Patent Application
20230404980
The present disclosure relates to MIF inhibitors and specifically to their use in treating spondyloarthritis or symptoms thereof.
Spondyloarthritis (SpA) is a chronic rheumatic disease characterized by severe inflammation in distinct anatomical sites and abnormal new bone formation (NBF) in the entheses of the spine and peripheral joints (1). Uncontrolled inflammation with mechanical stimulation facilitates NBF via endochondral ossification (ECO), and eventual ankylosis (2), contributing to severe pain and restriction in physical activities. Although inhibitors of tumor necrosis factor (TNF) and interleukin (IL)-17 are approved for treatment, up to 40% of SpA patients do not adequately respond to any therapeutic modality (3, 4). Furthermore, there is a challenge remaining in adequately controlling various SpA-associated extra-articular symptoms including psoriasis, colitis, and uveitis with currently available therapy.
MIF is an upstream immuno-regulatory cytokine that promotes inflammation and influences the differentiation of the adaptive immune response (5). Serum levels of MIF are increased in a number of inflammatory conditions including ankylosing spondylitis (AS), a subset of SpA (6). Autoantibodies directed against the MIF cognate receptor CD74 are also present in the serum of SpA patients((7-9). Moreover, CD4+ T cells from SpA patients produce more inflammatory cytokines in response to recombinant CD74 stimulation than those from rheumatoid arthritis (RA) or healthy donors (10).
AS patients have significantly higher levels of MIF in serum, synovial fluid, or gut tissues compared to healthy individuals or osteoarthritis disease controls (6). Baseline MIF levels in the serum independently predict radiographic progression of AS patients (6). Interestingly, overexpression of MIF did not induce clear SpA pathologies in wild type C57BL/6 or BALB/c mice.
The major contributors towards inflammation and NBF in SpA are type 3 immune cells including T helper 17 (Th17) lineage cells and group 3 innate lymphoid cells (ILC3s) that produce IL-17A A and IL-22 in both axial and peripheral joint tissues (11, 12). It is well-acknowledged that T cells undergo polarized differentiation from naïve CD4+ T cells into subpopulations such as Th1, Th2 and Th17, an outcome highly dependent on the cytokine microenvironment present during T cell activation (13, 14). It is also well-established that naïve CD4+ T cells can differentiate into CD25+Foxp3+ regulatory T cells (Tregs). In RA, Th17 cells with arthritogenic and autoreactive properties can arise from Tregs (15).
The SKG strain of mice, with a Zap70 point mutation on the BALB/c genetic background, develops chronic arthritis, enthesitis, sacroiliitis, spinal inflammation and extra-articular manifestations through T cell activation (16, 17); thus, the SKG mouse is a well-established mouse model of SpA (18, 19).
Inhibitors of tumor necrosis factor (TNF) and interleukin (IL)-17 are approved for treatment. However, up to 40% of SpA patients do not adequately respond to any therapeutic modality (3, 4) and available treatments do not uniformly control SpA-associated extra-articular symptoms including psoriasis, colitis, and uveitis. Treatments such as methotrexate, Leflunomide, sulfasalazine, inhibitors of IL-6 signaling (tocilizumab) and B cells (rituximab) as well as blockers of T cell co-stimulation (abatacept) effective in RA treatment, are not useful in SpA.
Accordingly, there is a need for additional treatments for SpA as well as its associated extra-articular symptoms.
As demonstrated herein, MIF inhibitors have been shown to inhibit Spondyloarthritis (SpA) and associated extra-articular manifestations. the. The inventors found that the expression of MIF and its receptor CD74 were significantly increased in blood and target tissues of curdlan-treated SKG mice, as compared to control SKG mice. Overexpression of MIF in vivo was sufficient to recapitulate major SpA clinical manifestations, whereas genetic deletion of MIF using Mif knockout (KO) SKG mice or antagonist blockade suppressed SpA-related pathology. Using these mouse models, the inventors found that MIF plays a critical role in expansion of Th17 cells, ILC3s and inflammatory macrophages. Neutrophils were found to be substantially expanded and to express MIF in curdlan-treated SKG mice, and to be sufficient to induce SpA pathology in Mif KO SKG mice. Strikingly, MIF boosted acquisition of a Th17 cell-like phenotype from Tregs in both mice and humans, including the upregulation of RORγt and IL-17A in vitro.
An aspect of the present disclosure provides a method of treating SpA comprising administering a MIF inhibitor to a subject in need thereof.
A further aspect provides use of a MIF inhibitor for treating SpA in a subject in need thereof.
A further aspect provides use of a MIF inhibitor in the manufacture of a medicament for treating SpA in a subject in need thereof.
Also provided is a pharmaceutical composition comprising a MIF inhibitor for treating SpA in a subject in need thereof.
Other features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the disclosure are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.
An embodiment of the present disclosure will now be described in relation to the drawings in which:
The following is a detailed description provided to aid those skilled in the art in practicing the present disclosure. Unless otherwise defined, 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. The terminology used in the description herein is for describing particular embodiments only and is not intended to be limiting of the disclosure. All publications, patent applications, patents, figures and other references mentioned herein are expressly incorporated by reference in their entirety.
The term “spondyloarthritis” or SpA as used herein refers to a family of related autoinflammatory diseases, including ankylosing spondylitis (AS) (which is also referred to as radiographic axial spondyloarthritis), non-radiographic axial SpA (nr-axSpA), reactive arthritis (ReA), psoriatic arthritis (PsA), IBD-related SpA, juvenile-onset idiopathic arthritis (JIA) and undifferentiated SpA (USpA). Broadly SpA may be divided into axial and peripheral SpA (AxSpA and perSpA) based on the predominant manifestations being back pain or peripheral joint symptoms respectively. Patients with SpA can have a variety of symptoms such as lower back pain, joint inflammation and/or radiologic findings such as inflammation and evidence of NBF or fusion. Patients can be in remission, be experiencing at least one SpA articular or extra-articular (e.g. inflammatory bowel disease (IBD) (e.g. ulcerative colitis or Crohn's diseases), psoriasis, iritis, dermatitis, or uveitis) symptom and/or have periods of flares.
The term “ankylosing spondylitis” or “AS” also referred to as “radiographic axial spondyloarthritis” as used herein, refers to a disease featured by chronic inflammatory arthritis primarily affecting the axial joints typically including involvement of the sacroiliac (SI) joints and in severe cases leading to vertebral fusion. Extra-articular symptoms can include one or more of uveitis or iritis which is present in about 20-40% of AS patients, psoriasis/dermatitis, and inflammatory bowel disease.
The term “early SpA” as used herein refers to a disease stage prior to radiographic findings appearing on X-rays in the sacroiliac joints (SIJs) that fulfill the modified New York (mNY) criteria for AS. Early SpA can include chronic back pain for example lasting less than 1 year, with MRI evidence of inflammation, but no X-ray changes, in the spine. HLA-B27 is a gene seen in 80% of patients with AS.
The term “late SpA” as used herein refers to a disease stage after new bone formation (NBF) results in extensive spinal fusion (at least two adjacent vertebrae fused) and/or more than grade 3 SIJs changes according to mNY criteria, assessed by X-rays
The term “extra-articular symptom” as used herein refers to a symptom or associated condition with SpA or a subtype thereof. Examples include uveitis or iritis, psoriasis, and/or inflammatory bowel disease.
