Novel chitosan-il-36ra conjugates for adjustable, sustained release and tissue distribution

Abstract

The present disclosure describes novel compounds disclosed include modified IL-36 receptor antagonist peptides for prolonged time release and consistent tissue distribution. Also disclosed herein are methods for treating diseases characterized by an IL-36 receptor antagonist deficiency such as osteoarthritis or a failure or a defect in regeneration of a damage organ or tissue.

Description

Novel Chitosan-IL-36Ra Conjugates for Adjustable, Sustained Release and Tissue DistributionREFERENCE TO SEQUENCE LISTING

[0001] The Sequence Listing associated with this application is provided in XML format in lieu of paper copy, and is hereby incorporated by reference into the specification. The name of the XML file containing the Sequence Listing is “SeqListing 42960-392608. xml”. The XML file is 7.87 KB, was created on July 26, 2023, and is being submitted electronically via Patent Center, concurrent with the filing of this specification.CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application Serial No. 63 / 516,131 filed on July 27, 2023, which is hereby incorporated by reference in its entirety.TECHNICAL FIELD

[0003] The present disclosure describes novel compounds disclosed include modified IL-36 receptor antagonist peptides for prolonged time release and consistent tissue distribution. Also disclosed herein are methods for treating diseases characterized by an IL-36 receptor antagonist (IL-36Ra) deficiency such as osteoarthritis or a failure or a defect in regeneration of a damage organ or tissue.BACKGROUND

[0004] Osteoarthritis (OA) is a debilitating disease causing cartilage damage. Other conditions such as osteophytosis (bone spurs), subchondral bone sclerosis (thickening of the bones under cartilage in joints), and synovitis (inflammation of the connective tissue around joints) may also occur with OA. Patients experience a range of painful symptoms including joint tenderness, joint stiffness, and pain. Current management of OA is limited by the lack of therapeutic interventions that alter the rate of progression of the disease.

[0005] Although pro-inflammatory cytokines have been historically associated with advanced OA, they now appear to also have regulatory roles in early stages of OA. See, D. T. Felson, (2006), incorporated by reference with regard to such background teaching. The TGF-cytokine superfamily has garnered attention with respect to joint development and OA. Mice with prenatally inactivated TGF-p type 2 receptor ( Tgfbr2) in osteochondral progenitors do not undergo joint formation. See A. Spagnoli, (2007), incorporated by reference with regard to such background teaching. TGF-p signaling is also required to maintain adulthood cartilage homeostasis as disrupted TGF-p signaling is evident during OA progression. See K. W. Finnson, et. al. (2012), incorporated by reference with regard to such background teaching. Previous studies have indicated that systemically delivered TGF-|3 is capable of attenuating surgically induced OA progression. See L. Xie, et. al., (2016), incorporated by reference with regard to such background teaching. However, TGF-p signaling has multifaceted nichedependent roles within joints, meaning systemic actions outside the joint with other transcriptional regulation targets have a high risk for causing multi-organ side effects. Therefore, effective TGF-p modulation for OA prevention must be accomplished using relevant local and downstream signaling mechanisms.

[0006] One of the TGF-p downstream cytokine signaling network members shown to have upregulation in OA mouse models and OA patient chondrocytes, is IL-36a. IL-36a is one of several IL-1 cytokine subfamilies and includes three activating ligands (IL-36a, IL-36|3, and IL- 36y), a receptor antagonist (named IL-36Ra in mice and IL-36RN in humans), and their common subfamily-specific receptor (IL-36R). Inhibition of IL-36a signaling activity by IL-36Ra has been shown to attenuate these pathological changes and reduced articular cartilage degeneration in different animal models of OA. See T. Li, et. al., (2019), incorporated by reference with regard to such background teaching. Furthermore, a deficiency in IL-36Ra has been reported to delay the healing responses, and IL-36Ra has been reported to play a role in myocardial healing after ischemia. See, J. El-Awaisi, et. al. (2022), incorporated by reference with regard to such background teaching. With the identification of IL-36Ra as a chondroinductive and chondroprotective factor, there is a need for treatment strategies to shift from the concept of blocking the external arthritic causes to creating replacement therapy to promote proper signaling for joint homeostasis in patients with chondroinductive deficiencies.

