Methods for determiing drug response and disease progression in amytrophic lateral sclerosis

Abstract

Methods for determining ALS patient responsiveness to a Mas agonist, are disclosed. The method includes determining the levels of at least two metabolites selected from the group consisting of citrate, fumarate, aconitate, creatinine and glutamine in a sample collected from an ALS patient who has been administered a Mas Agonist. Methods for determining disease progression in an ALS patient are also provided. The method include measuring the levels of at least two or more of citrate, fumarate, aconitate, creatinine and glutamine in a sample collected from the ALS patient at a time which serves to provide baseline levels and subsequently determining the levels of the at least two or more of citrate, fumarate, aconitate, creatinine and glutamine in a sample collected from the ALS patient, where a decrease in the levels of the at least two or more of citrate, fumarate, aconitate, creatinine and glutamine is indicative of worsening disease progression. The sample can be any biological sample collected from the patient such as a serum sample, muscle biopsy, CSF (cerebrospinal fluid) etc.

Description

[0001] ATTORNEY DOCKET NO. UA 24-001 PCT

[0002] METHODS FOR DETERMINING DRUG RESPONSE AND DISEASE PROGRESSION IN AMYTROPHIC LATERAL SCLEROSIS CROSS REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of and priority to U.S.S.N. 63 / 658,750 filed June 11, 2024 and which is incorporated by referenced herein in its entirety.

[0004] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0005] This invention was made with government support under W81XH-22-1-0074 awarded by the U.S. Army Medical Research and Development Command. The government has certain rights in the invention.

[0006] FIELD OF THE INVENTION

[0007] The disclosed invention is generally in the field of ALS and specifically in the area of methods for determining drug efficacy and disease progression.

[0008] BACKGROUND OF THE INVENTION

[0009] Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that is characterized by the progressive degeneration of motor neurons in the brain and spinal cord. ALS is an orphan disease that is diagnosed in 1-2 individuals per 100,000 each year in most countries and currently, there is no cure for ALS.

[0010] ALS is particularly hard to treat effectively due to the difficulties in diagnosis and heterologous underlying pathophysiology. In some cases, patient with suspected ALS can take up to between 12-18 months to get an accurate diagnosis (Segura et al. 2023; Paganoni et al. 2014). This delay from symptomatic onset to an accurate diagnosis leads to delays in treatment, ultimately affecting therapeutic efficacy as currently available treatments are more effected when started earlier in the disease course (Brooks 1999). The challenges in providing a quick and accurate diagnosis lies in the heterogeneity of symptomatic onset and clinical similarities with other, more common diseases. When diagnosing a patient, clinicians undergo a “rule-out” approach. This method is used mainly due to the lack of reliable biomarkers available.

[0011] The renin angiotensin system (RAS) consists of a family of peptide hormones that act at several G-Protein Coupled Receptors. The pathological arm is known to cause vasoconstriction and increases in OS and inflammation. The regenerative or protective arm (ACE2 / angiotensin (1-7) [A(l-7)] / Mas) induces vasodilation and regeneration and reduces OS, and inflammation. A(l-7) was previously shown to improve survival in a mouse model of ALS. Despite the physiological effects of Mas activation via A(l-7), the translation of these results to treating human diseases has been hampered by the properties of the peptide.

[0012] 45739246.1 1ATTORNEY DOCKET NO. UA 24-001 PCT

[0013] There are no known reliable biomarkers for ALS progression and for biomarkers of effect for Mas agonists. Given the actions of Mas and the hypermetabolic state found in ALS patients, unbiased biomarkers are needed to determine patient response to Mas agonists as well as disease progression.

[0014] It is an object of the present invention to provide methods for determining responsiveness to a Mas agonist in a ALS patient.

[0015] It is an also objection of the present invention to provide methods for determining disease progression in an ALS patient.

[0016] BRIEF SUMMARY OF THE INVENTION

[0017] Methods for determining ALS patient responsiveness to a Mas agonist, are disclosed. The method includes determining the levels of at least two metabolites selected from the group consisting of citrate, fumarate, aconitate, creatinine and glutamine in a sample collected from an ALS patient who has been administered a Mas Agonist. In some forms the Mas Agonist is RASRxl902.

[0018] In some forms, the method comprises determining the levels of two or more of citrate, fumarate, aconitate, creatinine and glutamine in a sample collected from the ALS subject prior to administration of the Mas agonist.

[0019] In some forms, the method comprises determining the levels of at least two or more of citrate, fumarate, aconitate, creatinine and glutamine in a sample collected from the ALS subject after administration of the Mas agonist. A patient is considered responsive to treatment with the mas agonist where there is no decrease in the levels of the at least two or more of citrate, fumarate, aconitate, creatinine and glutamine following treatment with the Mas agonist i.e., where the Mas agonist stabilizes the levels of the at least two or more of citrate, fumarate, aconitate, creatinine and glutamine.

[0020] The sample can be any biological sample collected from the patient such as a serum sample, muscle biopsy, CSF (cerebrospinal fluid) etc.

[0021] Methods for determining disease progression in an ALS patient are also provided. The method include measuring the levels of at least two or more of citrate, fumarate, aconitate, creatinine and glutamine in a sample collected from the ALS patient at a time which serves to provide baseline levels and subsequently determining the levels of the at least two or more of citrate, fumarate, aconitate, creatinine and glutamine in a sample collected from the ALS patient, where a decrease in the levels of the at least two or more of citrate, fumarate, aconitate, creatinine and glutamine is indicative of worsening disease progression.

[0022] 45739246.1 2ATTORNEY DOCKET NO. UA 24-001 PCT

[0023] In some forms, a 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 or 0.9 fold decrease in the levels of at least two or more of citrate, fumarate, aconitate, creatinine and glutamine in a sample collected from the ALS patient

[0024] In some forms, the subject has not been administered any therapeutic agent to slow down disease progression.

[0025] Additional advantages of the disclosed method and compositions will be set forth in part in the description which follows, and in part will be understood from the description, or can be learned by practice of the disclosed method and compositions. The advantages of the disclosed method and compositions will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention as claimed.

[0026] BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The accompanying drawings illustrate several embodiments of the disclosed method and compositions and together with the description, serve to explain the principles of the disclosed method and compositions.

[0028] FIG. 1 is a line graph showing RASRxl902 protects against muscle function loss. SOD1 mice were treated one daily with 2 mg / kg RASRxl902. Rotarod measurements started after symptom onset. Treatment with RASRxl902 delayed loss of muscle function.

[0029] FIG. 2 is a collection of graphs showing that the depletion of metabolites as disease progresses correlate with clinical scores.