The term “MIF inhibitor” includes any molecule that binds MIF (macrophage migration inhibitory factor), particularly human MIF, binds CD74, particularly human CD74, inhibits MIF-CD74 signal transduction (e.g. by inhibiting, or interfering with MIF-CD74 receptor binding), particularly human MIF-CD74 signal transduction, and/or inhibits MIF tautomerase activity, including for example MIF inhibitors described in WO2010021693 and U.S. Pat. No. 9,643,922 (MIF Modulators), each of which are herein incorporated by reference, Ibudilast (MN166), 2-methyl-1-(2-propan-2-ylpyrazolo[1,5-a]pyridin-3-yl)propan-1-one), CPSI-2705, CPSI-1306 (US20050250826; PCT/US11/21721 or national phase entry U.S. application Ser. No. 13/574,240, each of which are incorporated by reference in their entirety, Cytokine Pharmasciences), isoxazoline (ISO-1) (TalBiochem) as well as an anti-MIF antibody or a binding fragment thereof, such as the anti-MIF monoclonal antibody described in (Leng et al., J Immunol 186, 527-38 (2011)) herein incorporated by reference or imalumab (Takeda Pharmaceuticals), or an anti-CD74 antibody or binding fragment thereof, that for example inhibits CD74 and MIF binding or inhibits CD74-MIF signalling, for example anti-CD74 humanized monoclonal antibody Milatuzumab. The MIF inhibitor can for example bind MIF and/or CD74 and inhibit MIF-CD74 signal transduction. MIF-CD74 signal transduction can be assessed for example using the assay described in MIF- signal transduction initiated by binding to CD74 (Leng et al., J Exp Med 197,1467-1476 (2003)) (Ranganathan et al., Arthritis Rheumatol 69, 1796-1806 (2017)). MIF tautomerase activity can be assessed in a tautomerase assay that monitors the keto/enol interconversion for p-hydroxyphenylpyruvate (HPP) catalyzed by MIF (Stamps, S. L., (2000), Mechanism of the Phenylpyruvate Tautomerase Activity of Macrophage Migration Inhibitory Factor Properties of the P1G, P1A, Y95F, and N97A Mutants Biochemistry 39, 9671-9678). The level of signal transduction or tautomerase activity inhibition can for example be at least 60%, at least 70%, at least 80% or at least 90% compared to vehicle. Other examples of MIF inhibitors include, 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. Although inhibition of tautomerase enzymatic activity is not linked to inhibiting MIF-CD74 interaction, likely due to the proximity of this site to the MIF-CD74 interaction, those inhibitors that bind to the tautomerase site can effectively inhibit CD74 mediated MIF action (Kok et al., Drug Discov Today 23, 1910-1918 (2018)). The MIF inhibitors contemplated are for example, directed to interrupting extracellular MIF activation of CD74.
For example, the MIF inhibitor may comprise a compound of Formula I in WO2010021693 and U.S. Pat. No. 9,643,922 (MIF Modulators) that inhibits MIF as described herein , e.g. inhibits MIF-CD74 signal transduction, and/or inhibits MIF tautomerase activity such compound having a chemical structure of (I):
For example, the MIF inhibitor may comprise a compound of Formula II in WO2010021693 and U.S. Pat. No. 9,643,922 (MIF Modulators) that inhibits MIF as described herein, e.g. inhibits MIF-CD74 signal transduction, and/or inhibits MIF tautomerase activity, such compound having a chemical structure of (II):
For example, the MIF inhibitor may comprise a compound of Formula IIA in WO2010021693 and U.S. Pat. No. 9,643,922 (MIF Modulators) that inhibits MIF as described herein, e.g. inhibits MIF-CD74 signal transduction, and/or inhibits MIF tautomerase activity, such compound having a chemical structure of (IIA):
RYC2 is H, halogen, cyano, an optionally substituted C1-C8 alkyl, alkene or alkyne group (preferably RYC2 is an optionally substituted C1-C3 group when RYC1 is an optionally substituted C1-C3 group), an optionally substituted C1-C7 acyl group, an optionally substituted C2-C8 ester or carboxyester group, an optionally substituted C1-C10 alkoxy group, an optionally substituted C2-C8 ether group, an optionally substituted C1-C7 amido or carboxamido group, a C1-C7 urethane or urea group, an optionally substituted (CH2)j-phenyl group or an optionally substituted (CH2)m-heterocyclic (preferably heteroaryl) group, or together with RYC1 forms a ═O (keto) or ═C group, which is optionally substituted with a C1-C6 alkyl group, an optionally substituted (CH2)j-phenyl group or an optionally substituted (CH2)m-heterocyclic (preferably heteroaryl) group, or an optionally substituted carbonyl phenyl or an optionally substituted carbonyl heteroaryl group;
As a further example, the MIF inhibitor may comprise a compound of Formula B in U.S. Pat. No. 9,643,922 that inhibits MIF as described herein, e.g. inhibits MIF-CD74 signal transduction, and/or inhibits MIF tautomerase activity, such compound having a chemical structure of B:
For example, the compound according formula B can have a chemical structure of:
As another example, the compound of formula B can have a chemical structure of:
As a further example, the MIF inhibitor can comprise a compound selected from the following compounds and pharmaceutically acceptable salts thereof: 3-benzyl-5-fluorobenzoxazol-2-one; 3-(2-benzyloxybenzyl)-5-methylbenzoxazol-2-one; 3-(3-cyanobenzyl)-5-chlorobenzoxazol-2-one; 3-(2,3-dimethoxybenzyl)-5-hydroxybenzoxazol-2-one; 3-(2,3-dimethoxybenzyl)-5-methylbenzoxazol-2-one; 3-(2-ethoxybenzyl)-5-methylbenzoxazol-2-one; 3-(3,5-dimethoxybenzyl)-5-methylbenzoxazol-2-one; 3-(3-ethoxybenzyl)-5-methylbenzoxazol-2-one; 3-(3-ethoxy-5-hydroxybenzyl)-5-methylbenzoxazol-2-one; 5-ethyl-3-(3-hydroxybenzyl)benzoxazol-2-one; 5-ethyl-3-(3-methoxybenzyl)benzoxazol-2-one; 3-(3-fluorobenzyl)-5-methylbenzoxazol-2-one; 3-(4-fluorobenzyl)-5-methylbenzoxazol-2-one; 5-fluoro-3-(3-hydroxybenzyl)benzoxazol-2-one; 6-fluoro-3-(3-hydroxybenzyl)benzoxazol-2-one; 5-fluoro-3-(2-methoxybenzyl)benzoxazol-2-one; 5-fluoro-3-(3-methoxybenzyl)benzoxazol-2-one; 5-fluoro-3-(4-methoxybenzyl)benzoxazol-2-one; 6-fluoro-3-(3-methoxybenzyl)benzoxazol-2-one; 3-(3-hydroxybenzyl)-5-methoxybenzoxazol-2-one; 3-(3-hydroxybenzyl)-5-methylbenzoxazol-2-one; 3-(3-hydroxybenzyl)-6-methylbenzoxazol-2-one; 3-(4-hydroxybenzyl)-5-methylbenzoxazol-2-one; 5-hydroxy-3-(3-hydroxybenzyl)benzoxazol-2-one; 5-hydroxy-3-(2-methoxybenzyl)benzoxazol-2-one; 5-hydroxy-3-(3-methoxybenzyl)benzoxazol-2-one; 6-hydroxy-3-(2-methoxybenzyl)benzoxazol-2-one; 6-hydroxy-3-(4-methoxybenzyl)benzoxazol-2-one; 5-(hydroxymethyl)-3-(3-methoxybenzyl)benzoxazol-2-one; 3-(2-methoxybenzyl)-5-methylbenzoxazol-2-one; 3-(3-methoxybenzyl)-5-methylbenzoxazol-2-one; 3-(3-methoxybenzyl)-6-methylbenzoxazol-2-one; 3-(3-methoxybenzyl)-5,6-dimethylbenzoxazol-2-one; and 5-methoxy-3-(3-methoxybenzyl)benzoxazol-2-one. The compounds as described in WO2010021693 and U.S. Pat. No. 9,643,922 (MIF Modulators) can be prepared as described therein.
The term “MIF098” as used herein refers to the compound
or a pharmaceutically acceptable salt, enantiomer, solvate or polymorph thereof for example as described in U.S. Pat. No. 9,643,922. Methods of making are described therein.
The term MIF098 analog as used herein includes any one of
or a pharmaceutically acceptable salt, enantiomer, solvate or polymorph thereof or combinations thereof as described for example in U.S. Pat. No. 9,643,922. Methods of making are described therein.
