodiments, the alkyl group comprises 1 to 20 carbon atoms. In some embodiments, the alkyl group comprises 1 to 10 carbon atoms or 1 to 6 carbon atoms, while in other embodiments, the alkyl group comprises 1 to 4 carbon atoms. In still other embodiments, the alkyl group comprises 1 or 2 carbon atoms. Branched alkyl groups include at least 3 carbon atoms, and typically include 3 to 7, or in some embodiments, include 3 to 6 carbon atoms. Alkyl groups with 1 to 6 carbon atoms may be referred to as (C 1 -C 6 ) alkyl, and an alkyl group having 1 to 4 carbon atoms may be referred to as (C 1 -C 4 )alkyl. This nomenclature can also be used for alkyl groups with different numbers of carbon atoms. The term "alkyl" can also be used when the alkyl group is a further substituted substituent, in which case the bond between the second hydrogen atom and the C atom of the alkyl substituent is replaced by a bond to another atom such as, but not limited to, a halogen or an O, N, or S atom. For example, the group -O-(C 1 -C 6 Alkyl)-OH will be identified as a group in which the -O atom is adjacent to the C 1 -C 6 Alkyl bonded to C 1 -C 6 One of the H atoms bonded to the C atom of the alkyl group is replaced by a bond to the O atom of the -OH group. As another example, the group -O-(C 1 -C 6 alkyl)-O-(C 1 -C 6 Alkyl) will be identified as a group in which the -O atom is adjacent to the first C 1 -C 6 Alkyl bonded to the first C 1 -C 6 One of the H atoms bonded to the C atom of the alkyl group is separated from the second C 1 -C 6 The bond to which the alkyl group is bonded is replaced by the bond to which the second O atom is attached.

[0354] "Alkenyl" refers to an unsaturated branched or straight-chain hydrocarbon group having at least one carbon-carbon double bond derived from a single carbon atom of a parent olefin by removing a hydrogen atom. The group can be in the form of Z or E (cis or trans) around one or more double bonds. Typical alkenyls include, but are not limited to, vinyl; propenyl, such as prop-1-ene-1-yl, prop-1-ene-2-yl, prop-2-ene-1-yl (allyl) and prop-2-ene-2-yl; butenyl, such as but-1-ene-1-yl, but-1-ene-2-yl, 2-methyl-prop-1-ene-1-yl, but-2-ene-1-yl, but-2-ene-1-yl, but-2-ene-2-yl, but-1,3-diene-1-yl and but-1,3-diene-2-yl; etc. In certain embodiments, alkenyl has 2 to 20 carbon atoms, and in other embodiments, has 2 to 6 carbon atoms. Alkenyl groups having 2 to 6 carbon atoms may be referred to as (C 2 -C 6 ) alkenyl.

[0355] "Alkynyl" refers to an unsaturated branched or straight chain hydrocarbon group having at least one carbon-carbon triple bond derived from a single carbon atom of a parent alkyne by removing a hydrogen atom. Typical alkynyl groups include, but are not limited to, ethynyl, propynyl, butynyl, 2-pentynyl, 3-pentynyl, 2-hexynyl, 3-hexynyl, etc. In certain embodiments, the alkynyl group has 2 to 20 carbon atoms, and in other embodiments, has 2 to 6 carbon atoms. The alkynyl group having 2 to 6 carbon atoms may be referred to as -(C 2 -C 6 )alkynyl.

[0356] "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 include 1 to 10 carbon atoms, 1 to 6 carbon atoms, or 1 to 4 carbon atoms in the R group. Alkoxy groups including 1 to 6 carbon atoms may be designated as -O-(C 1 -C 6 )alkyl or -O-(C 1 -C 6 In some embodiments, the alkoxy group may include 1 to 4 carbon atoms and may be named -O-(C 1 -C 4 )alkyl or -O-(C 1 -C 4 alkyl) group.

[0357] "Aryl" refers to a monovalent aromatic hydrocarbon radical derived from a single carbon atom of a parent aromatic ring system by removing one hydrogen atom. Aryl encompasses monocyclic carbocyclic aromatic rings, such as benzene. Aryl also encompasses bicyclic carbocyclic aromatic ring systems in which each ring is aromatic, such as naphthalene. Thus, aryl can include fused ring systems in which each ring is a carbocyclic aromatic ring. In certain embodiments, aryl includes 6 to 10 carbon atoms. Such radicals may be referred to as C 6 -C 10 Aryl. However, aryl does not in any way encompass or overlap with heteroaryl as defined separately below. Thus, if one or more carbocyclic aromatic rings are fused to an aromatic ring that includes at least one heteroatom, the resulting ring system is a heteroaryl as defined herein, rather than an aryl.

[0358] "Carbonyl" refers to the group -C(O) or a -C(=O) group.

[0359] "Carboxy" refers to the group -C(O)OH.

[0360] "Cyano" refers to the radical -CN.

[0361] "Cycloalkyl" refers to a saturated cycloalkyl group derived from a single carbon atom of a parent cycloalkane by removing a hydrogen atom. Typical cycloalkyl groups include, but are not limited to, groups derived from cyclopropane, cyclobutane, cyclopentane, cyclohexane, cycloheptane, cyclooctane, and the like. Cycloalkyl groups may be described by the number of carbon atoms in the ring. For example, a cycloalkyl group having 3 to 7 ring members may be referred to as a (C 3 -C 7 ) cycloalkyl, and a cycloalkyl having 4 to 7 ring members may be referred to as (C 4 -C 7 In certain embodiments, the cycloalkyl group may be (C 3 -C 10 )cycloalkyl, (C 3 -C 8 )cycloalkyl, (C 3 -C 7 )cycloalkyl, (C 3 -C 6 )cycloalkyl or (C 4 -C 7 ) cycloalkyl, and these may be referred to using alternative language as C 3 -C 10 Cycloalkyl, C 3 -C 8 Cycloalkyl, C 3 -C 7 Cycloalkyl, C 3 -C 6 Cycloalkyl or C 4 -C 7 Cycloalkyl.

[0362] "Heterocyclyl" refers to a cyclic group including at least one saturated or unsaturated but non-aromatic ring. The heterocyclyl includes at least one heteroatom as a ring member. Typical heteroatoms include O, S and N and are independently selected. Heterocyclyl includes monocyclic and bicyclic systems. Bicyclic heterocyclyl includes at least one non-aromatic ring with at least one heteroatom ring member, which can be fused to a cycloalkyl ring or can be fused to an aromatic ring, wherein the aromatic ring can be a carbocyclic ring or can include one or more heteroatoms. The attachment point of the bicyclic heterocyclyl can be on the non-aromatic ring including at least one heteroatom or on another ring of the heterocyclyl. For example, a heterocyclyl derived from one of the 9-membered heterocyclic compounds shown below can be attached to the rest of the molecule on a 5-membered ring or a 6-membered ring.

[0363]

[0364] In some embodiments, the heterocyclyl includes 5 to 10 ring members, of which 1, 2, 3 or 4 or 1, 2 or 3 are heteroatoms independently selected from O, S or N. In other embodiments, the heterocyclyl includes 3 to 7 ring members, of which 1, 2 or 3 heteroatoms are independently selected from O, S or N. In such 3-7 membered heterocyclyls, when the ring includes only 3 members, only 1 of the ring atoms is a heteroatom, and when the ring includes 4 members, it includes 1 or 2 heteroatoms. In some embodiments, the heterocyclyl includes 3 or 4 ring members, of which 1 is a heteroatom selected from O, S or N. In other embodiments, the heterocyclyl includes 5 to 7 ring members, of which 1, 2 or 3 are heteroatoms independently selected from O, S or N. Typical heterocyclic groups include, but are not limited to, groups derived from epoxides, aziridine, azetidine, imidazolidine, morpholine, piperazine, piperidine, hexahydropyrimidine, 1,4,5,6-tetrahydropyrimidine, pyrazolidine, pyrrolidine, quinuclidine, tetrahydrofuran, tetrahydropyran, benzimidazolone, pyridone, and the like. Substituted heterocyclyl also includes 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, benzo[d]oxazol-2(3H)-onyl, 3,4-dihydroisoquinolin-1(2H)-onyl, indol-onyl, 1H-imidazo[4,5-c]pyridin-2(3H)-onyl, 7H-purin-8(9H)-onyl, imidazolidin-2-onyl, 1H-imidazol-2(3H)-onyl, 1,1-dioxo-1-thiomorpholinyl, and the like.

[0365] "Halo," "halo," or "halogen" refers to a fluoro, chloro, bromo, or iodo radical.

[0366] "Haloalkyl" refers to an alkyl group in which at least one hydrogen is replaced by a halogen. Therefore, the term "haloalkyl" includes monohaloalkyl (alkyl substituted by a halogen atom) and polyhaloalkyl (alkyl substituted by two or more halogen atoms). Representative "haloalkyl" groups include difluoromethyl, 2,2,2-trifluoroethyl, 2,2,2-trichloroethyl, etc. Unless otherwise indicated, the term "perhaloalkyl" means 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.

[0367] "Heteroaryl" refers to a monovalent heteroaromatic group derived from a single atom of a parent heteroaromatic ring system by removing a hydrogen atom. Heteroaryl groups typically include 5 to 14 members, but more typically include 5 to 10 membered aromatic monocyclic, bicyclic and tricyclic rings, which contain 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, and the remaining ring atoms are carbon. In monocyclic heteroaryl groups, the monocyclic ring is aromatic and includes at least one heteroatom. In some embodiments, the monocyclic heteroaryl group may include 5 or 6 ring members, and may include 1, 2, 3 or 4 heteroatoms, 1, 2 or 3 heteroatoms, 1 or 2 heteroatoms, or 1 heteroatom, wherein the one or more heteroatoms are independently selected from O, S or N. In bicyclic aromatic rings, both rings are aromatic. In bicyclic heteroaryl groups, at least one of these rings must include a heteroatom, but it is not necessary that both rings include heteroatoms, although this is allowed. For example, the term "heteroaryl" includes a 5- to 7-membered heteroaromatic ring fused to a carbocyclic aromatic ring or fused to another heteroaromatic ring. In a tricyclic aromatic ring, all three rings are aromatic and at least one of these rings includes at least one heteroatom. For fused bicyclic and tricyclic heteroaryl ring systems in which only one ring contains one or more heteroatoms, the point of attachment can be located on the ring including at least one heteroatom or on the 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 or overlap aryl as defined above. Examples of heteroaryl groups include, but are not limited to, groups derived from acridine, carbazole, cinnoline, furan, imidazole, indazole, indole, indolizine, isobenzofuran, isochromene, isoindole, isoquinoline, isothiazole, 2H-benzo[d][1,2,3]triazole, isoxazole, naphthyridine, oxadiazole, oxazole, perimidine, phenanthridine, phenanthroline, phenazine, phthalazine, pteridine, purine, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine, pyrrole, pyrrolizine, quinazoline, quinoline, quinolizine, quinoxaline, tetrazole, thiadiazole, thiazole, thiophene, triazole, etc. In certain embodiments, the heteroaryl group can be a 5-20 membered heteroaryl group, for example, a 5-14 membered or 5-10 membered heteroaryl group. In certain embodiments, heteroaryl groups may be those derived from thiophene, pyrrole, benzothiophene, 2H-benzo[d][1,2,3]triazole, benzofuran, indole, pyridine, quinoline, imidazole, benzimidazole, oxazole, tetrazole, and pyrazine.

[0368] As described herein, various embodiments of the present invention compounds, compositions and methods are described herein. The various embodiments described are intended to provide various illustrative examples and should not be construed as descriptions of alternative 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 present technology.

[0369] 5.16 Examples of Non-Limiting Aspects of the Disclosure

[0370] The aspects (including embodiments) of the subject matter of the present invention described above may be beneficial alone or in combination with one or more other aspects or embodiments. Without limiting the foregoing description, certain non-limiting aspects of the present disclosure numbered 1-52 are provided below. As will be apparent to one skilled in the art upon reading this disclosure, each of the separately numbered aspects may be used in combination with any of the previously or subsequently separately numbered aspects. This is intended to provide support for all combinations of these aspects, but is not limited to the combinations of the aspects explicitly provided below:

[0371] Aspect 1. A method of reducing blood-brain barrier (BBB) ​​permeability in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an apelin receptor agonist to reduce BBB permeability.