[0030] FIG. 3 is an enrichment analysis of pathways using the metabolites that correlated with disease progression as indicated by the animal’s clinical score.

[0031] FIG. 4 is a heat map showing depletion of metabolites as disease progresses correlate with clinical score.

[0032] FIG. 5 is an enrichment analysis showing depletion of metabolites as disease progresses correlate with clinical score.

[0033] FIG. 6 is a PCA plot of the metabolic profiles between 155 ALS (bulbar onset and limb onset) and 30 healthy controls.

[0034] FIG. 7 is an enrichment analysis using the significantly increased metabolites in plasma from ALS patients compared to healthy control samples. The significantly enriched pathways are relating to bile acid biosynthesis, arginine and proline metabolism, urea cycle, purine metabolism, sphingolipid metabolism, nicotinate and nicotinamide metabolism, and aspartate metabolism.

[0035] 45739246.1 3ATTORNEY DOCKET NO. UA 24-001 PCT

[0036] FIG. 8 is an enrichment analysis using the significantly decreased metabolites in plasma from ALS patients compared to healthy control samples. The significantly enriched pathways are relating to alpha linolenic acid and linoleic acid metabolism, fatty acid biosynthesis, and urea cycle.

[0037] FIGs. 9A and 9B: Heatmap of metabolite changes as disease progresses. The left side indicates a higher clinical score (healthier patients) with decreasing score (worsening prognosis) as the graph moves right. FIG. 9A represents the top lipid changes with disease progresses. FIG. 9B represents the non-lipid metabolites that changes with disease progression.

[0038] FIG. 10: NAD signaling pathway from RNAseq taken from TDP-43 iMNs [[WHAT DOES “IMNS” REFER TO]]. The comparison is between ALS patients vs healthy isogenic controls. Blue represents a predicted downregulation in the pathway while orange represents a predicted upregulation. A green color indicated a measured decrease in the gene while a red indicated a measured increase in the gene.

[0039] FIG. 11: NAD signaling pathway from RNAseq taken from TDP-43 iMNs. The comparison is between ALS patients iMNs treated with RASRxl902 for 24hrs vs untreated ALS iMNs. Blue represents a predicted downregulation in the pathway while orange represents a predicted upregulation. A green color indicated a measured decrease in the gene while a red indicated a measured increase in the gene.

[0040] FIG. 12: The mitochondrial function pathway from RNAseq taken from the motor cortex at symptomatic onset. The comparison is between RASRxl902 treated SOD1G93Amice vs vehicle control treated mice. Blue represents a predicted downregulation in the pathway while orange represents a predicted upregulation. A green color indicated a measured decrease in the gene while a red indicated a measured increase in the gene.

[0041] FIGs. 13A and 13B are tables showing metabolite changes over time within healthy WT mice and SOD1G93A mice (both RASRxl902 treated and vehicle treated controls). Metabolites are divided up by sub pathways glycolysis and TCA cycle. Five metabolites significantly increase in WT as the animals age. The vehicle treated animals have a depletion in 11 of the 14 metabolites of interest. RASRxl902 treated mice had a stabilization in their metabolite levels with time.

[0042] FIGs. 14A and 14B are tables showing metabolite changes over time within healthy WT mice and SOD1G93A mice (both RASRxl902 treated and vehicle treated controls). Metabolites are divided up by sub pathways long chain fatty acids and fatty acid metabolism. Two metabolites significantly increase in WT as the animals age. The vehicle treated animals have a

[0043] 45739246.1 4ATTORNEY DOCKET NO. UA 24-001 PCT

[0044] depletion in 8 of the 11 metabolites of interest. RASRxl902 treated mice had a stabilization in their metabolite levels with time.

[0045] FIG. 15 shows the mitochondrial function pathway from RNAseq taking from SOD1G93A WBC at symptomatic onset. The comparison is between RASRxl902 treated SOD1G93A mice vs vehicle control treated mice. Blue represents a predicted downregulation in the pathway while orange represents a predicted upregulation. A green color indicated a measured decrease in the gene while a red indicates a measured increase in the gene.

[0046] DETAILED DESCRIPTION OF THE INVENTION

[0047] The disclosed method and compositions can be understood more readily by reference to the following detailed description of particular embodiments and the Example included therein and to the Figures and their previous and following description.

[0048] I. DEFINITIONS

[0049] The term “biological sample” refers to a tissue (e.g., tissue biopsy), organ, cell, cell lysate, or body fluid from a subject. Non-limiting examples of body fluids include blood, urine, plasma, serum, tears, lymph, bile, cerebrospinal fluid, interstitial fluid, aqueous or vitreous humor, colostrum, sputum, amniotic fluid, saliva, anal and vaginal secretions, perspiration, semen, transudate, exudate, and synovial fluid.

[0050] The term “assay” refers to an in vitro procedure for analyzing a sample to determine the presence, absence, or quantity of one or more analytes of interest.

[0051] The term "contact" as used herein in connection with the disclosed methods refers to placement in direct physical association; for example, in solid and / or liquid form. For example, contacting can occur in vitro with one or more primers and / or probes and a biological sample (such as a sample including nucleic acids) in solution.

[0052] The terms “subject”, and “patient” are used interchangeably herein, and refer to a mammal, including, but not limited to, humans, rodents such as mice and rats, and other laboratory animals.

[0053] The term “biomarker” refers to an organic molecule produced by an organism that is indicative or correlative of a disease state. Biomarkers include, but are not limited to protein, metabolites, post-translationally modified proteins, etc.

[0054] IL METHIDS OF DETERMINING RESPONSIVENESS TO MAS AGONIST

[0055] The disclosed methods are based on the determination of biomarkers that are indicative of drug efficacy, in ALS patients.

[0056] According to the FDA, a biomarker is defined as a characteristic that is measured as an indicator of either normal biological processes, pathogenic processes, or biological response to 45739246.1 5ATTORNEY DOCKET NO. UA 24-001 PCT

[0057] an exposure or intervention, including therapeutic interventions. Biomarkers can range depending on the outcome of interest, including risk biomarkers, diagnostic biomarkers, prognostic biomarkers, and safety biomarkers. To develop a reliable biomarker, the biomarker should be both sensitive and specific to the disease of interest (Strimbu and Tavel 2010). An ideal biomarker would be one with the ability to bridge across species, allowing preclinical measures to predict clinical outcomes. Biomarkers play an integral role in the drug development process, improving study endpoints, stratifying patients for precision medicine, and demonstrating target engagement for drug studies (McMackin et al. 2023; Strimbu and Tavel 2010).

