Macrophage migration inhibitory factor blockade to treat atherosclerosis

Abstract

The second most common CHIP mutation, Ten eleven translocase 2 (TET2), is known to produce worse atherosclerosis in models of atheroprone mice, potentially through an inflammatory mechanism. Disclosed herein is an important modulator in the initiation and progression of atherosclerosis formation in the setting of TET2 inactivation. Macrophage migration inhibitory factor (MIF), a chemokine, was Identified as a highly expressed signaling pathway in single cell analysis of TET2 CHIP patients. In a large population, a MIF single nucleotide variant (SNV) resulting in increased MIF production was correlated with increased coronary disease in the setting of TET2 inactivation. Moreover, MIF inhibition was found to reduce the atherosclerotic burden in atheroprone mice with TET2 inactivation.

Description

TH Docket No. 222230-2260MACROPHAGE MIGRATION INHIBITORY FACTOR BLOCKADE TO TREAT ATHEROSCLEROSISCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of U.S. Provisional Application No. 63 / 586,048, filed September 28, 2023, which is hereby incorporated herein by reference in its entirety. STATEMENT OF GOVERNMENT INTEREST

[0002] This invention was made with Government Support under Grant Nos. OD029586 and HL138291 awarded by the National Institutes of Health. The Government has certain rights in the invention.BACKGROUND OF THE INVENTION

[0003] Clonal Hematopoiesis of Indeterminate Potential (CHIP) is a hematologic condition which confers increased risk of cardiovascular disease. Among asymptomatic individuals, individuals with CHIP have a greater burden of subclinical coronary atherosclerosis compared to those without.SUMMARY OF THE INVENTION

[0004] The second most common CHIP mutation, Ten eleven translocase 2 (TET2), is known to produce worse atherosclerosis in models of atheroprone mice, potentially through an inflammatory mechanism. Disclosed herein is an important modulator in the initiation and progression of atherosclerosis formation in the setting of TET2 inactivation. Macrophage migration inhibitory factor (MIF), a chemokine, was Identified as a highly expressed signaling pathway in single cell analysis of TET2 CHIP patients. In a large population, a MIF single nucleotide variant (SNV) resulting in increased MIF production was correlated with increased coronary disease in the setting of TET2 inactivation. MIF inhibition was found to reduce the atherosclerotic burden in atheroprone mice with TET2 inactivation.

[0005] Therefore, disclosed herein is a method for a treating cardiometabolic disease, including peripheral artery disease, non-alcoholic fatty liver disease (NAFLD) or atherosclerosis in a subject, that involves administering to the subject a therapeutically effective amount of a Macrophage migration inhibitory factor (MIF) or oxidized MIF (oxMlF) inhibitor, such as ISO-1 or Imalumab.

[0006] Also disclosed is a personalized method for treating or preventing cardiometabolic disease in a subject that involves first detecting in a sample from the subject a clonal hematopoiesis (CHIP) mutation; and then administering to the subject a therapeutically effective amount of a Macrophage migration inhibitory factor (MIF) or oxMlF inhibitor.TH Docket No. 222230-2260

[0007] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.BRIEF DESCRIPTION OF FIGURES

[0008] FIG. 1 shows enhanced Macrophage Migration Inhibitory Factor (MIF) production in TET2 is correlated with atherosclerosis.

[0009] FIG. 2 shows MIF inhibition reduces atherosclerosis in Ten eleven translocase 2 (TET2) mice.DETAILED DESCRIPTION

[0010] Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to particular embodiments described, and as such may, of course, 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 be limiting, since the scope of the present disclosure will be limited only by the appended claims.

[0011] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0012] 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. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described.

[0013] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such publication by virtueTH Docket No. 222230-2260 of prior disclosure. Further, the dates of publication provided could be different from the actual publication dates that may need to be independently confirmed.

[0014] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

[0015] Embodiments of the present disclosure will employ, unless otherwise indicated, techniques of chemistry, biology, and the like, which are within the skill of the art.

[0016] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to perform the methods and use the probes disclosed and claimed herein. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C, and pressure is at or near atmospheric. Standard temperature and pressure are defined as 20 °C and 1 atmosphere.

[0017] Before the embodiments of the present disclosure are described in detail, it is to be understood that, unless otherwise indicated, the present disclosure is not limited to particular materials, reagents, reaction materials, manufacturing processes, or the like, as such can vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting. It is also possible in the present disclosure that steps can be executed in different sequence where this is logically possible.Definitions

[0018] It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.

[0019] The term “atherosclerotic vascular disease (also referred to as atherosclerosis, arteriosclerosis, atheromatous vascular disease, arterial occlusive disease) as used herein, refers to a cardiovascular disease characterized by plaque accumulation on vessel walls and vascular inflammation. The plaque consists of accumulated intracellular and extracellular lipids, smooth muscle cells, connective tissue, inflammatory cells, and glycosaminoglycans. Inflammation occurs in combination with lipid accumulation in the vessel wall, and vascular inflammation is with the hallmark of atherosclerosis disease process.TH Docket No. 222230-2260

[0020] The terms “inhibiting”, “inhibit” or “inhibition” are used herein to refer to reducing the amount or rate of a process, to stopping the process entirely, or to decreasing, limiting, or blocking the action or function thereof. Inhibition may include a reduction or decrease of the amount, rate, action function, or process of a substance by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%. The terms “further inhibiting”, “further inhibit” or “further inhibition are used herein to refer to reducing the amount or rate of a second process, to stopping the second process entirely, or to decreasing, limiting, or blocking the action or function thereof in addition to reducing the amount or rate of a first process, to stopping the first process entirely, or to decreasing, limiting, or blocking the action or function thereof. The term “inhibitory profile” as used herein refers to the characteristic pattern of reduction of the amount or rate or decrease, blocking or limiting of the action of more than one protein or enzyme. The terms “substantially inhibiting”, “substantially inhibit”, “substantially inhibited”, or “substantially inhibition” are used here to refer to inhibition of kinase activity by at least 65%.