[0372] Aspect 2. A method of treating a disorder associated with increased BBB permeability in a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of an apelin receptor agonist.

[0373] Aspect 3. A method of treating a neurodegenerative or neurodegenerative disease in a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of an apelin receptor agonist.

[0374] Aspect 4. A method of reducing neuroinflammation in a subject, the method comprising administering a therapeutically effective amount of an apelin receptor agonist to a subject in need thereof.

[0375] Aspect 5. A method of treating a neurodegenerative disease in a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of an apelin receptor agonist.

[0376] Aspect 6. The method according to any one of Aspects 1-6, wherein the subject exhibits cognitive impairment.

[0377] Aspect 7. The method of any one of Aspects 1-7, wherein the subject suffers from age-related cognitive impairment.

[0378] Aspect 8. The method of any one of Aspects 1-8, wherein the subject suffers from dementia (eg, acute, chronic, or progressive dementia).

[0379] Aspect 9. The method of any one of aspects 1-9, wherein the subject suffers from neurodegeneration.

[0380] Aspect 10. The method of any one of aspects 1-9, wherein the subject suffers from cognitive impairment.

[0381] Aspect 11. The method of any one of Aspects 1 to 10, wherein the subject suffers from acute cognitive impairment (eg, cognitive impairment associated with acute inflammation).

[0382] Aspect 12. The method of any one of aspects 1 to 11, wherein the subject suffers from postoperative cognitive dysfunction (POCD).

[0383] Aspect 13. The method of any one of Aspects 1 to 12, wherein the subject suffers from traumatic brain injury (TBI).

[0384] Aspect 14. The method of any one of Aspects 1 to 13, wherein the subject suffers from intensive care unit (ICU) delirium, postoperative delirium, delirium caused by trauma, or delirium caused by a fracture (eg, a hip fracture).

[0385] Aspect 15. The method of Aspect 14, wherein the postoperative delirium is following cardiovascular surgery.

[0386] Aspect 16. The method of any one of Aspects 1 to 15, wherein the subject is on a ventilator.

[0387] Aspect 17. The method of any one of Aspects 1 to 16, wherein the subject suffers from neuroinflammation (eg, peripheral inflammation).

[0388] Aspect 18. The method of any one of aspects 1-4, wherein the subject is in need of treatment for a neurodegenerative disease.

[0389] Aspect 19. The method of any one of Aspects 1 to 17, wherein the subject suffers from a neurodegenerative disease selected from the group consisting of Alzheimer's disease (AD), vascular dementia (VaD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), stroke, Huntington's disease (HD) and multiple sclerosis (MS).

[0390] Aspect 20. The method of any one of aspects 1-3, 4, and 6-17, wherein the subject does not suffer from a neurodegenerative disease.

[0391] Aspect 21. The method of any one of aspects 1 to 20, wherein the subject is a human and is at least 40 years old.

[0392] Aspect 22. The method of aspect 21, wherein the subject is at least 50 years old.

[0393] Aspect 23. The method of aspect 22, wherein the subject is at least 60 years old.

[0394] Aspect 24. The method of aspect 23, wherein the subject is at least 65 years old.

[0395] Aspect 25. The method of aspect 24, wherein the subject is at least 70 years old.

[0396] Aspect 26. The method of aspect 25, wherein the subject is at least 75 years old.

[0397] Aspect 27. The method of aspect 26, wherein the subject is at least 80 years old.

[0398] Aspect 28. The method of any one of aspects 1 to 27, wherein the subject has or is identified as having low circulating levels of apelin.

[0399] Aspect 29. The method of any one of Aspects 1 to 28, wherein the apelin receptor agonist has formula (I) or (II):

[0400]

[0401] 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,

[0402] in:

[0403] R 1 is unsubstituted pyridyl, pyridone or pyridine N-oxide, or is substituted by 1, 2, 3 or 4 R 1a a pyridyl, pyridonyl or pyridine N-oxide substituted with a substituent;

[0404] R 1a is independently selected from -F, -Cl, -Br, -I, -CN, -C 1 -C 6 Alkyl, -C 1 -C 6 Haloalkyl, -C 1 -C 6 Perhaloalkyl, -OH, -O-(C 1 -C 6 Alkyl), -O-(C 1 -C 6 Haloalkyl), -O-(C 1 -C 6 Perhaloalkyl), -C2 -C 6 Alkenyl, -O-(C 1 -C 6 Alkyl)-OH, -O-(C 1 -C 6 alkyl)-O-(C 1 -C 6 Alkyl), -O-(C 1 -C 6 Haloalkyl)-OH, -O-(C 1 -C 6 (C 1 -C 6 Alkyl), -O-(C 1 -C 6 Perhaloalkyl)-OH, -O-(C 1 -C 6 Perhaloalkyl)-O-(C 1 -C 6 Alkyl), -NH 2 、-NH(C 1 -C 6 Alkyl), -N(C 1 -C 6 alkyl) 2 、-C(=O)-(C 1 -C 6 alkyl), -C(=O)OH, -(C=O)-O-(C 1 -C 6 alkyl), -C(=O)NH 2 、-C(=O)NH(C 1 -C 6 alkyl), -C(=O)N(C 1 -C 6 alkyl) 2 , phenyl, -C(=O)-(heterocyclyl) or heterocyclyl, wherein the heterocyclyl of the -C(=O)-(heterocyclyl) or the heterocyclyl is a 3- to 7-membered ring containing 1, 2 or 3 heteroatoms selected from N, O and S;

[0405] R 2 Selected from -H and C 1 -C 4 Alkyl, or, in compounds of formula II, is absent;

[0406] R 3 Selected from unsubstituted C 1 -C 10 Alkyl, 1, 2 or 3 R 1a Substituent-substituted C 1 -C 10 Alkyl, formula -(CR3b R 3c )-Q, a group of formula -NH-(CR 3b R 3c )-Q group, the formula -(CR 3b R 3c )-C(=O)-Q, a group of the formula -(CR 3d R 3e )-(CR 3f R 3g )-Q group, the formula -(CR 3b =CR 3c )-Q and -(heterocyclyl)-Q, wherein the heterocyclyl of -(heterocyclyl)-Q has 5 to 7 ring members and is unsubstituted or substituted with 1, 2 or 3 R3 h Substituents are substituted, wherein 1, 2 or 3 ring members are heteroatoms selected from N, O and S;

[0407] R 1a is independently selected from -F, -Cl, -CN, -OH, -O-(C 1 -C 6 Alkyl), -O-(C 1 -C 6 Haloalkyl), -O-(C 1 -C 6 Perhaloalkyl), -O-(C 1 -C 6 Alkyl)-OH, -O-(C 1 -C 6 alkyl)-O-(C 1 -C 6 Alkyl), C 2 -C 6 Alkenyl, C 2 -C 6 Alkynyl, -NH 2 、-NH(C 1 -C 6 Alkyl) and -N(C 1 -C 6 alkyl) 2 ;

[0408] R 3b and R 3c Independently selected from -H, -F, -Cl, -CN, -C 1 -C 6 Alkyl, -C 1 -C 6 Haloalkyl, -C 1 -C 6 Perhaloalkyl, -OH, -O-(C 1 -C6 Alkyl), -O-(C 1 -C 6 Haloalkyl), -O-(C 1 -C 6 Perhaloalkyl), -O-(C 1 -C 6 Alkyl)-OH, -O-(C 1 -C 6 alkyl)-O-(C 1 -C 6 Alkyl), -NH 2 、-NH(C 1 -C 6 Alkyl) and -N(C 1 -C 6 alkyl) 2 ;

[0409] R 3d and R 3e Independently selected from -H, -F, -Cl, -CN, -C 1 -C 6 Alkyl, -C 1 -C 6 Haloalkyl, -C 1 -C 6 Perhaloalkyl, -OH, -O-(C 1 -C 6 Alkyl), -O-(C 1 -C 6 Haloalkyl), -O-(C 1 -C 6 Perhaloalkyl), -O-(C 1 -C 6 Alkyl)-OH, -O-(C 1 -C 6 alkyl)-O-(C 1 -C 6 Alkyl), -NH 2 、-NH(C 1 -C 6 Alkyl) and -N(C 1 -C 6 alkyl) 2 ;

[0410] R 3f and R 3g Independently selected from -H, -F, -Cl, -CN, -C 1 -C 6 Alkyl, -C 1 -C 6 Haloalkyl, -C 1 -C 6Perhaloalkyl, -OH, -O-(C 1 -C 6 Alkyl), -O-(C 1 -C 6 Haloalkyl), -O-(C 1 -C 6 Perhaloalkyl), -O-(C 1 -C 6 Alkyl)-OH, -O-(C 1 -C 6 alkyl)-O-(C 1 -C 6 Alkyl), -NH 2 、-NH(C 1 -C 6 Alkyl) and -N(C 1 -C 6 alkyl) 2 ;

[0411] R 3h is independently selected from -F, -Cl, -CN, -C 1 -C 6 Alkyl, -C 1 -C 6 Haloalkyl, -C 1 -C 6 Perhaloalkyl, -OH, -O-(C 1 -C 6 Alkyl), -O-(C 1 -C 6 Haloalkyl), -O-(C 1 -C 6 Perhaloalkyl), -O-(C 1 -C 6 Alkyl)-OH, -O-(C 1 -C 6 alkyl)-O-(C 1 -C 6 Alkyl), -NH 2 、-NH(C 1 -C 6 Alkyl), -N(C 1 -C 6 alkyl) 2 and oxo;

[0412] Q is a monocyclic or bicyclic C 6 -C 10 Aryl, monocyclic or bicyclic heteroaryl with 5 to 10 ring members containing 1, 2 or 3 heteroatoms selected from N, O or S, C 3 -C 8Cycloalkyl, or a 3- to 7-membered heterocyclic group containing 1, 2 or 3 heteroatoms selected from N, O or S, wherein the C 6 -C 10 The aryl, heteroaryl, cycloalkyl and heterocyclyl groups are unsubstituted or substituted with 1, 2, 3 or 4 R Q Substituent substitution;

[0413] R Q is independently selected from -F, -Cl, -Br, -I, -CN, -C 1 -C 6 Alkyl, -C 1 -C 6 Haloalkyl, -C 1 -C 6 Perhaloalkyl, -C 2 -C 6 Alkenyl, -C 2 -C 6 Alkynyl, -OH, -O-(C 1 -C 6 Alkyl), -O-(C 1 -C 6 Haloalkyl), -O-(C 1 -C 6 Perhaloalkyl), -NH 2 、-NH(C 1 -C 6 Alkyl), -N(C 1 -C 6 alkyl) 2 、-C(=O)-(C 1 -C 6 alkyl), -C(=O)OH, -C(=O)-O-(C 1 -C 6 alkyl), -C(=O)NH 2 、-C(=O)NH(C 1 -C 6 alkyl), -C(=O)N(C 1 -C 6 alkyl) 2 、-S(=O) 2 -(C 1 -C 6 alkyl), phenyl and heteroaryl, and the Q heterocyclic group may be substituted with 1 oxo R Q Substituent substitution;

[0414] R 4 Selected from monocyclic or bicyclic C 6 -C 10aryl, monocyclic or bicyclic heteroaryl having 5 to 10 ring members containing 1, 2 or 3 heteroatoms independently selected from N, O and S, and monocyclic or bicyclic heterocyclyl having 5 to 10 ring members and containing 1, 2, 3 or 4 heteroatoms independently selected from N, O and S, wherein the C 6 -C 10 The aryl group, the heteroaryl group or the heterocyclic group is unsubstituted or substituted by 1, 2 or 3 R 4a Substituent substitution;

[0415] R 4a is independently selected from -F, -Cl, -Br, -I, -CN, -C 1 -C 6 Alkyl, -C 1 -C 6 Haloalkyl, -C 1 -C 6 Perhaloalkyl, -OH, -O-(C 1 -C 6 Alkyl), -O-(C 1 -C 6 Haloalkyl), -O-(C 1 -C 6 Perhaloalkyl), -NH 2 、-NH(C 1 -C 6 Alkyl), -N(C 1 -C 6 alkyl) 2 、-C(=O)-(C 1 -C 6 alkyl), -C(=O)OH, -C(=O)-O-(C 1 -C 6 alkyl), -C(=O)NH 2 、-C(=O)NH(C 1 -C 6 alkyl) and -C(=O)N(C 1 -C 6 alkyl) 2 , and the heterocyclic group R 4 may be further substituted with 1 oxo substituent; and

[0416] Further wherein:

[0417] If R 4 is an unsubstituted or substituted benzene ring and R 3 It is the formula -(CR 3b =CR 3c )-Q, then at least one of the following is true:

[0418] a) R4 By at least one -O-(C 1 -C 6 Alkyl) group substitution;

[0419] b) Q is not oxadiazole;

[0420] c) R 3b Not -H;

[0421] d) R 3c Not -H;

[0422] e) R1 is not 2-pyridyl; or

[0423] f)R 4 By two or more -O-(C 1 -C 6 alkyl) group.