The term “treatment” or “treating” as used herein means administering to a subject a therapeutically effective amount of a compound or composition, and may consist of a single administration, or alternatively comprise a series of administrations. As well understood in the art, “treatment” or “treating” is an approach for obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results can include, but are not limited to alleviation or amelioration of one or more symptoms or conditions, diminished extent of disease, stabilized (i.e. not worsening) state of disease, preventing spread of disease, reversal of disease, amelioration or palliation of the disease state, and remission (whether partial or total and optionally temporary). Beneficial or desired clinical results can also include reduction in frequency or intensity of flares. Treatment may result in stabilization of disease, an improvement in disease status, or normalization of ongoing inflammation. For example, treatment response can be the improvement or resolution of one or more disease features, including but not limited to inflammatory back pain, SIJ inflammation, and/or decreased flare up frequency or intensity of pain or inflammation in eyes, gut or skin. It can also include improvement in an articular or extra-articular symptom.
As used herein, “contemporaneous administration” and “administered contemporaneously” means for example in reference to two substances (e.g. two compounds, two compositions etc.) that the two substances are administered to a subject such that they are both biologically active in the subject at the same time. The exact details of the administration will depend on the pharmacokinetics of the two substances in the presence of each other, and can include administering one substance within 24 hours of administration of the other, if the pharmacokinetics are suitable. In particular embodiments, the substances (e.g. two or more compounds or compositions etc.) will be administered substantially simultaneously, i.e. within minutes of each other, or in a single composition that comprises both substances.
As used herein, the phrase “effective amount” or “therapeutically effective amount” means an amount effective, at dosages and for periods of time necessary to achieve a desired result.
The term “flares” as used herein refers to clinical exacerbations of clinical disease activity usually involving increase in symptoms and signs. Flares are typically followed by temporary periods of remission when symptoms subside.
The term “antibody” as used herein is intended to include monoclonal antibodies including chimeric and humanized monoclonal antibodies, polyclonal antibodies, humanized antibodies, human antibodies, and chimeric antibodies. Single chain antibodies are also contemplated. The antibody may be from recombinant sources and/or produced in transgenic animals. An antibody includes an antibody of any class, such as IgG, IgA, or IgM (or sub-class thereof), and the antibody need not be of any particular class. Depending on the antibody amino acid sequence of the constant domain of its heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.
The term “antibody fragment” as used herein is intended to include Fab, Fab′, F(ab′)2, scFv, dsFv, ds-scFv, dimers, minibodies, diabodies, and multimers thereof and bispecific antibody fragments. Antibodies can be fragmented using conventional techniques. For example, F(ab′)2 fragments can be generated by treating the antibody with pepsin. The resulting F(ab′)2 fragment can be treated to reduce disulfide bridges to produce Fab′ fragments. Papain digestion can lead to the formation of Fab fragments. Fab, Fab′ and F(ab′)2, scFv, dsFv, ds-scFv, dimers, minibodies, diabodies, bispecific antibody fragments and other fragments can also be synthesized by recombinant techniques.
Additional examples of antigen-binding fragments include an antigen-binding fragment of an IgG (e.g., an antigen-binding fragment of IgG1, IgG2, IgG3, or IgG4) (e.g., an antigen-binding fragment of a human or humanized IgG, e.g., human or humanized IgG1, IgG2, IgG3, or IgG4); an antigen-binding fragment of an IgA (e.g., an antigen-binding fragment of IgA1 or IgA2) (e.g., an antigen-binding fragment of a human or humanized IgA, e.g., a human or humanized IgA1 or IgA2); an antigen-binding fragment of an IgD (e.g., an antigen-binding fragment of a human or humanized IgD); an antigen-binding fragment of an IgE (e.g., an antigen-binding fragment of a human or humanized IgE); or an antigen-binding fragment of an IgM (e.g., an antigen-binding fragment of a human or humanized IgM).
The term “composition” as used herein, refers to a mixture comprising two or more compounds or components. For example, composition is a composition of two or more distinct compounds. In a further embodiment, a composition can comprise two or more “forms” of the compounds, such as, salts, solvates, or, where applicable, stereoisomers of the compound in any ratio. A person of skill in the art would understand that a compound in a composition can also exist as a mixture of forms. For example, a compound may exist as a hydrate of a salt. All forms of the compounds disclosed herein are within the scope of the present disclosure.
The term “subject” also referred as patient, as used herein includes all members of the animal kingdom including mammals, and suitably refers to humans.
The terms “pharmaceutically acceptable carrier,” “pharmaceutically acceptable diluent” and “pharmaceutically acceptable excipient” include any and all solvents, co-solvents, complexing agents, dispersion media, coatings, isotonic and absorption delaying agents and the like which are not biologically or otherwise undesirable. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic formulations is contemplated. Supplementary active ingredients can also be incorporated into the formulations. In addition, various adjuvants such as are commonly used in the art may be included. These and other such therapeutic agents are described in the literature, e.g., in the Merck Index, Merck & Company, Rahway, N.J. Considerations for the inclusion of various components in pharmaceutical formulations are described, e.g., in Gilman et al. (Eds.) (2010); Goodman and Gilman's: The Pharmacological Basis of Therapeutics, 12th Ed., The McGraw-Hill Companies.
As used herein, a reference to a drug's international nonproprietary name (INN) is to be interpreted as including generic, bioequivalent and biosimilar versions of that drug, including but not limited to any drug that has received abbreviated regulatory approval by reference to an earlier regulatory approval of that drug. Additionally, all drugs disclosed herein optionally include the pharmaceutically acceptable salts and solvates of the drugs thereof, unless expressly indicated otherwise.
The term “compound” as used herein, unless otherwise indicated, refers to any specific chemical compound disclosed herein and includes tautomers, regioisomers, geometric isomers as applicable , and also where applicable, optical isomers (e.g. enantiomers) thereof, as well as pharmaceutically acceptable salts thereof. Within its use in context, the term compound generally refers to a single compound, but also may include other compounds such as stereoisomers, regioisomers and/or optical isomers (including racemic mixtures) as well as specific enantiomers or enantiomerically enriched mixtures of disclosed compounds as well as diastereomers and epimers, where applicable in context. The term also refers, in context to prodrug forms of compounds which have been modified to facilitate the administration and delivery of compounds to a site of activity.
In understanding the scope of the present disclosure, the term “comprising” and its derivatives, as used herein, are intended to be open ended terms that specify the presence of the stated features, elements, components, groups, integers, and/or steps, but do not exclude the presence of other unstated features, elements, components, groups, integers and/or steps. The foregoing also applies to words having similar meanings such as the terms, “including”, “having” and their derivatives.
All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms.
All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.
All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.
The term “consisting” and its derivatives, as used herein, are intended to be closed ended terms that specify the presence of stated features, elements, components, groups, integers, and/or steps, and also exclude the presence of other unstated features, elements, components, groups, integers and/or steps.
Further, terms of degree such as “substantially”, “about” and “approximately” as used herein mean a reasonable amount of deviation of the modified term such that the end result is not significantly changed. These terms of degree should be construed as including a deviation of at least ±5% of the modified term if this deviation would not negate the meaning of the word it modifies.
More specifically, the term “about” means plus or minus 0.1 to 50%, 5-50%, or 10-40%, 10-20%, 10%-15%, preferably 5-10%, most preferably about 5% of the number to which reference is being made.
As used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural references unless the content clearly dictates otherwise. Thus, for example, a composition containing “a compound” includes a mixture of two or more compounds. It should also be noted that the term “or” is generally employed in its sense including “and/or” unless the content clearly dictates otherwise.
The definitions and embodiments described in particular sections are intended to be applicable to other embodiments herein described for which they are suitable as would be understood by a person skilled in the art.
The recitation of numerical ranges by endpoints herein includes all numbers and fractions subsumed within that range (e.g. 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.90, 4, and 5). It is also to be understood that all numbers and fractions thereof are presumed to be modified by the term “about.”
Further, the definitions and embodiments described in particular sections are intended to be applicable to other embodiments herein described for which they are suitable as would be under-stood by a person skilled in the art. For example, in the following passages, different aspects of the disclosure are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.
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, examples of methods and materials are now described.
Type 3 immunity-mediated inflammatory arthritis, represented by spondyloarthritis (SpA), is a systemic rheumatic disease that primarily affects the joints, spine, gut, skin and eyes.
Traditionally there were only a limited number of treatments available for spondyloarthritis (SpA) and the available treatments did not resolve extra-articular symptoms of SpA.