[0021] The term “sample from a subject” refers to a tissue (e.g., tissue biopsy), organ, cell (including a cell maintained in culture), cell lysate (or lysate fraction), biomolecule derived from a cell or cellular material (e.g. a polypeptide or nucleic acid), 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.

[0022] The term “subject” refers to any individual who is the target of administration or treatment. The subject can be a vertebrate, for example, a mammal. Thus, the subject can be a human or veterinary patient. The term “patient” refers to a subject under the treatment of a clinician, e.g., physician.

[0023] The phrase “subject in need of such treatment” is used to refer to a patient who has or will suffer from or is at risk of developing the disorder.

[0024] As used herein, the phrase “therapeutic agent” refers to any agent that has a therapeutic effect and / or elicits a desired biological and / or pharmacological effect, when administered to a subject.

[0025] As used herein, the term “therapeutically effective amount” refers to an amount of a therapeutic protein which confers a therapeutic effect on the treated subject, at a reasonable benefit / risk ratio applicable to any medical treatment. The therapeutic effect may be objectiveTH Docket No. 222230-2260(i.e. , measurable by some test or marker) or subjective (i.e. , subject gives an indication of or feels an effect). In particular, the “therapeutically effective amount” refers to an amount of a therapeutic protein or composition effective to treat, ameliorate, or prevent a desired disease or condition, or to exhibit a detectable therapeutic or preventative effect, such as by ameliorating symptoms associated with the disease, preventing or delaying the onset of the disease, and / or also lessening the severity or frequency of symptoms of the disease. A therapeutically effective amount is commonly administered in a dosing regimen that may comprise multiple unit doses. For any particular therapeutic protein, a therapeutically effective amount (and / or an appropriate unit dose within an effective dosing regimen) may vary, for example, depending on route of administration, on combination with other pharmaceutical agents. Also, the specific therapeutically effective amount (and / or unit dose) for any particular patient may depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific pharmaceutical agent employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and / or rate of excretion or metabolism of the specific fusion protein employed; the duration of the treatment; and like factors as is well known in the medical arts.

[0026] The term “treatment” refers to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder. In addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder.

[0027] The term “passenger mutation” as used herein refers to a somatic mutation in a cell that does not alter clonal fitness but occurs in a cell that coincidentally or subsequently acquires a driver mutation.

[0028] The term “driver mutation” as used herein refers to a somatic mutation in a cell that confers a selective growth advantage to the cell, i.e. it increases clonal fitness.TH Docket No. 222230-2260

[0029] As used herein, the term “MIF” refers to macrophage migration inhibitory factor or active fragments thereof. Accession number EMBL Z23063 describes the nucleic acid sequence encoding human MIF (Bernhagen et al., Biochemistry 33:14144-14155 (1994)). An active fragment of MIF may comprise a fragment or a portion of the MIF protein encoding the tautomerase enzymatic activity of MIF, or a fragment that is capable of binding CD74.

[0030] As used herein a “MIF antagonist” refers to any agent that attenuates, inhibits, opposes, counteracts, or decreases the biological activity of MIF. A MIF antagonist may be an agent that inhibits or neutralizes MIF activity (including, without limitation, small molecules and anti-MIF antibodies); an agent that inhibits or decreases the expression of MIF (including, without limitation, an antisense molecule); an agent that inhibits or decreases the expression of the CD44 receptor (including, without limitation, an antisense molecule or an RNAi molecule); an agent that prevents the binding of MIF to CD74 (including, without limitation, an anti-CD74 antibody or an anti-MIF antibody or a fragment thereof); an agent that prevents the interaction between CD74 and CD44 (such as an anti-CD74 antibody or an anti-CD44 antibody or a fragment thereof); or an agent that prevents the interaction between CD74 and CD44. Examples of such molecules are fragments of CD74 and CD44, such as soluble fragments of such receptors. Examples of MIF antagonists have been disclosed previously, see, e.g., U.S. Pat. No. 6,774,227, Bernhagen et al., Nature 365, 756-759 (1993), Senter et al., Proc Natl Acad Sci USA 99:144-149 (2002); Dios et al., J. Med. Chem. 45:2410-2416 (2002); Lubetsky et al., J Biol Chem 277:24976-24982 (2002), which are hereby incorporated by reference.Clonal Hematopoiesis of Indeterminate Potential (CHIP) Mutations

[0031] Clonal hematopoiesis of indeterminate potential (CHIP) defines patients who have detectable somatic clonal mutations in genes recurrently mutated in hematologic malignancies, but who lack a known hematologic malignancy or other clonal disorder. Under this definition, CHIP encompasses cytopenic patients with concurrent cancer-associated mutations who do not meet diagnostic criteria for MDS, as well as those with normal peripheral blood counts. CHIP would not include clearly described clonal conditions such as paroxysmal nocturnal hemoglobinuria, MBL, or MGUS. In general as a working definition, the mutant allele fraction must be >2% in the peripheral blood, because with deep enough sequencing, a mutation can be found in every individual, and current outcomes data are based on a minimum variant allele fraction of >2% in peripheral blood. Variants present below this threshold are not known to carry increased risk of adverse outcomes. A copy number variant resulting from a chromosomal rearrangement involving a chromosomal region where hematologic neoplasia- associated genes are encoded is also consistent with CHIP.TH Docket No. 222230-2260

[0032] CHIP is predominantly defined by the presence of somatic mutations, where the most frequently mutated genes include, for example, DNMT3A, TET2, ASXL1 , SF3B1 , and GNB1.

[0033] In any of the embodiments described herein, CHIP mutations include missense mutations, splice-site mutations, stop-gain mutations, start-loss mutations, stop-loss mutations, frameshift mutations, in-frame indel mutations, and / or mutations that results in a truncated polypeptide or a polypeptide having a partial loss-of-function, a complete loss-of-function, a predicted partial loss-of-function, or a predicted complete loss-of- function.

[0034] Genotyping hematopoietic cells and / or detection, identification and / or quantitation of the mutations can utilize sequencing. Sequencing can be accomplished using high-throughput systems using nucleic acids described herein such as genomic DNA, cDNA derived from RNA transcripts or RNA as a template. Sequencing may comprise massively parallel sequencing.