[0424] Aspect 30. The method according to aspect 29, wherein R 1 is unsubstituted pyridyl or substituted with 1 or 2 R 1a The substituent is substituted pyridinyl.

[0425] Aspect 31. The method according to aspect 29 or 30, wherein R 1a is independently selected from -CH 3 、-CH 2 CH 3 、-F、-Cl、-Br、-CN、-CF 3 、-CH=CH 2 、-C(=O)NH 2 、-C(=O)NH(CH 3 )、-C(=O)N(CH 3 ) 2 、-C(=O)NH(CH 2 CH 3 ), -OH, -OCH 3 、-OCHF 2 、-OCH 2 CH 3 、-OCH 2 CF 3 、-OCH 2 CH 2 OH, -OCH 2 C(CH 3 ) 2 OH, -OCH 2 C(CF 3 ) 2 OH, -OCH 2 CH 2 OCH3 、-NH 2 、-NHCH 3 、-N(CH 3 ) 2 , phenyl and a group of the formula

[0426]

[0427] When drawn across a bond, the symbol Indicates the point of attachment to the rest of the molecule.

[0428] Aspect 32. The method of any one of aspects 29 to 31, wherein R 1 Selected from

[0429]

[0430]

[0431] When drawn across a bond, the symbol Indicates the point of attachment to the rest of the molecule.

[0432] Aspect 33. The method of any one of aspects 29 to 32, wherein R 2 Yes -H.

[0433] Aspect 34. The method of any one of aspects 29 to 33, wherein R 4 is phenyl, azophenyl, pyrimidinyl, isoxazolyl, indolyl, naphthyl or pyridinyl, any of which may be unsubstituted or substituted with 1, 2 or 3 R 4a Substituent substitution.

[0434] Aspect 35. The method of aspect 34, wherein R 4 Is 1 or 2 R 4a Substituents: phenyl substituted.

[0435] Aspect 36. The method of aspect 35, wherein the 1 or 2 R 4a The substituent is -O-(C 1 -C 2 alkyl) group.

[0436] Aspect 37. The method of any one of aspects 29 to 36, wherein R 4a is independently selected from -CH 3 、-F、-Cl、-Br、-CN、-CF 3 、-OCH 3 、-OCHF 2 、-OCH 2 CH 3、-C(=O)OCH 3 、-C(=O)CH 3 or -N(CH 3 ) 2 .

[0437] Aspect 38. The method of any one of aspects 29 to 37, wherein R 3 Select from the formula -(CR 3b R 3c )-Q, a group of formula -NH-(CR 3b R 3c )-Q group, the formula -(CR 3b R 3c )-C(=O)-Q, a group of the formula -(CR 3d R 3e )-(CR 3f R 3g )-Q group, the formula -(CR 3b =CR 3c )-Q or a group of the 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 are substituted, 1, 2 or 3 of the ring members are heteroatoms selected from N, O or S.

[0438] Aspect 39. A method as described in any one of aspects 29 to 38, wherein Q is selected from pyrimidinyl, pyridinyl, isoxazolyl, thiazolyl, imidazolyl, phenyl, tetrahydropyrimidonyl, cyclopropyl, cyclobutyl, cyclohexyl, morpholinyl, pyrrolidinyl, pyrazinyl, imidazo[1,2-a]pyridinyl, pyrazolyl or oxetanyl, any of which may be unsubstituted or replaced by 1, 2 or 3 R Q Substituent substitution.

[0439] Aspect 40. The method of any one of aspects 29 to 39, wherein Q is a monocyclic heteroaryl having 5 or 6 ring members containing 1 or 2 heteroatoms selected from N, O or S, and Q is unsubstituted or substituted with 1 or 2 R Q Substituent substitution.

[0440] Aspect 41. The method of any one of aspects 29 to 40, wherein R 3 It is the formula -(CR 3d R 3e )-(CR 3f R 3g )-Q group.

[0441] Aspect 42. The method of any one of aspects 29 to 41, wherein R 3 With the following formula

[0442]

[0443] When drawn across a bond, the symbol Indicates the point of attachment to the rest of the molecule.

[0444] Aspect 43. A method as described in any of Aspects 29 to 42, wherein the apelin 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-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.

[0445] Aspect 44. A method as described in Aspect 43, wherein the apelin 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-pyrimidinyl)-2-butanesulfonamide or a pharmaceutically acceptable salt thereof.

[0446] Aspect 45. The method of any one of aspects 1 to 44, wherein the apelin receptor agonist is administered intravenously or intrathecally.

[0447] Aspect 46. The method of any one of aspects 1 to 45, wherein the apelin receptor agonist is administered orally.

[0448] Aspect 47. The method of any one of Aspects 1 to 46, wherein the dose is administered daily.

[0449] Aspect 48. The method of any one of aspects 1 to 47, wherein the dose is administered in a plurality of equally or unequally divided sub-doses.

[0450] Aspect 49. The method of any one of Aspects 1 to 48, wherein the doses are administered at different dosing intervals.

[0451] Aspect 50. The method of any one of Aspects 1 to 49, wherein the dose is 200 mg.

[0452] Aspect 51. The method of any one of Aspects 1 to 50, further comprising assessing cognitive function after administration.

[0453] Aspect 52. The method of aspect 51, wherein the cognitive function is assessed at least one day after administration (eg, at least one week or at least one month after administration).

[0454] 6. Examples

[0455] Below is an example of a specific embodiment to implement 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. The accuracy of the numbers used (e.g., amounts, temperatures, etc.) has been ensured as much as possible, but of course some experimental errors and deviations should be allowed.

[0456] The practice of the present invention will employ, unless otherwise indicated, conventional methods of protein chemistry, biochemistry, recombinant DNA techniques and pharmacology, which are within the skill of the art. Such techniques are fully explained in the literature.

[0457] 6.1 Example 1: Bioinformatics analysis identifies the relationship between apelin and cognitive decline in a human healthy aging cohort

[0458] Based on survival modeling, using unpublished clinical outcome data and proteomic data generated on archival samples, a survival prediction model was used to examine the relationship between serum levels of apelin and risk of future cognitive decline in a human healthy aging cohort. Cox proportional hazards models were used with hazard ratios and associated p-values ​​generated for apelin.

[0459] like Figure 2A - As shown in Figure 2C, the human aging platform proposed by the inventors revealed a protective association between higher circulating apelin protein levels and healthy lifespan outcomes. The graph showing the probability of a good outcome indicates that serum apelin concentrations are associated with a higher probability of grip strength retention and lifespan over decades of follow-up. The graph showing the retention of cognitive function indicates that the upper and lower 10th percentiles of serum apelin concentrations are associated with the retention of cognitive function (as assessed by a decrease in CASI scores) over 10 years of follow-up. Based on the Cognitive Ability Screening Inventory (CASI) score, the hazard ratio for cognitive decline was determined to be 0.85 (p=0.0292). Based on this result, an exemplary apelin receptor agonist was evaluated in vivo in BBB permeability and neurodegenerative disease models.

[0460] 6.1.15.1.1 Analysis

[0461] The change (Δ) in apelin levels between examination 3 and examination 4 in the Hawaiian cohort was calculated using proteomics data. It was found that the change in apelin levels (ΔAPLN) was associated with a higher probability of a decrease in CASI, as shown by survival analysis (COX proportional hazards model). The COXPH model was adjusted for age at examination 3 (Vagex3), smoking status (pack years - PACKYRX3), alcohol intake (ALCX3), and CASI score at examination 4 (CASIX4). The coefficients of the model are shown below, where the second column (exp(coef)) represents the hazard ratio, and the rightmost column shows the corresponding p-value.

[0462]

[0463] Figure 2A - Figure 2C shows the Kaplan-Meier curves of the bioinformatics analysis. Figure 2A-2B The Y-axis represents the probability of a CASI decrease. Figure 2A-2B The X-axis represents years (10 years). The graph represents the cohort participants whose APLN levels are in the lower 10% of the cohort (Coefficient (Apelin) lower) and the participants with the highest APLN levels (Coefficient (Apelin) upper) (upper 10%). The middle line (Coefficient (Apelin) middle) is between 10%-90%.

[0464] 6.2 Example 2: BGE-105 rescues BBB breakdown under LPS-induced BBB permeability

[0465] The activity of BGE-105 on BBB permeability was evaluated in 12 month old mice challenged with lipopolysaccharide (LPS), which induces increased BBB permeability. Lipopolysaccharide (LPS) induces inflammation and BBB disruption in mice.

[0466] 6.2.15.2.1 Methods

[0467] Mice were handled in accordance with the guidelines and regulations set forth in the Guide for the Care and Use of Laboratory Animals and were supervised by the Institutional Animal Care and Use Committee (IACUC) of the BioAge Lab. Mice were divided into three experimental groups:

[0468] Group 1: (Vehicle Control). Vehicle was administered PO to mice.

[0469] Group 2: (Vehicle+LPS). Mice were administered vehicle PO for 1 week followed by LPS on day 8; and

[0470] Group 3: (Treatment with BGE-105) Mice were administered BGE-105 PO for 1 week followed by LPS administration on day 8.

[0471] Note that the vehicle solution for LPS was sterile saline. The vehicle solution for BGE-105 was 2% HPMC and 1% Pluronic F68. A schematic diagram of the experimental protocol is shown in Figure 3AIn. The 3rd group of mice was administered BGE-105 at 50 mg / kg twice a day (BID) for one week (7 days), and then exposed to LPS stimulation on the 8th day. The healthy control group of mice received PO vehicle (BID) for one week (7 days), followed by an intraperitoneal injection of normal saline. The PO vehicle BID or BGE-105 BID was retained until the endpoint Evans blue was determined to measure BBB permeability. BID represents twice daily administration of vehicle or BGE-105.

[0472] 23 hours after LPS or saline injection, mice were injected intravenously with Evans blue (EB) (2%, 75ul / 30gBW). One hour later, blood samples were collected via retro-orbital bleeding under isoflurane anesthesia. Mice were euthanized via cardiac perfusion under isoflurane anesthesia, and then the whole brain was removed to perform microdissection on specific brain regions (olfactory bulb, hippocampus) from each hemisphere. Brain slices were pooled, quickly frozen in liquid nitrogen and stored at -80°C until further use.

[0473] Frozen olfactory bulb (OB) and hippocampus (HPF) slices were put into 200 μ l extraction buffer (50% trichloroacetic acid in formamide) for 24 hours. After mechanical homogenization, the sample was rotated at 12,000 g for 30 min at 4 ° C. Each sample of 30 μ l was added to microplate in duplicate together with the Evans blue standard dilution series, and all wells were diluted with 90 μ l of 95% ethanol to ensure that the optical path was consistent. End point fluorescence (620 nm excitation, 680 nm emission) was recorded with SpectraMax iD5 (Molecular Devices), normalized to tissue weight, and the relative LPS-related changes (measurement of blood-brain barrier leakage) of fluorescence compared to the mice processed with vehicle or BGE-105 were calculated.