[0058] Methods for determining ALS patient responsiveness to a Mas agonist such as a Mas receptor agonist, based on two or more biomarkers, are disclosed.

[0059] The method includes determining the levels of at least two metabolites selected from the group consisting of citrate, fumarate, aconitate, creatinine and glutamine in a sample collected from an ALS patient who has been administered a Mas receptor Agonist.

[0060] A wide range of physiological and pathophysiological conditions are related to the reninangiotensin system (RAS), which is an important regulator of arterial blood pressure and involves the formation and actions of several angiotensin peptides. The angiotensin peptides and the related enzymes and receptors play key roles in the cardiovascular system, the renal system, the hematopoietic system, the hepatobiliary system, the pulmonary system, the gastrointestinal system, the nervous system, and in many other critical physiological and pathophysiological pathways, in part, through stimulation of stem cell activity. Renin acts on angiotensinogen to form angiotensin I (Angl), which is cleaved by angiotensin converting enzyme (ACE) to form the octapeptide, angiotensin II (Angll), and by neutral endopeptidases to form the heptapeptide, angiotensin (Ang) (1-7), which is also produced from Angll via cleavage by ACE2. The three G-protein coupled receptors (GPCR) that mediate many of the actions of the angiotensin peptides are the Angll type 1 receptor (AT1R), the Angll type 2 receptor (AT2R), and the Mas receptor (Mas) known as the native receptor for Ang(l-7). The native receptor for Ang(l-7) is the GPCR, Mas. “Mas agonist” is used herein interchangeably with “Mas Receptor Agonist”.

[0061] In some forms the Mas receptor Agonist is RASRxl902. RASRxl902 is disclosed in U.S. Patent No. 10301298 (compound 7), whose structure is shown below.

[0062] 45739246.1 6ATTORNEY DOCKET NO. UA 24-001 PCT

[0063]

[0064] ' (RASRxl902).

[0065] However, the Mas agonist can be any of the Mas receptor agonist compounds disclosed in U.S. Patent No. 10301298, incorporated herein by reference, discussed further briefly, below.

[0066] Thus, the Mas receipt agonist can have the general formula 1 and includes salts thereof:

[0067]

[0068] wherein:

[0069] ring A is a five-membered or six-membered heteroaryl or heterocyclyl ring containing either a combination of two non-adjacent nitrogen or oxygen atoms, or a combination of three or four nitrogen or oxygen atoms;

[0070] ring B is a five-membered or six-membered heteroaryl ring that contains at least one nitrogen atom;

[0071] ring C is an optionally substituted aryl ring;

[0072] A1, A2, A3, A4are independently selected from a group consisting of =N — , — C(=O) — , — C(Ra)=, =C(Rb)— , — C(Rc)(Rd)— N(Re)— , — C(Rc)(Rd)— O— , or — [C(Rc)(Rd)]n— with n being 1 or 2;

[0073] X1— X2is (R6)C— N, N— C(R6), N— N, N— O, O— N, N— S or S— N;

[0074] X3is (R7)C=C(R8), O, S, or N(R9);

[0075] Z is O, NH or a bond to R5;

[0076] Raand Rbare independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, halo, hydroxy, hydroxyalkyl, alkoxyalkyl, alkoxy, aryloxy, formyl, acyl, acylamido or carboxy, provided that Raand Rbcan also join to form a ring of up to 6 atoms;

[0077] 45739246.1 7ATTORNEY DOCKET NO. UA 24-001 PCT

[0078] Rcand Rdare independently selected from a group consisting of hydrogen, alkyl, aryl, or heteroaryl, provided that Rcand Rdcan also join to form a ring of up to 6 atoms;

[0079] Reis hydrogen, alkyl, aryl, heteroaryl, acyl, alkoxyacyl, aminoacyl, dialkylaminoacyl, or dialkylaminoacyl;

[0080] R1, R3, R4, R6, R7, and R8are independently selected from a group consisting of hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, arylmethyl, heteroarylmethyl, fluoro, bromo, iodo, cyano, hydroxy, amino, alkylamino, alkoxy, aryloxy, alkoxyalkyl, or aryloxyalkyl;

[0081] R2is alkyl, alkenyl, alkynyl, aryl, heteroaryl, arylmethyl, heteroarylmethyl, alkoxy, trifluoromethoxy, perfluoroalkoxy, aryloxy, alkoxyalkyl, or aryloxyalkyl;

[0082] R5is alkyl, aryl, heteroaryl, hydroxyalkyl, carboxyalkyl, alkoxyalkyl, or aryloxyalkyl; and

[0083] R9is hydrogen, alkyl, aryl, heteroaryl, acyl, alkoxyacyl, aminoacyl, dialkylaminoacyl, or dialkylaminoacyl.

[0084] In some forms, ring A is selected from a group consisting of:

[0085]

[0086] 45739246.1 8ATTORNEY DOCKET NO. UA 24-001 PCT

[0087] wherein:

[0088] R10and Rnare independently selected from a group consisting of hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, halo, hydroxy, hydroxyalkyl, alkoxyalkyl, alkoxy, aryloxy, formyl, acyl, acylamido and carboxy,

[0089] or R10and R11, together with ring A to which they are attached, form:

[0090]

[0091] wherein

[0092] Rf, Rs, Rh, and R1, are independently selected from hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, arylmethyl, heteroarylmethyl, fluoro, bromo, iodo, hydroxy, amino, alkylamino, alkoxy, aryloxy, alkoxyalkyl, and aryloxyalkyl;

[0093] R12is hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, halo, hydroxy, hydroxyalkyl, alkoxyalkyl, alkoxy, aryloxy, or acylamido;

[0094] R13is hydrogen, alkyl, aryl or heteroaryl;

[0095] R14is hydrogen, alkyl, aryl, heteroaryl, acyl, alkoxyacyl, aminoacyl, dialkylaminoacyl, or dialkylaminoacyl; and

[0096] ring B is selected from a group consisting of:

[0097] 45739246.1 9ATTORNEY DOCKET NO. UA 24-001 PCT

[0098]

[0099] or a pharmaceutically acceptable salt thereof.

[0100] Exemplary compounds include, but are not limited to:

[0101] 45739246.1 10ATTORNEY DOCKET NO. UA 24-001 PCT

[0102] /

[0103]

[0104] In some forms, the method comprises determining the levels of two or more of citrate, fumarate, aconitate, creatinine and glutamine in a sample collected from the ALS subject prior to administration of the Mas agonist.