[0035] In some embodiments, high-throughput sequencing involves the use of technology available by Helicos BioSciences Corporation (Cambridge, Massachusetts) such as the Single Molecule Sequencing by Synthesis (SMSS) method. In some embodiments, high- throughput sequencing involves the use of technology available by 454 Lifesciences, Inc. (Branford, Connecticut) such as the Pico Titer Plate device which includes a fiber optic plate that transmits chemiluminescent signal generated by the sequencing reaction to be recorded by a CCD camera in the instrument. This use of fiber optics allows for the detection of a minimum of 20 million base pairs in 4.5 hours.

[0036] In some embodiments, high-throughput sequencing is performed using Clonal Single Molecule Array (Solexa, Inc.) or sequencing-by-synthesis (SBS) utilizing reversible terminator chemistry. These technologies are described in part in US Patent Nos. 6,969,488; 6,897,023; 6,833,246; 6,787,308; and US Publication Application Nos. 200401061 30; 20030064398; 20030022207; and Constans, A, The Scientist 2003, 17(13):36.

[0037] In some embodiments, high-throughput sequencing of RNA or DNA can take place using AnyDot.chips (Genovoxx, Germany), which allows for the monitoring of biological processes (e.g., miRNA expression or allele variability (SNP detection). In particular, the AnyDot-chips allow for 10x - 50x enhancement of nucleotide fluorescence signal detection. Other high-throughput sequencing systems include those disclosed in Venter, J., et al. Science 16 February 2001 ; Adams, M. et al, Science 24 March 2000; and M. J, Levene, et al. Science 299:682-686, January 2003; as well as US Publication Application No.20030044781 and 2006 / 0078937. The growing of the nucleic acid strand and identifying the added nucleotideTH Docket No. 222230-2260 analog may be repeated so that the nucleic acid strand is further extended and the sequence of the target nucleic acid is determined.

[0038] The methods disclosed herein may comprise amplification of DNA. Amplification may comprise PCR-based amplification. Alternatively, amplification may comprise nonPCR- based amplification. Amplification of DNA may comprise using bead amplification followed by fiber optics detection as described in Marguiles et al. "Genome sequencing in microfabricated high- density pricolitre reactors", Nature, doi: 10.1038 / nature03959; and well as in US Publication Application Nos.20020012930; 20030058629; 20030100102; 20030148344 ; 20040248161 ; 20050079510,20050124022; and 20060078909. Amplification of the nucleic acid may comprise use of one or more polymerases. The polymerase may be a DNA polymerase. The polymerase may be a RNA polymerase. The polymerase may be a high fidelity polymerase. The polymerase may be KAPA HiFi DNA polymerase. The polymerase may be Phusion DNA polymerase. Amplification may comprise 20 or fewer amplification cycles. Amplification may comprise 20, 19, 18, 17, 16, 15, 14, 13, 12, 11 , 10, or 9 or fewer amplification cycles. Amplification may comprise 18 or fewer amplification cycles. Amplification may comprise 16 or fewer amplification cycles. Amplification may comprise 15 or fewer amplification cycles.MIF Inhibitors

[0039] Disclosed herein are methods for treating atherosclerosis in a subject using a MIF inhibitor. MIF is a homotrimeric protein in which three monomers associate to form a symmetrical trimer. Each MIF trimer has three tautomerase active sites at the interfaces of the monomer subunits. Characteristic for this family, MIF has a N-terminal proline (Pro-1), which is located within a hydrophobic pocket. The residue Pro-1 was shown to be conserved between MIF and its bacterial homologues. Moreover, other invariant residues were identified as being clustered around the N-terminal proline. The evolutionary preservation of this region suggests its importance in the biological function of MIF. Despite the lack of a known physiological substrate, it was shown that D-dopachrome (a stereoisomer of naturally occurring L- dopachrome), phenylpyruvate and p-hydroxyphenylpyruvate are accepted as substrates by MIF. A crystal structure of MIF in complex with p-hydroxyphenylpyruvate demonstrated that Pro-1 functions as a catalytic base in the tautomerase reaction. It is well recognized that MIF’s currently defined substrates either do not exist naturally in vivo, or do not exist at significant concentrations required for biological activity. Nevertheless, small molecule modulators of MIF tautomerase activity may have an impact on MIF cytokine activity due to modulation of its conformation and / or ability to interact with other proteins.TH Docket No. 222230-2260

[0040] MIF inhibitors are known in the art and can be used in the disclosed methods.For example, MIF inhibitors are listed in Table 1 below.TH Docket No. 222230-2260TH Docket No. 222230-2260

[0041] In some embodiments, the MIF inhibitor is a compound described in U.S. Patent No. 11 ,584,717, which is incorporated by reference it its entirety for the teaching of these compounds.

[0042] Because of the essential involvement of MIF in the progression of numerous disorders with an inflammatory component, it is not surprising that attempts were made to find MIF directed therapeutics. One line of development is the application of biologicals, such as anti-MIF antibodies, as novel therapeutics. This approach has been used in many proof of concept studies. The other line of development is to generate MIF-binding small molecules with the aim to interfere with MIF functions. To develop MIF inhibitors, many studies resort to evaluating interference with MIF tautomerase activity. In this perspective, the evaluation of MIF tautomerase inhibitors for their interference with MIF cytokine functions in relevant disease models is highly important.