[0007] There are several challenges to the development of therapies that target joint cartilage. The avascular nature of joint cartilage makes systemic delivery of drugs inefficient. Utilizing intra-articular (i.a.) injection may be effective due to local bioavailability and less systemic exposure. However, the efficacy of an i.a. drug depends on the “dwell time,’’ that is the time that the molecule is cleared from the joint space by the lymphatic system. Because the lymphatic system is highly efficient, the dwell time of proteins is short, with a t1 / 2 in the order of 0.1 -2 hours, making promising short-term effects of i.a. drugs not sustainable over time. Usingparticle-based systems with sustained-release to increase i.a. drug residence time have showed limitations such as poor ability to maintain joint fluidity due to mechanical friction, poor stability in the synovial fluid and often unpredictable release, due to relying on local joint processing to become active. Additionally, i.a. therapies often struggle to reach the articular chondrocytes as the only way through the abundance of the extracellular matrix (ECM) and its components such as glycosaminoglymays (GAGs) is through diffusion.

[0008] To this end, the present disclose addresses the need for a deliverable therapy which sustainably and predictably releases IL-36 receptor antagonist peptides for the inactivation of TGF-p signaling and attenuation of pathological changes in OA. The disclosed compounds and methods include compositions of modified IL-36a peptides that have been altered to prolong time release and allow consistent tissue distribution for the treatment of diseases characterized by an IL-36 receptor antagonist deficiency such as OA, or a failure, or a defect, in regeneration of a damage organ or tissue.SUMMARY

[0009] In some embodiments, the present disclosure includes compounds for the treatment of diseases characterized by an IL-36 receptor antagonist deficiency comprising a polypeptide comprising the amino acid sequence of SEQ ID No: 1 or a sequence which is at least 70% homologous thereto; and a chitosan nanoparticle.

[0010] In some aspects, the compounds described herein comprises a chitosan nanoparticle covalently linked by an amine group to a carboxyl group of a polypeptide.

[0011] In some aspects, the compounds described herein comprise a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7.

[0012] In some aspects, the compounds described herein comprise a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a sequence which is at least 80% homologous thereto, or an analogue, derivative, fragment, variant, or peptidomimetic thereof.

[0013] In some aspects, the compounds described herein comprise a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or a sequence which is at least 80% homologous thereto, or an analogue, derivative, fragment, variant, or peptidomimetic thereof.

[0014] In some aspects, the compounds described herein comprise a polypeptide comprising the amino acid sequence of SEQ ID NO: 3 or a sequence which is at least 80% homologous thereto, or an analogue, derivative, fragment, variant, or peptidomimetic thereof.

[0015] In some aspects, the compounds described herein comprise a polypeptide comprising the amino acid sequence of SEQ ID NO: 4 or a sequence which is at least 80% homologous thereto, or an analogue, derivative, fragment, variant, or peptidomimetic thereof.

[0016] In some aspects, the compounds described herein comprise a polypeptide comprising the amino acid sequence of SEQ ID NO: 5 or a sequence which is at least 80% homologous thereto, or an analogue, derivative, fragment, variant, or peptidomimetic thereof.

[0017] In some aspects the compounds described herein comprise a polypeptide comprising the amino acid sequence of SEQ ID NO: 6 or a sequence which is at least 80% homologous thereto, or an analogue, derivative, fragment, variant, or peptidomimetic thereof.

[0018] In some aspects, the compounds described herein comprise a polypeptide comprising the amino acid sequence of SEQ ID NO: 7 or a sequence which is at least 80% homologous thereto, or an analogue, derivative, fragment, variant, or peptidomimetic thereof.

[0019] In some aspects, the compounds described herein comprise a nanoparticle comprising beta-cholanic acid-chitosan.

[0020] In some aspects, the compounds described herein comprise a nanoparticle further comprising a cholanic acid core.

