Prevention of heterotopic ossifications

Abstract

The invention pertains to the use of glucocorticoid receptor antagonists to prevent heterotopic ossification. Provide herein are methods and uses related to the prophylaxis of heterotopic ossification (HO) in subjects that have experienced an injury or trauma that rely on the administration of a glucocorticoid receptor antagonist. The glucocorticoid receptor antagonist may be an antagonist to cortisol binding the glucocorticoid receptor, such as mifepristone or relacorilant, or a pharmaceutically acceptable salt, derivative, prodrug or solvate thereof.

Description

PREVENTION OF HETEROTOPIC OSSIFICATIONSTechnical Field

[0001] The present invention broadly relates to method of preventing heterotopic ossification.Background of the Invention

[0002] Heterotopic ossification (HO) is a pathological condition involving bone formation in soft tissue, which typically causes pain and restricted movement. In some cases, HO arises due to a rare genetic condition known as Fibrodysplasia Ossificans Progressiva (FOP). More common, however, is acquired HO. Acquired HO may occur as a result of traumatic injury, usually soft tissue injury, and may occur following neurologic injury. HO arising from neurologic injury is commonly known as neurogenic heterotopic ossification (NHO).

[0003] NHOs are abnormal extra- skeletal lamellar bone masses usually forming in periarticular muscles, that typically form after severe central nervous system (CNS) insults such as spinal cord injury (SCI), traumatic brain injury (TBI), or stroke (Meyers et al., 2019; Ohlmeier et al., 2017). NHOs were first identified in soldiers during World War I (Schurch and Dollfus, 1998) and are still prevalent today with an incidence of 10-23% in patients with TBI, 10-53% in SCI, and up to 68% in army personel who recieve combat blast injuries involving the spine (Citak et al., 2012; Forsberg et al., 2009; Reznik et al., 2014; van Kuijk et al., 2002; Wittenberg et al., 1992). Recently, NHOs have been observed to be highly prevalent in mechanically ventilated SARS-Cov-2 patients treated with dexamethasone.

[0004] NHOs typically develop in peri-articular muscles, most frequently in the hip, elbow, knee and shoulder (Genet et al., 201 lb). Due of their common periarticular location, NHO can be very incapacitating, mainly due to their size (up to 2kg), often causing significant pain and progresses to complete ankylosis of the affected joint. NHOs exacerbate functional disabilities by increasing difficulty in sitting, eating and dressing (Vanden Bossche and Vanderstraeten, 2005). NHO can also cause nerve and blood vessel compression, further increasing patient morbidity (Bradleigh et al., 1992; Saiga et al., 2014).

[0005] Previous studies have shown that SCI can exacerbate and prolong macrophage-mediates inflammatory responses in injured muscles (Genet et al., 2015 and Tseng H-W et al., 2022), but the signals which exacerbate inflammation and drive NHO in injured muscles remain unknown (Alexander et al., 2020). All previous studies in SCI / TBI patients have been being retrospective; such studies do not investigate the early initiating cellular and molecular events which drive NHO pathogenesis, and the mechanism underlying acquired HO remains unclear.

[0006] Treatment of NHOs is still limited to surgical resection of mature NHO (Genet et al., 2012; Genet et al., 2011b; Genet et al., 2009; Vanden Bossche and Vanderstraeten, 2005); however, NHOs recur in ~6% patients (Genet et al., 2011a). The development of improved treatments forNHO has been slow and trials of pharmacological interventions have continued to show limited effectiveness (Haran et ah, 2004), reflecting the current limited knowledge on the etiology and pathophysiology of NHO (Alexander et al., 2020). There remains a need for effective treatments which reduce HO, especially NHO, development.Summary of the Invention

[0007] According to a first aspect, the present disclosure provides a method of prophylaxis of heterotopic ossification, comprising administering to a subject in need thereof an effective amount of a glucocorticoid receptor antagonist. In some embodiments, the heterotopic ossification is acquired heterotopic ossification.

[0008] In some embodiments, the subject has experienced an injury or trauma. In some embodiments, the injury or trauma comprises a soft tissue injury or trauma. In some embodiments, the injury or trauma comprises an injury or trauma to muscle or connective tissue. In some embodiments, the injury or trauma comprises a non-neurologic injury or trauma. In some embodiments, the injury or trauma comprises a burn, a blast injury, an amputation, an arthroplasty, an injury or trauma resulting from a fall or crush, or an injury or trauma associated with artificial ventilation, immobilization and / or muscle spasticity. In some embodiments, the glucocorticoid receptor antagonist is administered to the subject within 7 days of the injury or trauma, or within 2 days of the injury or trauma.

[0009] In some embodiments, the heterotopic ossification is neurogenic heterotopic ossification and the subject has further experienced a neurologic injury or trauma. In some embodiments, the neurologic injury is a spinal cord injury, a traumatic brain injury, a stroke or cerebral anoxia. In some embodiments, the glucocorticoid receptor antagonist is administered to the subject within 7 days of the neurologic injury or trauma, or within 2 days of the neurologic injury or trauma.

[0010] In some embodiments, the subject has, is currently, or is expected to experience artificial ventilation, immobilization and / or muscle spasticity.

[0011] In some embodiments, the glucocorticoid receptor antagonist is mifepristone or relacorilant, or a pharmaceutically acceptable salt, derivative, prodrug or solvate thereof.

[0012] According to a second aspect, the present disclosure provides use of a glucocorticoid receptor antagonist in the manufacture of a medicament for the prophylaxis of heterotopic ossification.

[0013] According to a third aspect, the present disclosure provides a glucocorticoid receptor antagonist for use in a method of prophylaxis of heterotopic ossification.Brief description of the drawings

[0014] Exemplary embodiments of the present disclosure are described herein, by way of nonlimiting example only, with reference to the following drawings.

[0015] Fig. 1 shows mass spectrometry measurements of plasma concentrations of corticosterone (CORT) (A), its inactive precursor l ip-deoxycorticosterone (DCORT) (B), and catecholamine catabolites 3-methoxy tyramine (3MT) (C), metadrenaline (MET) (D) and normetadrenaline (NorMET) (E) in C57BL / 6 female mice that underwent SCI, Sham injury, alone or in combination with CDTX muscle injury.

[0016] Fig. 2 shows NHO volumes via pCT at 7 days post-CDTX with and without corticosterone (Cort) or dexamethasone (Dex) administration. (A) NHO volumes via pCT at 7 days post-CDTX muscle injury in C57BL / 6 female mice with or without corticosterone (Cort) in drinking water from day -2 to +7 post-CDTX intramuscular injection or day +2 to +7 post-CDTX intramuscular injection, with three representative pCT images / treatment group. Red arrow heads show HO in hamstring muscles. (B) NHO volumes via pCT at 7 days post-CDTX administered saline or lOmg / kg / day dexamethasone (Dex) until 7 days post-CDTX, with two representative pCT images / treatment group. (C) NHO volumes via pCT at 7 days post-CDTX administered saline or lOmg / kg / day dexamethasone (Dex) administered either saline or lOmg / kg / day dexamethasone either immediately (Ohr) or between -lhr and +72hr post-CDTX muscular injury as indicated. Each dot represents one mouse.

[0017] Fig. 3 shows immunohistochemistry of serial muscle sections at 21 days post CDTX muscle injury. (A) Immunohistochemistry of serial muscle sections at 21 days post-CDTX muscle injury in C57BL / 6 female mice with or without corticosterone (CORT) in drinking water. (B) Isotype-matched IgG controls confirming specificity of staining for F4 / 80 (RatIgG2b) and osterix (Rabbit IgG). # symbols mark the same anatomical location.

[0018] Fig. 4 shows the in vitro effect of cortisol (CORT) and dexamethasone (DEX) on calcium mineralization of PDGFRa+ CD56- mesenchymal progenitor cells sorted from muscles surrounding human NHO and cultured in osteogenic conditions. Dots represent results from each different biopsy, and lines link results from the same biopsy. Columns are the average.

[0019] Fig. 5 shows postnatal deletion of the glucocorticoid receptor gene Nr3cl prevents NHO development. (A) NHO volumes via pCT at 7 and 21 days after spinal cord and CDTX-muscle injury (SCI+CDTX) in Rosa26CreERT2; Nr3clWT / WTcontrol mice (R26Cre / WT) or Nr3clA mice with a tamoxifen-inducible postnatal deletion of the glucocorticoid receptor gene (Rosa26CreERT2; Nr3clflox / flox), with three representative pCT images / treatment group / timepoint. (B) Immunohistochemistry of serial muscle sections at 21 days post SCI+CDTX in control mice or mice with a postnatal deletion of the glucocorticoid receptor. Each dot represents one mouse.

[0020] Fig. 6 shows NHO volumes via pCT at 7 and 21 days post-injury in female mice which underwent SCI + CDTX intramuscular injection and were orally gavaged daily with vehicle (black dots) or 50 mg / kg / day mifepristone (red dots) from day 0-7 post-surgery. (A) NHO volumes viau CT at 7 and 21 days after SCI+CDTX in mice which were orally gavaged daily with vehicle (VEH) or 50 mg / kg / day GR antagonist mifepristone (MIF) from day 0-7 post-surgery. (B) Representative pCT images / treatment group / timepoint. (C) NHO volumes via pCT at 7 days postsurgery in female C57BL / 6 SCI+CDTX mice treated with either vehicle (VEH) or 50 mg / kg / day mifepristone (MIF) from day 0-2, or (D) or day 3-5 post- injury, or (E) MIF treatment initiated at +5, 10, 24 or 48 hours post-surgery, or (F) short-term mifepristone treatment (day 0-2 post-injury) in male mice. (G) Runx2 mRNA expression in injured muscles by qRT-PCR at days 2 and 4 post- SCI and muscle injury in female mice after day 0-2 treatment with vehicle or mifepristone. Each dot represents one mouse.

[0021] Fig. 7 shows mRNA expression of the glucocorticoid receptor and progesterone receptor in mouse leukocytes and muscle cells. Each dot represents one mouse.

[0022] Fig. 8 shows mouse NHO volumes via pCT at 7 and 21 days post-surgery following treatment with vehicle (black dots) or relacorilant (60mg / kg twice daily, red dots) from day 0-2 post surgery (A); and representative pCT images at 7 and 21 days post-surgery (B). Each dot represents one mouse.

[0023] Fig. 9 shows immunohistochemistry on serial muscle sections at 21 days, in vehicle-treated mice, compared to CDTX-injured muscles from relacorilant treated (A); and isotype-matched IgG controls confirming specificity of staining for F4 / 80 (RatIgG2b) and osterix (Rabbit IgG) in CDTX-injured muscles in mice with SCI and CDTX-muscle injury treated with vehicle or relacolirant (B). # symbols in (B) mark the same anatomical location as in (A).