[0043] Inhibitors containing a chromen-4-one scaffold were identified in 2001 and their Kj values range between 0.04 and 7.4 pM. The most potent compound of this class is Orita-13 with a Ki of 0.04 pM. However, a later investigation reported values in the range of 13-22 pM for Orita-13. The phenol functionality in Orita-13 is also found in the MIF tautomerase substrates D-dopachrome and p-hydroxyphenylpyruvate and proved to be a succesfull design motif for MIF inhibitors (Table 1).Isoxazolines as MIF inhibitors

[0044] The most frequently used reference inhibitor for MIF tautomerase activity is ISO- 1 , which was discovered in 2002. This inhibitor of the isoxazoline class was reported to inhibit MIF tautomerase activity in a dose-dependent manner. It binds at the same position as the substrate p-hydroxyphenylpyruvate with an ICso of about 7 pM. A later study described a of 24 pM for inhibition of MIF tautomerase activity. A MIF-CD74 binding study reported a maximum of 40% inhibition at 10 pM (Table 1). Further studies showed that inhibition of MIF by ISO-1 in a mouse model significantly reduced prostate cancer, colon cancer, and blocked melanoma cell growth. Another study in a mouse model showed that this MIF inhibitor blocks corticosteroidinsensitive lung inflammation. In addition, ISO-1 was also reported to inhibit MIF activity in a mouse model of type 1 diabetes and to result in the delayed onset of this disease. Altogether, ISO-1 is a valuable compound that is widely used as a reference inhibitor in the initial validation of small molecule MIF tautomerase inhibitors as potential therapeutics in diseases with an inflammatory component.

[0045] Another small molecule MIF tautomerase inhibitor with an isoxazoline scaffold is CPSI-1306 (Table 1). This inhibitor lacks the characteristic phenol functionality, which isTH Docket No. 222230-2260 advantageous for applications in vivo. Phenol functionalities are generally considered to be nondruglike because they are prone to phase II bioconjugation reactions thus resulting in quick inactivation and excretion in in vivo experiments. Oral administration of this inhibitor resulted in less severe symptoms in a mouse model for multiple sclerosis as compared to untreated mice. Further small molecules with the same scaffold were synthesized and evaluated for MIF- inhibitory activity. The IC50 of the most active compound, Alam-4b (Table 1), was 7.3 pM in a MIF tautomerase assay and this compound was shown to be nontoxic in a cell viability assay. Subsequently, in 2014, another small molecule inhibitor of isoxazoline class, ISO-66 (Table 1), was reported. Its IC50 in the MIF tautomerase assay was 1.5 pM and long-term administration of ISO-66 in a mouse model of colon cancer or melanoma was shown to be nontoxic and to decrease tumor burden significantly. Thus, studies on MIF inhibitors with an isoxazoline scaffold demonstrate that the development and application of small molecule MIF inhibitors has potential to provide novel therapeutics.1 ,2,3-triazoles as MIF inhibitors

[0046] In 2010, 1 ,2,3-triazole derivatives were reported as inhibitors of MIF. The most potent compounds, Jorgensen-3g and Jorgensen-3h (Table 1), showed IC50 values of about 1 pM for MIF tautomerase activity and MIF-CD74 binding. Subsequently, in 2015, improvements were made by synthesis of several optimized biaryltriazoles. This provided potent compounds with a phenolic hydroxyl group that bind to the MIF tautomerase active site. Neverthless, some compounds had limited water solubility. The activity of this class of compounds was further improved by the addition of a fluorine atom adjacent to the phenolic hydroxyl group to enhance the hydrogen bond interaction with residue Asn-97 of MIF. This yielded the most potent compound, Dziedzic-3bb (Table 1), having a value of 0.057 pM and a solubility that is in the normal range for orally avialable drugs.

[0047] The synthesis of fluorescently-labeled MIF inhibitors with a biaryltriazole scaffold was described in 2016. These inhibitors were used in a fluorescence polarization assay to assess the direct binding of inhibitors to the active site of MIF. The two most potent inhibitors, denoted Cisneros-3i and Cisneros-3j, were reported to have ’s of 0.057 and 0.034 pM in the tautomerase assay and Kd’s of 0.071 and 0.063 pM in the fluorescence polarization-based binding assay, respectively (Table 1).Covalent MIF inhibitors

[0048] The specific reactivity of the proline in the MIF active site provides opportunities to develop covalent inhibitors. In 2008, a compound of the phenyl-pyrimidine class, 4-IPP (Table 1), was reported to inactivate the MIF catalytic function by dehalogenation and formation of aTH Docket No. 222230-2260 covalent bond between C-4 of pyrimidine and the N-terminal nitrogen of Pro-1 in the MIF tautomerase active site. This compound also interferes with the biological functions of MIF as it was reported to irreversibly inhibit lung adenocarcinoma cell migration and anchorageindependent growth. Later on, it was described that 4-IPP inhibits the growth of thyroid cancer cells by inducing apoptosis and mitotic cell death. In 2009, isothiocyanates were discovered as irreversible MIF tautomerase inhibitors. The isothiocyanate BITC (Table 1) was shown to covalently modify the Pro-1 residue in the MIF active site. This drastically alters the MIF tertiary structure and results in loss of its tautomerase activity and in inhibition of MIF binding to CD74.

[0049] The Woodward’s reagent K is a classical heterocyclic electrophile with a specific reactivity. Taking advantage of the specific reactivity of Woodward's Reagent K, covalent MIF inhibitors were developed. These inhibitors were shown to react with the active site Pro-1 of MIF and were applied for covalent labeling of MIF that proved to be selective. The covalent inhibitors were used as probes for labeling and imaging of MIF activities in living cells. These examples demonstrate that it is possible to develop covalent active site-directed inhibitors of MIF that bind with a reasonable level of selectivity. Such inhibitors have great potential for labeling and imaging of enzyme activity in vitro and in vivo. In contrast to their application in imaging, covalent inhibitors are not preferred in pharmacotherapy due to concerns about their off-target effects.Other type of MIF inhibitors

[0050] Apart from the isoxazolines and 1 ,2,3-triazoles, other types of reversible MIF inhibitors were also developed. In some embodiments, the MIF inhibitor is a CD74 or CD44 antagonist. In 2006, MIF inhibitors with a benzoxazinone scaffold were described and patented. The most potent compound, NVS-2, was reported to have an IC50 of 0.020 pM (Table 1). A later assay reported a similar value for the Ki of 0.027 pM in the tautomerase assay and a Kd of 0.055 pM in the fluorescence polarization-based binding assay. Subsequently, in 2010, substituted benzoxazol-2-ones were discovered as MIF antagonists (Table 1). One potent inhibitor from this class, MIF098 (Alissa-5), showed noncovalent inhibition in the MIF tautomerase assay, with an IC50 of around 0.010 pM. This inhibitor was further reported to attenuate MIF-dependent ERK phosphorylation in human synovial fibroblasts, which demonstrates possible use of MIF inhibition as therapy in rheumatoid arthritis.