[0021] In some aspects, the compounds described herein comprise a polypeptide covalently coupled to the surface of the nanoparticle.

[0022] In some aspects, the compounds described herein comprise a nanoparticle that has amino or amine functionality for attachment of a polypeptide to the surface of the nanoparticle.

[0023] In some aspects are a pharmaceutical composition comprising the compound of the disclosure and a pharmaceutically acceptable carrier.

[0024] In some aspects are a pharmaceutical composition comprising a therapeutically effective amount of the compound of the disclosure and one or more pharmaceutically acceptable carriers or vehicles.

[0025] Another embodiment described herein is a method of treating or lessening the severity in a subject of a disease characterized by an IL-36 receptor antagonist deficiency comprises administering a therapeutically effective amount of any of the compounds described herein or a pharmaceutical composition comprising any of the compounds described herein.

[0026] In some aspects, the method includes the diseases osteo-arthritis, myocardial infarction, fibrotic diseases and disorders, injury-induced organ damage, or injury-induced tissue damage.

[0027] In some aspects, the method of administration comprises administration to a tissue. In some aspects, the tissue is cartilage.

[0028] In some aspects, the method includes administration to a knee joint, shoulder joint, or hip joint.

[0029] In some aspects of the method, the subject that is a mammal.

[0030] In some aspects, any of the compounds described herein, or a pharmaceutical composition comprising the any of the compounds described herein, for use in a method for treating osteo-arthritis in a subject.

[0031] In some aspects, any of the compounds described herein or a pharmaceutical composition comprising any of the compounds described herein for use in treating or lessening the severity of a disease or condition in a subject characterized by a deficiency in IL-36 receptor antagonist.

[0032] In some aspects, any of the compounds described herein or a pharmaceutical composition comprising any of the compounds described herein for use as a medicament.

[0033] In some aspects, any of the compounds described herein or a pharmaceutical composition comprising any of the compounds described herein for use in the manufacture of a medicament for the treatment of disease associated with an IL-36 receptor antagonist deficiency.BRIEF DESCRIPTION OF THE FIGURES

[0034] FIG. 1. illustrates the components of the protein-conjugated nanoparticles.

[0035] FIG. 2 illustrates the modifications to the human IL-36RN sequence by mutating to insert aspartic acid residues (Mutant 1) and the heparin binding consensus sequence motif, namely XBBBXXBX, where B is a basic amino acid exposed on one side and X is a neutral or hydrophobic amino acid, to either the C-terminus of the protein without (Mutant 2) or in tandem with aspartic acids residues (Mutant 3).

[0036] FIG. 3 illustrates the modifications to the mouse IL-36Ra sequence by mutating to insert aspartic acid residues (Mutant 4) and the heparin binding consensus sequence motif, namely XBBBXXBX, where B is a basic amino acid exposed on one side and X is a neutral or hydrophobic amino acid, to either the C-terminus of the protein without (Mutant 5) or in tandem with aspartic acids residues (Mutant 6).

[0037] FIG. 4 illustrates the strategy used to obtain purified HGC-IL-36 receptor antagonist conjugates with the fractions collected throughout the preparation. Starting solution (F1 ), after loading-evaporation (F2), unloaded “free” fraction (F3) and loaded fraction (F4).

[0038] FIG. 5 illustrates the size distribution and morphology of (A) HGC nanoparticles, (B) HGC-BSA nanoparticles, and (C) the HGC-mlL-36Ra (wt) nanoparticles determined by DSL and TEM respectively.

[0039] FIG. 6 illustrates the tissue distribution of HGC-BSA nanoparticles i.a. injected into joints. Limbs from E15.5 old mice were i.a. (interphalangeal joint) injected with HGC-BSA (A) or HGC (B) or left non-injected (C). CBR-3BA staining (A-C) was performed on frozen sections, IHC for BSA was performed on adjacent frozen sections (DF). 100X magnifications are depicted.