[0474] 6.2.25.2.2 Results

[0475] like Figure 3B As shown, the LPS challenge group (Group 2) increased BBB permeability in the OB (+17%) and HPF (+39%; p<0.01, one-way ANOVA with Tukey's multiple comparisons) compared to the healthy control group 1 (control vs. LPS). Pretreatment with BGE-105 (Group 3) significantly reversed the LPS-induced increase in BBB permeability in the OB and HPF of treated mice (p<0.05, one-way ANOVA with Tukey's multiple comparisons) (LPS vs. LPS+BGE-105).

[0476] The activity of Exemplary Compound 3 (BGE-105; eg, as described herein) was evaluated according to the methods described above.

[0477] 6.3 Example 3: BGE-105 rescues BBB permeability in naturally aged mice

[0478] break down

[0479] Next, the activity of BGE-105 on BBB permeability was evaluated in naturally aged mice, which exhibit an age-related increase in BBB permeability.

[0480] 6.3.1 Methods

[0481] Mice were handled in accordance with the guidelines and guidelines established by the Guide for the Care and Use of Laboratory Animals and were supervised by the Institutional Animal Care and Use Committee (IACUC) of BioAgeLab. Mice were routinely housed in an animal room with a constant temperature (20°C-22°C) and humidity (40%-60%), with a 12 / 12h light / dark cycle and free access to food and water.

[0482] Figure 4A The development of an aging mouse model for age-related increases in BBB permeability is shown. Figure 4A As shown, BBB permeability was increased in both the olfactory bulb and hippocampus regions of the brain in aged mice (22 months old) compared to young (3 months old) or adult (13 months old) mice, as measured using an Evans blue (EB) staining assay (e.g., as described herein).

[0483] To evaluate BGE-105 activity, an age-dependent BBB permeability model was established using 26-month-old female C57BL / 6 mice. Mice were randomly divided into two groups (vehicle or BGE-105, n=8) according to body weight and body condition scores. Mice were PO-administered with vehicle (control) or BGE-105 via PO at 50 mg / kg (BID) for one week (7 days). For a schematic diagram of the protocol, see Figure 4B . BBB permeability was measured via Evans blue assay. Mice were injected intravenously with Evans blue (2%, 75ul / 30g BW). One hour later, blood samples were collected via retro-orbital bleeding under isoflurane anesthesia. Mice were euthanized via cardiac perfusion under isoflurane anesthesia, and the whole brain was removed for microdissection of specific brain regions (olfactory bulb, hippocampus) from each hemisphere. Brain sections were pooled, snap frozen in liquid nitrogen and stored at -80°C until further use.

[0484] Frozen olfactory bulbs and hippocampi were placed in 200 μl extraction buffer (50% trichloroacetic acid in formamide) for 24 hours. After mechanical homogenization, the samples were rotated at 12,000 g for 30 min at 4 ° C. 30 μl of each sample was added to a microplate in duplicate together with a standard dilution series of Evans blue, and all wells were diluted with 90 μl of 95% ethanol to ensure consistent light path. Endpoint fluorescence (620 nm excitation, 680 nm emission) was recorded with SpectraMax iD5 (Molecular Devices), normalized to tissue weight, and the relative treatment-related changes in fluorescence compared to untreated aging mice (a measure of blood-brain barrier leakage) were calculated.

[0485] 6.3.2 Results

[0486] like Figure 4C As shown, pretreatment with BGE-105 significantly reversed the age-induced increase in BBB permeability in OB (-28%; p < 0.01, Mann-Whitney U test) (aging vs. aging + BGE-105). The activity of exemplary compound 3 (BGE-105; e.g., as described herein) was evaluated according to the above method. Based on these results, it was found that BGE-105 restored BBB integrity in aging mice.

[0487] 6.4 Example 4: Efficacy of BGE-105 in Age-Dependent Peripheral Inflammation

[0488] Next, the activity of BGE-105 on age-dependent peripheral inflammation was evaluated in aged mice, where age-related neuroinflammation was hypothesized to play a role in age-related BBB permeability and neurodegeneration.

[0489] method

[0490] Mice were treated according to the guidelines and guidelines of the "Guide to the Care and Use of Laboratory Animals" and supervised by the Institutional Animal Care and Use Committee (IACUC) of BioAgeLab. Mice were routinely housed in a constant temperature (20°C-22°C) and humid (40%-60%) animal room with a 12 / 12h light / dark cycle and free access to food and water. According to body weight and physical condition scores, 26-month-old mice were randomly divided into two groups (vehicle or BGE-105, n=8). 4-month-old female mice were used as another control group (vehicle, n=4). Vehicle or BGE-105 was administered PO to mice at 50mg / kg (BID) for one week. After administration was completed, blood samples were collected in K2-EDTA tubes via retroorbital bleeding under isoflurane anesthesia to prevent coagulation. Mice were euthanized via cardiac perfusion under isoflurane anesthesia. Whole blood was centrifuged to separate plasma, and plasma was stored at -80°C until further experiments.

[0491] For qualitative analysis of 40 different cytokines, plasma samples were evaluated using the Mouse Cytokine Array Panel (R&D Systems) according to the manufacturer's instructions. The array was developed using chemiluminescent detection and imaged using the ChemiDoc XRS+ system (BioRad). The optics of each point on the array acted as a relative measure of cytokine abundance (e.g., more intensely stained points indicated more cytokines in the sample) and were analyzed using ImageJ software (NIH).

[0492] For quantitative analysis, the cells were stained with antibodies against mouse CXCL1 and CXCL13 according to the manufacturer’s instructions. Plasma samples were evaluated by enzyme-linked immunosorbent assay (ELISA) (R&D Systems). The optical density of the completed ELISA was imaged using SpectraMaxiD5 (Molecular Devices), sample concentrations were calculated compared to a standard curve, and relative changes under aging + / - BGE-105 treatment were calculated using young mouse plasma as a standard.

[0493] result:

[0494] In the cytokine array, 10 (25%) of all 40 cytokines evaluated were detectable (CXCL1, CXCL10, CXCL12, CXCL13, IFN-γ, IL-1Rαα, IL-6, IL-23, TIMP-1, TNF-α). Of these cytokines, treatment with BGE-105 reduced the optical density by an average of 28% ( Figure 5 In ELISA, the CXCL1 concentration in the plasma of aged mice under BGE-105 treatment was reduced by 35% (one-way ANOVA with Tukey's multiple comparison test; *, p<0.05) ( Figure 6 ).like Figure 6 As shown, the CXCL13 concentration in the plasma of aged mice treated with BGE-105 was decreased by 26% (one-way ANOVA with Tukey's multiple comparison test; *, p<0.05) ( Figure 6 ).

[0495] Figure 5 and Figure 6 The results showed that treatment with BGE-105 reduced peripheral inflammation.

[0496] Figure 5 and Figure 6The results showed that treatment with BGE-105 showed that BGE-105 reduced circulating levels of two cytokines (CXCL1 / 13) associated with mortality, neutrophil recruitment, and propagation of inflammation.

[0497] 6.5 Example 5: Efficacy of BGE-105 in Age-Dependent Hippocampal Brain-Derived Neurotrophic Factor (BDNF) Expression

[0498] Next, the activity of BGE-105 on age-dependent hippocampal BDNF expression was evaluated in aged mice. BDNF belongs to the neurotrophic factor family. BDNF plays an important role in the survival and growth of neurons, acts as a neurotransmitter regulator, and is involved in neuronal plasticity, which is essential for learning and memory and is associated with cognitive function.

[0499] method:

[0500] Mice were treated according to the criteria and guidelines formulated in the "Guidelines for the Care and Use of Laboratory Animals" and supervised by the Institutional Animal Care and Use Committee (IACUC) of BioAgeLab. Mice were routinely housed in a constant temperature (20°C-22°C) and humid (40%-60%) animal room with a 12 / 12h light / dark cycle and free access to food and water. According to body weight and physical condition scores, 26-month-old mice were randomly divided into two groups (vehicle or BGE-105, n=8). Vehicle or BGE-105 was administered PO to mice at 50mg / kg (BID) for one week. After administration was completed, mice were euthanized via cardiac perfusion under isoflurane anesthesia, the whole brain was excised, and the entire hippocampus was extracted, which was concentrated and quickly frozen in liquid nitrogen, and then stored at -80°C.

[0501] The hippocampus samples were homogenized with 30 volumes of radioimmunoprecipitation immunoassay (RIPA) buffer (Thermofisher) with Halt protease and phosphatase inhibitors, then spun at 16,000 x g for 20 minutes at 4°C, and the supernatant was transferred to a new labeled tube. The total protein concentration of each sample was analyzed using the Pierce BCA protein assay kit (Thermofisher) according to the manufacturer's instructions, and all samples were then diluted to 0.2 μg / μL with RIPA buffer.

[0502] For quantitative analysis of hippocampal BDNF expression, total BDNF was expressed in 4% BDNF cells according to the manufacturer's instructions. Tissue samples were evaluated by enzyme-linked immunosorbent assay (ELISA) (R&D Systems). The optical density of the completed ELISA was imaged using SpectraMaxiD5 (Molecular Devices), sample concentrations were calculated compared to a standard curve, and relative changes under aging + / - BGE-105 treatment were calculated using young mouse plasma as a standard.

[0503] result:

[0504] like Figure 7 As shown, oral treatment with BGE-105 increased total BDNF concentrations in the hippocampus of aged mice by 20% in a BDNF ELISA measuring free BDNF and BDNF bound to its receptor TrkB (unpaired two-sided t-test; *, p<0.05).

[0505] The results showed that oral administration of BGE-105 increased the production / release of the pro-neuronal survival factor BDNF, which is normally reduced with age and neurodegeneration.

[0506] 6.6 Example 6: Effects of BGE-105 on neuroinflammation

[0507] Neuroinflammation is associated with neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis (ALS). Astrocytes are known to express the apelin receptor (Aplnr) and are the major cell type expressing APJ in the CNS. Astrocytes undergo inflammatory transformation following infection, acute injury, and chronic neurodegenerative diseases. Astrocytes are key components of the neurovascular unit that support the function of the blood-brain barrier (BBB). Pathological transformation of astrocytes into a reactive state may be protective or detrimental to BBB function.

[0508] The activity of BGE-105 and its activity at apelin receptors were evaluated to attenuate the effects of inflammatory stimuli on cultured brain-specific immune cells and to prolong the potential effects of BGE-105 on brain-specific cells.

[0509] In vitro experiments were performed in mouse or human astrocytes and microglia (fresh primary cells or transformed stable cell lines) to explore the effects of BGE-105 on inflammation.

[0510] Reactive astrocyte mixture (RAC) drives the reactivity of neurotoxic astrocytes, as seen in the aged and diseased brain. Degenerated or aged astrocytes can be recapitulated by RAC in vitro. Therefore, RAC-induced astrocytes are used as a model of neurotoxicity. Table 1 lists the common factors in RAC.

[0511]

[0512] To demonstrate the effects of BGE-105 on astrocytes, control astrocytes or RAC-induced neurotoxic astrocytes were treated with one of the following test compounds: (a) BGE-105, an agonist of apelin receptors, (b) Pyr (1) - apelin-13, a ligand for the apelin receptor, or (c) BAY-11-7082, an NF-κdB inhibitor.

[0513] Methods and Materials

[0514] Mouse astrocytes were seeded in 24-well plates at a low density of approximately 20k astrocytes per well.