[0051] In 2012, an allosteric MIF tautomerase inhibitor p425 was identified in a high- throughput screening of a library consisting of 230,000 small molecules. However, this compound is a sulfonated azo compound (also known as pontamine sky blue), which has poor druglike properties. Another study, in 2016, reported inhibitor K664-1 (Table 1) with aTH Docket No. 222230-2260 pyrimidazole scaffold as a novel MIF inhibitor with an IC50 of 0.16 pM in the MIF tautomerase assay. This inhibitor provided protection to p-cells from cytokine-triggered apoptosis in a mouse model, which demonstrates its potential for the prevention of diabetes progression.

[0052] In 2016, compound T-614 (also known as iguratimod) was found to selectively inhibit MIF in vitro and in vivo. The compound has synergic effects with glucocorticoids to slow disease progression in a mouse model of multiple sclerosis. The IC50 of that compound in the MIF tautomerase assay was 6.81 pM (Table 1). Recently, novel types of MIF inhibitors were discovered using substitution-oriented screening (SOS). Inspired by the known chromen-4-one inhibitor Orita-13, a focused collection of compounds with a chromene scaffold was screened for MIF binding. In this study, Kok-10 and Kok-17 (Table 1) provided IC50 values in the low micromolar range (18 and 6.2 pM, respectively) in the MIF tautomerase assay. The binding proved to be reversible and the enzyme kinetics suggested no direct interaction of these compounds with the substrate binding pocket.

[0053] MIF inhibitors are also described in U.S. Patent Nos. 7,230,106; 7,312,221 ; 7,365,200; 8,742,173; 8,618,147; 9,050,334; 10,899,739; 10,968,198; in U.S. Patent Publication No. 2022 / 093817; and in International Patent Publication No. WO / 2022 / 093817, which are incorporated by reference in their entireties for the teaching of these inhibitors.Screening Assay

[0054] In some embodiments, the subject being treated has a mutation in Ten eleven translocase 2 (TET2). In some embodiments, the TET2, the mutation is selected from the group consisting of Frameshift / nonsense / splice-site, missense mutations in catalytic domains (p.1104- 1481 and 1843-2002).

[0055] Methods for detecting TET2 mutations in a subject are described in U.S. Patent Publication No. 2022 / 0170100, which is incorporated by reference in its entirety for the teaching of this mutation and methods for detecting it in a sample from a subject.

[0056] There are many methods known in the art for determining the genotype of a patient and for identifying or analyzing whether a given DNA sample contains a particular somatic mutation.

[0057] Any method for determining genotype can be used for determining TET2 genotypes. Such methods include, but are not limited to, amplimer sequencing, DNA sequencing, fluorescence spectroscopy, fluorescence resonance energy transfer (or “FRET”)- based hybridization analysis, high throughput screening, mass spectroscopy, nucleic acid hybridization, polymerase chain reaction (PCR), RFLP analysis and size chromatography (e.g., capillary or gel chromatography), all of which are well known to one of skill in the art.TH Docket No. 222230-2260

[0058] Whole exome sequencing and targeted amplicon sequencing have commercial applications in diagnostic kits for the detection of the somatic mutations in patients. In a particular advantageous embodiment, a companion diagnostic for use in the disclosed methods may comprise testing for a TET2 genotype.

[0059] In some embodiments, probes are immobilized on a solid or flexible support, such as paper, nylon or other type of membrane, filter, chip, glass slide, microchips, microbeads, or any other such matrix. The probe of this form is now called a “DNA chip”. These DNA chips can be used for analyzing TET2 somatic mutations.Pharmaceutical Compositions

[0060] Pharmaceutical compositions including MIF inhibitors are provided. Pharmaceutical compositions containing the compounds can be formulated for enteral or parenteral (intramuscular, intraperitoneal, intravenous (IV) or subcutaneous injection, intravitreal) routes of administration, topical administration, or using bioerodible inserts and can be formulated in dosage forms appropriate for each route of administration.

[0061] In some embodiments, the amount of MIF inhibitor in the formulation can be from 1.0 mg / mL to 100 mg / mL, about 1.0 mg / mL to about 50 mg / mL protein, about 5.0 mg / mL to about 25 mg / mL. In another embodiment, the amount of MIF inhibitor in the formulation can be about 10 mg / mL, about 15 mg / mL, about 20 mg / mL, about 25 mg / mL, about 30 mg / mL, about 35 mg / mL, about 40 mg / mL, about 45 mg / mL, about 50 mg / mL, about 55 mg / mL, about 60 mg / mL, about 65 mg / mL, about 70 mg / mL, about 75 mg / mL, about 80 mg / mL, about 85 mg / mL, about 90 mg / mL, about 95 mg / mL, or about 100 mg / mL. The composition can be formulated to achieve a concentration of about 9 mg / mL.

[0062] In some embodiments, the disclosed MIF inhibitor compositions are administered in an aqueous solution, by parenteral injection, typically by oral administration. The formulation may also be in the form of a suspension or emulsion. In general, pharmaceutical compositions are provided including effective amounts of MIF inhibitor, and optionally include pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants and / or carriers. Such compositions optionally include one or more for the following: diluents, sterile water, buffered saline of various buffer content (e.g., Tris-HCI, acetate, phosphate), pH and ionic strength; and additives such as detergents and solubilizing agents (e.g., TWEEN 20 (polysorbate-20), TWEEN 80 (polysorbate-80)), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite), and preservatives (e.g., Thimersol, benzyl alcohol) and bulking substances (e.g., lactose, mannitol). Examples of non-aqueous solvents or vehicles are propylene glycol, polyethylene glycol, vegetable oils, such as olive oil and corn oil, gelatin, and injectable organicTH Docket No. 222230-2260 esters such as ethyl oleate. The formulations may be lyophilized and redissolved / resuspended immediately before use. The formulation may be sterilized by, for example, filtration through a bacteria retaining filter, by incorporating sterilizing agents into the compositions, by irradiating the compositions, or by heating the compositions.