Macrophage migration inhibitory factor (MIF) is an immune-regulatory cytokine. As demonstrated below, the expression of MIF and its receptor CD74 are increased in blood, spleen, gut, sacroiliac and ankle joints of curdlan-treated SKG mice, a mouse model of SpA. It is further shown that delivery of a MIF-enhanced episomal vector EEV in vivo to overexpress MIF is sufficient to induce SpA-like clinical manifestations in SKG mice including expanded populations of T helper 17 (Th17) cells, group 3 innate lymphoid cells and inflammatory macrophages, with decreased regulatory T cells (Tregs) in the inflamed joints. In contrast, Mif-knockout (Mif KO) SKG mice and SKG mice treated with a MIF antagonist prevent or attenuate these manifestations with substantial reduction of type 3 immunity. Further, neutrophils are demonstrated to expand and produce MIF in the disease.
As used herein, PMN-MDSCs and neutrophils are used interchangeably, and mMDSCs and monocytes are used interchangeably.
Cell adoptive transplantation of neutrophils into Mif KO SKG mice induces a SpA-like phenotype, while blocking the function of neutrophils with anti-Gr-1 antibody suppresses the induced SpA-like phenotype. Without wishing to be bound by theory, mechanistically, MIF enhances acquisition of a Th17 cell-like phenotype and suppresses expansion of Tregs from naïve CD4+ T cells. It is also demonstrated that MIF boosts both human and mouse Treg acquisition of a Th17 cell-like phenotype, including the upregulation of RORγt and IL-17A in vitro. These results indicate that MIF is a crucial regulator of type 3 immunity-mediated inflammation and therapeutic target in SpA.
Accordingly, provided herein are methods, compositions and uses for treating SpA.
An aspect is directed to a method of treating SpA in a subject comprising administering a MIF inhibitor to a subject in need thereof.
In one embodiment the SpA is early SpA. Patients can be administered the MIF inhibitor upon diagnosis. As demonstrated in the examples, the MIF inhibitors provided were able to inhibit progression of ankylosing spondylitis (AS) before radiologic changes were detectable.
In another embodiment, the SpA is late SpA. As demonstrated in the Examples, the MIF inhibitors were also able to inhibit late stage radiologic changes when joint damage was visible.
The subject may comprise one or more symptoms associated with SpA, optionally one or more articular or extra-articular symptoms. In one embodiment, the subject is treated during a flare. In another embodiment, the subject is treated while in remission. For example, the subject may be treated when one or markers suggest that inflammation is worsening such as CRP (C-reactive protein) or ESR (erythrocyte sedimentation rate). Alternatively, the subject may have increased pain or other symptom of SpA without elevation of CRP and/or ESR.
In one embodiment, the SpA is ankylosing spondylitis.
In another embodiment, the subject is a patient with a higher likelihood of progression (e.g. those with elevated inflammatory parameters ESR/CRP, baseline existing NBF and/or smokers).
Remarkably and as demonstrated in the Examples, the MIF inhibitors also resolved extra-articular symptoms.
Also provided is a method of inhibiting new bone formation in a subject with SpA comprising administering a MIF inhibitor to a subject with a higher likelihood of progression (e.g. those with elevated inflammatory parameters ESR/CRP, baseline existing NBF and smokers).
Accordingly, in another aspect the method is for treating an extra-articular symptom and/or condition associated with SpA.
In one embodiment, the extra-articular symptom and/or condition associated with SpA is an eye manifestation, optionally uveitis or iritis.
In another embodiment, the extra-articular symptom and/or condition associated with SpA is a skin manifestation, optionally psoriasis.
In another embodiment, the extra-articular symptom and/or condition associated with SpA is a gut manifestation such as IBD.
The MIF inhibitor can be any of the inhibitors described herein. The MIF inhibitor can be an inhibitor described in WO2010021693 and U.S. Pat. No. 9,643,922 (MIF Modulators), each of which are herein incorporated by reference.
In another embodiment, the MIF inhibitor is compound MIF098.
In one embodiment, the MIF inhibitor is a MIF098 analog, salt or derivative thereof.
In one embodiment, the MIF inhibitor is Ibudilast or an analog, salt or derivative thereof.
In another embodiment, the MIF inhibitor is anti-MIF antibody or binding fragment thereof that inhibits MIF activity by binding to its active site or by inhibiting its binding to the receptor CD74 and/or the complex CD74/CXCR2/CXCR4/CXCR7. For example, MIF098 prevents MIF-CD74 signaling by binding to the active enzymatic site of MIF that interferes with its interaction with CD74 through stearic hindrance. Ibudilast, binds adjacent to the active site and inhibits the tautomerase enzymatic activity. Other MIF inhibitors which interfere including anti-MIF antibodies or anti-CD74 antibodies, are also useful. For instance, Milatuzumab, a humanized monoclonal antibody (hLL1/IMMU-115) can be used for SpA. Derivatives of known anti-MIF or anti CD74 antibodies can also be used, for example, derivatives such as a single chain antibody, and/or modified form such as a fusion protein thereof having binding specificity for MIF or CD74 as the unmodified form.
In an embodiment, the MIF inhibitor is an anti-MIF antibody or antigen-binding portion thereof. In particular, the anti-MIF antibody can any antibody that inhibits interaction with CD74 or produces steric hindrance such that the function of MIF-CD74 complex is inhibited.
Treatment can for example improve pain, fatigue and/or disease progression.
Disease progression may be monitored by assessing any imaging changes including MRI and conventional X-rays, assessing sites or new sites of NBF, optionally neo-ossification in SIJ and/or axial joints, and/or ankylosis of the spine.
The MIF inhibitor can be suitably formulated into pharmaceutical compositions for administration to human subjects in a biologically compatible form suitable for administration in vivo.
The composition can comprise a pharmaceutically acceptable carrier, pharmaceutically acceptable diluent or pharmaceutically acceptable excipient.
In one embodiment, the dosage form is a solid dosage form. The MIF inhibitors described in WO2010021693 and U.S. Pat. No. 9,643,922 (MIF Modulators), and in particular MIF098 or MIF098 analogs, salts or derivatives thereof, can be formulated as solid dosage form, for example for oral administration
In one embodiment, the dosage form is a liquid dosage form. Anti-MIF or anti-CD74 antibody or binding fragments thereof, can for example be formulated for IV injection.
Suitable vehicles are described, for example, in Remington's Pharmaceutical Sciences (2003—20th Edition). On this basis, the compositions include, albeit not exclusively, solutions of the substances in association with one or more pharmaceutically acceptable vehicles or diluents, and contained in buffered solutions with a suitable pH and iso-osmotic with the physiological fluids.
The compositions described herein can be prepared by per se known methods for the preparation of pharmaceutically acceptable compositions that can be administered to subjects, such that an effective quantity of the active substance is combined in a mixture with a pharmaceutically acceptable vehicle.
The inhibitors described herein can also be administered contemporaneously with a SpA therapy. For example, the SpA therapy can be a TNF inhibitor such as adalimumab, certolizumab, etanercept, golimumab or infliximab. The SpA therapy can optionally be an IL-17 inhibitor such as secukinumab or ixekizumab.
Pharmaceutical compositions include, without limitation, lyophilized powders or aqueous or non-aqueous sterile injectable solutions or suspensions, which may further contain antioxidants, buffers, bacteriostats and solutes that render the compositions substantially compatible with the tissues or the blood of an intended recipient. Other components that may be present in such compositions include water, surfactants (such as Tween), alcohols, polyols, glycerin and vegetable oils, for example. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, tablets, or concentrated solutions or suspensions.
Suitable pharmaceutically acceptable carriers include essentially chemically inert and nontoxic compositions that do not interfere with the effectiveness of the biological activity of the pharmaceutical composition. Examples of suitable pharmaceutical carriers include, but are not limited to, water, saline solutions, glycerol solutions, ethanol, N-(1(2,3-dioleyloxy)propyl)N,N,N-trimethylammonium chloride (DOTMA), diolesylphosphotidyl-ethanolamine (DOPE), and liposomes. Such compositions should contain a therapeutically effective amount of the compound, together with a suitable amount of carrier so as to provide the form for direct administration to the patient.
The compositions described herein can be administered for example, by parenteral, intravenous, subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intraspinal, intracisternal, intraperitoneal, or oral administration.