[0105] In some forms, the method comprises determining the levels of at least two or more of citrate, fumarate, aconitate, creatinine and glutamine in a sample collected from the ALS subject after administration of the Mas agonist. A patient is considered responsive to treatment with the Mas agonist where there is no decrease in the levels of the at least two or more of citrate, fumarate, aconitate, creatinine and glutamine following treatment with the Mas agonist i.e., where the Mas agonist stabilizes the levels of the at least two or more of citrate, fumarate, aconitate, creatinine and glutamine.

[0106] In some forms, method includes determining the levels of at least three metabolites selected from the group consisting of citrate, fumarate, aconitate, creatinine and glutamine in a sample collected from an ALS patient who has been administered a Mas Agonist.

[0107] In some forms, method includes determining the levels of at least four metabolites selected from the group consisting of citrate, fumarate, aconitate, creatinine and glutamine in a sample collected from an ALS patient who has been administered a Mas Agonist.

[0108] In some forms, the method includes determining the levels of citrate, fumarate, aconitate, creatinine and glutamine in a sample collected from an ALS patient who has been administered a Mas Agonist.

[0109] 45739246.1 11ATTORNEY DOCKET NO. UA 24-001 PCT

[0110] In some forms, the method includes determining the levels of two or more of citrate, fumarate, aconitate, creatinine and glutamine in a sample collected from the ALS subject prior to administration of the Mas agonist to the ALS patient.

[0111] In some forms, the method includes determining the levels of three or more of citrate, fumarate, aconitate, creatinine and glutamine in a sample collected from the ALS subject prior to administration of the Mas agonist.

[0112] In some forms, the method includes s determining the levels of four or more of citrate, fumarate, aconitate, creatinine and glutamine in a sample collected from the ALS subject prior to administration of the Mas agonist.

[0113] In some forms, the method includes determining the levels of citrate, fumarate, aconitate, creatinine and glutamine in a sample collected from the ALS subject prior to administration of the Mas agonist.

[0114] The sample can be any biological sample collected from the patient such as a serum sample, muscle biopsy, CSF fluid etc.

[0115] Methods of measuring citrate, fumarate, aconitate, creatinine and glutamine levels in a sample are known in the art and can be determined as exemplified in the Examples herein.

[0116] Citrate assay kits are commercially available, for example from Sigma, (Catalog Number MAK333). Citrate levels were determined in ethylenediaminetetraacetic acid (EDTA) anticoagulated plasma samples using nuclear magnetic resonance (NMR) spectroscopy (Garcia, et al., Pract Lab Med. 2021 Mar 18;25:e00213. doi: 10.1016 / j.plabm.2021.e00213). Fumarate assay kits are commercially available; EnzyChrom™ Fumarate Assay Kit is available from https: / / bioassaysys.com / fumarate-assay-kit / . Imam, et al. I Pharm Biomed Anal, 30:146:109-116 discloses Bioanalysis of monomethyl fumarate in human plasma by a sensitive and rapid LC-MS / MS method. See also, Shi, et al., Bioanalysis, 2016;8(7):661-75. Several methods can be used to determine aconitate levels in a sample, such as plasma: Liquid Chromatography-Mass Spectrometry / Mass Spectrometry (LC-MS / MS): This highly specific and sensitive method involves precipitating aconitate from the sample. The precipitated aconitate is then separated using a Liquid Chromatography (LC) column. Finally, the separated aconitate is detected and quantified using tandem Mass Spectrometry (MS / MS) (Rathod, et al.,

[0117] doi: 10.3390 / metabol0030103) Targeted Metabolomics: This approach focuses on identifying and quantifying specific metabolites, including aconitate, in a sample such as plasma. It often utilizes mass spectrometry-based techniques like LC-MS / MS to identify and quantify aconitate. Nanda, et al (Int J Nanomedicine, 2015 10(Spec Iss):93-99 ) disclose measurement of creatinine in human plasma using a functional porous polymer structure sensing motif. Darmoun, 45739246.1 12ATTORNEY DOCKET NO. UA 24-001 PCT

[0118] et al. (Analytical Biochemistry Volume 147, Issue 1, 92-102) discloses a method for measuring both glutamine and glutamate levels and stable isotopic enrichments.

[0119] III. METHODS OF DETERMINING DISEASE PROGRESSION

[0120] Methods for determining disease progression in an ALS patient are also provided.

[0121] The method include measuring the levels of at least two or more of citrate, fumarate, aconitate, creatinine and glutamine in a sample collected from the ALS patient at a time which serves to provide baseline levels and subsequently determining the levels of the at least two or more of citrate, fumarate, aconitate, creatinine and glutamine in a sample collected from the ALS patient, where a decrease in the levels of the at least two or more of citrate, fumarate, aconitate, creatinine and glutamine is indicative of worsening disease progression.

[0122] In some forms, a 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 or 0.9 fold decrease in the levels of at least two or more of citrate, fumarate, aconitate, creatinine and glutamine in a sample collected from the ALS patient compared to their baseline level is indicative of disease progression.

[0123] In some forms, the subject has not been administered any therapeutic agent to slow down ALS disease progression.

[0124] In some forms, the method includes determining the levels of three or more of citrate, fumarate, aconitate, creatinine and glutamine in a sample collected from the ALS patient.

[0125] In some forms, the method includes s determining the levels of four or more of citrate, fumarate, aconitate, creatinine and glutamine in a sample collected from the ALS patient prior to administration of the Mas agonist.

[0126] In some forms, the method includes determining the levels of citrate, fumarate, aconitate, creatinine and glutamine in a sample collected from the ALS patient.

[0127] The disclosed methods will be further understood in view of the following non limiting paragraphs and examples.

[0128] 1. A method for determining ALS patient responsiveness to a Mas agonist, comprising determining the levels of at least two metabolites selected from the group consisting of citrate, fumarate, aconitate, creatinine and glutamine in a sample collected from an ALS patient before and after administration of a Mas Agonist.

[0129] 2. The method of paragraph 1, wherein the Mas agonist is RASRxl902.

[0130] 3. The method of any one of paragraphs 1-2, wherein the sample is selected from the group consisting of a serum sample, muscle biopsy sample, and CSF (cerebrospinal fluid).

[0131] 4. The method of any one of paragraphs 1-3, comprising determining the levels of at least three metabolites selected from the group consisting of citrate, fumarate, aconitate,

[0132] 45739246.1 13ATTORNEY DOCKET NO. UA 24-001 PCT

[0133] creatinine and glutamine in a sample collected from an ALS patient before and after administration of a Mas Agonist.

[0134] 5. The method of any one of paragraphs 1-4, comprising determining the levels of at least four metabolites selected from the group consisting of citrate, fumarate, aconitate, creatinine and glutamine in a sample collected from an ALS patient before and after administration of a Mas Agonist.