[0063] In some embodiments, the Ml F inhibitor is incorporated into or encapsulated by a nanoparticle, microparticle, micelle, synthetic lipoprotein particle, or carbon nanotube. For example, the compositions can be incorporated into a vehicle such as polymeric microparticles which provide controlled release of the active agent(s). In some embodiments, release of the drug(s) is controlled by diffusion of the active agent(s) out of the microparticles and / or degradation of the polymeric particles by hydrolysis and / or enzymatic degradation. Suitable polymers include ethylcellulose and other natural or synthetic cellulose derivatives. Polymers which are slowly soluble and form a gel in an aqueous environment, such as hydroxypropyl methylcellulose or polyethylene oxide may also be suitable as materials for drug containing microparticles. Other polymers include, but are not limited to, polyanhydrides, poly (ester anhydrides), polyhydroxy acids, such as polylactide (PLA), polyglycolide (PGA), poly(lactide-co- glycolide) (PLGA), poly-3-hydroxybut rate (PHB) and copolymers thereof, poly-4- hydroxybutyrate (P4HB) and copolymers thereof, polycaprolactone and copolymers thereof, and combinations thereof. In some embodiments, both agents are incorporated into the same particles and are formulated for release at different times and / or over different time periods. For example, in some embodiments, one of the agents is released entirely from the particles before release of the second agent begins. In other embodiments, release of the first agent begins followed by release of the second agent before all of the first agent is released. In still other embodiments, both agents are released at the same time over the same period of time or over different periods of time.

[0064] In one embodiment, the extended release composition includes microparticles having a diameter from about 1 pm to about 10 pm. The microparticles can have a diameter of 1 pm, 2 pm, 3 pm, 4 pm, 5 pm, 6 pm, 7 pm, 8 pm, 9 pm, or 10 pm. In another embodiment, the extended release compositions include nanoparticles have a diameter ranging from 10 nm to 950 nm. The nanoparticles can have a diameter of 10 nm, 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, or 950 nm.

[0065] In some embodiments, the MIF inhibitor composition releases the MIF inhibitor at a steady rate for 30, 60, 90, or 120 days after administration.TH Docket No. 222230-2260

[0066] Some embodiments provide formulations for oral administration. Suitable oral dosage forms include tablets, capsules, solutions, suspensions, syrups, and lozenges. Tablets can be made using compression or molding techniques well known in the art. Gelatin or nongelatin capsules can be prepared as hard or soft capsule shells, which can encapsulate liquid, solid, and semi-solid fill materials, using techniques well known in the art.

[0067] Examples of suitable coating materials include, but are not limited to, cellulose polymers such as cellulose acetate phthalate, hydroxypropyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate and hydroxypropyl methylcellulose acetate succinate; polyvinyl acetate phthalate, acrylic acid polymers and copolymers, and methacrylic resins that are commercially available under the trade name Eudragit® (Roth Pharma, Westerstadt, Germany), Zein, shellac, and polysaccharides.

[0068] Additionally, the coating material may contain conventional carriers such as plasticizers, pigments, colorants, glidants, stabilization agents, pore formers and surfactants.

[0069] Optional pharmaceutically acceptable excipients present in the drug-containing tablets, beads, granules or particles include, but are not limited to, diluents, binders, lubricants, disintegrants, colorants, stabilizers, and surfactants. Diluents, also termed “fillers,” are typically necessary to increase the bulk of a solid dosage form so that a practical size is provided for compression of tablets or formation of beads and granules. Suitable diluents include, but are not limited to, dicalcium phosphate dihydrate, calcium sulfate, lactose, sucrose, mannitol, sorbitol, cellulose, microcrystalline cellulose, kaolin, sodium chloride, dry starch, hydrolyzed starches, pregelatinized starch, silicone dioxide, titanium oxide, magnesium aluminum silicate and powder sugar.

[0070] Binders are used to impart cohesive qualities to a solid dosage formulation, and thus ensure that a tablet or bead or granule remains intact after the formation of the dosage forms. Suitable binder materials include, but are not limited to, starch, pregelatinized starch, gelatin, sugars (including sucrose, glucose, dextrose, lactose and sorbitol), polyethylene glycol, waxes, natural and synthetic gums such as acacia, tragacanth, sodium alginate, cellulose, including hydorxypropylmethylcellulose, hydroxypropylcellulose, ethylcellulose, and veegum, and synthetic polymers such as acrylic acid and methacrylic acid copolymers, methacrylic acid copolymers, methyl methacrylate copolymers, aminoalkyl methacrylate copolymers, polyacrylic acid / polymethacrylic acid and polyvinylpyrrolidone.

[0071] Lubricants are used to facilitate tablet manufacture. Examples of suitable lubricants include, but are not limited to, magnesium stearate, calcium stearate, stearic acid, glycerol behenate, polyethylene glycol, talc, and mineral oil.TH Docket No. 222230-2260

[0072] Disintegrants are used to facilitate dosage form disintegration or “breakup” after administration, and generally include, but are not limited to, starch, sodium starch glycolate, sodium carboxymethyl starch, sodium carboxymethylcellulose, hydroxypropyl cellulose, pregelatinized starch, clays, cellulose, alginine, gums or cross linked polymers, such as crosslinked PVP (Polyplasdone XL from GAF Chemical Corp).

[0073] Stabilizers are used to inhibit or retard drug decomposition reactions which include, by way of example, oxidative reactions.