[0040] FIG. 7 illustrates that hlL-36RN is decreased in human cartilage affected by degenerative OA. (A) In the left panel human cartilage with histologically section samples from subjects with Grade 0 (without) to grade 3 (with OA) were Safranin O and Fast Green-stained. The right panel was subjected to IHC of hlL-36RNa. Dashed and black frame squares indicate representative and comparable regions in the superficial zone and the middle zone, n = 4 donors per group. Scale bars, 100 pm. (B) RT-PCR results for hlL-36RN are shown using tissues were collected from human tissue donors graded with the Collins method and from patients with end-stage OA. n = 4 donors per group. Data are means ± SEM. *p < 0.05 and **p < 0.01 (one-way ANOVA and Tukey’s multiple comparison post test). (C) RT-PCR results for hlL-36RN are shown using human tissues with and without impact 2 and 14 days after impact occurred.

[0041] FIG. 8 illustrates that mlL-36Ra (wt) attenuates injury-induced OA in mice. Mice (DMM and sham) had daily i.a. injections of mlL-36Ra (wt) (250ng / dose) or PBS started after DMM / Sham for 5 days. Euthanasia after 20 weeks. n=5.DETAILED DESCRIPTION

[0042] The present disclosure relates to nanoparticle conjugates and methods of using the targeted nanoparticle conjugates to deliver a therapeutic amount of modified IL-36 receptor antagonist to a targeted cell or tissue of a subject or mammal. The modified IL-36 receptor antagonist nanoparticle conjugates may distribute across tissues with an abundance of extracellular matrix including those high in glycosaminoglymans. The particle-based drugdelivery also allows the sustained and adjustable release of therapeutic agent to the targeted tissue.

[0043] While various embodiments of the disclosure have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the disclosure. It should be understood that various alternatives to the embodiments of the disclosure described herein may be employed.

[0044] Unless otherwise defined, all terms of art, notations, and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this application pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art.

[0045] As used herein, the articles "a," "an," and "the" are used herein to 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" may mean one element or more than one element.

[0046] As used herein, the term "about" or "approximately" refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 % to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length. In some embodiments, the terms "about" or "approximately" when preceding a numerical value indicates the value plus or minus a range of 10%, 5%, or 1%.

[0047] As used herein, "an effective amount" refers to an amount that causes relief of symptoms of a disorder or disease as noted through clinical testing and evaluation, patient observation, and / or the like. An "effective amount" may further designate a dose that causes a detectable change in biological or chemical activity. The detectable changes may be detected and / or further quantified by one skilled in the art for the relevant mechanism or process. Moreover, an "effective amount" may designate an amount that maintains a desired physiological state, i.e., reduces or prevents significant decline and / or promotes improvement in the condition of interest. An "effective amount" may further refer to a “therapeutically effective amount”.

[0048] As used herein, the terms “protein,” “polypeptide,” “peptide” and “amino acid sequence” and their grammatical equivalents as used herein, refer to a polymer of amino acid residues covalently linked by an amide bond and may be used interchangeably herein toregardless of length or post-translational modification (e.g., glycosylation or phosphorylation). The polypeptide may optionally include glycosylation or other modifications typical for a given protein in a given cellular environment. Polypeptides and proteins disclosed herein (including functional portions and functional variants thereof) may comprise synthetic amino acids in place of one or more naturally-occurring amino acids. Such synthetic amino acids are known in the art, and include, for example, aminocyclohexane carboxylic acid, norleucine, a-amino n-demayoic acid, homoserine, S-acetylaminomethyl-cysteine, trans-3- and trans-4-hydroxyproline, 4- aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, [3- phenylserine [3-hydroxyphenylalanine, phenylglycine, a-naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-2-carboxylic acid, 1 ,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N’-benzyl-N’-methyl-lysine, N’,N’-dibenzyl- lysine, 6-hydroxylysine, ornithine, a-aminocyclopentane carboxylic acid, a-aminocyclohexane carboxylic acid, a-aminocycloheptane carboxylic acid, a-(2-amino-2-norbornane)-carboxylic acid, a,y-diaminobutyric acid, a,|3-diaminopropionic acid, homophenylalanine, and a-tert-butylglycine.