[0135] 6. The method of any one of paragraphs 1-5, determining the levels of citrate, fumarate, aconitate, creatinine and glutamine in a sample collected from an ALS patient before and after administration of a Mas Agonist.

[0136] 7. The method of any one of paragraphs 1-6, wherein the patient is considered responsive to treatment with the Mas agonist where there is no decrease in the levels of the at least two or more of citrate, fumarate, aconitate, creatinine and glutamine following treatment with the Mas agonist i.e., where the Mas agonist stabilizes the levels of the at least two or more of citrate, fumarate, aconitate, creatinine and glutamine.

[0137] 8. A method for determining disease progression in an ALS patient comprising measuring the levels of at least two or more of citrate, fumarate, aconitate, creatinine and glutamine in a sample collected from the ALS patient at first a time which serves to provide baseline levels and subsequently determining the levels of the at least two or more of citrate, fumarate, aconitate, creatinine and glutamine in a sample collected from the ALS patient at a second time point, wherein a decrease in the levels of the at least two or more of citrate, fumarate, aconitate, creatinine and glutamine is indicative of worsening disease progression.

[0138] 9. The method paragraph 8, wherein a decrease of at least 0.5 fold in the levels of at least two or more of citrate, fumarate, aconitate, creatinine and glutamine in a sample collected from the ALS patient is indicative of worsening disease progression.

[0139] 10. The method of any one of paragraphs 1-9, comprising administering a Mas agonist to the patient.

[0140] 11. The method of paragraph 10, wherein the Mas agonist is RASRxl902.

[0141] 12. A method for treating an ALS patient, comprising determining the levels of at least two metabolites selected from the group consisting of citrate, fumarate, aconitate, creatinine and glutamine in a sample collected from an ALS patient administering oa Mas Agonist to the patient.

[0142] 13. The method of paragraph 12, wherein the Mas agonist is RASRxl902.

[0143] 14. The method of paragraph 12 or 13, wherein the sample is selected from the group consisting of a serum sample, muscle biopsy sample, and CSF (cerebrospinal fluid).

[0144] 45739246.1 14ATTORNEY DOCKET NO. UA 24-001 PCT

[0145] 15. The method of any one of paragraphs 12-14, comprising determining the levels of at least three metabolites selected from the group consisting of citrate, fumarate, aconitate, creatinine and glutamine in a sample collected from an ALS patient before and after administration of a Mas Agonist.

[0146] 16. The method of any one of paragraphs 12-15, comprising determining the levels of at least four metabolites selected from the group consisting of citrate, fumarate, aconitate, creatinine and glutamine in a sample collected from an ALS patient before and after administration of a Mas Agonist.

[0147] 17. The method of any one of paragraphs 12-16, determining the levels of citrate, fumarate, aconitate, creatinine and glutamine in a sample collected from an ALS patient before and after administration of a Mas Agonist.

[0148] 18. The method of any one of paragraphs 12-17, comprising measuring the levels of the at least two metabolites after administration of the Mas agonist, wherein the patient is considered responsive to treatment with the Mas agonist where there is no decrease in the levels of the at least two or more of citrate, fumarate, aconitate, creatinine and glutamine following treatment with the Mas agonist.

[0149] Examples

[0150] Materials and Methods

[0151] Animals

[0152] SOD1G93A mice were purchased from the Jackson Laboratory (Cat. #002726; B6SJL-Tg (SODl*G93A)lGur)[Bar Harbor, ME, USA]. Wild type (WT) littermates were used as experimental controls. SOD1 G93A mice were randomized into 6 groups with both males and females (n=5 group / sex). Animals were kept on a 12-hour light / dark cycle and food and water were available ad libitum. All procedures were approved by the University of Arizona’ s Institutional Animal Care and Use Committee (IACUC).

[0153] Dosing

[0154] SOD1G93A mice were dosed daily with either vehicle control (saline with Tween- 20, SQ) or RASRxl902 (2mg / kg SQ) starting at 8 weeks of age until necropsied.

[0155] Necropsy

[0156] Animals were necropsied at three different time points throughout times of disease progression; day 75 (presy mptomatically), day 96 (disease onset), and day 117 (disease end stage). Whole blood was collected via cardiac puncture. The blood was centrifuged at 1200rpm for 10 minutes and the plasma was collected, snap frozen, and stored in -80C for analysis.

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[0158] Sample Acquisition and Selection

[0159] Archived plasma from 155 ALS patients were obtained by the VA biorepository (Durham NC, USA). Due to the available supply, plasma only from Caucasian men were used to minimize potential variability due to sex and ethnicity differences. All patients were negative for any confounding comorbidities past medical history (such as stroke, traumatic brain injury, cancer, diabetes, cardiovascular diseases) at the time of plasma collection. Thirty age, gender and ethnicity matched control samples from non- ALS patients were graciously supplied by Dr. Daniel Nation (University of Southern California, USA). The control samples were from healthy individuals with no major past medical history.

[0160] Metabolomic Quantification

[0161] All plasma samples were sent to Metabolon Inc. (Morrisville, USA) and run using their global metabolic profiling platform, which has a total of 1,641 biochemicals, with 1,291 compounds of a known identity (named biochemicals) and 350 compounds of unknown structural identity (unnamed biochemicals). Metabolite quantification and data normalization was performed at Metabolon.

[0162] Data Analysis

[0163] Pathway and enrichment analysis were performed using MetaboAnalyst, a web-based platform dedicated for comprehensive metabolomic data analysis. Only metabolites with matching HMDB identifiers were included in the pathway enrichment analysis. All analysis was completed using version 6.0. Heatmaps were generated using Rstudio. Linear regressions were performed to assess metabolite changes as a function of disease severity, as indicated by the patient’s ALSFRS-R score, in GraphPad PRISM (version 10.0).

[0164] Statistical Analysis

[0165] A two-way ANOVA was used to measure statistical significance between groups of interest. A p- value of less than 0.05 was used to indicate statistical significance.

[0166] Bulk RNA Sequencing (RNA-seq)

[0167] RNA isolation and sequencing were performed at VANTAGE (Vanderbilt University; Nashville, TN). RNA was isolated from white blood cells (WBCs) and motor cortex samples from mice necropsied at three different timepoints (presymptomatic, disease onset, and disease end stage). Library preparation of cDNA were done using a stranded mRNA (poly(A)-selected) sample preparation kit. Sequencing was performed using a NovaSeq600, targeting 30 million reads per sample.