[0074] Surfactants may be anionic, cationic, amphoteric or nonionic surface active agents. Suitable anionic surfactants include, but are not limited to, those containing carboxylate, sulfonate and sulfate ions. Examples of anionic surfactants include sodium, potassium, ammonium of long chain alkyl sulfonates and alkyl aryl sulfonates such as sodium dodecylbenzene sulfonate; dialkyl sodium sulfosuccinates, such as sodium dodecylbenzene sulfonate; dialkyl sodium sulfosuccinates, such as sodium bis-(2-ethylthioxyl)-sulfosuccinate; and alkyl sulfates such as sodium lauryl sulfate. Cationic surfactants include, but are not limited to, quaternary ammonium compounds such as benzalkonium chloride, benzethonium chloride, cetrimonium bromide, stearyl dimethylbenzyl ammonium chloride, polyoxyethylene and coconut amine. Examples of nonionic surfactants include ethylene glycol monostearate, propylene glycol myristate, glyceryl monostearate, glyceryl stearate, polyglyceryl-4-oleate, sorbitan acylate, sucrose acylate, PEG- 150 laurate, PEG-400 monolaurate, polyoxyethylene monolaurate, polysorbates, polyoxyethylene octyl phenylether, PEG-1000 cetyl ether, polyoxyethylene tridecyl ether, polypropylene glycol butyl ether, POLOXAMER© 401, stearoyl monoisopropanolamide, and polyoxyethylene hydrogenated tallow amide. Examples of amphoteric surfactants include sodium N-dodecyl-.beta.-alanine, sodium N-lauryl-. beta. -iminodipropionate, myristoamphoacetate, lauryl betaine and lauryl sulfobetaine.

[0075] If desired, the tablets, beads granules or particles may also contain minor amount of nontoxic auxiliary substances such as wetting or emulsifying agents, dyes, pH buffering agents, and preservatives.

[0076] The compositions disclosed herein can also be administered in controlled release formulations. Controlled release polymeric devices can be made for long term release systemically following implantation of a polymeric device (rod, cylinder, film, disk) or injection (microparticles). The matrix can be in the form of microparticles such as microspheres, where the agent is dispersed within a solid polymeric matrix or microcapsules, where the core is of a different material than the polymeric shell, and the peptide is dispersed or suspended in the core, which may be liquid or solid in nature. Unless specifically defined herein, microparticles,TH Docket No. 222230-2260 microspheres, and microcapsules are used interchangeably. Alternatively, the polymer may be cast as a thin slab or film, ranging from nanometers to four centimeters, a powder produced by grinding or other standard techniques, or even a gel such as a hydrogel.

[0077] Either non-biodegradable or biodegradable matrices can be used for delivery of the disclosed compositions, although in some embodiments biodegradable matrices are preferred. These may be natural or synthetic polymers, although synthetic polymers are preferred in some embodiments due to the better characterization of degradation and release profiles. The polymer is selected based on the period over which release is desired. In some cases, linear release may be most useful, although in others a pulse release or “bulk release” may provide more effective results. The polymer may be in the form of a hydrogel (typically in absorbing up to about 90% by weight of water), and can optionally be crosslinked with multivalent ions or polymers.

[0078] The matrices can be formed by solvent evaporation, spray drying, solvent extraction and other methods known to those skilled in the art. Bioerodible microspheres can be prepared using any of the methods developed for making microspheres for drug delivery, for example, as described by Mathiowitz and Langer, J. Controlled Release, 5:13-22 (1987); Mathiowitz, et al., Reactive Polymers, 6:275-283 (1987); and Mathiowitz, et al., J. Appl. Polymer Sci., 35:755-774 (1988).

[0079] The devices can be formulated for local release to treat the area of implantation or injection — which will typically deliver a dosage that is much less than the dosage for treatment of an entire body — or systemic delivery.

[0080] In some in vivo approaches, the Ml F inhibitor compositions disclosed herein are administered to a subject in a therapeutically effective amount. As used herein the term “effective amount” or “therapeutically effective amount” means a dosage sufficient to treat, inhibit, or alleviate one or more symptoms of the disorder being treated or to otherwise provide a desired pharmacologic and / or physiologic effect. The precise dosage will vary according to a variety of factors such as subject-dependent variables (e.g., age, immune system health, etc.), the disease, and the treatment being effected.

[0081] For the disclosed MIF inhibitor compositions, as further studies are conducted, information will emerge regarding appropriate dosage levels for treatment of various conditions in various patients, and the ordinary skilled worker, considering the therapeutic context, age, and general health of the recipient, will be able to ascertain proper dosing. The selected dosage depends upon the desired therapeutic effect, on the route of administration, and on the durationTH Docket No. 222230-2260 of the treatment desired. For the disclosed MIF inhibitor compositions, generally dosage levels of 0.001 to 20 mg / kg of body weight daily are administered to mammals.Methods of Treatment

[0082] Methods are disclosed herein for treating atherosclerosis and / or decreasing arterial endothelial inflammation in a subject. Methods are also disclosed for reducing monocyte adhesion to blood vessels, inhibiting inflammation in blood vessels, and / or protecting the endothelial permeability barrier in blood vessels in the subject. Methods are also disclosed for treating a subject with atherosclerosis, a stroke, peripheral artery disease, and / or or a myocardial infarction.

[0083] The disclosed methods include administering to the subject a therapeutically effective amount of an MIF inhibitor. Administration can be systemic or local. Systemic administration may be by any suitable route, such as, but not limited to, parenteral administration (intravenously, intramuscularly, intraperitoneally, or subcutaneously), or oral administration. In some embodiments, the MIF inhibitor is administered locally to a blood vessel of the subject. Local administration can include administration to a specific lesion, such as an atherosclerotic plaque. In specific, non-limiting embodiments, the MIF inhibitor is administered to the subject in a stent.

[0084] In any of the disclosed methods, the subject can be a human or veterinary subject. Veterinary subjects include domesticated animals or household pets, such as dogs, cats, horses, cows, and pigs. Non-human primates and wild animals can also be treated. The subject (such as a human subject) can be any age, such as a child or an adult, for example, a younger adult, middle- aged adult, or older adult. Most typically, a human subject is a middle- aged adult or older adult, and thus is at least 40 years of age, such as at least 45 years of age, such as at least 50, 55, 60, 65, or 70 years of age.