[0515] Stimulated reactive astrocytes. To stimulate astrocytes into a reactive state found in aging and neurodegenerative diseases according to previous publications (Liddelow et al., 2017; Barbar et al., 2020), a combination of cytokines TNF-α and IL-1α and complement factor C1q was added to astrocyte culture medium at 30 ng / mL, 3 ng / mL, and 400 ng / mL, respectively, to make a “reactive astrocyte mixture” (RAC) culture medium. In the presence or absence of different concentrations of BGE-105 (final concentrations of 10 nM, 50 nM, 250 nM), (Pyr 1 Astrocytes were cultured in RAC medium for 24 hours in the presence of 50 nM (50 nM; Bachem, catalog number 4029110), BAY-11-7082 (50 μM; Abcam, catalog number ab141228) or an equal volume of vehicle (0.01% DMSO in PBS) (hereinafter referred to as 'RAC treatment'). Cells in groups 3-6 were treated with RAC for 24 hours in the presence or absence of test compounds (n=4 per group).

[0516] Group 1: Control [CON]

[0517] Group 2: CON+BGE-105(50nM)[CON+105]

[0518] Group 3: Reactive controls treated with RAC alone [RAC]

[0519] Group 4: RAC+BGE-105 (50 nM) [RAC+105]

[0520] Group 5: RAC+Pyr (1)-Apelin-13 (50nM)

[0521] Group 6: RAC + BAY-11-7082 (NF-κB inhibitor; 50 μM) [RAC + BAY]

[0522] Culture medium from each test cell group was collected for cytokine release analysis. Cells were lysed for RNA collection, and RT-PCR was performed under standard conditions to synthesize cDNA. Next, transcriptome analysis was performed to determine the expression levels of astrocyte genes or astrocyte response genes. The changes in expression were normalized to β-actin ( Figure 8 and Fig. 9 ). Exemplary biomarkers include CCL2, CXCL1θ, CXCL11, CXCL1, CXCL2, CXCL3, CXCL8, EDN1, SERPINA3, C1S, C1RL, C3, CFB, VEGF, and IL-6.

[0523] Mice were treated in accordance with the guidelines and guidelines established by the Guide for the Care and Use of Laboratory Animals and supervised by the Institutional Animal Care and Use Committee (IACUC) of BioAgeLab. Primary mouse astrocytes were isolated and cultured in 6-well or 12-well tissue culture plates coated with 0.01% poly-L-lysine at 200,000 or 120,000 cells / well, respectively, in astrocyte growth medium as previously described in (Lundquist et al. 2022. Knockdown of Astrocytic Monocarboxylate Transporter 4in the Motor Cortex Leads to Loss of Dendritic Spines and a Deficit in Motor Learning. Mol Neurobiol 59, 1002-1017). Embryonic rat neurons were purchased from ScienCell (Catalog No. R1550) and cultured in neuronal culture medium (ScienCell, Catalog No. 1521) according to the manufacturer's protocol with slight modifications. Briefly, neurons were thawed and resuspended in neuronal culture medium supplemented with 1 μg of laminin (Sigma, catalog number L2020) per 1 mL of culture medium, and then plated at 60,000 cells / well in 96-well tissue culture plates coated with 0.01% poly-L-lysine. All cells were cultured under standard mammalian cell culture conditions (37°C, 5% CO 2 ) and all experiments were performed using two separate preparations and at least three replicates. Unless otherwise stated, all statistical analyses and graphs were performed in Prism (version 9; GraphPad).

[0524] Cytokine Secretion. For analysis of cytokine secretion by astrocytes, culture medium was collected after RAC treatment, centrifuged at 10,000 RPM for 5 minutes to pellet any debris, and the supernatant was transferred to a fresh tube and either assayed immediately or frozen at -80°C.

[0525] To evaluate a panel of cytokines and chemokines, cell culture supernatants were diluted 1:10 and assayed using the Proteome Analyzer Mouse Cytokine Array Kit Set A (R&D Systems, catalog number ARY006) according to the manufacturer's protocol. Dot blot analysis was performed in Fiji (Schindelin et al. 2012. Fiji: an open-source platform for biological-image analysis. Nat Methods 9, 676-682), and transcription factor enrichment was performed using Enrichr (Chen et al. 2013. Enrichr: interactive and collaborative HTML5 gene listenenrichment analysis tool. BMC Bioinformatics 14, 128).

[0526] Biomarker concentrations. For analysis of CXCL1 or IL-6 concentrations alone, cell culture supernatants were diluted 1:50 or 1:10, respectively, and assessed using mouse-specific ELISA kits (R&D Systems, catalog numbers DY453 and DY406) according to the manufacturer's protocol.

[0527] Astrocyte protein expression. To analyze astrocyte protein expression, RAC-treated cells were washed with PBS and lysed with neuronal protein extraction reagent (Thermo Scientific, catalog number 87792), centrifuged to precipitate the debris, and the supernatant was transferred to a new tube. Total protein concentration was measured by BCA protein assay (Thermo Scientific, catalog number PI23227). 10 μg of protein from each sample was loaded onto a 10% Bis-Tris gel (Thermo Scientific, catalog number NP0301) and run for 2 hours at 100 V, then dry transferred to a PVDF membrane (Invitrogen, catalog number IB401031). The membranes were blocked in 2% bovine serum albumin (BSA) in TBS with 0.05% Tween-20 (TBS-T) for 1 hour at room temperature and then incubated overnight at 4°C with primary antibodies diluted as follows: rabbit anti-β-actin (1:5000, Genetex, catalog number G109639), rabbit anti-APJ (1:500, Invitrogen, catalog number 702069), rabbit anti-phospho-AKT (Ser473; 1:1000, Cell Signaling, catalog number 4060), rabbit anti-AKT (1:1000, Cell Signaling, catalog number 4685), rabbit anti-NF-κB p65 (1:1000, Cell Signaling, catalog number 8242S), rabbit anti-phospho-IκBα (Ser32, 1:1000, Cell Signaling, catalog number Signaling, catalog number 2859T) and rabbit anti-IκBα (1:1000, Cell Signaling, catalog number 4812S). The blot was washed with TBS-T and probed with goat anti-rabbit secondary antibody (1:5000, Jackson ImmunoResearch, catalog number 111-035-144) in 2% BSA in TBS-T at room temperature for 1 hour. The blot was washed with TBS-T, developed in enhanced chemiluminescent substrate (Thermo Scientific, catalog number 34095) for 1 minute, and imaged on a ChemiDoc XRS+ molecular imager (Bio-Rad, catalog number 1708265). The blot was analyzed in Fiji.

[0528] Astrocyte gene expression. To analyze astrocyte gene expression, RAC-treated cells were washed with PBS and processed for RNA extraction according to the manufacturer's protocol (Zymo Research, catalog number R1050). Total RNA purity and concentration were assessed using NanoDrop (ThermoFisher). Using High-Capacity cDNA Reverse Transcription Kit (Fisher Scientific, catalog number 43-749-66), 500 ng RNA was used for cDNA synthesis, and the total volume of cDNA was diluted to a final concentration of 2 ng / μl. Gene expression was assessed using an RT analyzer PCR array (Qiagen, catalog number 330231) specific for mouse NF-κB signaling targets according to the manufacturer's protocol.

[0529] Astrocyte glutamate uptake. To analyze astrocyte glutamate uptake, RAC-treated cells were washed with PBS and then with Hank's balanced salt solution (HBSS). Fresh HBSS was added to each well, and 100 μM glutamate (Sigma, catalog number G1251) was added to each well. Empty wells without cells were filled with HBSS and 100 μM glutamate as negative controls. The cells were incubated at room temperature for 3 hours, and then HBSS was collected and glutamate concentration was determined by colorimetric assay (Sigma, catalog number MAK004). The relative change in glutamate uptake was calculated compared to the negative control wells.

[0530] Neuronal survival. To analyze neuronal survival, culture medium was collected after RAC treatment and added directly to rat neurons (described previously above) for 24 hours. After exposure, neuronal viability was assessed using ApoTox-Glo Triplex assay (Promega, catalog number G6320) according to the manufacturer's protocol.

[0531] result

[0532] Treatment with BGE-105 showed a reduction in inflammatory biomarkers in astrocytes in vitro (such as RAC-induced astrocytes as described below). The data demonstrate that administration of BGE-105 to astrocytes stimulated with reactive astrocyte mixture (RAC) reduces the pro-inflammatory reactivity signature.

[0533] Figure 8 Results from gene expression profiling analysis of groups 1-4 are shown. BGE-105 attenuated RAC-induced CXCL1, CD3, and IL-6 gene expression. CXCL1, CD3, and IL-6 are usually driven by IL-1 / TNF-mediated NF-κB activation. Figure 8As shown, the expression of biomarkers CXCL1, C3 and IL-6 is low in control (CON) astrocytes and BGE-105 (CON+105), and BGE-105 has no effect or little effect on the expression of biomarkers in astrocytes. CXCL1 is a chemokine that is usually upregulated in neurodegenerative diseases (such as Alzheimer's disease). When compared with the control (CON) group, CXCL1 expression in RAC-induced reactive astrocytes (RAC) was significantly upregulated by about 1200-1600 times (p < 0.0001) (left small figure). When compared with the RAC group, treatment with BGE-105 (RAC+105) significantly reduced the expression of CXCL1 (p = 0.0204).

[0534] C3 is a biomarker for reactive astrocytes. When compared to the control (CON), C3 expression in RAC-induced reactive astrocytes (RAC) was significantly upregulated by about 20-30 times (p < 0.0001) (middle panel). Compared to the RAC group (RAC), treatment with BGE-105 (RAC+105) significantly reduced the expression of C3 by 10-20 times (p = 0.0015). The results show that BGE-105 effectively reduces the neurotoxicity of reactive astrocytes.

[0535] Reactive astrocytes in pathological conditions adversely affect endothelial integrity via secreted proteins such as VEGF or IL-6. Figure 8 The right panel shows that IL-6 was upregulated by about 10-15 fold in RAC-induced reactive astrocytes (RAC) (p<0.0001). Treatment with BGE-105 (RAC+105) significantly reduced the expression of IL-6 by about 5-10 fold (p<0.0001).

[0536] Fig. 9 Results of biomarker measurements in culture medium obtained from Groups 1-4 and Group 6 are shown. BGE-105 (CON+105) had little or no effect on the concentrations of CXCL1 and IL-6 detected in culture medium of astrocytes when compared to control (CON). Fig. 9 The yields of Groups 1-4 and 6 are presented, but it is noted that the yield of Group 6 was very low, too low to be processed together with Groups 1-4.

[0537] Fig. 9The left panel shows that BGE-105 treatment (RAC+105, Group 4) greatly reduces the release of CXCL1 from astrocytes, and thus reduces the concentration of CXCL1 (p=0.0032). When compared with control astrocytes (CON, Group 1), reactive astrocytes (RAC, Group 3) increase the release of CXCL1 by about 30000-40000 times (p<0.0001). Similar results were observed in Group 6 (RAC+BAY) by treating with NF-κB inhibitors (RAC+BAY, p<0.0001).

[0538] Fig. 9 The right panel shows that the reactive astrocytes (RAC) (RAC+BGE-105, the 4th group) treated with BGE-105 reduce the IL-6 release of astrocytes, and thus reduce the concentration of IL-6 (p=0.0008). When compared with control astrocytes (CON), reactive astrocytes (RAC) increase the release of IL-6 by about 1600-2400 times (p<0.0001). Similar results of the 6th group (RAC+BAY) were observed with NF-κB inhibitor treatment (RAC+BAY, p<0.0001).

[0539] Figures 10A-10D BGE-105 is shown to activate apelin receptor (APJ) signaling in astrocytes. Protein expression of APJ, p-AKT, t-AKT, and β-actin in control astrocytes, cells treated with reactive astrocyte cocktail (RAC), and RAC cells treated with BGE-105 (RAC+BGE-105 [50 nM]) is shown. Fig. 10A The expression of APJ was shown to be comparable in all three experimental groups (control (CON), RAC, RAC + BGE-105). When normalized to β-actin expression, no significant fold change in expression was detected ( Fig. 10B ). Fig. 10C The expression of p-AKT in the RAC+BGE-105 group was shown to be increased, while the expression of t-AKT in all three experimental groups remained comparable. Compared with t-AKT, and normalized with β-actin expression, the expression level of p-AKT increased significantly by about 2-fold in response to RAC+BGE-105 treatment ( Fig. 10D ). p-AKT: phosphorylated AKT.