[0049] The present disclosure further contemplates that expression of polypeptides or proteins described herein in an engineered cell may be associated with post-translational modifications of one or more amino acids of the polypeptide or protein. Non-limiting examples of post-translational modifications include phosphorylation, acylation including acetylation and formylation, glycosylation (including N-linked and O-linked), amidation, hydroxylation, alkylation including methylation and ethylation, ubiquitylation, addition of pyrrolidone carboxylic acid, formation of disulfide bridges, sulfation, myristoylation, palmitoylation, isoprenylation, farnesylation, geranylation, glypiation, lipoylation and iodination.

[0050] As used herein, the term “amino acids” are referred to herein by either their commonly known three-letter symbols or by the one-letter symbols recommended by IUPACIUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single letter codes.

[0051] As used herein, the term “expression,” “recombinantly expressed,” and “over expression” includes any step involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, and post-translational modification. In some embodiments, the term also encompasses secretion of the polypeptide from a cell.

[0052] The terms “variant”, “homologue” or “derivative” in relation to a nucleotide sequence include any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) nucleic acid from or to the sequence. The terms “variant”, “homologue” or“derivative” in relation to a polypeptide sequence include any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) amino acid from or to the sequence, and “a functional variant” means a variant of an polypeptide sequence which has one or more of the aforementioned changes to the reference sequence but still retains full or part of the functions of the reference sequence.

[0053] The terms “percent similarity,” “percent identity,” and “percent homology” when referring to a particular sequence are used as set forth in the University of Wisconsin GCG software program BestFit. Other algorithms may be used, e.g. BLAST, psiBLAST or TBLASTN (which use the method of Altschul et al. (1990) J. Mol. Biol. 215: 405-410), FASTA (which uses the method ofPearson and Lipman (1988) PNAS USA 85: 2444-2448).

[0054] As used herein, the term “nanoparticle” refers to a structure comprising one or more polymers that have been collapsed into a stable sub-micron sized particle.

[0055] As used here, the term “receptor antagonist” includes both the IL-36 receptor antagonists found in mice and humans. This includes the IL-36Ra, mouse IL-36 receptor antagonist, and the IL-36RN human IL-36 receptor antagonist. The terms “IL-36Ra”, “mlL-36Ra”, and “mouse IL-36 receptor antagonist” may be used interchangeably throughout the present disclosure. The terms “IL-36RN”, “hlL-36RN”, and “human IL-36 receptor antagonist” may be used interchangeably throughout the present disclosure.

[0056] As used herein, "treatment", "treat", and "treating" refer to reversing, alleviating, mitigating, or slowing the progression of, or inhibiting the progress of, a disorder or disease or symptoms associated with such disorder or disease, and as described in more detail herein.

[0057] As used herein, the term "individual", "subject", or “patient” are often used interchangeably and refer to any human or domestic animal that may be treated with the methods disclosed herein. Suitable subjects (e.g., patients) include humans and domestic animals or pets (such as a cat or dog). Non-human primates and human patients are included. In one embodiment, subjects may include human patients that have been diagnosed with osteoarthritis. As used herein, the term "patient" refers to a subject that may receive a treatment of a disease or condition.

[0058] For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1 , 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.

[0059] The present disclosure describes novel compounds comprising a hydrophobic chitosan nanoparticle conjugated to modified IL-36 receptor antagonist (IL-36Ra) peptides withimproved conjugation. Also described herein are pharmaceutical compositions and methods of using the compounds described herein. The compounds prolong time release and biological actions. Interleukin 36 receptor antagonist is also known as IL-36Ra in mice and IL-36RN in humans. GenBank ID: 26525 (human) and 54450 (mouse).

[0060] In some embodiments, compounds of the present disclosure may be used for the treatment of diseases characterized by an IL-36 receptor antagonist deficiency including a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 , a wild-type IL-36Ra peptide. In other aspects, the polypeptide described herein comprises an amino acid sequence having at least 70% identity to to SEQ ID NO: 1 , conjugated to a chitosan nanoparticle.