[0085] In some embodiments, the method includes selecting a subject for treatment. In certain non-limiting examples, a subject is selected that has atherosclerosis, a stroke, peripheral artery disease, and / or or myocardial infarction. In some embodiments, the subject has been determined to be at risk for cardiovascular disease based on risk factors, such as, but not limited to, Framingham risk factors, or guidelines jointly issued by the American Heart Association and American College of Cardiology. In specific non-limiting examples, the method can include evaluating a subject to determine if the subject is at risk for cardiovascular disease using Framingham risk factors. These risk factors include age, gender, whether the subject smokes, blood pressure, total cholesterol level, and high-density lipoprotein cholesterol level.TH Docket No. 222230-2260

[0086] The Framingham Risk Score is a gender-specific algorithm used to estimate the 10-year cardiovascular risk of a subject using specific factors. The Framingham Risk Score was first developed based on data obtained from the Framingham Heart Study, to estimate the 10- year risk of developing coronary heart disease (See, Third Report of the National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III) final report, Circulation 2002 Dec 17; 106(25):3143-421 , incorporated herein by reference). The method can include evaluation of a subject to determine if the subject is at risk for cardiovascular disease using risk factors, such as, but not limited to, Framingham risk factors and / or guidelines jointly issues by the American Heart Association and American College of Cardiology.

[0087] Framingham risk factors include age, gender, low density lipoprotein (LDL) cholesterol level, whether the subject smokes, blood pressure (and whether the subject is receiving pharmacological treatment for hypertension), total cholesterol level, and high-density lipoprotein (HDL) cholesterol level. Programs for this evaluation are available on the internet, such as at the U.S. National Heart, Lung, and Blood Institute (NHLBI) website. The disclosed methods can include (a) selecting a subject for treatment based on the Framingham risk factor and / or (b) evaluating the Framingham risk factors as part of the treatment protocol.

[0088] In some embodiments, the disclosed methods are of use to treat a subject with atherosclerosis. The subject can have atherosclerotic heart disease. These subject can be selected for treatment. In some embodiments the subject also be administered a therapeutically effective amount of a statin, niacin, a fibrate, a bile acid binding resin, a cholesterol absorption inhibitor, a PCSK9-targeting drug, an LDL-targeting drug or an HDL-targeting drug.

[0089] In some embodiments, the disclosed methods are of use to treat a subject who has a myocardial infarction, or previously had a myocardial infarction. Generally, these subjects have cardiac tissue death caused by ischemia. Acute myocardial infarction (AMI), or a "heart attack," occurs when localized myocardial ischemia causes the development of a defined region of tissue death. AMI is most often caused by rupture of an atherosclerotic lesion in a coronary artery. This causes the formation of a thrombus that plugs the artery, stopping it from supplying blood to the region of the heart that it supplies. These subjects can be selected for treatment.

[0090] The disclosed methods are of use to treat a subject that has cardiac ischemia. Severe and prolonged ischemia produces a region of necrosis spanning the entire thickness of the myocardial wall. Such a transmural infarct usually causes ST segment elevation. Less severe and protracted ischemia can arise when coronary occlusion is followed by spontaneous reperfusion; an infarct- related artery is not completely occluded; occlusion is complete, but anTH Docket No. 222230-2260 existing collateral blood supply prevents complete ischemia; or the oxygen demand in the affected zone of myocardium is smaller. Under these conditions, the necrotic zone may be mainly limited to the subendocardium, typically causing non-ST segment elevation Ml. A subject with any of these changes can be selected for treatment.

[0091] In other embodiments the disclosed methods are of use to treat a subject that has a vascular disorder, thrombotic stroke, peripheral vascular disease, restenosis, acute coronary syndrome, or reperfusion myocardial injury.

[0092] The subject can have a myocardial infarction or cardiac ischemia, and can also be administered a therapeutically effective amount of an antiplatelet agent, an anti coagulation agent, a lipid or blood pressure regulating agent, or an anti-oxidant. Exemplary lipid regulating agents are statin, niacin, PCSK9-targeting drug, bile acid binding resin, or HDL-cholesterol targeting drug.

[0093] In any embodiment disclosed herein, the subject can be administered a therapeutically effective amount of an antioxidant, such as N-acetylcysteine, vitamin C, beta carotene, or vitamin E. In a specific non-limiting example, the subject can be administered a therapeutically effective amount of N-acetylcysteine

[0094] A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.EXAMPLESExample 1 :

[0095] FIG. 1 shows enhanced Macrophage Migration Inhibitory Factor (MIF) production in TET2 is correlated with atherosclerosis.

[0096] FIG. 2 shows MIF inhibition reduces atherosclerosis in Ten eleven translocase 2 (TET2) mice.

[0097] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed invention belongs. Publications cited herein and the materials for which they are cited are specifically incorporated by reference.

[0098] 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 invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

TH Docket No. 222230-2260CLAIMS1. A method for treating cardiometabolic disease in a subject, comprising administering to the subject a therapeutically effective amount of a Macrophage migration inhibitory factor ( IF) or oxidized MIF (oxMlF) inhibitor or CD74 inhibitor.

2. The method of claim 1 , wherein the MIF inhibitor or oxMlF inhibitor is ISO-1 or Imalumab.

3. The method of claim 1 or 2, wherein the cardiometabolic disease comprises peripheral artery disease, non-alcoholic fatty liver disease (NAFLD), or atherosclerosis.

4. A personalized method for treating or preventing cardiometabolic disease in a subject, comprising(a) detecting in a sample from the subject a clonal hematopoiesis (CHIP) mutation; and(b) administering to the subject a therapeutically effective amount of a Macrophage migration inhibitory factor (MIF), oxMlF inhibitor or CD74 inhibitor.

5. The method of claim 4, wherein the MIF inhibitor is ISO-1 or Imalumab.

6. The method of claim 4 or 5, wherein the cardiometabolic disease comprises peripheral artery disease, non-alcoholic fatty liver disease (NAFLD), or atherosclerosis.

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