[0540] Figures 11A-11D BGE-105 was shown to reduce cytokine release in a dose-dependent manner. Fig.11ARelative fold changes in expression of various cytokines are shown. The legend on the right ranges from 0-fold change (blue) to 1-fold change (or no change, marked in white) to 4+-fold change (dark red). Fig.11A Shown is a panel of cytokines and chemokines release detected in astrocytes treated with reactive astrocyte mixture (RAC) and different doses of BGE-105 (RAC+BGE-105 [10 nM, 50 nM, 250 nM]) compared to RAC. Fig. 11B Transcription factor enrichment showing the following biomarkers: IKBKB, IRF1, STAT6, NF- K B1 and RELA. Fig. 11C Fold changes in cytokine and chemokine release detected from different treatments (RAC, RAC+105(10 nM), RAC+105(50 nM), RAC+105(250 nM)) are shown. Fig. 11C It was shown that the number of cytokines that were downregulated (or <1-fold change) increased with increasing doses of BGE-105. Treatment with BGE-105 (RAC + BGE-105 [50 nM and 250 nM]) significantly reduced the release of cytokines and chemokines. Fig.11D Protein bands of cytokines and chemokines quantified by Western blotting and presented in dot blots are shown.

[0541] Fig.12 showed that treatment of reactive astrocytes with BGE-105 reduced CXCL1 and IL-6 release from astrocytes. Fig.12 The left panel shows that the concentration / release of CXCL1 of astrocytes treated with reactive astrocyte mixture (RAC) increased by about 40,000-60,000 pg / ml compared to the control (CON) group. BGE-105 (RAC+BGE-105[50nM, 250nM]) significantly reduced the concentration / release of CXCL1 to about 40,000 pg / ml (50nM) or about 20,000-40,000 pg / ml (250nM). It should be noted that no significant difference in CXCL1 concentration / release was detected between the BGE-105 (RAC+BGE-105[50nM]) treated group and the apelin peptide (APL) (RAC+APL[50nM]) treated group. Fig.12The right panel shows that treatment with RAC increased the concentration / release of IL-6 by about 10,000 pg / ml compared to the control (CON) group. BGE-105 (RAC+BGE-105[50 nM, 250 nM]) significantly reduced the concentration / release of IL-6 to about 5,000-10,000 pg / ml (50 nM) or about 10,000 pg / ml (250 nM). It should be noted that no significant difference in IL-6 concentration / release was detected between the BGE-105 (RAC+BGE-105[50 nM]) treated group and the Apelin (APL) (RAC+APL[50 nM]) treated group.

[0542] Nuclear factor-κB (NF-κB) consists of a family of transcription factors that play key roles in inflammation, immunity, cell proliferation, differentiation, and survival. Inducible NF-κB activation is dependent on the NF-κB protein inhibitors (IκB, e.g. K Bα, K Bβ) phosphorylation-induced proteosome degradation, which retains inactive NF-κB dimers in the cytosol of unstimulated cells. Several studies have shown that signaling through NF-κB in astrocytes contributes to proinflammatory responses after injury, and inhibition of NF-κB in astrocytes can promote functional recovery (Dresselhaus EC, Meffert MK. Cellular Specificity of NF-κB Function in the Nervous System. Front Immunol. 2019 May 9; 10: 1043). Figure 13A-13C BGE-105 was shown to inhibit NF- K B activation and I K Bα phosphorylation. Fig.13A The expression of NF-κB in control astrocytes, astrocytes treated with reactive astrocyte cocktail (RAC), and astrocytes treated with BGE-105 (RAC+BGE-105 [50 nM]) was shown. K B. pI K Bα、tI K Protein expression of Bα and β-actin. Fig. 13B NF-κB normalized to β-actin is shown. K B p65 expression. Compared with the control (CON), RAC increased NF- K However, treatment with BGE-105 (RAC+BGE-105 (50 nM)) significantly reduced the expression of NF- K B Expression of p65. Fig. 13C Shown is the pI normalized to β-actinK Bα / tI K The ratio of Bα expression. RAC has increased pI K Bα / tI K The ratio of Bα expression was significantly reduced by BGE-105 treatment (RAC+BGE105). K Bα / tI K The ratio of Bα expression.

[0543] Figure 14A-14B showed that reactive astrocytes treated with BGE-105 were responsive to NF- K B signal transduction transcriptional response has a dose-dependent effect. Fig.14A is a heat map showing the expression of NF- K B. Expression of biomarkers related to the signal transduction pathway and in response to the treatment of BGE-105 (RAC+BGE-105 [50 nM, 250 nM]). The legend shows the fold change of expression compared to the control group (not shown), ranging from -10-fold change (blue) to 0-fold change (or no change, marked in white), up to 30-fold change (dark red). Fig. 14B Shows relative to the control, from Fig.14A Fold changes in the expression of an exemplary subset of biomarkers (Rela, Il2, Cxcl3, and Csf3) of RACs were shown. In response to treatment of reactive astrocytes (RAC+BGE-105 [250 nM]) with a higher dose of BGE-105 (250 nM), downregulation of 2 pro-inflammatory genes (Rela [decreased by 182%], Cxcl3 [decreased by 55%]) and upregulation of 2 anti-inflammatory / neurotrophic genes (Il2 increased by 184%], Csf3 [increased by 16%]) were observed.

[0544] Fig.15 BGE-105 was shown to improve insufficient glutamate clearance in RAC astrocytes. Glutamate clearance in RAC astrocytes (RAC) was significantly reduced, which may contribute to glutamate excitotoxicity in neurodegeneration / aging. BGE-105 (RAC+BGE-105 [10nM, 50nM, 250nM]) and apelin (APL) (RAC+APL [50nM]) significantly and statistically increased the percentage of glutamate uptake.

[0545] Figure 16A-16BReactive astrocytes treated with BGE-105 showed increased cell viability after challenge with RAC conditioned medium. The percentage change in viability of astrocytes relative to RAC in control (RAC) and BGE-105 (RAC+BGE105 [50 nM, 250 nM]) and apelin (APL) (RAC+APL [50 nM]) treated groups is shown. Fig.16A It was shown that BGE-105 (RAC+BGE105 [250 nM]) significantly and statistically increased cell viability by about 30% compared to RAC.

[0546] To understand whether BGE-105 could act directly on neurons to exert its protective effects, or whether any protective mechanism would require acting on astrocytes, cells were treated with exogenous BEG-105 (i.e., BGE-105 was added directly to neurons in addition to the toxic RAC medium). This approach is in contrast to typical experimental conditions, in which BGE-105 is added to astrocytes while they are being stimulated with RAC components. Fig. 16B As shown, treatment with exogenous BGE-105 (RAC+ex105[50nM]) did not provide statistical changes in cell viability relative to RAC. The results indicate that exogenous BGE-105 does not directly protect neurons from RAC-induced neurotoxicity.

[0547] In summary, these data indicate that apelin receptors are abundantly expressed in astrocytes and activated by BGE-105. The data indicate that BG5-105 attenuates the astroglial inflammatory response after mouse astrocytes are exposed to RAC. In addition, the data indicate that BGE-105 limits RAC-induced astroglial inflammation by modifying NF-κB signaling. Overall, the data indicate that BGE-105 improves the cellular function of astrocytes and protects neurons from RAC-induced cell death. The results indicate that BG5-105 effectively reduces the neurotoxicity of degenerated or aged astrocytes and can therefore be used to rescue BBB permeability in patients suffering from or suspected of suffering from neurodegenerative diseases.

[0548] 6.7 Example 7: Effects of BGE-105 on neuroinflammation in aged mice in a traumatic brain injury (TBI) model

[0549] This study evaluated the effects of BGE-105 on attenuating the effects of CNS damage and inflammation and accelerating functional recovery in a mouse model of TBI.

[0550] BGE-105 was orally administered to aged mice at different possible doses for a period of time before and / or after a controlled cortical impact (a mouse TBI model); during the experimental period, the mice underwent various behavioral assessments to understand whether BGE-105 treatment would affect motor and cognitive functions known to be impaired by TBI. Afterwards, the brains of the mice were analyzed for neuroinflammatory markers by various histological and biochemical methods.

[0551] result

[0552] Treatment with BGE-105 caused a reduction in neuroinflammatory markers and suppressed neuroinflammation in aged mice with TBI.

[0553] 6.8 Example 8: Effects of BGE-10S on Neural Stem Cells from Patient-Derived Induced Pluripotent Stem Cells (iPSCs)

[0554] This study evaluated the effects of BGE-105 (or mediators from other brain cells such as microglia and astrocytes exposed to BGE-105) in human patients, iPSC-derived neurons improved various functions associated with beneficial neuronal outcomes, including those listed below:

[0555] i. Proliferation and survival

[0556] ii. Autophagy

[0557] iii. Synaptic plasticity

[0558] iv. ROS generation

[0559] result

[0560] Treatment with BGE-105 improves overall survival, differentiation, and cellular function of stem cell-derived neurons in humans. The improvements were assessed by comparison with neural stem cells derived from iPSCs that were not treated with BGE-105 (control).

[0561] 6.9 Example 9: Effects of BGE-105 on astrocyte apelinergic signaling and motor neuron function in an ALS mouse model

[0562] This study evaluated the effects of BGE-105 on astrocyte apelin peptidergic signaling and motor neuron function. G93A Anti-inflammatory and neurotrophic apelin peptidergic signaling events in mouse astrocytes. This study characterizes the effects of BGE-105 on wild-type and SOD1-derived G93AIn vitro effects on primary mouse astrocytes. This included measuring effects on gene expression, inflammatory cytokine secretion, and glutamate uptake. In addition, this study used SOD1 G93A Astrocytes and wild-type motor neurons were co-cultured to determine the role of apelin signaling in the rescue of neuronal survival and function using measurements of viability, neuronal homeostasis, and morphological endpoints.

[0563] The aim of this study was to demonstrate the effects of BGE-105 on astrocyte apelinergic signaling and motor neuron function. Astrocyte reactivity plays a causal role in motor neuron loss, decreased motor function, and shortened lifespan in a preclinical ALS mouse model (REF). Astrocyte dysfunction in ALS is driven by microglia-derived proinflammatory signals including IL-1α, TNFα, and C1q. In turn, this IL-1α / TNFα / C1q stimulation promotes proinflammatory NFκB-mediated signaling, leading to impaired astrocyte support and ultimately neuronal death. BGE-105 is a potent agonist of the apelin receptor APJ, which is known to regulate NFκB activation (REF), and attenuates inflammatory damage in vitro and in vivo. This study demonstrates that in SODl from ALS, astrocytes are activated and activated. G93A In primary cultures of astrocytes and neurons in mouse models, BGE-105 attenuated astroglial IL-1α / TNFα / C1q-mediated neurotoxicity and improved overall astrocyte and neuronal function. These data provide a mechanistic rationale for the effects of enhanced apelin peptidergic signaling in the CNS on BGE-105 treatment in a well-validated ALS mouse model.

[0564] 6.10 Example 10: Study of primary SOD1 G93A Anti-inflammatory and neurotrophic apelin peptidergic signaling events in mouse astrocytes.

[0565] This study characterized the effects of BGE-105 on wild-type and SOD1 G93A In vitro effects on primary mouse astrocytes. This included measuring effects on gene expression, inflammatory cytokine secretion, and glutamate uptake.

[0566] method

[0567] In this study, SOD1 G93AMouse model Primary mouse astrocyte and neuronal cultures were generated as previously described (Lundquist et al. 2019. Exercise induces region-specific remodeling of astrocyte morphology and reactive astrocyte gene expression patterns in male mice. Journal of Neuroscience Research, 97(9), 1081-1094), with some modifications. Briefly, SOD1 was expressed from PND0-4 (postnatal) G93A+ The entire spinal cord was removed from pups and wild-type littermates. The spinal cords were enzymatically and mechanically treated, and astrocytes were positively selected using MACS (magnetic activated cell sorting) with ACSA2-conjugated magnetic beads (Miltenyi Biotec) (REF). Astrocytes were cultured in T75 flasks under serum-free conditions for 7 days and then divided into 6-well or 12-well plates for final experiments.