[0024] Fig. 10 shows results of SCI + CDTX injection carried out on female mice and treatment from day 0-5 post-surgery with either saline (black dots) or dexamethasone (lOmg / kg / day, red dots).Detailed description of the invention

[0025] The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

[0026] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, typical methods and materials are described.

[0027] Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or" comprising", will be understood to imply the inclusion of a stated step or element or integer or group of steps or elements or integers, but not the exclusion of any other step or element or integer or group of elements or integers. Thus, in the context of this specification, the term "comprising" means "including principally, but not necessarily solely".

[0028] In the context of this specification, the terms "a" and "an" refer to one or to more than one (i.e. to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0029] In the context of this specification, the term "about" is understood to refer to a range of numbers that a person of skill in the art would consider equivalent to the recited value in the context of achieving the same function or result.

[0030] In the context of this specification, reference to a range of numbers disclosed herein (for example, 1 to 10) also incorporates reference to all rational numbers within that range (for example, 1, 1.1, 2, 3, 3.9, 4, 5, 6, 6.5, 7, 8, 9 and 10) and also any range of rational numbers within that range (for example, 2 to 8, 1.5 to 5.5 and 3.1 to 4.7) and, therefore, all sub-ranges of all ranges expressly disclosed herein are hereby expressly disclosed. These are only examples of what is specifically intended and all possible combinations of numerical values between the lowest value and the highest value enumerated are to be considered to be expressly stated in this application in a similar manner.

[0031] As used herein, the term "and / or" means "and" or "or" or both.

[0032] The term “subject” as used herein refers to a vertebrate subject, particularly a mammalian subject, for whom therapy or prophylaxis is desired. Suitable subjects include, but are not limited to, primates; avians (birds); livestock animals such as sheep, cows, horses, deer, donkeys and pigs; laboratory test animals such as rabbits, mice, rats, guinea pigs and hamsters; companion animals such as cats and dogs; and captive wild animals such as foxes, deer and dingoes. In particular, the subject is a human.

[0033] As used herein the terms "treating", “treatment”, "treating", “reduce”, “reducing”, “prevent” "preventing", "prevention" and “prophylaxis” and the like refer obtaining a desired pharmacologic and / or physiologic effect. The effect may be to remedy, or otherwise hinder, retard, reverse the progression of or arrest the development of a condition or disease or at least one symptom of a condition or disease, including reducing the severity of a condition or disease. Thus, the terms “treat”, "treating", “treatment”, do not necessarily imply that a subject is treated until complete elimination of or recovery from the condition or disease. Similarly, the terms “prevent”, "preventing", “prevention”, “prophylaxis” and the like refer to any and all applications that may preserve the health of the subject and prevent the establishment of a condition or disease orotherwise delay the onset of a condition or disease or reduce or inhibit the progression, development or severity of a disease or condition or one or more of its symptoms. It will be understood that the aforementioned terms do not imply that symptoms are present at the time of prophylaxis or thereafter, in that a method of prophylaxis may preserve the health of the subject by preventing the emergence of symptoms, or the emergence of a disease or disorder before symptoms are identifiable.

[0034] As used herein in the context of the invention, a method of prophylaxis of heterotopic ossification and the like, may, for example, prevent the formation of bone tissue in the soft tissue of a subject, or otherwise reduce or inhibit the progression, development or severity of the formation of bone tissue in the soft tissue of a subject, or may reduce or inhibit the progression, development or severity one or more symptoms of heterotypic ossification. In other examples, the method of prophylaxis of heterotopic ossification as described herein may prevent the emergence of symptoms, or the exacerbation of symptoms, in a subject that has been determined to have heterotopic ossification, or may prevent, reduce, or inhibit the formation of bone tissue in additional soft tissue sites in a subject already experiencing heterotopic ossification in a particular area of soft tissue.

[0035] As used herein, the phrase “inhibit the development / severity / progress! on of’ refers to a prophylactic treatment which increases the resistance of a subject to developing the disease or condition or, in other words, decreases the likelihood that the subject will develop the disease or condition as well as a treatment after the disease or condition has begun in order to reduce or eliminate it altogether or prevent it from becoming worse. This phrase also includes within its scope preventing the disease or condition from occurring in a subject which may be predisposed to the disease or condition but has not yet been diagnosed as having it.

[0036] The terms “reduce”, “inhibit”, “suppress”, “decrease”, “prevent”, and grammatical equivalents when used in reference to the level of a substance and / or phenomenon in a first sample relative to a second sample, mean that the quantity of substance and / or phenomenon in the first sample is lower than in the second sample by any amount that is statistically significant using any art-accepted statistical method of analysis. When these terms are used to refer to the action of a molecule or agent, the first sample may be a sample following action of the molecule or agent and the second sample may be a comparative sample without the molecule or agent. In one embodiment, the reduction may be determined subjectively, for example when a patient refers to their subjective perception of disease symptoms, such as pain, fatigue, motor symptoms, etc. In another embodiment, the reduction may be determined objectively, for example when heterotopic ossification is lower than in comparable subjects. In another embodiment, the quantity of substance and / or phenomenon in the first sample is at least 10% lower than the quantity of the same substanceand / or phenomenon in a second sample. In another embodiment, the quantity of the substance and / or phenomenon in the first sample is at least 25% lower than the quantity of the same substance and / or phenomenon in a second sample. In yet another embodiment, the quantity of the substance and / or phenomenon in the first sample is at least 50% lower than the quantity of the same substance and / or phenomenon in a second sample. In a further embodiment, the quantity of the substance and / or phenomenon in the first sample is at least 75% lower than the quantity of the same substance and / or phenomenon in a second sample. In yet another embodiment, the quantity of the substance and / or phenomenon in the first sample is at least 90% lower than the quantity of the same substance and / or phenomenon in a second sample. Alternatively, a difference may be expressed as an “n- fold” difference. By analogy, terms “increase”, “promote” and “heighten”, and grammatical equivalents when used in reference to the level of a substance and / or phenomenon in a first sample relative to a second sample, mean that the quantity of substance and / or phenomenon in the first sample is higher than in the second sample by any amount that is statistically significant using any art-accepted statistical method of analysis.

[0037] The term "optionally" is used herein to mean that the subsequently described feature may or may not be present or that the subsequently described event or circumstance may or may not occur. Hence the specification will be understood to include and encompass embodiments in which the feature is present and embodiments in which the feature is not present, and embodiments in which the event or circumstance occurs as well as embodiments in which it does not.

[0038] As used herein the terms "effective amount” and "effective dose" include within their meaning a non-toxic but sufficient amount or dose of a compound to provide the desired effect. The exact amount or dose required will vary from subject to subject depending on factors such as the species being treated, the age and general condition of the subject, the severity of the condition being treated or potential condition being prevented, the particular compound being administered and the mode of administration and so forth. Thus, it is not possible to specify an exact “effective amount” or "effective dose". However, for any given case, an appropriate “effective amount” or "effective dose" may be determined by one of ordinary skill in the art using only routine experimentation.

[0039] The term “antagonist” and grammatical equivalents thereof as used herein refers to a molecule that partially or completely inhibits, by any mechanism, an effect of another molecule such as a receptor or intracellular mediator. In the context of the present invention, the term “glucocorticoid receptor antagonist” refers to a molecule that is an antagonist, especially a direct antagonist that binds to or otherwise interacts with a glucocorticoid receptor (GR). In particular embodiments, the molecule partially or completely inhibits a glucocorticoid receptor from interacting with a glucocorticoid, or partially or completely inhibits the downstream effectspotentiated by a glucocorticoid receptor from interacting with a glucocorticoid. Antagonism of a glucocorticoid receptor typically inhibits or reduces one or more glucocorticoid receptor activities and / or functions.

[0040] “ Glucocorticoid receptor activity”, “glucocorticoid receptor function” and variants thereof are used herein to refer to the biological activity and / or function of the glucocorticoid receptor and includes interaction of the glucocorticoid receptor with glucocorticoids, glucocorticoid receptor activation and downstream effects.

[0041] By ‘ ‘derivative” is meant a molecule that has been derived from the basic molecule by modification, for example by conjugation or complexing with other chemical moieties as would be understood in the art. The term “derivative” also includes within its scope alterations that have been made to a parent molecule including substitution of functional groups that provide for functionally equivalent molecules.

[0042] As used herein, the term “dosage unit form” refers to physically discrete units suited as unitary dosages for the subject to be treated, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required pharmaceutically acceptable vehicle.

[0043] By “pharmaceutically acceptable carrier” is meant a pharmaceutical vehicle comprised of a material that is not biologically or otherwise undesirable, i.e., the material may be administered to a subject along with the selected active agent without causing any or a substantial adverse reaction. Carriers may include excipients and other additives such as diluents, fillers, detergents, coloring agents, wetting or emulsifying agents, pH buffering agents, preservatives and the like.

[0044] By "pharmaceutically acceptable salt" it is meant those salts which, within the scope of sound medical judgement, are suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit / risk ratio. Reference to a compound herein shall be understood to include its pharmaceutically acceptable salts unless specified otherwise or otherwise understood from context. Similarly, a “pharmacologically acceptable” derivative, ester, amide, prodrug or solvate of a compound as provided herein is a derivative, ester, amide, prodrug or solvate that this not biologically or otherwise undesirable.

[0045] As used herein, the terms “salts” and “prodrugs” include any pharmaceutically acceptable salt, ester, hydrate or any other compound which, upon administration to the recipient, is capable of providing (directly or indirectly) a glucocorticoid receptor antagonist molecule used in the present invention, or an active metabolite or residue thereof. The term “pharmaceutically acceptable salts” refers without limitation to derivatives of the disclosed compounds wherein the parent compound is modified by converting an existing acid or base moiety to its salt form (e.g.by reacting the free base group with a suitable organic acid). Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3 -phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate and valerate salts, and the like. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like, as well as nontoxic ammonium, quaternary ammonium, and amine cations, including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like. Pharmaceutically acceptable salts include the conventional non-toxic salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. The pharmaceutically acceptable salt can be synthesized from the parent compound which contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, nonaqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. Lists of suitable salts are found in, for example, Remington (1985) Remington’s Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 17th edition; Stahl and Wermuth (2002) Pharmaceutical Salts: Properties, Selection, and Use, Wiley-VCH; and Berge etal. (1977) Journal of Pharmaceutical Science, 66: 1-19, each of which is incorporated herein by reference in its entirety

[0046] Each embodiment described herein is to be applied mutatis mutandis to each and every embodiment unless specifically stated otherwise.

[0047] The present invention is predicated on the present inventors’ surprising finding that administration of glucocorticoid receptor (GR) antagonists to subjects following spinal cord injury (SCI) and muscle injury may be used to prevent or hinder the progression of heterotopic ossification (HO) in said subjects. In particular, the present inventors have surprisingly found that the spike in adrenal glucocorticoid (GC) corticosterone secretion following SCI drives neurogenic heterotopic ossification (NHO) development in injured muscles; in fact, exogenousglucocorticoid administration has been found to be sufficient to cause HO even when no SCI has taken place, and treatment with a glucocorticoid receptor agonist exacerbates NHO development. Further, the present inventors have surprisingly found that timely administration of two structurally unrelated selective GR antagonists inhibited NHO formation after SCI, and that glucorticoid antagonists may therefore be used in the prophylaxis of HO, to prevent HO onset and / or progression.