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[0169] Differentially Expressed Genes (DEGs)

[0170] Transcripts, provided by VANTAGE, were mapped to mouse cDNA (ensembl release 111) using Salmon (v.1.9.0). Tximport (vl.26.1) andDESeq (v.1.38.3) were used to generate and normalize the counts, respectively. DEGs were obtained by taking the average normalized counts from one group and dividing them by the second group of interest.

[0171] Ingenuity Pathway Analysis (IP A)

[0172] Qiagen’s IPA software was utilized to investigate significant conical pathway changes using differentially expressed genes (DEGs), obtained as previously mentioned. The DEGs previously obtained were uploaded and processed using the core analysis feature of IPA. Only DEGs with a p- value of less than 0.05 were considered statistically significant. Canonical pathways were then identified based on an enrichment analysis of statistically significant gene changes. The outputs are predictive pathway changes described by changes in the gene expression linked to a specific pathway. The predicted activation or inhibition of pathways of interest are determined by KEGG networks and other available sources that were compiled and within Ingenuity’s database.

[0173] Results

[0174] ALS patients exhibit distinct global metabolomic changes compared to non-ALS patients A principal component analysis (PCA) was used to visualize the similarity of different groups and understand population variability (Figure 6). There was considerable overlap in the ALS populations, regardless of the disease onset (limb vs bulbar onset). This indicates similar biochemical profiles between the two ALS groups, and therefore, were grouped into one “ALS” group. Additionally, there is separation between the ALS patients and the control samples, suggesting a unique biochemical profile between the disease state.

[0175] In total, 1,291 compounds with a known identity were analyzed. Overall, there were 696 total metabolites that were statistically significant different in the ALS patient samples compared to healthy control patients (p-value < 0.05). Of these 696 metabolites, 334 were significantly higher while 362 were significantly lower when compared to the control samples (Table 1).

[0176] Table 1: Metabolite changes between ALS patients and healthy control plasma samples. Green indicates a decrease in metabolite levels and a red represents an increase.

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[0178] Statistical Comparisons

[0179] Human Plasma

[0180] ALS ANOVA Contrasts

[0181] CONTROL

[0182] Total biochemicals p<0.05 696

[0183] Biochemicals (Tl) 3341362 Total biochemicals 0.05<p<0.10 106

[0184] Biochemicals (t|) 55151

[0185]

[0186] Increased Metabolites

[0187] Of the 334 metabolites that were significantly elevated in ALS, 215 metabolites had a known HMDB identifier that could be used for pathway enrichment analysis. The metabolites without an HMDB ID are those metabolites that are not well characterized, xenobiotics, or unnamed metabolites. Since these metabolites are not well characterized, they were excluded in the analysis. Additionally, when the list of metabolites with available HMDB IDs were entered into the online MetaboAnalyst portal, only 139 metabolites had matching records. The metabolites without matching IDs were excluded from the analysis.

[0188] The pathway enrichment analysis using the significantly increased metabolites show dysfunction in a variety of key metabolic pathways. The top 25 enriched pathways are shown in Figure 7. The pathways with significance are pathways in the first 7 pathways (bile acid biosynthesis, arginine and proline metabolism, urea cycle, purine metabolism, sphingolipid metabolism, nicotinate and nicotinamide metabolism, and aspartate metabolism).

[0189] Decreased Metabolites

[0190] Of the 362 total metabolites that were significantly reduced in the ALS patient samples, 208 metabolites had a known HMDB ID. As mentioned before, 167 metabolites had a matching HMDB name in the MetaboAnalyst portal and were therefore used for the enriched pathway analysis. Of the top 25 pathways (Figure 8), alpha linolenic acid and linoleic acid metabolism, fatty acid biosynthesis, and urea cycle were significantly enriched.

[0191] Metabolite levels correlate with disease progression

[0192] Considering ALS is a progressive disease, a linear regression was performed to see if metabolites correlated to disease severity, as indicated by the patient’s ALSFRS-R score. Overall, there are 124 metabolites that correlated with disease severity. Of these 124 45739246.1 18ATTORNEY DOCKET NO. UA 24-001 PCT

[0193] metabolites, 86 further decrease as ALS progresses. Of these decreased metabolites, key pathways relating to energy utilization are enriched, such as metabolites involved in the TCA cycle and fatty acid oxidation. The top metabolites correlating to disease progression are shown and broken up between lipid metabolites and non-lipid metabolites (Figures 9A and 9B).

[0194] RASRxl902 prevents ALS derived iMNs from shifting from glucose metabolism to fatty acid oxidation.

[0195] Considering ALS patients experience a hypermetabolic phenotype, to overcome this energy demand, patients switch from primarily glycolysis to fatty acid beta oxidation. This shift is in part due to the depletion of glycolytic metabolites, where the body utilizes alternative fuel sources to meet energy demand. To test this in patients, iMNs were derived from ALS patients and transcriptional changes were analyzed using IPA pathway analysis. In ALS motor neurons, there is a shift away from glycolysis with an upregulation in fatty acid metabolism (Figure 10).

[0196] To investigate the changes associated with RASRxl902 treatment, cell cultures were treated for 24 hours. After 24 hours with RASRxl902 exposure, there is a reversal of this shift (Figure 11). RASRxl902 is preserving the motor neuron’s ability to utilize glucose to maintain their energy demand. This protection is associated with a decrease in the reliance in fatty acid metabolism and lipid metabolism.

[0197] RNAseq changes in SOD1G93A mice suggest mitochondrial benefits with RASRxl902 treatment

[0198] Glycolysis and energy synthesis is done within the mitochondria. Therefore, in SOD1G93A animals, RNAseq was performed on the motor cortex at symptomatic onset. With RASRxl902 treatment, there is an increase in mitochondrial function, primarily in complex I and ATP synthase (Figure 12). Additionally, there is a measured decrease in calpain (which are activated by intracellular calcium). This decrease increases mitophagy and mitochondrial biosynthesis. Similar changes are also seen in the WBC of SOD1G93A mice at this same time point (FIG. 16). This suggests a system wide effect on mitochondrial function.

[0199] Changes in metabolites in FFA and glycolysis are reversed with RASRxl 902 treatment in SOD1G93A mice.

[0200] In evaluating the effects of RASRxl902 on metabolite levels, the administration of drug stabilized metabolite utilization and delayed progression of disease. When comparing metabolite levels in the vehicle treated control SOD1 mice, metabolites in the glycolysis / TCA cycle significantly dropped from presymptomatic levels to end stage levels (Figure 13B), whereas the mice treated with RASRxl902 did not have significant decreases in these metabolites. Similar effects were seen in fatty acid levels in the plasma (data not shown). There was a reduction over 45739246.1 19ATTORNEY DOCKET NO. UA 24-001 PCT

[0201] time in free fatty acids in vehicle treated mice and treatment RASRxl902 stabilized these metabolites, possibly protecting against the shift from glycolysis to b-oxidation, a characteristic typically seen in hypermetabolic ALS patients.