The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersion and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that easy syringability exists.
The MIF inhibitor can be for administration daily or twice daily.
Ibudilast for example, which has undergone clinical trials for asthma and multiple sclerosis has shown minimal toxicity. Similarly inhibition by MIF antibodies have also not resulted in major opportunistic infections or other limiting side effects. In an embodiment, the amount administered is an effective amount. For example, a single 30-mg dose followed by 14 days of 30 mg b.i.d was found to be generally safe in healthy adults (Rolan et al., Br J Clin Pharmacol 66,792-801(2008)) (ClinicalTrials.gov Identifier: NCT03489850). The dose may also be higher for example up to a 60 mg dose.
Also provided is a package comprising a MIF inhibitor or a CD74 inhibitor and a package insert. In one embodiment, the package insert indicates that the inhibitor, is indicated for the treatment of adults with early SpA. In another embodiment, the package insert indicated that inhibitor is indicated for the treatment of adults with moderate to severe active SpA, optionally adults who have had an inadequate response to conventional therapy.
As further expanded on in the Examples, a role for MIF in the initiation and progression of SpA through the modulation of type 3 immunity was demonstrated in a SpA mouse model. Substantial increases of MIF in blood and various tissues of curdlan-treated SKG mice were found. Furthermore, all observed SpA-like pathologies including spinal and peripheral arthritis, psoriasis-like dermatitis, blepharitis, ileitis, and NBF were successfully attenuated by targeting MIF, demonstrating that pharmacologic MIF blockade impacts most SpA disease manifestations. As further demonstrated, MIF inhibition is advantageous for example in axial SpA treatment over the current type 3 immunity cytokine blocking therapies, IL-23 inhibition being ineffective and IL-17A blockade showing limited efficacy (e.g., no benefit with colitis or iritis) (20)(21).
MIF is expressed upstream of multiple inflammatory cytokines (5), and without wishing to be found by theory, MIF inhibition may achieve more effective control of the cytokine-driven manifestations in different tissues. In addition, the data herein described shows that MIF possesses site-specific cytokine production regulatory activity. The data presented demonstrate that MIF is a key upstream molecule that site-specifically regulate cytokine production for example by modulating type 3 immune cells though direct and indirect mechanisms.
Interestingly, overexpression of MIF did not induce clear SpA pathologies in wild type C57BL/6 or BALB/c mice.
Overall, the data described herein demonstrates the importance of MIF in the induction and progression of SpA.
The above disclosure generally describes the present application. A more complete understanding can be obtained by reference to the following specific examples. These examples are described solely for the purpose of illustration and are not intended to limit the scope of the application. Changes in form and substitution of equivalents are contemplated as circumstances might suggest or render expedient. Although specific terms have been employed herein, such terms are intended in a descriptive sense and not for purposes of limitation.
The following non-limiting examples are illustrative of the present disclosure:
Spondyloarthritis (SpA) is a chronic rheumatic disease characterized by severe inflammation in the spine, peripheral joints, intestine, skin and eyes. Although current treatment modalities including tumor-necrosis-factor (TNF) and interleukin (IL)-17 blockers could control inflammation, up to 40% of SpA patients don't adequately respond to any medications or lose their efficacies, resulting in severe pain, increased cardiovascular risk and deteriorating mental health. Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine that exhibits pro-inflammatory effects. MIF has functions in the regulation of immune responses and has been implicated in various inflammatory conditions. We recently discovered that serum levels of MIF were significantly elevated in Ankylosing Spondylitis (AS) patients compared to healthy controls. However, the specific role of MIF in SpA is largely unknown.
Methods: Curdlan (β-glucan) or MIF-plasmid (mini-circle) treated SKG mice (8-10 weeks) were used as SpA mouse models. The expression of MIF in serum or tissues was measured by ELISA, quantitative PCR (qPCR), western blotting, immunohistochemistry (IHC) and/or immunofluorescence (IF). MIF knockout (KO) SKG mice were generated as MIF deficiency mice. MIF inhibitor (MIF098) was used to block the function of MIF in SpA mouse models to assess the therapeutic or prophylactic effects in a curdlan-treated SpA mouse model. Populations of immunological cells were assessed by flow cytometry. Anti-Gr-1 monoclonal antibody (mAb) or isotype control mAb was used to block the function of neutrophils or monocytes. Clinical scores, histopathology and microCT imaging were used to assess the severity of inflammation in the various tissues of the mouse models.
Results: The expression of MIF and its receptor CD74 were significantly up-regulated in serum, spleen, ileum, sacroiliac and ankle joints of curdlan-treated SKG mice. MIF-overexpressed SKG mice injected with MIF-plasmid remarkably induced major SpA clinical features including colitis, psoriasis and arthritis in the axial and peripheral joints, while MIFKO SKG mice or blocking the function of MIF with MIF inhibitor (MIF098) dramatically suppressed or attenuated these manifestations, with decreased populations of Th17 and increased regulatory T (Treg) cells. We have also identified the cell populations (neutrophils) substantially producing MIF in the disease condition. Interestingly, adoptive transfer of these cells into non-disease control mice clearly exhibits SpA phenotype including arthritis, blepharitis and psoriasis. Furthermore, blocking the function of those cells with anti-Gr-1 antibody suppresses the SpA phenotype. Further details are provided in Example 2.
#1. Animal model, treatments and clinical scoring: Female and male SKG mice (age 8-10 weeks) were injected with PBS, curdlan (3 mg/mouse, i.p.), MIF-plasmid (EEV, 5 μg/mouse, i.v., from systemic biosciences), or control-plasmid (5 μg/mouse, i.v., from systemic biosciences) to induce SpA phenotype and weekly observed the clinical manifestations of inflammation and NBFs over 8 weeks (n=10 mice/group). C57BL/6 and BALB/c mice (age 8-10 weeks) were also subjected to MIF- or control-plasmid injection to observe clinical symptoms. MIF- or control plasmid was administered by HDD tail vein injection as previously described (11). We also generated Mif−/− (KO) SKG mice by crossing SKG mice and BALB/c Mif KO mice (22).
After 1 or 4 weeks post-curdlan treatment, we injected MIF antagonist (MIF098; 40 mg/kg, twice/day, i.p.) (23) or control vehicle [PEG400 (Sigma, cat#91893) and HP-P-P-CD (Sigma, cat#C0926)] to assess the prophylactic and therapeutic effects of the MIF098 on the inflammation and NBFs in the SpA mouse model until 8 weeks post-curdlan treatment. Anti-mouse Gr-1 antibody (100 μg/mouse, Life Technologies) or isotype IgG monoclonal antibodies (100 μg/mouse, Life Technologies) were administered on day 3, 6, 9, 12 and 15 post-curdlan treatment.
Clinical scores for SpA-related manifestations were assessed based on severity scales (Table 1); arthritis, maximum 6 points; dermatitis, maximum 2 points; blepharitis, maximum 2 points). The scores were evaluated at least 2 independent scorers in a blinded fashion and final scores were the average of the observations. At the endpoint, ankle joints, spleen and lymph nodes (PLNs and MLNs) were dissected for FACS, one ankle was dissected prior to storage in RNA later for qPCR unless used for FACS, the other ankle was kept intact and fixed in 10% neutral buffered formalin for histopathological assessments. The upper tail spine, pelvis, eyes and ileum were dissected and fixed in 10% neutral buffered formalin for histopathology. For ankle digestion, skin was peeled off and toes (at distal phalanges) and tibia (˜0.5 cm above tibia) were cut off. After flushing bone marrow, the tissue was digested with RPMI culture media containing hyaluronidase and collagenase type VIII for 1 h at 37° C. in incubator. Following passing through 70 μm of cell strainer and RBC lysis treatment, cells were centrifuged and used for FACS analysis.
Ankle joints, tail spine, and pelvis were fixed in 10% neutral buffered formalin for at least 72 h, decalcified in 10% EDTA (BioShop) for 14-21 days and embedded in paraffin. Eyes and ileum were fixed in formalin for at least 72 h without decalcification. Serial sections (4 μm) were stained with hematoxylin and eosin (H&E; Fisher Scientific). To assess endochondral ossification, safranin O/ fast green staining was also applied to NBF in distal tibia as previously described (24, 25). For histological scores, multiple sections (three sections approximately 40-80 μm apart) per joint sample were evaluated by 2 independent scorers in a blinded fashion according to histological assessments as previously reported (11, 24). Final scores were the average of the observations, as previously reported55.