[0061] Additional sequences of the present disclosure are described in Table 1 , with the underlined portions indicating modified portions of the sequences. In some aspects, the modified sequences comprise a poly-aspartic acid tail of 1 -10 residues. In other aspects, the modified sequences comprise Heparin binding consensus sequence motifs, where B represents a basic amino acid exposed on one side, and X is a neutral or hydrophobic amino acid. Thus, in one aspect, the compounds of the present disclosure includes a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, or polypeptide sequences comprising the amino acid sequence of SEQ ID Nos: 1 - 7 that are at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or at least about 100% homologous to the sequence, as described in Table 1 .

[0062] In some aspects, the compounds of the disclosure includes a polypeptide comprising the amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or at least about 100% homologous to the sequence set forth in SEQ ID NO: 1 . In other aspects, the polypeptide may comprise an analogue, derivative, fragment, variant or peptidomimetic of the amino acid sequence set forth in SEQ ID NO: 1 .

[0063] In some aspects, the compounds of the disclosure includes a polypeptide comprising the amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or at least about 100% homologous to the sequence set forth in SEQ ID NO: 2. In other aspects, the polypeptide may comprise an analogue, derivative, fragment, variant or peptidomimetic of the amino acid sequence set forth in SEQ ID NO: 2.

[0064] In some aspects, the compounds of the disclosure includes a polypeptide comprising the amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or at least about 100% homologous to the sequence set forth in SEQ ID NO: 3. In other aspects, the polypeptide may comprise an analogue, derivative, fragment, variant or peptidomimetic of the amino acid sequence set forth in SEQ ID NO: 3.

[0065] In some aspects, the compounds of the disclosure includes a polypeptide comprising the amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or at least about 100% homologous to the sequence setforth in SEQ ID NO: 4. In other aspects, the polypeptide may comprise an analogue, derivative, fragment, variant or peptidomimetic of the amino acid sequence set forth in SEQ ID NO: 4.

[0066] In some aspects, the compounds of the disclosure includes a polypeptide comprising the amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or at least about 100% homologous to the sequence set forth in SEQ ID NO: 5. In other aspects, the polypeptide may comprise an analogue, derivative, fragment, variant or peptidomimetic of the amino acid sequence set forth in SEQ ID NO: 5.

[0067] In some aspects, the compounds of the disclosure includes a polypeptide comprising the amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or at least about 100% homologous to the sequence set forth in SEQ ID NO: 6. In other aspects, the polypeptide may comprise an analogue, derivative, fragment, variant or peptidomimetic of the amino acid sequence set forth in SEQ ID NO: 6.

[0068] In some aspects, the compounds of the disclosure includes a polypeptide comprising the amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or at least about 100% homologous to the sequence set forth in SEQ ID NO: 7. In other aspects, the polypeptide may comprise an analogue, derivative, fragment, variant or peptidomimetic of the amino acid sequence set forth in SEQ ID NO: 7.

[0069] The use of chitosan nanoparticles is supported by in vivo studies reporting safety profiles for chitosans with high biodegradability, no immunogenicity, and absence of local or systemic side effects. See Li, T. et. al. J. Control. Release, 2010, 144(1), 101. Notably, the FDA considers chitosan as GRAS (Generally Recognized As Safe) and has approved its use in foods and drugs. However, its clinical use is limited by its insolubility at the physiological pH of 7.4. Hydrophobically modified glycol chitosan nanoparticles made of hydrophilic glycol chitosan conjugated with hydrophobic beta-cholanic acid resolves this issue. See Park, KM. Tissue Eng. Regen. Med. 2020, 17(1), 91 . The fabricated beta cholanic acid chitosan nanoparticles self-aggregate in aqueous solution, are soluble at a neutral pH, and are capable of encapsulating peptides for continuous release.