[0568] First, the wild-type and SOD1 G93A Astrocytes of mice were studied to compare transcriptomic profiles and cytokine release profiles. Next, vehicle or 250nM BGE-105 (dissolved in 0.01% DMSO in saline) was added to wild-type or SOD astrocytes for 24 hours. Then, either of the two culture media was collected, and the cells were lysed for transcriptomic and protein analysis, or the cells were incubated with 100 μM glutamate in HBSS (Hank's balanced salt solution) for three hours to monitor glutamate uptake using a commercially available colorimetric kit (Sigma). The release of cytokines and neurotrophic factors from ACM (astrocyte conditioned medium) was analyzed using a mouse proteome analyzer array (R&D Systems) and a commercially available ELISA (for selecting cytokines from protein arrays). Astrocyte gene expression was analyzed by RT-qPCR, and astrocyte protein expression was analyzed by Western blotting as previously described (Lundquist et al., 2022).

[0569] result

[0570] SOD1 treated with BGE-105 G93A Astrocytes released fewer proinflammatory cytokines into astrocyte-conditioned medium (ACM). These cytokines were regulated by NF-κB stabilization and were in response to SOD1 in untreated and BGE-105-treated cells. G93AGenetic analysis of astrocytes revealed changes in NF-κB signaling activity, resulting in less cytokine production.

[0571] 6.11 Example 11: Effect of Apelin Signaling on Rescue of Neuronal Survival and Function.

[0572] In this study, SOD1 G93A Co-culture experiments with astrocytes and wild-type motor neurons were performed to determine the role of apelin signaling in the rescue of neuronal survival and function. Co-culture experiments were performed by measuring viability, neuronal homeostasis, and morphological endpoints.

[0573] 7. Methods

[0574] Neurons were isolated from mouse spinal cords as previously described in Example 12 above. Non-astroglial flow-throughs following column-based cell separation (MACS) were collected and cultured in serum-free, neuron-selective medium in 12-well or 96-well plates for at least 14 days prior to the experiment. To specifically select spinal motor neurons, wild-type C57BL / 6J mouse PND0 or adult spinal cords were processed using published methods (Beaudet, 2015).

[0575] Neurons were cultured for at least 14 days before the start of the experiment. First, ACM from wild-type or SOD astrocytes exposed to 250nM BGE-105 (or vehicle) was added to neurons seeded in 96-well plates for 6, 12, 24, or 48 hours, followed by analysis of neuronal survival using the ApoTox-Glo Triplex kit (Promega) to determine the optimal exposure time. Next, ACM from wild-type or SOD astrocytes exposed to 250nM BGE-105 (or vehicle) was added to neurons seeded in 12-well plates, and neuronal protein lysates were then collected to analyze synaptic protein markers synaptophysin and PSD-95 by Western blotting as previously described (Lundquist et al., 2022).

[0576] Next, SODl G93Aor wild-type astrocytes were added to neurons seeded in 96-well plates and grown together for the next 10 days. Independent astrocyte-neuron co-cultures were treated with 250 nM BGE-105 continuously, fixed with 4% PFA (paraformaldehyde) on the first, third, fifth and tenth days, stained with fluorescent antibodies against synaptic and axonal markers (synaptic markers-synaptophysin, PSD95, Bassoon, Homer; axonal markers-MAP2, β-III tubulin), and imaged using a high-content imager (CX5, Thermofisher) to assess morphological endpoints.

[0577] The overall male / female ratio of mouse litters could not be controlled. To account for the possible role of sex as a biological variable, the sex of all mice in each litter was tracked to ensure that equal numbers of male and female pups of either genotype were used in experiments across litters. All primary mouse cell culture experiments were repeated using at least three independent preparations from several litters.

[0578] result

[0579] Use SOD1 G93A ACM treatment of neurons resulted in higher cell death, whereas pretreatment with BGE-105 resulted in SOD1 G93A Astrocytes attenuated cell death. In addition, when wild-type neurons were treated with SOD1 G93A BGE-105 preserves synaptic protein expression and neuronal structural integrity when cultured together with astrocytes.

[0580] 7.1 Example 12: Effect of BGE-105 in ALS Mouse Model

[0581] This study investigated the effects of BGE-105 in a mouse model of ALS. This study determined the pharmacokinetic / pharmacodynamic profile of intracerebroventricularly (ICV) administered BGE-105 in wild-type C57BL / 6J mice. Multiple doses across multiple time points were tested to characterize the drug within the central nervous system (CNS). SOD1 was delivered using an Alzet minipump connected to an ICV cannula. G93A Mice were administered BGE-105.

[0582] Astrocyte reactivity driven by IL-1α / TNFα / C1q-mediated neurotoxicity contributes to spinal motor neuron death and motor dysfunction in a mouse model of ALS. G93ATriple transgenic knockout of IL-1α / TNFα / C1q in a mouse model significantly extended lifespan and motor neuron survival (Guttenplan, 2020). Preliminary in vitro evidence, as shown in Example 6, suggests that administration of BGE-105 attenuates IL-1α / TNFα / C1q-mediated astrocyte reactivity, which is prominently manifested by proinflammatory cytokine release and impaired glutamate cycling. This study first validated the intracerebroventricular administration of BGE-105 to wild-type mice to determine CNS-specific drug properties and guide optimal dose selection. Next, in the SOD1 of ALS G93A The efficacy of CNS delivery of BGE-105 to minimize motor neuron death, improve motor function, and extend lifespan was tested in a mouse model. For in vivo experiments, an Alzet minipump was used to deliver SOD1 G93A Mice were administered BGE-105. Alzet minipumps were implanted at 4 months of age and BGE-105 was administered continuously for 1 month.

[0583] Analysis measuring locomotion, behavior, and survival endpoints. Brain, spinal cord, and skeletal muscle were dissected postmortem for histochemical and transcriptomic analyses to determine target engagement and mechanism of action.

[0584] Results: SOD1 in ALS G93A In a mouse model, treatment of mice with BGE-105 minimized motor neuron death, improved motor function and prolonged lifespan.

[0585] Determine the pharmacokinetic and pharmacodynamic properties of intracerebroventricular (ICV) administration of BGE-105 in wild-type mice. BGE-105 was administered to the lateral ventricles of 4-month-old C57BL / 6J mice via stereotactic surgery with 1, 5 or 25 mg BGE-105 / kg body weight, followed by tissue collection at 1, 8 and 24 hours (n=27 mice [3 mice / dose / time point]). Alternative delivery routes (such as direct CNS delivery of BGE-105) were used to achieve adequate exposure throughout the spinal cord. Blood and CSF (cerebrospinal fluid) were extracted by retroorbital bleeding and cerebellomedullary cistern puncture, respectively, and then ice-cold saline was perfused into mice, and the whole brain and spinal cord were removed, divided into two equal parts and quickly frozen in liquid nitrogen. Whole blood was spun to separate and collect plasma; plasma, CSF, and half of each tissue sample from each mouse were sent to Quintara Discovery, Inc (Hayward, CA) for drug exposure measurements by LC-MS (liquid chromatography-mass spectrometry). The other half of the brain and spinal cord were processed for protein analysis by mechanical digestion in N-PER extraction reagent (Thermofisher) with Halt protease / phosphatase inhibitors (Thermofisher), followed by Western blot analysis of phosphorylated AKT (Gi -PCR activity) to determine APJ engagement throughout the CNS.

[0586] Study on the effect of ICV administration of BGE-105 on SOD1 in ALS G93A Efficacy in mouse models. Continuous CNS delivery of BGE-105 was performed using an Alzet minipump connected to an intraventricular cannula, similar to previous studies on APJ agonism in the CNS (Zhu et al. 2020. Apelin-36 mediates neuroprotective effects by regulating oxidative stress, autophagy and apoptosis in MPTP-induced Parkinson's disease model mice, Brain Research, Vol. 1726, 2020, 146493, ISSN 0006-8993). G93A Mice (C57BL / 6J background; strain number 004435, Jackson Laboratory) (n = 12 / group; 6 males, 6 females) were randomized to vehicle or two separate treatment groups and then underwent baseline motor behavior assessments, including open field, grid hanging, balance beam walking, and accelerating rotarod. Next, bilateral cannulas were targeted to the lateral ventricles using mouse stereotaxic coordinates (Paxinos and Franklin, 2019) and fixed to the skull with dental cement. Alzet minipumps (model 2004) were filled with two doses (low and high doses, based on the results of the above PK / PD studies) of BGE-105, or the vehicle was implanted subcutaneously along the back and connected to the cannula via a catheter.

[0587] Following surgical recovery, mice were housed individually in cages with wireless, continuously monitored running wheels for 28 days. Following 28 days of BGE-105 administration, mice were subjected to repeated motor behavior assessments (open field, grip strength, balance beam walking, and accelerating rotarod) and then euthanized for molecular and histological evaluations.

[0588] For molecular endpoints (n = 6 / group; 3 males, 3 females), mice were anesthetized with isoflurane, perfused with ice-cold saline and cervical dislocation, and then their whole brains and spinal cords were removed independently, weighed, and snap-frozen in liquid nitrogen. Samples were processed for protein analysis by mechanical digestion in N-PER extraction reagent (Thermofisher) with Halt protease / phosphatase inhibitors (Thermofisher) and then analyzed by Western blotting. Markers of APJ activation (phosphorylated AKT), synaptogenesis (synaptophysin, PSD95), and neuroinflammation (IBA1, GFAP) were assessed in the brain and spinal cord.

[0589] For histological evaluation (n=6 / group; 3 males, 3 females), mice were anesthetized with isoflurane and perfused with ice-cold saline followed by ice-cold 4% PFA. The whole brain and spinal cord were removed independently and transferred to 4% PFA to fix overnight at 4°C, followed by incubation in 30% sucrose at 4°C until the tissue sank. Separately, the gastrocnemius muscle was dissected and transferred to ice-cold 1% PFA at 4°C overnight, followed by incubation in 30% sucrose until the tissue sank.

[0590] Tissues were snap frozen in chilled isopentane and stored at -80°C, then embedded in OCT and sectioned on a cryostat for histological analysis. Whole brain sections were sectioned at 30 μm thickness, transferred to gelatin-coated slides, and stored at -80°C until further use. Spinal cord and gastrocnemius muscle sections were sectioned at 14 μm thickness, transferred to gelatin-coated slides, and stored at -80°C until further use.

[0591] Brain slices were used for measurements of astrocyte and microglial reactivity (GFAP and IBA1, respectively) as previously described (Lundquist et al., 2019) and for measurements of glial and microglial reactivity (GFAP and IBA1, respectively) as previously described ( et al., 2011. Corticospinal motor neurons and related subcerebral projection neuronsundergo early and specific neurodegeneration in hSOD1G 93 A transgenic ALSmice. J Neurosci. 2011 Mar 16;31(11):4166-77; Lundquist et al., 2022) Measurement of motor cortical cell density via Nissl staining, imaging, and relative glial reactivity and cortical thickness by counter blinded to treatment conditions.

[0592] Spinal cords were stained for motor neuron density via Nissl staining as previously described (Lundquist et al., 2022). Briefly, frozen sections were rehydrated, immersed in 0.1% cresyl violet solution for 5 minutes, and immediately washed in distilled water, then dehydrated in 100% ethanol and cleared in xylene. Slides were covered with coverslips, sealed, imaged, and analyzed by a counter blinded to conditions to assess motor neuron density.