[0048] This prophylactic treatment for HO, and the discovery that glucocorticoids exacerbate HO, is in direct contrast to the practice in the art of administering glucocorticoids to subjects with Fibrodysplasia Ossificans Progressiva (FOP), a rare genetic form of HO, to manage immune system flare-ups and pain prior to initiation of HO. The present inventors’ finding is also contrary to the well-established anabolic and catabolic effects of agonistic glucocorticoids (GCs) in the skeletal system (Hardy et al., 2018; Klein, 2015). Basal GC levels are required for the development of skeletal bones in the cranium during fetal development (Zhou et al., 2009), whereas chronic excessive GCs is detrimental to skeletal bone health, as excessive and continuous GR agonism can result in GC-induced osteoporosis by reducing bone formation by osteoblasts and increasing bone degradation by osteoclasts (Hardy et al., 2018).

[0049] Accordingly, provided herein is a method of prophylaxis of heterotopic ossification comprising administering to a subject in need thereof an effective amount of a glucocorticoid receptor antagonist.

[0050] Any suitable glucocorticoid receptor (GR) antagonist, including their pharmaceutically acceptable salts, derivatives, prodrugs or solvates, may be used in methods of the present disclosure.

[0051] In embodiments of the invention, the GR antagonist is a direct GR antagonist that partially or completely prevents the binding of a glucocorticoid, preferably a lipophilic glucocorticoid, to the GR. In these embodiments of the invention, the prevention of binding of the GR by the antagonist is by way of the antagonist physically blocking the binding of the glucocorticoid to the GR (i.e., by the antagonist binding the GR). In embodiments of the invention, the GR antagonists may be an indirect antagonist that partially or completely prevents the binding of a lipophilic glucocorticoid to the GR by down-regulating or blocking the synthesis of the lipophilic glucocorticoid. In other embodiments, the GR antagonist partially or completely prevents the binding of cortisol to the GR, such that the GR antagonist is an antagonist to cortisol binding the GR. It would be understood that by partially or completely preventing the binding of a glucocorticoid, such as, for example, cortisol, to a GR, either by blocking the binding or the synthesis of cortisol, the associated confirmational change and activation said binding wouldordinarily trigger in the GR would be partially or completely inhibited, thus effecting the downstream effects associated with GR activation.

[0052] Reference to GR antagonists herein, unless specified otherwise or derivable from context, includes reference to their pharmaceutically acceptable salts, derivatives, prodrugs and solvates. Suitable GR antagonists will be familiar to a person skilled in the art. By way of example only, potential glucocorticoid receptor antagonists include mifepristone (RU-486), relacorilant (CORT125134), RU-43044, onapristone (ZK-89299), AL086D06, ORG-34517, ORG-34850, ketaconazole and ORG-34116 and their pharmaceutically acceptable salts, derivatives, prodrugs and solvates.

[0053] In embodiments of the invention, the GR antagonist is an antagonist to cortisol binding the GR, and said antagonist is selected from the group consisting of mifepristone (RU-486), relacorilant (CORT125134)

[0054] In some embodiments, the GR antagonist comprises mifepristone or relacorilant, or a pharmaceutically acceptable salt, derivative, prodrug or solvate thereof. Relacorilant is a nonsteroid selective GR antagonist which is, at the time of filing, in clinical trials to treat Cushing syndrome and adrenal carcinomas (Hunt et al., 2018; Hunt et al., 2017). The structures of mifepristone and relacorilant are shown below, together with the structure of three GR agonists (corticosterone, dexamethasone and cortisol).

[0055] The methods of the present disclosure are used for the prophylaxis of HO. Preferably, the HO is acquired HO (i.e. not genetic HO such as FOP).CortisoiMifepristone Refecoriiant

[0056] In particular embodiments, the subject has experienced an injury or trauma. The terms “injury” and “trauma”, and “physical insult” and “physical trauma”, are readily understood in the art to refer to some sort of physical insult to the body, whether this be due to illness or accident or due to treatment or surgery, or some other cause. Said injury or trauma may be expected to give rise to HO, or to more severe HO, in the absence of prophylactic treatment. HO following an injury or trauma may be termed “traumatic HO”. Where said injury or trauma, such as a non-neurologic injury or trauma, occurs together with (although not necessarily at the same time as) a neurologic injury or trauma, the resultant HO may be termed “neurogenic HO”, as discussed below.

[0057] In particular embodiments, the injury or trauma may comprise a soft tissue injury or trauma. In some embodiments the injury or trauma comprises a muscle injury or trauma or a connective tissue injury or trauma, for example a tendon injury or trauma. In some embodiments, the injury or trauma is a joint injury or trauma. In some embodiments, the injury or trauma may be a periarticular muscle or tissue injury or trauma. In some embodiments, the injury or trauma may be a wound, such as an extended wound, such as a deep burn.

[0058] In some embodiments the injury or trauma may comprise a burn, for example severe body burns. In particular embodiments, the burns may be deep burns that may reach the muscle layer under the dermis and connective tissue. In some embodiments, the injury or trauma may comprise, for example, a blast injury, for example a combat-related blast injury. In some embodiments the injury or trauma may comprise an amputation. In some embodiments, the subject may have sustained injuries following a crush or fall. In some embodiments, the injury or trauma may comprise arthroplasty. In some embodiments, the injury or trauma may be a multi-trauma.

[0059] In some embodiments, the subject may have an injury / injuries with an injury severity score of >16. In some embodiments, the injury or trauma may be associated with artificial ventilation, immobilization and / or muscle spasticity. For example, injury or trauma may comprise muscle tears resulting from active or passive moment during immobilisation and / or muscle spasticity. In some embodiments, the subject may not have experienced any neurologic injury or trauma.

[0060] In some particularly preferred embodiments, the HO is neurogenic HO (NHO), and, in addition an injury or trauma such as those described above, the subject has further experienced a neurologic injury or trauma. Said neurologic injury or trauma may comprise a central nervous system (CNS) injury, for example a spinal cord injury (SCI), a traumatic brain injury (TBI) or other brain injury, a stroke or cerebral anoxia. In some embodiments, the neurologic injury or trauma may comprise peripheral nerve injury and / or peripheral denervation; in some embodiments, the peripheral nerve injury may compromise central nervous system (CNS) integrity.

[0061] In embodiments wherein the HO is neurogenic HO, the subject will typically have experienced a (typically non-neurologic) trauma or injury, i.e. “another injury or trauma”, as described above, as well as the neurologic injury or trauma. The injury or trauma may comprise, for example a soft tissue injury or trauma. In some embodiments the injury or trauma comprises a muscle injury or trauma or a connective tissue injury or trauma, for example a tendon injury or trauma. In some embodiments, the injury or trauma is a joint injury or trauma. In some embodiments, the injury or trauma may be a periarticular muscle or tissue injury or trauma. In some embodiments, the injury or trauma may be a wound, such as an extended wound, such as a deep burn. The injury or trauma may be, in some embodiments, an injury or trauma as described above. Said injuries or traumas may be non-neurologic in nature and occur as well as a neurologic injury or trauma in cases of anticipated neurogenic HO. Said injuries or trauma may occur at the same time as, or due to the same event as, the neurologic injury or trauma, or may occur before or after the neurologic injury or trauma.

[0062] As such, in preferred embodiments, the HO is neurogenic HO and the subject has experienced both a neurologic injury or trauma and a further injury or trauma, wherein the further injury or trauma may be, for example, a soft tissue injury or trauma, for example a muscle or connective tissue injury or trauma, and / or may be a non-neurologic injury or trauma. The further injury or trauma is typically a non-neurologic injury or trauma.

[0063] For example, neurogenic HO is known to occur in humans wherein the neurologic injury or trauma is a stroke or cerebral anoxia and the sufferer falls during the stroke or cerebral anoxia, causing further injury, thus giving rise to HO. Similarly, neurogenic HO has been observed in instances of a severe crush of the body (such as an accident) which causes brain or spinal cord injuries as well as damaging soft tissues such as muscles and joints. Without wishing to be bound by theory, it is thought that joints and periarticular muscles and tissues are particularly vulnerable in such uncontrolled falls and crushes, and as such a large proportion of observed neurogenic HO following such events are located in the periarticular muscles or tissues. Accordingly, in someembodiments, the subject has experienced any of the aforementioned injuries or traumas or combinations thereof.

[0064] Where a subject has experienced an injury or trauma (with or without an additional neurologic injury or trauma), such as a soft tissue injury or trauma, HO is typically expected to develop at the site of the injury or trauma.

[0065] In some embodiments the subject may have experienced, is experiencing or is expected to experience artificial ventilation, immobilization and / or muscle spasticity. Such artificial ventilation, immobilization and / or muscle spasticity may occur, for example, following a neurologic injury or trauma.

[0066] In some embodiments, the subject may have experienced or is experiencing a respiratory condition such as COVID- 19. Such a subject, according to some embodiments, may have experienced, is experiencing or is expected to experience artificial ventilation, immobilization and / or muscle spasticity.

[0067] In particular embodiments, the HO is HO occurring in periarticular muscles or tissues.

[0068] Whilst in many instances the subject has experienced an injury, in some embodiments acquired HO may be anticipated wherein the subject has not undergone an apparent injury as such. For example, it has been observed that subjects with severe cases of Sars-Cov-2 who are mechanically ventilated in an induced coma exhibit growth of mineralised HO. Whilst the exact cause of HO in such subjects is not yet known, it is anticipated that said cause might not be considered a traditional “injury” but, nonetheless, such subjects may benefit from the prophylactic treatment of the present invention. However, it will be appreciated that further study may show that such patients have in fact sustained a form of injury.

[0069] It will be appreciated that the terms “acquired HO”, “traumatic HO” and “neurogenic HO” may be used with some degree of interchangeability in the art, but that the meaning of each is readily understood from their context of use.

[0070] In preferred embodiments, the GR antagonist is administered to the subject soon after the neurologic or other injury or trauma. In preferred embodiments, the GR antagonist is administered as soon as possible following the neurologic or other injury or trauma. In some preferred embodiments, the GR antagonist is administered to the subject within 7 days following the neurologic or other injury or trauma. In some embodiments, the GR antagonist is administered to the subject within 4 days following the injury or trauma. In some embodiments, the GR antagonist is administered within 2 days following the neurologic or other injury or trauma. The precise time frame in which the GR antagonist is optimally administered following injury or trauma may vary depending on various factors, including the species of the subject.