[0202] Similar to ALS patients, SOD1G93A mice experience a significant reduction in glucose, pyruvate, and TCA cycle intermediates. This change is not seen in healthy WT mice, indicating a disease specific phenotype (Figures 13A and 13B). However, with RASRxl902 treatment, these metabolites are unchanged as disease progresses.

[0203] Considering ALS is associated with a shift from glycolysis to FFA utilization, these metabolites were also measured. In the WT mice, there is a preservation in metabolite levels, indicating an energy balance with age. However, in the SOD1G93A mutant mice, there is a complete depletion, indicating an increase utilization to maintain energy production. With RASRxl902 treatment, these levels are also preserved over time, suggesting a maintenance in mitochondrial function and a decreased reliance in FFAs to maintain energy homeostasis (Figures 14A and 14B).

[0204] There are no known reliable biomarkers for ALS progression and for biomarkers of effect for Mas agonists. Given the actions of Mas and the hypermetabolic state found in ALS patients, unbiased biomarker work was initially conducted with Metabolon Global Metabolic Profiling platform. In this set of studies, approximately 1100 metabolites were measured in mouse plasma. In this study, changes in mouse weight were followed. Treatment with RASRx 1902 (aka CAP 1902) mitigated weight loss in the superoxide dismutase type 1 (SOD1) mouse model and loss of rotarod performance (Figure 1).

[0205] Within the SOD1 animals, 468 metabolites correlated with ALS clinical score, when using an 8-point scoring system (0=No evidence of disease, l=tremor or splaying of hindlimbs when suspended by the tail, 2=change in the animals gait, 2.5=inability to dorsiflex, 3=extreme weakness in both hindlimbs, 3.5=functional paralysis in one hindlimb, 4=functional paralysis in both hindlimbs, 5=unable to right themselves to the sternum within 20s after being placed on the animal’s side). All of these metabolites were reduced as disease progressed, highlighting the complete depletion of key processes within this model. Figure 2 shows the results for the linear regression for key metabolites for glycolysis, including glucose, fumarate, pyruvate, and citrate . All metabolites had a significant association with a decrease as the disease progressed.

[0206] An enrichment analysis using the metabolites associated with clinical progression reveals stress on multiple pathways. Twenty-one pathways are significantly associated with the metabolites of interest in the SOD1 ALS model (Figure 3, Table 2).

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[0208] Table 2

[0209] total expected hits Raw p Alpha Linolenic Acid and Linoleic Acid Metabolism 17 1.56 10 3.02E-07 Urea Cycle 28 2.57 10 8.61E-05 Glycine and Serine Metabolism 59 5.42 15 0.000125 Aspartate Metabolism 35 3.21 11 0.000142 Ammonia Recycling 31 2.85 10 0.00023 Arginine and Proline Metabolism 52 4.77 13 0.000448 Gluconeogenesis 33 3.03 9 0.0019 Alanine Metabolism 17 1.56 6 0.00275 Glutamate Metabolism 48 4.41 11 0.00277 Warburg Effect 57 5.23 12 0.00381 Thiamine Metabolism 9 0.826 4 0.00586 Glycolysis 23 2.11 6 0.0142 Glycero lipid Metabolism 25 2.3 6 0.0214 Citric Acid Cycle 32 2.94 7 0.0218 Sphingolipid Metabolism 40 3.67 8 0.0243 Amino Sugar Metabolism 33 3.03 7 0.0256 Glutathione Metabolism 20 1.84 5 0.0299 Phenylalanine and Tyrosine Metabolism 27 2.48 6 0.0308 Valine, Leucine and Isoleucine Degradation 59 5.42 10 0.0365 Homocysteine Degradation 9 0.826 3 0.0419 Transfer of Acetyl Groups into Mitochondria 22 2.02 5 0.0438

[0210]

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[0212] Table 2:

[0213] Fold of Change

[0214] Control End 1902 End

[0215] Control 1902

[0216] Presymptomatic Presymptomatic

[0217] Biochemical Name

[0218] 1,5-anhydroglucitol (1,5-AG) 0.70 0.97

[0219] glucose 0.66 0.98

[0220] 3-phosphoglycerate 0.43 0.56

[0221] pyruvate 0.44 0.64

[0222] lactate 0.61 0.92

[0223] citrate 0.65 0.98

[0224] aconitate [cis or trans] 0.63 0.78

[0225] isocitric lactone 0.48 0.87

[0226] alpha-ketoglutarate 0.49 0.84

[0227] succinylcarnitine (C4-DC) 0.56 3.47

[0228] succinate 0.88 0.83

[0229] fumarate 0.69 0.79

[0230] malate 0.76 0.73

[0231] citraconate / glutaconate 0.65 0.98

[0232]

[0233] Differential Metabolites Correlate with Disease Severity

[0234] To assess whether certain metabolites correlate with clinical status, a linear regression was performed using the patient’s ALSFRS-R score at the time of plasma collection. Seventy nine metabolites were associated with a significant decrease as ALS clinical score worsened. A heatmap of the top 45 was created to show the metabolites with clinical score, with the healthier patients on the left (high ALSFRS-R to low) (Figure 4). The super pathways with the most changes were changes in lipids, amino acids, and energy metabolites.

[0235] Using MetaboAnalyst, an enrichment analysis of the metabolites that correlated with a reduction as ALS disease progressed reveals specific pathways affect most by these metabolites. Significant pathways include those involved with energy production and energy utilization, such as the citric acid cycle, mitochondrial acetyl group transfer, and the Warburg effect (Figure 5 and Table 3).

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[0237] Table 3:

[0238] total expected hits Raw p

[0239] Citric Acid Cycle 32 0.383 3 0.00536

[0240] Transfer of Acetyl Groups into 22 0.263 2 0.0266

[0241] Mitochondria

[0242] Warburg Effect 57 0.683 3 0.0266

[0243]

[0244] Discussion and Conclusions

[0245] Over half of ALS patients experience an increase in their resting energy expenditure (Jesus et al. 2019). This phenomenon is rather caused by the body’s increase in energy requirements rather than malnutrition (Bouteloup et al. 2009). The cause leading to the shift in rising resting energy expenditure remains elusive (Dupuis et al. 2004). This study shows transcriptional changes in ALS motor neurons that favor the utilization of beta oxidation and a decreased reliance in glycolysis for ATP synthesis. This could be a result from faulty mitochondrial function, a hallmark characteristic widely seen in ALS (Zhao et al. 2022).