[0593] As previously described (Guttenplan et al. 2020. Knockout of reactive astrocyte activating factors slows disease progression in an ALS mouse model. Nat Commun 11, 3753), with some modifications, gastrocnemius sections were processed using fluorescent immunohistochemistry to obtain the innervation of the neuromuscular junction. In brief, the sections were permeabilized with 0.1% Trition (Tris-buffered saline) in TBS at room temperature for 1 hour, followed by blocking in 10% normal goat serum for 2 hours at room temperature, and incubated overnight at 4°C with neurofilament heavy chain (NF-H) antibody (rabbit anti-NF-H, Abcam). The sections were washed in TBS and then incubated with goat anti-rabbit fluorescent secondary antibody and Alexa 488-conjugated α-bungarotoxin (Invitrogen) at room temperature for 2 hours. The sections were washed in TBS, covered with coverslips with VECTASHIELD nuclear counting staining mounting media (Vector), imaged, and analyzed by a counter to assess the innervation of the neuromuscular junction by blinding the conditions.

[0594] Mice were examined for health daily and weighed three times a week to assess animal welfare and survival, and catheter patency was checked daily.

[0595] result

[0596] The data demonstrate the feasibility and technical merits of developing the drug BGE-105 (and related compounds) as a safe and effective treatment to slow the progression and reduce the severity of amyotrophic lateral sclerosis (ALS).

[0597] 7.2 Example 13: Effects of BGE-105 on human immune biomarkers

[0598] The study described herein characterizes the effect of BGE-105 on human immune biomarkers in a Phase 1b clinical trial for the treatment of muscular atrophy. BGE-105 is a highly selective, potent, orally available small molecule agonist of the apelin receptor APJ. In healthy volunteers aged 65 years or older, after 10 days of strict bed rest, BGE-105 treatment resulted in statistically significant prevention of muscular atrophy relative to placebo. The data show changes in gene expression of immune biomarkers in response to BGE-105 treatment.

[0599] Study Design and Results

[0600] This double-blind, placebo-controlled trial evaluated the safety and pharmacodynamics of BGE-105. Twenty-one volunteers received an infusion of BGE-105 or a placebo while on bed rest for 10 days.

[0601] Biological samples from treated and placebo groups were collected on days 6 and 11. Fold changes in immune biomarker expression were measured and are summarized in Table 2.

[0602]

[0603] The drug was well tolerated in this study. These human data support the use of BGE-105 as a mechanism for restoring the BBB to reduce inflammation and extend to treating, preventing or alleviating disorders or conditions associated with BBB permeability, such as neurodegenerative diseases or related conditions, including Alzheimer's disease (AD), vascular dementia (VaD), delirium, cognitive impairment, Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), stroke, Huntington's disease (HD), multiple sclerosis (MS) and traumatic brain injury (TBI).

[0604] 8. EQUIVALENTS AND INCORPORATION BY REFERENCE

[0605] While the present invention has been particularly shown and described with respect to a preferred embodiment and various alternative embodiments, it will be understood by those skilled in the relevant art that various changes in form and details may be made therein without departing from the spirit and scope of the invention.

[0606] All references, issued patents, and patent applications cited in the text of this specification are hereby incorporated by reference in their entirety for all purposes.

Claims

1. A method of treating a disease or disorder associated with blood-brain barrier (BBB) ​​permeability in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an apelin receptor.

2. The method of claim 1, wherein the condition or disorder is associated with increased BBB permeability.

3. The method of claim 1 or 2, wherein the disease is a neurodegenerative disease.

4. The method of claim 3, wherein the neurodegenerative disease is selected from Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), stroke, Huntington's disease (HD), multiple sclerosis (MS).

5. The method of any one of claims 1 to 3, wherein the disorder is cognitive impairment (eg, postoperative cognitive dysfunction, age-related cognitive impairment).

6. The method of any one of claims 1 to 2, wherein the disorder is delirium.

7. The method of claim 6, wherein the subject suffers from intensive care unit (ICU) delirium, postoperative delirium, delirium caused by trauma, or delirium caused by trauma from hip fracture or cardiovascular surgery.

8. The method of any one of claims 1 to 2, wherein the disorder is chronic or progressive dementia.

9. The method of any one of claims 1 to 2, wherein the disorder is traumatic brain injury (TBI).

10. The method of any one of claims 1 to 9, wherein the subject suffers from acute cognitive impairment (eg, cognitive impairment associated with acute inflammation).

11. The method of claim 10, wherein the subject suffers from postoperative cognitive dysfunction (POCD).

12. The method of any one of claims 1 to 11, wherein the subject is on a ventilator.

13. The method of any one of claims 1 to 2, wherein the disorder is neurodegeneration.

14. The method of any one of claims 1 to 13, wherein the subject suffers from neuroinflammation.

15. The method of any one of claims 1 to 14, wherein the subject is a human and is at least 40 years old.

16. The method of claim 15, wherein the subject is at least 50 years old.

17. The method of claim 16, wherein the subject is at least 60 years old.

18. The method of claim 17, wherein the subject is at least 65 years old.

19. The method of claim 18, wherein the subject is at least 70 years old.

20. The method of claim 19, wherein the subject is at least 75 years old.

21. The method of claim 20, wherein the subject is at least 80 years old.

22. The method of any one of claims 1-27, wherein the subject has or is identified as having low circulating levels of apelin.

23. The method of any one of claims 1 to 22, wherein the apelin receptor agonist has 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, in: R 1 is unsubstituted pyridyl, pyridone or pyridine N-oxide, or is substituted by 1, 2, 3 or 4 R 1a a pyridyl, pyridonyl or pyridine N-oxide substituted with a substituent; R 1a is independently selected from the group consisting of -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)-(heterocyclyl) or heterocyclyl, wherein the heterocyclyl of -C(=O)-(heterocyclyl) or the heterocyclyl is a 3- to 7-membered ring containing 1, 2 or 3 heteroatoms selected from N, O and S; R 2 is selected from -H and C1-C4 alkyl, or, in the compound of formula II, is absent; R 3 Selected from unsubstituted C1-C 10 Alkyl, 1, 2 or 3 R 1a Substituent-substituted C1-C 10 Alkyl, formula -(CR 3b R 3c )-Q, a group of formula -NH-(CR 3b R 3c )-Q group, the formula -(CR 3b R 3c )-C(=O)-Q, a group of the formula -(CR 3d R 3e )-(CR 3f R 3g )-Q group, the formula -(CR 3b =CR 3c )-Q and groups of the 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 are substituted, wherein 1, 2 or 3 ring members are heteroatoms selected from N, O and S; R 1a is independently selected from the group consisting of -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 at each occurrence; 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; 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; 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; R 3h is independently selected from the group consisting of -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 at each occurrence; Q is a monocyclic or bicyclic C6-C 10 aryl, monocyclic or bicyclic heteroaryl with 5 to 10 ring members containing 1, 2 or 3 heteroatoms selected from N, O or S, C3-C8 cycloalkyl, or 3 to 7 membered heterocyclyl containing 1, 2 or 3 heteroatoms selected from N, O or S, wherein the C6-C8 10 The aryl, heteroaryl, cycloalkyl and heterocyclyl groups are unsubstituted or substituted with 1, 2, 3 or 4 R Q Substituent substitution; RQ 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, and the Q heterocyclyl may be substituted with 1 oxo RQ substituent; R 4 Selected from monocyclic or bicyclic C6-C 10 aryl, monocyclic or bicyclic heteroaryl having 5 to 10 ring members containing 1, 2 or 3 heteroatoms independently selected from N, O and S, and monocyclic or bicyclic heterocyclyl 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 The aryl group, the heteroaryl group or the heterocyclic group is unsubstituted or substituted by 1, 2 or 3 R 4a Substituent substitution; R 4a is independently selected from the group consisting of -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 heterocyclyl R 4 may be further substituted with 1 oxo substituent; and Further wherein: If R 4 is an unsubstituted or substituted benzene ring and R 3 It is the formula -(CR 3b =CR 3c )-Q, then at least one of the following is true: a) R 4 is substituted with 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 is not 2-pyridyl; or f)R 4 Substituted with two or more -O-(C1-C6 alkyl) groups.

24. The method of claim 23, wherein R 1 is unsubstituted pyridyl or substituted with 1 or 2 R 1a The substituent is substituted pyridinyl.

25. The method of claim 23 or 24, wherein R 1a is independently selected at each occurrence 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 radical of the formula When drawn across a bond, the symbol Indicates the point of attachment to the rest of the molecule.

26. The method of any one of claims 23 to 25, wherein R 1 Selected from When drawn across a bond, the symbol Indicates the point of attachment to the rest of the molecule.

27. The method of any one of claims 23 to 26, wherein R 2 Yes -H.

28. The method of any one of claims 23 to 27, wherein R 4 is phenyl, azophenyl, pyrimidinyl, isoxazolyl, indolyl, naphthyl or pyridinyl, any of which may be unsubstituted or substituted with 1, 2 or 3 R 4a Substituent substitution.

29. The method of claim 28, wherein R 4 Is 1 or 2 R 4a Substituents: phenyl substituted.

30. The method of claim 29, wherein the 1 or 2 R 4a The substituent is an -O-(C1-C2 alkyl) group.

31. The method of any one of claims 23 to 30, wherein R 4a is independently selected at each occurrence from -CH3, -F, -Cl, -Br, -CN, -CF3, -OCH3, -OCHF2, -OCH2CH3, -C(=O)OCH3, -C(=O)CH3 or -N(CH3)2.

32. The method of any one of claims 23 to 31, wherein R 3 Select from the formula -(CR 3b R 3c )-Q, a group of formula -NH-(CR 3b R 3c )-Q group, the formula -(CR 3b R 3c )-C(=O)-Q, a group of the formula -(CR 3d R 3e )-(CR 3f R 3g )-Q group, the formula -(CR 3b =CR 3c )-Q or a group of the 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 are substituted, 1, 2 or 3 of the ring members are heteroatoms selected from N, O or S.

33. The method of any one of claims 23 to 32, wherein Q is selected from pyrimidinyl, pyridinyl, isoxazolyl, thiazolyl, imidazolyl, phenyl, tetrahydropyrimidinyl, cyclopropyl, cyclobutyl, cyclohexyl, morpholinyl, pyrrolidinyl, pyrazinyl, imidazo[1,2-a]pyridinyl, pyrazolyl or oxetanyl, any of which may be unsubstituted or substituted with 1, 2 or 3 R Q Substituent substitution.

34. The method of any one of claims 23 to 33, wherein Q is a monocyclic heteroaryl having 5 or 6 ring members containing 1 or 2 heteroatoms selected from N, O or S, and Q is unsubstituted or substituted with 1 or 2 RQ substituents.

35. The method of any one of claims 23 to 34, wherein R 3 It is the formula -(CR 3d R 3e )-(CR 3f R 3g )-Q group.

36. The method of any one of claims 23 to 35, wherein R 3 With the following formula When drawn across a bond, the symbol Indicates the point of attachment to the rest of the molecule.

37. The method of any one of claims 23 to 37, wherein the apelin 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-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.

38. The method of claim 37, wherein the apelin 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-pyrimidinyl)-2-butanesulfonamide or a pharmaceutically acceptable salt thereof.

39. The method of any one of claims 1 to 38, wherein the apelin receptor agonist is administered intravenously or intrathecally.

40. The method of any one of claims 1-38, wherein the apelin receptor agonist is administered orally.

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

42. The method of claim 41, wherein the subject is at least 50 years old.

43. The method of claim 42, wherein the subject is at least 60 years old.

44. The method of claim 43, wherein the subject is at least 65 years old.

45. The method of claim 44, wherein the subject is at least 70 years old.

46. ​​The method of claim 45, wherein the subject is at least 75 years old.

47. The method of claim 46, wherein the subject is at least 80 years old.

48. The method of any one of claims 1-47, wherein the apelin receptor agonist is administered daily.

49. The method of any one of claims 1 to 48, wherein the apelin receptor agonist is administered in multiple equally or unequally divided sub-doses.

50. The method of any one of claims 1 to 49, wherein the apelin receptor agonist is administered at different dosing intervals.

51. The method of any one of claims 1 to 50, wherein the apelin receptor agonist is administered at a dose of 200 mg.

52. The method of any one of claims 1 to 51, further comprising assessing cognitive function after administration.

53. The method of claim 52, wherein the cognitive function is assessed at least one day after administration (e.g., at least one week or at least one month after administration).

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