[0071] Typically, neurologic and other injury or trauma will occur due to the same event (for example, an accident or stroke and fall) and thus occur at substantially the same time or close in time to one another, but in instances wherein, for example, a neurologic injury or trauma occurs before a further injury or trauma (for example where further injury or trauma occurs as a result of artificial ventilation, immobilization and / or muscle spasticity following a neurologic injury or trauma), timing of administration of the GR antagonists preferably refers to timing following the neurologic injury or trauma. In some alternative embodiments, timing of administration of the GR antagonists refers to timing following the other (for example non -neurologic and / or soft tissue) injury or trauma, or both the neurologic and other injury or trauma.

[0072] In particular embodiments, the GR antagonist is administered at least daily. In particular embodiments, the GR antagonist is administered every day for at least the first 2 days following the neurologic or other injury or trauma. In some embodiments, the GR antagonist is administered every day for at least the first 3 days following the neurologic or other injury or trauma, optionally every day for at least the first 4 days following the injury or trauma, or every day for at least the first 7 days following the neurologic or other injury or trauma. In some preferred embodiments, the GR antagonist is administered immediately following the neurologic or other injury or trauma or as soon as practicably possible thereafter. In some particular embodiments, the GR antagonist is administered at least daily for at least 2 days, for example at least 3 days, for example for 2 or 3 days. In particular embodiments, the GR antagonist is administered as soon as possible following the neurologic or other injury or trauma for at least 2 days, for example at least 3 days, for example 2 or 3 days.

[0073] In some embodiments, the GR antagonist is not administered beyond 2, 3, 4 or 7 days following the neurologic or other injury or trauma, for example not beyond 2 days following the injury or trauma or not beyond 7 days following the neurologic or other injury or trauma.

[0074] In some embodiments, for example wherein a subject has not experienced an apparent specific injury, or wherein the subject is suffering from COVID- 19, the GR antagonist is administered continuously for a period without reference to the time of a particular injury or trauma. For example, the GR antagonist is administered for a period of at least 2 days, for example at least 3 days, for example 2 days or 3 days to a subject for whom HO prevention is thought to be necessary. In some embodiments, the GR antagonist is administered throughout a period during which prevention of HO remains a concern, for example throughout a period in which a subject is mechanically ventilated or immobile or experiencing muscle spasticity.

[0075] In some embodiments, endogenous glucocorticoid levels of a patient are measured, for example following a neurologic or other injury or trauma, and the GR antagonist administered to the subject when glucocorticoid levels are elevated. Glucocorticoid levels may be measured andcompared to a predetermined threshold, such as that of a typical, comparable healthy individual, and a GR antagonist administered when the glucocorticoid level of the subject exceeds said threshold. In some embodiments, the GR antagonist may be administered, optionally in multiple doses, until the glucocorticoid level of the subject falls below the threshold. Such methods enable GR antagonists to be administered when most effective in preventing HO progression, whilst limiting unnecessary GR antagonist administration when less effective. Glucocorticoid levels of a subject may be measured by any means familiar to a person skilled in the art, for example by collection of a blood plasma sample from said subject and analysis of said plasma sample by mass spectrometry.

[0076] GR antagonists may be used in methods of the present disclosure in the form of a prodrug. Typically, prodrugs will be functional derivatives of GR antagonists which are readily converted in vivo to the required (active) GR antagonists. Typical procedures for the selection and preparation of prodrugs are known to those of skill in the art and are described, for instance, in H. Bundgaard (Ed), Design of Prodrugs, Elsevier, 1985.

[0077] GR antagonists, including their salts, may also be used in the form of solvates, in particular hydrates. In the context of the invention, solvates refer to those forms of compounds which, in the solid or liquid state, form a complex by coordination with solvent molecules. Hydrates are a specific form of the solvates in which the coordination is with water. Crystals of the present compounds may, for example, include the solvent used for crystallization. Different crystalline forms may be present.

[0078] GR antagonists for use in the methods of the present disclosure may be provided in a pharmaceutical composition and / or therapeutic formulation, that is, GR antagonists present together with a pharmaceutical acceptable carrier, excipient, diluent and / or vehicle. In general pharmaceutical formulations of GR antagonists for use in methods of the present disclosure may be prepared according to methods which are known to those of ordinary skill in the art and accordingly may include a pharmaceutically acceptable carrier, excipient, diluent, vehicle and / or adjuvant. The carriers, excipients, diluents, vehicles and adjuvants must be "acceptable" in terms of being compatible with the other ingredients of the formulation, and not deleterious to the recipient thereof. As will be appreciated by those skilled in the art, the choice of pharmaceutically acceptable carrier or diluent will be dependent on the route of administration and on the nature of the condition and subject to be treated. For example, for administration as an injectable solution or suspension, non-toxic parenterally acceptable diluents or carriers can include, Ringer's solution, isotonic saline, phosphate buffered saline, ethanol and 1, 2-propylene glycol. The particular carrier or delivery system and route of administration may be readily determined by a person skilled in the art.

[0079] The GR antagonists may be administered in any suitable form or method. Possible administrations forms and methods will be familiar to a person skilled in the art, and may depend, amongst other factors, on the age or size of the subject, the extent of any injuries sustained and their circumstances, and the type of GR antagonist used. Examples of suitable routes of administration, include, but are not limited to, oral, rectal, topical, intranasal, intraocular, transmucosal, intestinal, enteral, intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intracerebral, intravaginal, intravesical, intravenous or intraperitoneal administration. In particular embodiments, the GR antagonist is administered by oral ingestion. In some embodiments, the GR antagonist is administered intravenously.

[0080] The compounds may be administered in a local rather than systemic manner, such as by injection directly into a tissue, which is preferably subcutaneous or omental tissue, often in a depot or sustained release formulation. In other embodiments, the compound is systemically administered.

[0081] As a particular example, if a subject is in intensive care in a coma following serious injuries or some other illness or trauma, subjects are typically fed by way of a nasogastric bypass tube; medicines such as the GR antagonist may also be administered orally via the nasogastric bypass tube. Alternatively, GR antagonists may be administered intravenously.

[0082] Single or multiple administrations of the GR antagonists can be carried out with dose levels and patterns being selected by the treating physician. Regardless, the GR antagonists used in the methods of the present disclosure should provide a quantity of the GR antagonist, or derivative, salt, prodrug or solvate thereof sufficient for treatment or prophylaxis of HO.

[0083] One skilled in the art would be able, by routine experimentation, to determine an effective, non-toxic amount of the GR antagonists or prodrugs, derivatives, salts or solvates thereof, or pharmaceutical compositions used in the invention, which would be required for effective prophylaxis of HO.

[0084] By way of example only, GR antagonists may be administered in a dose of, for example, up to about 500 mg / kg / day, for example up to about 400 mg / kg / day, for example up to about 300 mg / kg / day, for example up to about 200 mg / kg / day, for example up to about 100 mg / kg / day, for example from about 10 to about 90 mg / kg / day, for example from about 20 to about 80 mg / kg / day, for example from about 30 to about 70 mg / kg / day, for example from about 30 to about 60 mg / kg / day, for example from about 40 to about 60 mg / kg / day, for example from about 50 to about 60 mg / kg / day, for example about 50 or about 60 mg / kg / day, based on the body weight of the subject.

[0085] In some embodiments, for example wherein the glucocorticoid receptor antagonist is mifepristone, the dose of glucocorticoid receptor antagonist administered to the subject does not exceed 20 mg / kg / day.

[0086] In some embodiments, for example wherein the subject is a human, for example an adult human, and / or for example wherein the glucocorticoid receptor antagonist is mifepristone, the glucocorticoid receptor antagonist is administered in a dose of from about 100 mg / day, for example from about 200 mg / day, for example from about 300 mg / day, for example from about 400 mg / day. In particular embodiments, the glucocorticoid receptor antagonist is administered in a dose of up to about 2500 mg / day, for example up to about 2000 mg / day. In some embodiments, the glucocorticoid receptor antagonist is administered in a dose of from about 300 mg / day to about 2000 mg / day, for example from about 400 mg / day to about 2000 mg / day, from about 400 mg / day to about 1200 mg / day, or from about 400 mg / day to about 600 mg / day. In some embodiments, the glucocorticoid receptor antagonist is administered in a dose of about 300 mg / day, about 400 mg / day, about 500 mg / day, about 600 mg / day, about 1000 mg / day, about 1200 mg / day, about 1500 mg / day, or about 2000 mg / day.

[0087] In some embodiments, for example wherein the subject is a human, for example an adult human, and / or for example wherein the glucocorticoid receptor antagonist is relacorilant, the glucocorticoid receptor antagonist is administered in a dose of from about 50 mg / day, for example from about 100 mg / day, for example from about 200 mg / day, for example from about 300 mg / day. In some embodiments, the glucocorticoid receptor antagonist is administered in a dose of up to about 500 mg / day, for example up to about 400 mg / day. In some embodiments, the glucocorticoid receptor antagonist is administered in a dose of from about 100 mg / day to about 400 mg / day.

[0088] A skilled person will be able to readily determine the amount of a prodrug, solvate, salt or derivative of a given GR antagonist required to be administered in order to provide a given biologically effective dose of GR antagonist.

[0089] The dosage and frequency will depend on the subject, the condition, type and potential extent of HO to be prevented and the route of administration. A skilled person will readily be able to determine suitable dosages and frequency of such dosages. For example, the GR antagonist may be administered at a frequency of, for example, once daily, or twice or three times daily. The treatment may be continued for multiple days, weeks, months or years, in particular for multiple days.

[0090] Any one of the methods described above may, in some embodiments, involve the administration of one or more further active agents, such as an anti-inflammatory agent, immunosuppressant, analgesic or therapy for neurological or cardiovascular or cerebrovascular disorders. In embodiments where the methods comprise administration of one or more additionalactive ingredients, the dosages and frequency of administration may be determined by reference to the usual dose and manner of administration of the said ingredients.

[0091] A skilled person would be aware of suitable assays used to evaluate the antagonism of a glucocorticoid receptor and / or an inhibition or reduction of glucocorticoid receptor activity or function. For example, the method may include contacting a cell expressing a glucocorticoid receptor with a compound and screening for the inhibition of binding of a ligand of a glucocorticoid receptor, such as a glucocorticoid (e.g. a ligand binding assay). Alternatively, the method may include screening for the inhibition of the activity, presence or expression of a downstream cellular target or product. Detecting such inhibition may be achieved utilizing techniques including, but not limited to, ELISA, a ligand binding assay (e.g. a radioligand binding assay or fluorescence binding assay), surface plasmon resonance, immunofluorescence, Western blots, immunoprecipitation, immunostaining, scintillation proximity assays or cell proliferation assays such as a WST-1 proliferation assay.