IHC was performed as previously described (24, 25). Specifically, 4 μm sections were deparaffinized in xylene followed by a graded series of alcohol washes. Following proteinase K treatment (10 μg/ml) for 15 min, endogenous peroxide was blocked using 3% H2O2 for 30 min. Non-specific IgG binding was blocked by incubating sections with bovine serum albumin (BSA 1%) in PBS for 30 min. Sections were then incubated with primary antibodies, for MIF (abcam, cat# ab226166; Dilution 1:330), CD74 (abcam, cat# ab202844; Dilution 1:330), Sox9 (abcam, cat#185966; Dilution 1:330), type X collagen (abcam, cat# ab182563; Dilution 1:330), MMP13 (abcam, cat# ab39012; Dilution 1:330), Gr-1 (Invitrogen, cat#14-5931-82; Dilution 1:330) or rabbit IgG (Invitrogen, cat#02-610; Dilution 1:330) as an isotype negative control in a humidified chamber overnight at 4° C. temperature. After washing twice in water, the slides were incubated with their respective biotinylated secondary antibodies for 30 min. Signal was amplified with HRP conjugated secondary antibody followed by Vectastain Elite ABC kit (Vector Laboratories), as per the manufacturer's protocol, and counterstained with hematoxylin (Fisher Scientific).
Similar to IHC, 4 μm sections were deparaffinized in xylene followed by a graded series of alcohol washes. Non-specific IgG binding was blocked by incubating sections with BSA 1% in PBS for 30 min. Sections were then incubated with primary antibodies, for MIF (abcam, cat# ab226166; Dilution 1:330), CD74 (abcam, cat# ab202844; Dilution 1:330) or rabbit IgG (Invitrogen, cat#02-610; Dilution 1:330) as an isotype negative control in a humidified chamber overnight at 4° C. temperature. After washing twice in water, the slides were incubated with secondary antibodies conjugated with either Texas Red (abcam, cat# ab6719) or Alexa fluor (abcam, cat# ab150113) for 30 min at room temperature. To test the expression of RORγt in ankle soft tissue, PE-conjugated primary antibody (BD, cat#562607) was used without secondary antibody. After washing, diluted DAPI solution was added to each well and incubated 2 minutes at room temperature. The slides were washed with PBS once and mounted with an anti-fade mounting media (DAKO). The slides were visualized using EVOS FL Imaging System (Life Technologies).
In vitro splenocytes (1×106/well) and ex vivo ankle soft tissue (0.5 g/well) were cultured in twelve-well plates with curdlan (1 μg/ml) or rmMIF (0, 10, 100 ng/ml) in RPMI or DMEM culture media containing 10% FBS and 1% Penicillin/Streptomycin at 37° C. in a humidified atmosphere of 5% CO2 and 95% air for 0.5, 1, 1.5 or 24 h. RNAs or proteins were then extracted for qPCR and/or western blotting analysis, respectively.
Fresh mouse naïve CD4+ T cells and Tregs (CD4+D25+) were isolated from spleen and PLNs of female SKG mice (8-10 weeks of age) using the mouse naïve CD4 (BioLegend, cat#480040) and Treg isolation kits (STEMCELL, cat#18783). The purity of CD4+CD25+T cells was 95.28%±0.11 (n=4, average±SEM). On day 0, a 96 well plate was coated with 50 μl of anti-mouse CD3ε (5 μg/ml, BioLegend, cat#100340) and incubated overnight in 4° C. After washing the plate with PBS on the next day, equal numbers of naïve CD4+ T cells or Tregs (2×105/well) were cultured in the 96 well plate in the complete IMDM containing anti-mouse CD28 (5 μg/ml, BioLegend, cat#102116) alone or in combination with rmMIF (50 ng/ml, BioLegend, cat#599504) for 4 days. Equal numbers of naïve CD4+ T cells (4×105/well) were also cultured with neutrophils (2×105/well) isolated from curdlan-treated SKG mice for 4 days. The cells and culture supernatant were used for further analysis.
Fresh antigen presenting cells (APCs) and CD4+CD25− T cells were isolated from spleen of female WT BALB/c mice (age: 8 weeks) using beads isolation kits (both are STEM CELLS; cat#18951 and cat#18783, respectively). The purity of CD4+CD25− T cells was 95.13%±0.19 (n=3, average±SEM). Fresh mouse Tregs (CD4+D25+) were isolated from either WT BALB/c, WT SKG, or Mif KO SKG mice (age: 8 weeks) as described above. Following the labelling with Cell Proliferation Dye eFluor450 (10 μM, eBioscience, cat#65-0842-85), equal amount of CD4+CD25− T cells (5×104 cells) were co-cultured with irradiated APCs (2×105 cells) and Tregs (10.0, 5.0, 2.5, or 1.25×104 cells) in RPMI containing 10% FBS and 1.0 μg/ml of anti-CD3 for 72 h. Cell proliferation after stimulation for 72 h was assessed by flow cytometry.
Human naïve CD4+ T cells and Tregs (CD4+CD25+CD127low) were isolated from PBMCs of healthy male controls without any history of back pain, arthritis, and joint injuries (age: 18-40, n=4 individuals in total) using the human naïve T cells isolation kit (STEM CELL, Cat #19555) and Treg isolation kit (STEM CELL, cat#18063), respectively. Isolated human Tregs (3×104 cells/well) were cultured in the complete IMDM culture media containing ImmunoCult™ Human CD3/CD28/CD2 T Cell Activator (25 μl/ml, STEMCELL, cat#10970), and rhIL-2 (100 IU/ml, BioLegend, cat#589102) alone or in combination with or without rhMIF (50 ng/ml, BioLegend, cat#599404), rhIL-1β (25 ng/ml, BioLegend, cat#579402) and rhIL-23 (100 ng/ml, BioLegend, cat#574102) for 12 days. Each culture media was replaced ever 2-3 days. The cells were used for further analysis.
Released cytokine in cell culture supernatant on day 12 was quantified using the LEGENDplex Human Th17 Cytokine Panel (BioLegend, cat#741032) according to the manufacturer's instructions.
Total Gr-1+ cells were isolated from bone marrow and spleen of curdlan-treated female SKG mouse at 8 weeks post curdlan using mouse CD11b+Gr1+ Isolation Kit (STEMCELL, catalog#19867). After the bead isolation, neutrophils (2×106 cell) were isolated using FACS Aria III cell sorter (BD). Neutrophils (2×106 cell) from female SKG mice treated with PBS were used as controls. After washing with PBS three time, the neutrophils or control neutrophilswere injected into Mif KO SKG mice at 1- and 2-week post-curdlan treatment through tail vein.
For all panels, single cell suspensions were first stained with a fixable live dead stain (L/D NIR, Invitrogen, cat#L10119) as directed by the manufacturers. Cells were blocked with FcX (BioLegend, cat#101320) or Monocyte Blocker (BioLegend, cat#426102) prior to staining with surface antibodies. For experiments in which transcription factors were stained, cells were fixed and permeabilized with True-Nuclear kit (BioLegend, cat#424401) as directed. For experiments in which cytokines were stained, cells were fixed with a PFA buffer and permeabilized with intracellular staining buffer (BioLegend, cat#420801 and cat#421002) as indicated. Brefeldin A (BioLegend, cat#420601) with or without PMA/ionomycin (BioLegend, cat#423302) were used for in vitro stimulations to detect intracellular cytokines. Data were acquired on LSR II or Canto II (BD) and analyzed with FlowJo (version 10.6, Becton Dickinson).