[0246] Considering the critical role mitochondrial play in the ALS pathophysiology, targeting and stabilizing mitochondrial function may be a promising therapeutic target.

[0247] One plausible target may be through targeting protective arm of the RAS via the mas receptor. Mas receptor activation has been shown to increase nitric oxide production, countering reactive oxygen species, which are detrimental to mitochondrial function (Valenzuela et al. 2021). A predicted increase in reactive oxygen species detoxification with RASRxl902 treatment in-vitro after 24 hours was demonstrated. Additionally, there is a predictive increase in mitophagy, a cellular process responsible for the removal of damaged mitochondria, and an increase in mitochondrial biosynthesis (Ding and Yin 2012). This stabilization in proper mitochondrial function, seen through RNAseq pathway analysis, could be one way in preserving mitochondrial function, and therefore, maintaining proper energy biosynthesis.

[0248] This stabilization in mitochondrial function could be allowing for adequate ATP formation, alleviating the energy deficit seen in ALS patients. In both ALS patients and SOD1G93A mice, there is a depletion in glycolytic metabolites, suggesting there is an increase meet energy demand. However, this increase is not enough to fully meet the energy demand, and therefore, the body relies on a shift to fatty acid oxidation. There is a decrease in the fatty acids as a result from an increase in fatty acid utilization in the SOD1G93A mice and humans.

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[0250] However, with RASRxl902 treatment, this depletion does not occur, indicating a prevention from FAO to meet the animal’s energy demand.

[0251] The bioenergetic crisis that exists in ALS demonstrates the importance mitochondria play in the disease pathology. Many drugs being repurposed for ALS are those that directly affect mitochondrial function and mitophagy. Another approach to stabilize mitochondrial performance is with the MasR activation. This study has shown that RASRx 1902 may be beneficial in ALS due to stabilizing bioenergetics. With RASRxl902 treatment, many of the translatable phenotypes, such as energy metabolite depletion and body weight decreases, are protected against. This stabilization in energy demand may be a mechanism of how RASRxl902 has been shown to delay symptomatic onset and extend life expectancy.

[0252] Metabolites were reduced in the plasma as disease progressed in both SOD1 mice and from human samples. There were three enriched pathways being shared in both the human samples and SOD1 mouse model, suggesting a shared bioenergetic dysfunction phenotype between species. These shared mechanisms could potentially be translatable for drug development. Similar to the human ALS patients, there is a stress on the metabolites associated with energy production and utilization. Treatment with RASRxl902 stabilized the metabolites within these pathways, suggesting that these may act as biomarkers of effect for this therapeutic intervention.

[0253] It is understood that the disclosed method and compositions are not limited to the particular methodology, protocols, and reagents described as these can vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.

[0254] Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, many equivalents to the specific embodiments of the method and compositions described herein. Such equivalents are intended to be encompassed by the following claims.

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Claims

ATTORNEY DOCKET NO. UA 24-001 PCTCLAIMSWe claim:

1. A method for determining ALS patient responsiveness to a Mas agonist, comprising determining the levels of at least two metabolites selected from the group consisting of citrate, fumarate, aconitate, creatinine and glutamine in a sample collected from an ALS patient before and after administration of a Mas Agonist.

2. The method of claim 1, wherein the Mas agonist is RASRxl902.

3. The method of claim 1, wherein the sample is selected from the group consisting of a serum sample, muscle biopsy sample, and CSF (cerebrospinal fluid).

4. The method of claim 1 , comprising determining the levels of at least three metabolites selected from the group consisting of citrate, fumarate, aconitate, creatinine and glutamine in a sample collected from an ALS patient before and after administration of a Mas Agonist.

5. The method of claim 1, comprising determining the levels of at least four metabolites selected from the group consisting of citrate, fumarate, aconitate, creatinine and glutamine in a sample collected from an ALS patient before and after administration of a Mas Agonist.

6. The method of claim 1 , determining the levels of citrate, fumarate, aconitate, creatinine and glutamine in a sample collected from an ALS patient before and after administration of a Mas Agonist.

7. The method of claim 1, wherein the patient is considered responsive to treatment with the Mas agonist where there is no decrease in the levels of the at least two or more of citrate, fumarate, aconitate, creatinine and glutamine following treatment with the Mas agonist.

8. A method for determining disease progression in an ALS patient comprising measuring the levels of at least two or more of citrate, fumarate, aconitate, creatinine and glutamine in a sample collected from the ALS patient at first a time which serves to provide baseline levels and subsequently determining the levels of the at least two or more of citrate, fumarate, aconitate, creatinine and glutamine in a sample collected from the ALS patient at a second time point, wherein a decrease in the levels of the at least two or more of citrate, fumarate, aconitate, creatinine and glutamine is indicative of worsening disease progression.

9. The method claim 8, wherein a decrease of at least 0.5 fold in the levels of at least two or more of citrate, fumarate, aconitate, creatinine and glutamine in a sample collected from the ALS patient is indicative of worsening disease progression.45739246.1 25ATTORNEY DOCKET NO. UA 24-001 PCT10. The method of any one of claims 1-9, comprising administering a Mas agonist to the patient.

11. The method of claim 10, wherein the Mas agonist is RASRxl902.

12. A method for treating an ALS patient, comprising determining the levels of at least two metabolites selected from the group consisting of citrate, fumarate, aconitate, creatinine and glutamine in a sample collected from an ALS patient administering oa Mas Agonist to the patient.

13. The method of claim 12, wherein the Mas agonist is RASRxl902.

14. The method of claim 12, wherein the sample is selected from the group consisting of a serum sample, muscle biopsy sample, and CSF (cerebrospinal fluid).

15. The method of claim 12, comprising determining the levels of at least three metabolites selected from the group consisting of citrate, fumarate, aconitate, creatinine and glutamine in a sample collected from an ALS patient before and after administration of a Mas Agonist.

16. The method of claim 12, comprising determining the levels of at least four metabolites selected from the group consisting of citrate, fumarate, aconitate, creatinine and glutamine in a sample collected from an ALS patient before and after administration of a Mas Agonist.

17. The method of claim 12, determining the levels of citrate, fumarate, aconitate, creatinine and glutamine in a sample collected from an ALS patient before and after administration of a Mas Agonist.

18. The method of claim 12, comprising measuring the levels of the at least two metabolites after administration of the Mas agonist, wherein the patient is considered responsive to treatment with the Mas agonist where there is no decrease in the levels of the at least two or more of citrate, fumarate, aconitate, creatinine and glutamine following treatment with the Mas agonist.45739246.1 26

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