[0092] Typically, potential GR agonists and antagonists are tested via either direct binding to the GR or via reporter assays. In particular embodiments, a reporter assay is used involving cell lines transduced with a luciferase or GFP cDNA under the control of a typical glucocorticoid response element (GRE) such as 5’ AGAACANNNTGTTCT (although others are known in the art). When an agonist is added to the cell line, luciferin is expressed and luminescence is produced in the presence of luciferin. To measure antagonist activity, the cell line is incubated with a typical agonist (eg dexamethasone) together with increasing doses of antagonists or molecules to screen for blocking of the expression of luciferase and GR antagonism.

[0093] Commercially available kits and / or products may also be used to screen for agonists and antagonists, such as the Human Glucocorticoid Receptor Reporter Assay System from Indigo Biosciences (https: / / www.caymanchem.com / product / 15733 / human-glucocorticoid-receptor- reporter-assay-system ).

[0094] The present disclosure further provides use of a GR antagonist in the manufacture of a medicament for the prophylaxis of heterotopic ossification, and to GR antagonists for use in methods of prophylaxis of heterotopic ossifications. The prophylaxis typically involves the methods described herein. Features described herein in relation to methods of prophylaxis of HO may also be applied to such uses and to GR antagonists for such use.

[0095] This invention may also be said broadly to consist in the parts, elements and features referred to or indicated in the specification of the application, individually or collectively, and any or all combinations of any two or more said parts, elements or features, and where specific integers are mentioned herein which have known equivalents in the art to which this invention relates, such known equivalents are deemed to be incorporated herein as if individually set forth.

[0096] The present disclosure will now be described with reference to the following specific examples, which should not be construed as in any way limiting the scope of the invention.ExamplesEXAMPLE 1: Spinal cord injury leads to increased glucocorticoid secretion SCI mouse model

[0097] An animal model of SCI-NHO in which mice undergo a spinal cord transection together with a muscle injury induced by the intramuscular injection of cardiotoxin (CDTX) was carried out (Genet et al, 2015). The model was carried out as described in Genet et al., 2015 in 5-6 week old female C57BL / 6 mice (Genet, 2015 #4552) or male mice by performing a spinal cord transection between vertebrae T11-T13 together with intramuscular (i.m.) injection of cardiotoxin (CDTX) at 0.32mg / kg purified from the venom of Naja pallida (Latoxan) under general anesthesia (lOOmg / kg Ketamine, 10 mg / kg xylazine and 1% isoflurane). Control animals were anesthetized and sham-operated with an incision in the dorsal skin and muscle. All mice received a subcutaneous injection of ciprofloxacin (lOmg / kg), Buprenorphine (0.075mg / kg) and recovered on a heat pad. As SCI causes paraplegia, mouse bladders were expressed manually by gentle massage of the bladder twice daily throughout the experiments. For these reasons all experiments were performed on female mice unless specified. Mice were given 800mg / L Bactrim (Roche) in drinking water following surgical procedures as prophylaxis for bladder infections.Blood collection for mass spectrometry

[0098] For plasma isolation, blood samples were taken from all groups by terminal cardiac puncture 1,2, 3, 4, 5, 6 and 7 days post-surgery under anesthesia (2-3% isoflurane). All surgeries and harvests were performed between 7:00-9:00am to avoid day to day circadian GC concentration fluctuations. Blood was collected in tubes containing EDTA (4 mM final for 1 mL blood collected) and plasma was isolated by centrifugation (1000g for 10 mins), plasma was subsequently transferred into new tubes and spun again (1000g for 10 mins) and plasma stored at -80°C until use.

[0099] Plasma free metadrenaline was measured by an automated on-line Solid Phase Extraction (SPE) coupled to a UPLC-MS / MS system. Plasma samples were diluted 1:1 with deuterated internal standards (d3-normetadrenaline, d3 metadrenaline and d4 3-methoxytyramine) in a zinc sulfate / acetonitrile solution in 2 mL 96 well plates. After centrifugation, a 25pL aliquot of the supernatant was injected into the online SPE equipped UPLC-MS / MS system. The online sample manager loads the sample onto an OASIS WCX cartridge (Waters Corporation, Milford MA, USA) which is subsequently washed to remove interferences and the purified extract is eluted directly on to the ACQUITY UPLC BEH Amide, 2.1 x 100 mm, 1.7 pm column. Quantification was achieved by monitoring two transitions for each analyte on a Waters Xevo TQD massspectrometer. The assay time between injections was 4.5 minutes. The analytical range of the assay was up to 50,000 pM. The Limit of quantitation (LOQ) with a CV of 20% was 20 pM for all 3 analytes. The inter-run imprecision across 3 levels for the 3 analytes were all < 8%.

[0100] Plasma Corticosterone and 11 P-deoxycorticosterone were also measured by an automated on-line Solid Phase Extraction (SPE) coupled to a UPLC-MS / MS system. Plasma samples were diluted 1:1 with deuterated internal standards (d8-corticosterone and d8-lip-deoxycorticosterone) in a zinc sulfate / acetonitrile solution in 2 mL 96 well plate. After centrifugation, a 20pL aliquot of the supernatant was injected into the online SPE equipped UPLC-MS / MS system. The online sample manager loads the sample onto an OASIS XBridge C18 cartridge (Waters Corporation, Milford MA, USA) which is subsequently washed to remove interferences and the purified extract is eluted directly on to the ACQUITY UPLC BEH C18, 2.1 x 50 mm, 1.7 pm column. Quantification was achieved by monitoring two transitions for each analyte on a Waters Xevo TQD mass spectrometer. The assay time between injections was 4.0 minutes. The analytical range of the assay was up to 500 and 50 nM for corticosterone and 11 P-deoxycorticosterone respectively. The Limit of quantitation (LOQ) with a CV of 20% was 0.5 nM for corticosterone and 0.05 nM for 11 P-deoxycorticosterone. The inter-run imprecision across 4 levels for the 2 analytes were all < 9%.Results

[0101] Fig. 1 shows mass spectrometry measurements of plasma concentrations of corticosterone (CORT) (A), its inactive precursor 11 P-deoxycorticosterone (DCORT) (B), as well as catecholamine catabolites (C) 3-methoxy tyramine (3MT), (D) metadrenaline (MET) and (E) normetadrenaline (NorMET) in the C57BL / 6 female mice that underwent SCI, Sham injury, alone or in combination with CDTX muscle injury (Mean ± SD, n=4 mice / surgical group / timepoint, one experiment. *p=0.018, **p=0.007, *p=0.018, **p=0.056 and p=0.0024 respectively, 2-way ANOVA with a Dunnett’s multiple comparison test).

[0102] Mass spectrometry analysis detected an elevation in plasma corticosterone (CORT, the physiological glucocorticoid (GC) in rodents; Fig. 1 A) and its precursor 11 P-deoxycorticosterone (DCORT; Fig. 1 B) at one day post-SCI in mice; no influence of CDTX-mediated muscle injury was observed. As for catecholamines, 3-methoxy tyramine levels (dopamine catabolite) were elevated post-surgery but not different between groups (Fig. 1 C). Metadrenaline levels (adrenaline catabolite) were similarly increased in all operated mice, but only at day 1 post-injury (Fig. 1 D); no significant changes in normetadrenaline (noradrenaline catabolic product) were seen in SCI mice (Fig. 1 E).

[0103] Together, these data suggest that SCI selectively and acutely activates the adrenal gland cortex, leading to increased GC secretion.EXAMPLE 2: Corticosterone promotes HO development in injured muscles in the absence of SCI Administration of CORT

[0104] To demonstrate a direct causal effect of corticosterone (CORT) secretion, CORT was then administered via the drinking water to mice with a CDTX-induced muscle injury but without SCI.

[0105] Mice were given drinking water containing either corticosterone (Merck, 200 mg / L in 1% ethanol and water) or control water (1% ethanol).

[0106] The following day all mice were anesthetized using an isoflurane vaporizer with 2-3% isoflurane and an oxygen flow of IL / min. All mice received an i.m. injection of CDTX at 0.3125mg / kg in the hamstring muscles and PBS as a control in the contralateral limb. Mice subsequently received a subcutaneous injection of buprenorphine (0.075mg / kg). Corticosterone - containing drinking water was replaced every 2 days.Tissue Collection for histology and immunohistochemistry

[0107] At 1-3 weeks post-surgery mice were euthanized by CO2 asphyxiation. Hind limbs were fixed in phosphate buffered saline (PBS) with 4% paraformaldehyde and decalcified and processed as previously described (Torossian et al 2017; Alexander et al, 2019; Tseng et al, 2020). Immunohistochemistry (IHC) was performed as described previously described (Genet et al, 2015; Torossian et al 2017; Alexander et al, 2019; Tseng et al, 2020). Primary antibodies used were: rat anti-mouse F4 / 80 monoclonal antibody (mAb), rabbit anti-mouse Osterix / Sp7 mAb, or relevant isotype-matched control antibodies; rabbit IgG or rat IgG2b (Fig. 3B and 9B discussed below). A 3-step procedure was employed using biotinylated F(ab)2 secondary antibodies (biotinylated goat anti-rat IgG and goat anti-rabbit IgG) and VECTASTAIN Elite ABC-Peroxidase Kit was used to detect primary antibodies. Slides were viewed using an BX50 microscope (Olympus, Japan) with an attached DP26 camera and imaged using Olympus CellSens standard 1.7 imaging software (Olympus). Antibodies and reagents used throughout the Examples are set out in Tables 1 and 2.Micro-computerized tomography (pCT) and NHO Volume Quantification

[0108] All NHO volumes were measured in vivo or ex vivo using the Inveon positron emission tomography / computed tomography (PET-CT) multimodality system (Siemens Medical Solutions Inc.) as previously described (Torossian et al 2017; Alexander et al, 2019; Tseng et al, 2020), except for Fig. 6D discussed below where NHO volumes were measured in vivo using the Molecubes -Cube and X-Cube pPET-CT system (Molecubes). To calculate NHO volumes, the region of interest (ROI) was drawn around the muscles containing NHO and carefully checked from three dimensions. After defining the ROI, the NHO region was defined by setting the threshold Hounsfield units (HU) to 450 HU.Results

[0109] HOs never developed in mice with muscle injury alone. In sharp contrast, 62% (18 out of 29) of mice which were administered corticosterone in their drinking water from day -2 to +7 developed non-neurogenic HOs (HO volume >0.5mm3) in injured muscles compared to mice administered control water as measured by micro-computerized tomography (pCT) (Fig. 2A). This different outcome in NHO development was significant by both Mann -Whitney test and Fisher’s exact test. Delaying the administration of corticosterone until 2 days post-CDTX muscle injury resulted in the development of smaller HOs in only a few mice (Fig. 2A), and HO volumes did not significantly differ from mice administered control water by Mann-Whitney or Fisher’s exact test (p=0.198).

[0110] As corticosterone administration in water is consumed ad libitum with inherent mouse-to- mouse variability in drinking patterns, a causal role of GCs was further investigated by injecting the synthetic and selective long-acting GC receptor (GR) agonist dexamethasone daily from day 0-7 post-CDTX muscle injury. Dexamethasone treatment induced HO development in CDTX- injured muscles in 9 out of 13 (69%) mice (Fig. 2B) similar to the effect of corticosterone in drinking water.