RNA concentrations were determined using NanoVue (GE Healthcare Life Science). Following RNA quantification, equal amounts of RNA (1000 ng) were converted to cDNA using the QuantiTect Reverse Transcription PCR Kit (Qiagen) for mRNA, as per the manufacturer's protocol. For qPCR reactions, 5 ng of RNA per well was used for gene expression with primers and SYBR Green Master Mix (BIO-RAD) with primers and SYBR Green Master Mix Kit (Qiagen) according to the manufacturer's protocol. The reactions were incubated in 96 well plates (BIO-RAD) and performed in duplicate. Specificity of the amplified qPCR product was assessed by performing melting curve analysis on the LightCycler® 480 Instrument (Roche). The relative expression of PCR products was calculated by the 2-ΔCt method. All primers were designed using Primer3 online software. Data were normalized to GAPDH for mRNA analyses. The reference genes showed highly stable expression compared to other candidates for reference genes as previously reported55,56.
The concentration of MIF in SKG mice or culture supernatant media and the concentration of IL-17A in the human Tregs culture media were assessed by mouse MIF ELISA kit (LEGEND MAX™ Mouse MIF ELISA Kit, BioLegend, cat# 44107) and Human IL-17A ELISA kit (LEGEND MAX™ Human IL-17A ELISA Kit, BioLegend, cat# 433917) were used, respectively. Samples were analyzed according to the manufacture's instruction.
Equal amount of cell lysates in RIPA buffer were applied to SDS-polyacrylamide gels (10%) for electrophoresis, as previously reported55,56. Separated protein was electroblotted onto polyvinylidene fluoride membranes. Membranes were blocked in 10 mM Tris-buffered saline (TBS) containing 5% skimmed milk and probed for 1.5 h with rabbit IgG primary antibodies (1:250) specific for MIF (abcam, cat# ab226166) and CD74 (abcam, cat# ab202844) or mouse monoclonal IgG for β-actin (1:1000; Sigma-Aldrich, catalog A1978) in blocking buffer. After washing the membranes with TBS containing 0.1% Tween-20 (TBS-T) 3 times, the membranes were incubated for 1 h at room temperature with HRP conjugated anti-rabbit (1:5,000; Sigma-Aldrich, catalog# SAB3700843) or anti-mouse (1:10,000; Sigma-Aldrich, cat#A2179) secondary antibodies in TBS containing 5% skimmed milk. Membranes were subsequently washed in TBS-T and protein bands were visualized with an enhanced chemiluminescence substrate (Clarify™ Western ECL Substrate, BIORAD and SuperSignal West Pico, Thermo Science) using a BIO-RAD Chemidocapparatus. Blots were scanned and signal intensity was quantified using Image J (National Institutes of Health, USA).
#14. Micro-CT: For assessments of bone formation and the temporal profile of bone structural changes, in vivo longitudinal micro-CT (SkyScan 1276, Bruker Corporation, Kontich, Belgium) were performed in curdlan-treated SKG mice, MIF PLM-injected SKG mice, MIF098-treated SKG mice or Mif−/− SKG mice accompanied by controls per group. At 8 weeks of curdlan or plasmid treatments, mice were euthanized with CO2 (1.3 L/min) in a cage and scans performed. All micro-CT scans were reconstructed with InstaRecon software (Champaign, Ill., USA) and screen captures taken of volume rendered CTvox (Bruker Corporation, Kontich, Belgium) images.
All statistical analysis performed with GraphPad Prism8 (San Diego, Calif., USA). Data tested for normality before statistical test selected. Statistical analysis comparing two treatment groups with parametric and non-parametric data were performed by two-tailed Student's T tests and Mann-Whitney U tests (unpaired) or Wilcoxon signed-rank tests (paired), respectively. Statistical analysis comparing multiple treatment groups with parametric were performed by one- or two-way analysis of variance followed by Tukey's post-hoc test. For statistical analysis comparing multiple treatment groups with paired or unpaired non-parametric were performed by Kruskal Wallis test or Friedman test followed by Dunn's multiple comparisons test, respectively. A value of P<0.05 were considered statistically significant for all comparison tests.
Mouse neutrophils, monocytes, B cells and T cells were isolated from bone marrow, spleen or PLNs of Mif+/+ or Mif−/− SKG mice and sorted by FACS. The number of each cell population (two million neutrophils, 0.22 million monocytes, 0.11 million B cells, and 0.11 million T cells per well) was determined based on the ration of inflamed ankle joint (
Fresh human neutrophils were isolated from blood in SpA or healthy volunteers using EasySep Human Neutrophil Isolation Kit (STEMCELL Technologies, cat#17957). Cells were cultured in a 96 well plate (two million cells per well) containing HBSS for 60 minutes with or without lipopolysaccharide (LPS, 0.1 μg/ml for 60 minutes) or curdlan (1 μg/ml for 60 minutes). The level of secreted MIF into the culture media was measured by ELISA as described below.
In line with previous reports (17), curdlan (β-glucan)-treated female SKG mice exhibited accelerated and more severe development of SpA-like clinical symptoms over 8 weeks, compared to male SKG mice; thus female SKG mice were primarily used for subsequent studies, unless indicated. Histological tissue sections showed evidence of severe inflammation of the ankle, sacroiliac joint, tail vertebrae, enthesis, ileum and skin of SKG mice at 8 weeks post-curdlan treatment, whereas there was no evidence of such clinically-relevant or histological features in saline (PBS)-treated SKG mice (
To investigate gene expression of inflammatory markers in response to curdlan in vitro, ankle soft tissues or splenocytes were isolated from healthy SKG mice and cultured with curdlan or PBS for 24 hours. An increase in gene expression of major SpA-related inflammatory markers (Il1b, Il6, Il23a, Tnfa, Il17a and Ccl2) was observed in both joint tissues and splenocytes cultured with curdlan compared to PBS treatment, with the exception of Il23a in splenocytes (
Abnormal NBF at entheseal sites (enthesophyte formation) following inflammation is also a cardinal feature of SpA. NBF was evident in the distal tibia of SKG mice at 8 weeks post-curdlan (
Consistent with SpA patients (6), the concentration of MIF in serum was increased in curdlan-treated SKG mice compared to PBS-treated SKG mice (
In vitro culture of the major immune cell lineages isolated from healthy SKG mice revealed that after 60 minutes of curdlan treatment, neutrophils (CD11b+Ly6G+Ly6Clo) substantially increased secretion of MIF, whereas monocytes (CD11b+Ly6G−Ly6Chi), CD19+ B cells, and CD3+ T cells showed milder increases (
The potential mechanism of how MIF is released from neutrophils in SKG mice was explored. It has been established that curdlan binds to the innate pattern recognition receptor Dectin-1 and promotes the expression of pro-inflammatory cytokines through phosphorylation of spleen tyrosine kinase (p-Syk), a downstream transducer of Dectin-1 (26-28). To determine the mechanism of MIF secretion in neutrophils, neutrophils were isolated from healthy SKG mice and treated the cells with or without curdlan in the presence or absence of anti-Dectin-1 neutralizing monoclonal antibody (anti-Dectin-1 mAb) or isotype control mAb in vitro. SKG neutrophils promptly secreted MIF into the culture media upon the stimulation with curdlan, whereas secretion was partially attenuated by anti-Dectin-1 mAb (
In addition to MIF secretion, confirming expansion of MIF-producing neutrophils in inflamed tissues is critical to substantiate neutrophils as key reposits of MIF that provoke inflammation in SKG mice. Gr-1+(Ly6G+/Ly6C+) cells, chiefly neutrophils and monocytes, were expanded in the ankle joints of curdlan-treated SKG mice compared to PBS-treated SKG mice (
To confirm that neutrophils were the dominant cells producing MIF in inflamed tissues, cells from ankle joints of curdlan-treated SKG mice were isolated and sorted into neutrophils, monocytes, B cells, and T cells. Among live cells, more than 60% were neutrophils, followed by 7% monocytes, and 3 to 4% B and T cells each (
Next, whether MIF was increased in tissues of SKG mice was tested. Increased proportions of cells positive for MIF or CD74 were observed in spleen, sacroiliac joints, distal tibia with NBF, and ileum of curdlan-treated SKG mice compared to PBS-treated SKG mice, as determined by immunofluorescence (IF) and immunohistochemistry (IHC) (
Although curdlan-treated SKG mice have SpA-like pathologies with increased expression of MIF, the potential contribution of MIF to these pathologies is unknown. Since curdlan-treated SKG mice also highly expressed other inflammatory markers (
Increased inflammation in the ankle, sacroiliac joints, spine, ileum and skin were identified with histopathological scorings of ankle arthritis and tail spinal inflammation in MIF PLM-injected SKG mice compared to CTL PLM-injected mice (
Since serum concentrations of MIF increased bimodally (initially peaking at 1 week and again increasing between 4-5 weeks post-MIF PLM injection in both female and male SKG mice;
As MIF-overexpressing SKG mice exhibited SpA-like features, whether MIF-overexpressing BALB/c or C57BL/6 mice injected with MIF PLM develop SpA-like pathologies was also tested. No clear evidence of SpA-like characteristics including arthritis and spinal inflammation was observed, indicating that SKG mice are uniquely predisposed to development of MIF-induced SpA-like pathologies.