[0111] SCI and CDTX muscle injury have to occur within 3 hours of each other to trigger NHO development, so whether a similar synchrony was required for dexamethasone treatment to induce HO formation in injured muscles was investigated. Indeed, by delaying the start of dexamethasone treatment to between 1 and 72 hrs after CDTX muscle injury, a similar temporal dependence was found, whereby GC administration was required within the first 6 hours of muscle injury to elicit significant HO formation (Fig. 2C).

[0112] Failed muscle regeneration and immature HO formation was also evident histologically in corticosterone-treated mice, with Masson’s stain showing abundant collagen I deposition amongst numerous F4 / 80+macrophages and osterix+osteolineage cells 21 days post- injury. Fig. 3 A shows immunohistochemistry of serial muscle sections at 21 days post CDTX muscle injury illustrating muscle is largely repaired in control water mice, with minimal F4 / 80+macrophage infiltration and no osterix+osteo-lineage cells. In CDTX-injured mice with drinking water containing CORT, injured muscle contained immature HO (#) with numerous F48O+macrophages (arrows) associated with osterix+osteo-lineage cells (arrow heads), around areas of dense collagen deposition (blue). Images 40X magnification, scale bar 20pm. Fig. 3B shows representative images showing matching isotype controls for immunohistochemistry outlined with respect to Fig. 3A, confirming specificity of staining for F4 / 80 (RatIgG2b) and osterix (Rabbit IgG) in CDTX- injured muscles in mice given drinking water with or without corticosterone (CORT) (all images taken at 40x magnification, scale bars represent 20pm).

[0113] Together, these results establish that administration of exogenous corticosterone is sufficient to trigger HO development in injured muscles in the absence of SCI.EXAMPLE 3: Validation in the human pathology

[0114] The results from mice discussed above in Example 2 were validated in the human pathology by culturing CD56 PDGRa+mesenchymal progenitor cells (MPCs) sorted from the muscles surrounding NHO from 5 different biopsies I patients and culturing them in osteogenic conditions with ascorbic acid, P -glycerophosphate and increasing concentrations of cortisol, the physiological adrenal GC in humans.Isolation of human PDGFRa+CD56- FAPs

[0115] Muscle surrounding NHOs was collected from NHO surgical waste following their excision from patients with brain injuries, spinal cord injuries or strokes. NHO resection surgeries were performed at Raymond Poincare Hospital (Garches, France). All samples were obtained with the informed consent of the patients, the approval from the people protection committee (CPP approval n°09025) and the approval from the National Commission for Informatics and Liberties (CNIL approval n° Eyo 10662111). Muscle fragments were minced using scalpel and small scissors, placed in a 50 ml Falcon tube and incubated in 1.5 mg / ml pronase (Sigma- Aldrich) in a- MEM, 45min in a 37°C water bath. After addition of a-MEM supplemented with 15% FCS and 1% antibiotics, the cell suspension was filtered through a 100 pm cell strainer followed by a 40 pm cell strainer (BD Falcon). Isolated muscle progenitor cells (MPCs) were maintained 10 days in a-MEM supplemented with 15% FCS, 1% antibiotics and 10 ng / ml basic fibroblast growth factor (bFGF) (R&D Systems). Human MPCs were trypsinized and incubated 30 min with antihuman PDGFRa-APC and anti-human CD56-PE monoclonal antibodies or with control isotypes IgGl-PE and mouse IgGl-APC in PBS 2% FCS, 2mM EDTA. Cells were washed and incubated 30 min with the viability dye 7-AAD (Sony). Cells were washed and filtered through a 30pm cell strainer (Sysmex) and sorted using a FACSAria III SORP sorter (BD Biosciences). PDGFRa+CD56- FAPs were seeded at 3,000 per cm2 in a-MEM supplemented with 20% FCS and 1% antibiotics.In vitro osteogenic differentiation and quantification of mineralization.

[0116] Human PDGFRa+CD56- FAPs were seeded in 24-well plates at 3,000 per cm2in a-MEM supplemented with 10% FCS and 1% antibiotics. After three days, medium was replaced by a- MEM supplemented with 10% FCS, 1% antibiotics, 12.8 pg / ml ascorbic acid, and 2.15 mg / ml B- glycerophosphate (Sigma- Aldrich) and either 10-7M dexamethasone (DI 179, Sigma), 10-6 to 10- 8M hydrocortisone (Sigma-Aldrich) or Mifepristone (Santa Cruz) to induce osteogenic differentiation. Cells were cultured for 14 days at 37°C in 5% CO2 atmosphere, and medium was changed twice a week. Quantification of mineralization was performed using Alizarin Red Sstaining. Cells were washed with PBS, fixed in 70% ethanol, quickly washed with distilled water and incubated 5 min in 20g / L Alizarin Red S, pH-4.2 (Sigma- Aldrich). Cells were washed with distilled water and dried. Alizarin Red S dye was extracted with 0.5N hydrochloric acid and 5% SDS and quantified by spectrophotometry at 405 nm.Results

[0117] Results are shown in Fig. 4. In particular, Fig. 4 shows the in vitro effect of cortisol (CORT) and dexamethasone (DEX) on calcium mineralization of PDGFRcC CD56’ MPCS sorted from muscles surrounding human NHO and cultured in osteogenic conditions. The five lines connect data from 5 different NHO patients. Dots represent results from each different biopsy, columns are the average for each dose of cortisol. P values were calculated using the non-parametric Friedman test with Dunn’s correction for multiple comparison.

[0118] The present inventors have previously established that these muscle MPCs are the cells of origin of NHO in both human and mice (Tseng HW et al., 2022). The present results show that treatment with cortisol significantly increased human muscle MPC mineralization suggesting that physiological adrenal GCs promote muscle mineralization in both species.EXAMPLE 4: Postnatal deletion of the glucocorticoid receptor gene prevents NHO development

[0119] To further demonstrate the key role of endogenous GCs in driving NHO development after SCI, mice were utilized to allow the deletion of the Nr3cl gene encoding the GC receptor (GR) post-natally, to avoid the perinatal lethality observed with germinal Nr3cl knock-out mice.

[0120] To this end, Nr3clfl / flmice were crossed with mice containing a tamoxifen-inducible recombinase CreERT2 knocked into the ROSA26 gene trap locus to allow efficient postnatal tamoxifen-inducible deletion of the floxed Nr3cl alleles in all cells. Both male and female NrScl^fl x ROSA26CreERT2mice and control Nr3clWT / WTx ROSA26CreERT2mice were gavaged daily with tamoxifen from between 4-7 weeks of age to delete the floxed Nr3cl alleles and then left to rest for an additional 2 weeks before undergoing SCI and CDTX muscle injury.

[0121] At 7 and 21 days post-SCI and CDTX muscle injury, none of tamoxifen-treated Nr3clfl / fl x ROSA26CreERT2 (Nr3clA) mice developed NHO whereas all tamoxifen-treated control Nr3clWT / WTx ROSA26CrerERT2mice did develop NHO (Fig. 5A). CDTX-injured muscles from Nr3clWT / WTx ROSA26CreERT2control mice, 21 days post-surgery contained numerous NHO foci, with F4 / 80+ macrophages intercalated amongst osterix+ osteo-lineage cells on NHO surfaces (Fig. 5B) as expected. In contrast, injured muscles from Nr3clA mice had no NHO foci, less F4 / 80+ expression and no osterix+ osteoblast lineage cell (Fig. 5B).

[0122] This demonstrates that GR-mediated signaling above a certain threshold is necessary for NHO formation post-SCI.EXAMPLE 5: GC receptor (GR) antagonist as a treatment for SCI-NHOMifepristone treatment

[0123] Mice underwent SCI and CDTX muscle injury as described above and were treated with mifepristone / RU-486, a potent dual antagonist of GR and the structurally related progesterone receptor (PGR) (Jung-Testas and Baulieu, 1983; Morgan et al., 2002) for 7 days post-surgery. Mice were administered Mifepristone (Sigma Aldrich) at 50 mg / kg once daily via oral gavage in vehicle (peanut oil, Sigma), or vehicle control (10% DMSO in peanut oil) from either day 0-7, day 0-2 or day 3-5 post-surgery.Relacorilant treatment

[0124] To definitively rule out PGR antagonism, the non- steroid selective GR antagonist relacorilant / CORT125134, currently in clinical trials to treat Cushing syndrome and adrenal carcinomas (Hunt et al., 2018; Hunt et al., 2017), was also administered. Mice were treated with relacorilant (ChemieTek) at 60 mg / kg bi-daily by oral gavage in vehicle (peanut oil), or vehicle control (12% DMSO in peanut oil) from day 0-2 post-surgery.NHO aggravation by GC agonist

[0125] Glucocorticoids are typically considered as immunosuppressive and ‘anti-inflammatory’ and stable synthetic GCs that agonize the GR are prescribed to treat chronic inflammatory disorders and acute inflammation (Hardy et al., 2020; Kadmiel and Cidlowski, 2013; Rhen and Cidlowski, 2005). High doses of agonistic GCs such as prednisone can reduce HO ‘flare-ups’ in patients with the rare genetic disorder fibrodysplasia ossificans progressiva, caused by activating mutations in the ACVR1 gene (Pignolo et al., 2011). Despite this established use of GCs in the management of HO, the present inventors theorized that agonistic GC treatment would likely aggravate NHO development.

[0126] Mice were administered with the synthetic and selective agonistic GC dexamethasone sodium phosphate at 10 mg / kg or saline control once daily via subcutaneous injection, from day 0-5 post SCI.Leukocyte isolation

[0127] Leukocytes were isolated from mouse hamstring muscles 4 days post-injury using a skeletal muscle dissociation kit (Miltenyi Biotech) as per manufacturer's instructions. Total muscle leukocytes were also sorted into multiple populations using a BD FACS Aria Fusion using the fluorescent mAbs specific for cell surface markers CD45, TER-119 / Erythroid Cells, CD45R / B220, CD3E, F4 / 80, CDl lb, Ly-6C, Ly-6G, CD48 and cell viability as previously described (Alexander et al, 2019). Muscle progenitor cells were isolated from hamstring muscles of naive C57BL / 6 muscles using the muscle dissociation kit as above. Populations were sorted using a BD FACS Aria Fusion according to the following phenotypes: Satellite cells (SC): CD45-lineage-(Terl 19, B220, CD3s, CDl lb, Grl)- CD31- CD34+ Scal-integrin a7+; fibro-adipogenic precursors (FAP) CD45- Lin- CD31- CD34+ Scal+ integrin al-. All populations were sorted directly into 1 mb Trizol LS (ThermoFisher) and frozen until extraction.RNA Extraction and qRT-PCR

[0128] For RNA isolation, frozen muscle samples and ovaries (positive controls for Pgr mRNA expression) were homogenized using a TissueRuptor (Qiagen), directly in Trizol (Life Technologies), and after chloroform separation, RNA was isolated from aqueous phase. mRNA was isolated from all sorted cells using chloroform separation followed by GeneJET RNA cleanup and concentration micro kit (ThermoFisher). Reverse transcription was performed using the SensiFAST™ cDNA Synthesis Kit (ThermoFisher) as per manufacturer's instructions. mRNA expression was analyzed using a single-step reverse transcription quantitative real-time polymerase chain reaction (qRT-PCR) Taqman system using TaqMan™ fast PCR Master Mix and TaqMan™ gene expression assay of interest (Supplementary table 2). Ct values were normalized by the expression of house-keeping gene Hprt and presented as ratio to house-keeping gene.Results

[0129] Mifepristone treatment decreased NHO volumes 4-fold at all endpoints (Fig. 6A, 6B). As blood corticosterone levels were highest one day post-SCI, mifepristone treatment was shortened to only day 0-2 post-surgery and NHO was found to be similarly attenuated (Fig. 6C). However, delaying mifepristone treatment to 3 days post-surgery, i.e. when blood corticosterone had returned to basal levels after SCI, had no impact on NHO development (Fig. 6D), suggesting that mifepristone treatment is only effective during a narrow therapeutic window post-SCI when corticosterone levels are highest.

[0130] By delaying the starting point of mifepristone after SCI and muscle injury, it was established that mifepristone treatment must be started within a 48-hour therapeutic window, with mifepristone effective to inhibit NHO formation when started 5, 10, 24 or 48 hours post-surgery (Fig. 6E).

[0131] As mifepristone is also a PGR antagonist, it was confirmed that the day 0-2 post-surgery treatment regimen was also effective in male mice (Fig. 6F), which have 40-fold lower progesterone blood concentrations compared to females.

[0132] Dexamethasone treatment upregulated Runx2 / Cbfal expression in CDTX-injured muscles, and RUNX2 is a transcription factor essential for osteoblast differentiation from mesenchymal progenitor cells. Interestingly, Runx2 was also significantly upregulated in SCI+CDTX groups between 2 and 4 days post-surgery, but significantly reduced after mifepristone treatment (Fig. 6G). Likewise, mifepristone treatment trended towards decreased STAT3 phosphorylation in injured muscles (data not shown), which is associated with NHOdevelopment. As it was previously demonstrated that blockade of STAT3 phosphorylation via administration of JAK1 / 2 tyrosine kinase inhibitor ruxolitinib attenuated NHO development, the efficacy of combining mifepristone with ruxolitinib was investigated. Ruxolitinib treatment alone resulted in a 20% reduction in NHO volumes at day 7 and 35% at day 21 but this did not reach statistical significance by one-way ANOVA (significant by Mann- Whitney test at day 21 p=0.037) (data not shown). There was also no significant difference between mifepristone alone vs mifepristone + ruxolitinib treatment groups at either timepoint (data not shown). Overall, this suggests that there is no additive contribution of further JAK1 / 2-STAT3 signaling to GR signaling in promoting NHO development as GR agonism already activates JAK1 / 2 STAT3 signaling.

[0133] Fig. 7 shows mRNA expression of the glucocorticoid receptor and progesterone receptor in mouse leukocytes and muscle cells. GR mRNA Nr3cl) itself was abundantly expressed by inflammatory monocyte / macrophage subsets and granulocytes within injured muscles at 4 days post-surgery, and also by muscle progenitor populations such as fibro-adipogenic progenitors and satellite cells isolated from naive muscles (Fig. 7 A). In contrast, Pgr was mostly absent and / or expressed at very low levels in all cell types above, compared to the ovary / uterus which are well known to abundantly express Pgr (Fig. 7 B). Overall, these suggest that the effect of mifepristone is via GR and not PGR antagonism. Each dot of Fig. 7 represents a separate mouse, bars represent mean ± SD, one experiment.

[0134] Similar to mifepristone, relacorilant significantly reduced NHO volumes (Fig. 8, A and B). Fig. 8 A shows NHO volumes via pCT at 7 and 21 days post-surgery following treatment with vehicle (black dots) or relacorilant (60mg / kg twice daily, red dots) from day 0-2 post surgery. Each dot represents one mouse, mean ± SD, experiment repeated twice. ****p=<0.0001, ***p=0.003 two-sided Mann- Whitney. Fig. 8B shows representative pCT images at 7 and 21 days postsurgery. Muscles from vehicle-treated mice contained numerous NHO foci with F4 / 80+macrophages intercalated amongst osterix+osteo-lineage cells on NHO surfaces 21 days postsurgery. In sharp contrast, relacorilant-treated mice had significantly fewer NHO foci, less F4 / 80+macrophages and mostly no osterix+osteolineage cells (Fig. 9A). As shown in Fig. 9A, immunohistochemistry on serial muscle sections at 21 days illustrate the formation of NHO foci within damaged muscles by Masson’s trichrome (blue, *), F4 / 80+macrophages (arrows) were closely associated with osterix+osteo-lineage cells (arrow heads) present on NHO foci in vehicle- treated mice. In contrast, CDTX-injured muscles from relacorilant treated mice had less NHO foci with reduced F4 / 80+macrophage infiltration and minimal osterix expression (boxed areas) (# symbols mark the same anatomical location. 40X magnification, scale bars 20pm). Fig. 9B shows isotype controls confirming specificity of staining for F4 / 80 (RatIgG2b) and osterix (Rabbit IgG) in CDTX-injured muscles in mice with SCI and CDTX-muscle injury treated with vehicle orrelacolirant, # symbols mark the same anatomical location as in Fig. 9A. All images taken at 40x magnification, scale bars represent 20pm.

[0135] Administration of the GR agonist dexamethasone from day 0-5 post SCI caused a significant increase in NHO volumes within CDTX-injected muscles (Fig. 10). Fig. 10 shows results of SCI + CDTX injection carried out on female mice and treatment from day 0-5 postsurgery with either saline (black dots) or dexamethasone (lOmg / kg / day, red dots). NHO volumes were measured by pCT at days 7 and 21 post-surgery. Each dot represents one mouse, mean ± SD, experiment repeated twice, **p=0.0015 and ***p=0.0006 two-sided Mann- Whitney. These results suggest that, contrary to established procedure in the art, ‘anti-inflammatory” treatment with GR agonists has the potential to drive and / or exacerbate NHO development post-SCI, which is consistent with the present inventors’ finding that endogenous adrenal CORT is sufficient to induce HO within injured muscles in the absence of SCI. The present inventors’ findings are also consistent with the recently observed high prevalence or peri-articular HOs developing in mechanically ventilated severe cases of Sars-Cov-2 (COVID- 19) who are treated with dexamethasone to alleviate severe respiratory syndrome (de 1'Escalopier et al., 2021; Meyer et al., 2020).

[0136] All experimental procedures in mice were approved by the Health Sciences Animal Ethics Committee of The University of Queensland and followed the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes. C57BL / 6 mice were obtained from Animal Resource Centre (Perth, Australia). All mice were housed at the Translational Research Institute, Biological Research Facility (Queensland, Australia), under specific pathogen free conditions. All mice were fed a standard diet chow (Specialty Feeds, Western Australia, Australia) with ad libitum water access and simulated diurnal cycle.

[0137] Data are presented as mean + standard deviation. Statistically significant differences were determined using either two-sided Mann -Whitney test for 2 population comparisons, or two-way ANOVA or non-parametric Friedman test for multiple comparisons using Prism software (version 8.0; GraphPad Software).