It was previously shown that serum MIF levels were significantly elevated in AS patients with rapid ankylosis progression compared to slow progressive AS or healthy controls (6); however, the specific role of MIF on NBF in SpA is unclear. In MIF PLM-injected SKG mice, NBF in the distal tibia at 8 weeks post-injection was clearly observed, as assessed by microCT imaging (
Similar to curdlan-treated SKG mice, NBF in MIF PLM-injected SKG mice likely developed through the process of ECO (
The expression of SpA-related inflammatory markers including Il1β, Il6, Il23a, Tnfa, Il17a and Mcp1 in the ankle soft tissues or spleen isolated from either MIF PLM- or CTL PLM-injected female SKG mice was evaluated. In the ankle soft tissues, a significant increase in the expression of Il1β, Il6, Il23a, Il17a and Mcp1, but not Tnfa was observed (
To further assess the expression of pro-inflammatory cytokines induced by MIF, CD4+ T cells from popliteal lymph nodes (PLNs), mesenteric lymph nodes (MLNs) and spleen in SKG mice were isolated and treated with either MIF PLM or CTL PLM. In line with the gene expression analysis, CD4+ cells with intracellular expression of IL-17A and IL-22 were significantly increased in PLNs of SKG mice injected with MIF PLM compared to SKG mice injected with CTL PLM (
After determining that MIF PLM-treated SKG mice had enhanced expression of select inflammatory markers, SpA-related immune cells in the PLNs and spleen were also evaluated. The percentage of Th17 lineage cells (CCR6+ and/or RORγt+ in CD4+ cells) was significantly increased in PLNs of MIF PLM-treated SKG mice compared to CTL PLM-treated SKG mice (
The percentage of Tregs (CD4+CD25hiFoxp3+) was reduced in PLNs of MIF PLM-injected SKG mice compared to CTL PLM-injected SKG mice (
Overexpression of MIF Increases Inflammatory Macrophages and Decreases Patrolling Macrophages in Ankle Tissues
Since macrophages are reported to be important in the development of SpA-like pathologies in SKG mice (30), and their plasticity between inflammatory and patrolling characteristics are indispensable during the development of arthritis (31), changes in the proportions of inflammatory and patrolling macrophages in SKG mice 8 weeks post-CTL PLM or MIF PLM injection were evaluated. It was found that the frequency of inflammatory macrophages (CD11b+CD11c−Ly6ChiCX3CR1loCCR2+) in ankle soft tissues was significantly higher in MIF PLM-injected SKG compared to CTL PLM-injected SKG. In contrast, the frequency of patrolling macrophages (CD11b+CD11c−Ly6CloCX3CR1hiCCR2−) was significantly decreased in MIF PLM-injected SKG compared to CTL PLM. These results indicate that MIF stimulation regulates macrophage populations by enhancing inflammatory macrophages and decreasing patrolling macrophages in the inflamed joint tissues, which may also play a pivotal role in the pathogenesis of arthritis of SKG mice, in addition to type 3 immune response.
Mif KO Suppresses the Severity of SpA-Like Pathologies Induced by Curdlan in SKG Mice
Since MIF-overexpressing SKG mice caused SpA-like pathologies, whether Mif KO (Mif−/−) SKG mice demonstrated a decreased severity of SpA phenotype compared to wild type (WT; Mif+/+) SKG mice following curdlan treatment was evaluated. Mif KO SKG mice were generated by crossing Mif−/− BALB/c mice with Mif+/+ SKG mice. Mif KO SKG mice showed approximately 10% lower body weight compared to WT SKG (
SpA-like clinical features assessed by clinical scoring were monitored for 8 weeks. Compared to WT or Het SKG mice, Mif KO SKG mice exhibited substantially lower scores for arthritis, dermatitis and blepharitis following curdlan treatment (
Gene expression of SpA-related inflammatory markers in ankle joints of WT SKG and Mif KO SKG mice at 8 weeks post-curdlan treatment was evaluated. Consistent with previous results, curdlan treatment significantly increased the expression of Il1β, Il6, IL17a, IL23a, Tnfa and Mcp1 in ankle soft tissues of WT SKG mice. In contrast, curdlan-induced expression of these inflammatory cytokines was attenuated in ankle tissues of Mif KO SKG mice (
With respect to SpA-related immune cells in PLNs, the curdlan-induced increase in the frequency of Th17 lineage cells in WT SKG mice was attenuated in Mif KO SKG mice, with reduced populations of IL17A and IL22 positive CD4+ cells in PLNs of Mif KO SKG mice compared to WT SKG mice post-curdlan treatment (
To assess the impact of pharmacologic MIF antagonism on SpA-like disease, a pre-clinical small molecule MIF antagonist (MIF098), which blocks the MIF/CD74 interaction (23), was injected into curdlan-treated female SKG mice. First, to test the prophylactic effect of MIF098, twice daily injections were started beginning one week post-curdlan treatment for 7 weeks (
Similar to Mif KO SKG mice, the frequency of Th17 lineage cells and ILC3s in PLNs were significantly deceased in curdlan-treated SKG mice injected with MIF098 compared to CTL (
The therapeutic effect of MIF098 in curdlan-treated SKG mice upon reaching moderate-to-severe clinical symptoms was also tested, which would support the application of pharmacologic MIF blockade in established disease. Thus, MIF098 was injected from 4 weeks to 8 weeks post-curdlan treatment in female SKG mice (
Since neutrophils were substantially expanded in curdlan-induced SKG mice and produced increased amount of MIF in vitro, adoptive transfer studies were performed using neutrophils (2×106 cells/injection) obtained from curdlan-treated SKG mice, where neutrophils were transferred into curdlan-treated Mif KO SKG mice at 1 and 2 weeks post-curdlan treatment (
Since curdlan-induced neutrophils transferred SpA-like pathologies, whether blocking the function of neutrophils could suppress disease progression in curdlan-treated SKG mice was tested. Either anti-Gr-1 or isotype IgG monoclonal antibody (mAb) was injected into curdlan-treated SKG mice every 3 days until 15 days post-curdlan treatment (
Since an imbalance in the ratio of Th17/Treg has been reported in SpA (32). Using flow cytometry, it was found that RORγt+Foxp3+CD4+ T cells were significantly expanded in curdlan-treated and MIF-overexpressing SKG mice, whereas this population was decreased with MIF098 or in Mif KO SKG mice (
Similar to mouse Tregs, human Tregs were isolated from healthy individuals and treated with or without rhMIF in the presence of IL1β and IL23 (
Anti-MIF antibody (IgG1, NIH IIID.9), a monoclonal antibody against MIF (anti-MIF mAb) (Leng et al., J Immunol 186, 527-38 (2011)) was administered to the mouse model described in Examples 1 and 2 and showed inhibition of arthritis (
While the present application has been described with reference to what are presently considered to be the preferred examples, it is to be understood that the application is not limited to the disclosed examples. To the contrary, the application is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.
All publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety. Specifically, the sequences associated with each accession numbers provided herein including for example accession numbers and/or biomarker sequences (e.g. protein and/or nucleic acid) provided in the Tables or elsewhere, are incorporated by reference in its entirely.
The scope of the claims should not be limited by the preferred embodiments and examples, but should be given the broadest interpretation consistent with the description as a whole.
This PCT application claims priority to U.S. Provisional Patent Application No. 63/106,859 filed Oct. 28, 2020, the contents of which are incorporated herein by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CA2021/051470 | 10/19/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63106859 | Oct 2020 | US |