[0138] Antibodies used throughout the study described by the above Examples are set out in Table 1 below:Table 1. Antibodies used in study 1) Flow cytometry (anti-mouse)2) Flow cytometry (anti-human)3) IHC (anti-mouse)Reagents used in the study are set out in Table 2 below:Table 2. Reagent listReferencesAlexander, K.A., H.-W. Tseng, M. Saiga, F. Genet, and J.-P. Levesque. 2020. When the Nervous System Turns Skeletal Muscles into Bones: How to Solve the Conundrum of Neurogenic Heterotopic Ossification. Current Osteoporosis Reports 18:666-676.Alexander KA, Tseng HW, Fleming W, Jose B, Saiga M, Kulina I, Millard SM, Pettit AR, Genet F, Levesque JP. Inhibition of JAK1 / 2 Tyrosine kinases reduces neurogenic heterotopic ossification after spinal cord injury. 2019 Front Immunol 10: Article 377. DOI: 10.3389 / fimmu.2019.00377Bradleigh, L.H., A. Perkash, S.H. Linder, G.H. Sullivan, H.K. Bhatt, and I. Perkash. 1992. Deep venous thrombosis associated with heterotopic ossification. Arch Phys Med Rehabil 73:293- 294.Citak, M., E.M. Suero, M. Backhaus, M. Aach, H. Godry, R. Meindl, and T.A. Schildhauer. 2012. Risk factors for heterotopic ossification in patients with spinal cord injury: a case-control study of 264 patients. Spine 37: 1953-1957. de 1'Escalopier, N., L. Mathieu, C. Duret, S. Banzet, F. Genet, and M. Saiga. 2021. Re: 'High prevalence of heterotopic ossification in critically ill patients with severe COVID-19' by Stoira et al. Clinical microbiology and infection : the official publication of the European Society of Clinical Microbiology and Infectious DiseasesForsberg, J.A., J.M. Pepek, S. Wagner, K. Wilson, J. Flint, R.C. Andersen, D. Tadaki, F.A. Gage, A. Stojadinovic, and E.A. Elster. 2009. Heterotopic Ossification in High-Energy Wartime Extremity Injuries: Prevalence and Risk Factors. J Bone Joint Surg Am 91: 1084-1091.Genet, F., C. Chehensse, C. lourdan, C. Lautridou, P. Denormandie, and A. Schnitzler. 2012. Impact of the operative delay and the degree of neurologic sequelae on recurrence of excised heterotopic ossification in patients with traumatic brain injury. J Head Trauma Rehabil 27:443- 448.Genet, F., C. Jourdan, C. Lautridou, C. Chehensse, K. Minooee, P. Denormandie, and A. Schnitzler. 2011a. The impact of preoperative hip heterotopic ossification extent on recurrence in patients with head and spinal cord injury: a case control study. PLoS ONE 6:e23129.Genet, F., C. Jourdan, A. Schnitzler, C. Lautridou, D. Guillemot, T. Judet, S. Poiraudeau, and P. Denormandie. 2011b. Troublesome heterotopic ossification after central nervous system damage: a survey of 570 surgeries. PLoS One 6:el6632.Genet, F., I. Kulina, C. Vaquette, F. Torossian, S. Millard, A.R. Pettit, N.A. Sims, A. Anginot, B. Guerton, I.G. Winkler, V. Barbier, J. -J. Lataillade, M.-C. Le Bousse-Kerdiles, D.W. Hutmacher, and J.-P. Levesque. 2015. Neurological heterotopic ossification following spinal cord injury is triggered by macrophage-mediated inflammation in muscle. J Pathol 236:229-240.Genet, F., J.L. Marmorat, C. Lautridou, A. Schnitzler, L. Mailhan, and P. Denormandie. 2009. Impact of late surgical intervention on heterotopic ossification of the hip after traumatic neurological injury. J Bone Joint Surg Br 91:1493-1498.Haran, M., T. Bhuta, and B. Lee. 2004. Pharmacological interventions for treating acute heterotopic ossification. Cochrane Database Sy st Rev Cd003321.Hardy, R.S., K. Raza, and M.S. Cooper. 2020. Therapeutic glucocorticoids: mechanisms of actions in rheumatic diseases. Nat Rev Rheumatol 16: 133-144.Hardy, R.S., H. Zhou, MJ. Seibel, and M.S. Cooper. 2018. Glucocorticoids and Bone: Consequences of Endogenous and Exogenous Excess and Replacement Therapy. Endocr Rev 39:519-548.Hunt, H., K. Donaldson, M. Strem, V. Zann, P. Leung, S. Sweet, A. Connor, D. Combs, andJ. Belanoff. 2018. Assessment of Safety, Tolerability, Pharmacokinetics, and Pharmacological Effect of Orally Administered CORT125134: An Adaptive, Double-Blind, Randomized, Placebo- Controlled Phase 1 Clinical Study. Clinical pharmacology in drug development 7 :408-421.Hunt, H.J., J.K. Belanoff, I. Walters, B. Gourdet, J. Thomas, N. Barton, J. Unitt, T. Phillips, D. Swift, and E. Eaton. 2017. Identification of the Clinical Candidate (R)-(l-(4-Fluorophenyl)-6- ((1-methyl- lH-pyrazol-4-yl)sulfonyl)-4, 4a, 5,6,7, 8-hexahydro-lH-pyrazolo[3,4-g]isoquinolin-4a- yl)(4-(trifluoromethyl)pyridin-2-yl)methano ne (CORT125134): A Selective Glucocorticoid Receptor (GR) Antagonist. J Med Chem 60:3405-3421.Jung-Testas, I., and E.-E. Baulieu. 1983. Inhibition of glucocorticosteroid action in cultured L-929 mouse fibroblasts by RU 486, a new anti-glucocorticosteroid of high affinity for the glucocorticosteroid receptor. Exp Cell Res 147:177-182.Kadmiel, M., and I.A. Cidlowski. 2013. Glucocorticoid receptor signaling in health and disease. Trends Pharmacol Sci 34:518-530.Klein, G.L. 2015. The effect of glucocorticoids on bone and muscle. Osteoporosis and sarcopenia 1:39-45.Komori, T., H. Yagi, S. Nomura, A. Yamaguchi, K. Sasaki, K. Deguchi, Y. Shimizu, R.T. Bronson, Y.H. Gao, M. Inada, M. Sato, R. Okamoto, Y. Kitamura, S. Yoshiki, and T. Kishimoto. 1997. Targeted disruption of Cbfal results in a complete lack of bone formation owing to maturational arrest of osteoblasts. Cell 89:755-764.Li, L., S. Xiang, B. Wang, H. Lin, S. Kihara, H. Sun, P.G. Alexander, and R.S. Tuan. 2020. TGF-01 plays a protective role in glucocorticoid-induced dystrophic calcification. Bone 136:115355.Meyer, C., M.A. Haustrate, J.F. Nisolle, and T. Deltombe. 2020. Heterotopic ossification in COVID- 19: A series of 4 cases. Ann Phys Rehabil Med 63:565-567.Meyers, C., J. Lisiecki, S. Miller, A. Levin, L. Fayad, C. Ding, T. Sono, E. McCarthy, B. Levi, and A.W. lames. 2019. Heterotopic Ossification: A Comprehensive Review. JBMR Plus 3:el0172.Morgan, B.P., A.G. Swick, D.M. Hargrove, I.A. LaFlamme, M.S. Moynihan, R.S. Carroll,K.A. Martin, E. Lee, D. Decosta, and I. Bordner. 2002. Discovery of Potent, Nonsteroidal, and Highly Selective Glucocorticoid Receptor Antagonists. J Med Chem 45:2417-2424.Nilsson, M E., L. Vandenput, A. Tivesten, A.-K. Norlen, M.K. Lagerquist, S.H. Windahi, A.E. Bdrjesson, H.H. Farman, M. Poutanen, A. Benrick, M. Maliqueo, E. Stener-Victorin, H. Ryberg, and C. Ohlsson. 2015. Measurement of a Comprehensive Sex Steroid Profile in Rodent Serum by High-Sensitive Gas Chromatography-Tandem Mass Spectrometry. Endocrinology 156:2492-2502.Ohlmeier, M., E.M. Suero, M. Aach, R. Meindl, T.A. Schildhauer, and M. Citak. 2017. Muscle localization of heterotopic ossification following spinal cord injury. Spine J 17: 1519-1522.Otto, F., A.P. Thornell, T. Crompton, A. Denzel, K.C. Gilmour, I.R. Rosewell, G.W. Stamp, R.S. Beddington, S. Mundlos, B.R. Olsen, P.B. Selby, and M.J. Owen. 1997. Cbfal, a candidate gene for cleidocranial dysplasia syndrome, is essential for osteoblast differentiation and bone development. Cell 89:765-771.Pignolo, R.J., E.M. Shore, and F.S. Kaplan. 2011. Fibrodysplasia ossificans progressiva: clinical and genetic aspects. Orphanet J Rare Dis 6:80.Reznik, J.E., E. Biros, R. Marshall, M. Jelbart, S. Milanese, S. Gordon, and M.P. Galea. 2014. Prevalence and risk-factors of neurogenic heterotopic ossification in traumatic spinal cord and traumatic brain injured patients admitted to specialised units in Australia. J Musculoskelet Neuronal Interact 14: 19-28.Rhen, T., and J.A. Cidlowski. 2005. Antiinflammatory action of glucocorticoids— new mechanisms for old drugs. N Engl J Med 353:1711-1723.Saiga, M., C. Jourdan, M.C. Durand, C. Hangard, P. Denormandie, R.Y. Carlier, and F. Genet. 2014. Sciatic nerve compression by neurogenic heterotopic ossification: use of CT to determine surgical indications. Skeletal RadiolTorossian F, Guerton B, Anginot A, Alexander KA, Desterke C, Soave S, Tseng HW, Arouche N, Boutin L, Kulina I, Saiga M, Je B, Pettit AR, Clay D, Rochet N, Vlachos E, Genet G, Debaud C, Denormandie P, Genet F, Sims NA, Banzet S, Levesque JP, Lataillade JJ, Le Bousse- Kerdiles MC. Macrophage-derived oncostatin M contributes to human and mouse neurogenic heterotopic ossifications. 2017 J CI Insight 2(ll):e96034.Tseng H-W, Girard D, Alexander KA, et al. Spinal cord injury reprograms muscle fibroadipogenic progenitors to form heterotopic bones within muscles. Bone Res. 2022;10(l):22.Tseng H-W, Kulina I, Girard D, et al. Interleukin- 1 is overexpressed in injured muscles following spinal cord injury and promotes neurogenic heterotopic ossification. J Bone Miner Res. 2022;37(3):531-546Tseng HW, Kulina, I, Saiga M, Fleming W, Vaquette C, Genet F, Levesque JP, Alexander KA. Neurogenic heterotopic ossification develops independently of granulocyte -colonystimulating factor and neutrophils. 2020 J Bone Miner Res 35(11): 2242-2251. DOI: 10.1002 / jbmr.4118Schurch, B., and P. Dollfus. 1998. The 'Dejerines': an historical review and homage to two pioneers in the field of neurology and their contribution to the understanding of spinal cord pathology. Spinal Cord 36:78-86. van Kuijk, A.A., A.C. Geurts, and H.J. van Kuppevelt. 2002. Neurogenic heterotopic ossification in spinal cord injury. Spinal Cord 40:313-326.Vanden Bossche, L., and G. Vanderstraeten. 2005. Heterotopic ossification: a review. J Rehabil Med 37: 129-136.Wittenberg, R.H., U. Peschke, and U. Botel. 1992. Heterotopic ossification after spinal cord injury. Epidemiology and risk factors. J Bone Joint Surg Br 74:215-218.Zhou, H., W. Mak, R. Kalak, J. Street, C. Fong-Yee, Y. Zheng, C.R. Dunstan, and M.J. Seibel. 2009. Glucocorticoid-dependent Wnt signaling by mature osteoblasts is a key regulator of cranial skeletal development in mice. Development 136:427-436.

Claims

The claims defining the invention are as follows:

1. A method of prophylaxis of heterotopic ossification (HO), comprising administering to a subject in need thereof an effective amount of a glucocorticoid receptor antagonist, wherein the subject has experienced an injury or trauma.

2. The method of claim 1, wherein the HO is acquired heterotopic ossification.

3. The method of claim 2, wherein the injury or trauma comprises a soft tissue injury or trauma.

4. The method of claim 3, wherein the injury or trauma comprises an injury or trauma to muscle or connective tissue.

5. The method of claim 3 or claim 4, wherein the injury or trauma comprises a non-neurologic injury or trauma.

6. The method of any one of claims 3 to 5, wherein the HO is neurogenic heterotopic ossification and the subject has further experienced a neurologic injury or trauma.

7. The method of claim 6, wherein the neurologic injury is a spinal cord injury, a traumatic brain injury, a stroke or cerebral anoxia.

8. The method of any one of claims 3 to 7, wherein the glucocorticoid receptor antagonist is administered to the subject within 7 days of the injury or trauma.

9. The method of claim 8, wherein the glucocorticoid receptor antagonist is administered to the subject within 2 days of the injury or trauma.

10. The method of claim 6, wherein the glucocorticoid receptor antagonist is administered to the subject within 7 days of the neurologic injury or trauma.

11. The method of claim 10, wherein the glucocorticoid receptor antagonist is administered to the subject within 2 days of the neurologic injury or trauma.

12. The method of any one of claims 1 to 11, wherein the glucocorticoid receptor antagonist partially or completely prevents the binding of a lipophilic glucocorticoid to the glucocorticoid receptor.

13. The method of claim 12, wherein the glucocorticoid receptor antagonist is an antagonist to cortisol binding the glucocorticoid receptor.

14. The method of any one of claims 1 to 13, wherein the glucocorticoid receptor antagonist is mifepristone or relacorilant, or a pharmaceutically acceptable salt, derivative, prodrug or solvate thereof.

15. Use of a glucocorticoid receptor antagonist in the manufacture of a medicament for the prophylaxis of HO in a subject that has experienced an injury or trauma.

16. A glucocorticoid receptor antagonist for use in a method of prophylaxis of HO in a subject that has experienced an injury or trauma

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