[0070] Chitosan may be obtained by alkaline deacetylation of chitin, which is a rich polysaccharide next to cellulose. Chitosan is positively charged by amine groups, making it suitable for binding to negatively charged molecules and to react with the carboxyl group of amino acids to therefore conjugate chitosan to the protein. Thus in some aspects, the compounds of the disclosure include a nanoparticle conjugated to the polypeptides described herein. In one aspect, the nanoparticle comprises chitosan. In other aspects, the compounds of the disclosure include a nanoparticle comprising beta-cholanic acid-chitosan. In further aspects,the compounds of the disclosure include a nanoparticle comprising a cholanic acid core. In some aspects, the compounds of the disclosure include a polypeptide covalently coupled to the surface of the nanoparticle. In other aspects, the compounds of the disclosure include a nanoparticle with amino or amine functionality for conjugation of the polypeptide to the nanoparticle to form the chitosan-IL-36Ra conjugates described in Figure 1 . In some aspects, the chitosan nanoparticle is covalently linked by an amine group to a carboxyl group of the polypeptide.Pharmaceutically Acceptable Compositions

[0071] As described herein, the disclosure provides compounds that are antagonists of IL- 36 receptor protein, and thus the present compounds, are useful for the treatment of diseases, disorders, and conditions including, but not limited to a disease characterized by an IL-36 receptor antagonist deficiency, such as osteo-arthritis, myocardial infarction, fibrotic diseases and disorders, injury-induced organ damage, injury-induced tissue damage, or tissue regeneration failure or defect.

[0072] As described herein, the pharmaceutically acceptable compositions of the disclosure additionally comprise a pharmaceutically acceptable carrier, adjuvant, or vehicle, which, as used herein, includes any and all solvents, diluents, or other liquid vehicle, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired. Remington’s Pharmaceutical Sciences, Sixteenth Edition, E. W. Martin (Mack Publishing Co., Easton, Pa., 1980) discloses various carriers used in formulating pharmaceutically acceptable compositions and known techniques for the preparation thereof. Except insofar as any conventional carrier medium is incompatible with the compounds of the disclosure, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutically acceptable composition, its use is contemplated to be within the scope of this invention. Some examples of materials which may serve as pharmaceutically acceptable carriers include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, or potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, wool fat, sugars such as lactose, glucose andsucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragamayth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil; safflower oil; sesame oil; olive oil; corn oil and soybean oil; glycols; such a propylene glycol or polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen- free water; isotonic saline; Ringer’s solution; ethyl alcohol, and phosphate buffer solutions, as well as other non-toxic compatible lubrimayts such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants may also be present in the composition, according to the judgment of the formulator.

[0073] In another aspect described herein is a pharmaceutical composition comprising any of the chitosan-IL-36Ra conjugate compounds described herein, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0074] In another aspect described herein is a pharmaceutical composition comprising a therapeutically effective amount of any of the chitosan-IL-36Ra conjugate compounds described herein, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers or vehicles.Methods of Use of Compounds and Pharmaceutically Acceptable Compositions

[0075] The compounds described herein may thus be useful for the treatment of disorders characterized by depletion of IL-36Ra. These disorders may thus be diverse in nature, and include, for example, arthritic disorders, inflammatory disorders and cardiovascular disorders, and may further include diverse tissue types. In some aspects, the disclosure includes a method of treating or lessening the severity in a subject of a disease characterized by an IL-36 receptor antagonist deficiency comprising administering a therapeutically effective amount of any of the chitosan-IL-36Ra conjugate compounds of the disclosure or a pharmaceutical composition.

[0076] In some aspects, the disease comprises one or more of osteo-arthritis, myocardial infarction, fibrotic diseases and disorders, injury-induced organ damage, or injury-induced tissue damage. In other aspect, the disease comprises osteo-arthritis.

[0077] Furthermore, without being bound by any theory, it is believed that the solubility characteristics of the compounds described herein make them well suited for tissues with rich extracellular matrices, including, for example, cartilage. Thus, in some aspects the administration of the compounds described herein is to cartilage tissues. In other aspects, theadministration of the compounds described herein is to a joint. Exemplary joints include any joint that may be compromised by inflammation or IL-36Ra depletion. In some aspects, administration is to a knee joint, shoulder joint, or hip joint.

[0078] In some aspects, the disclosure includes the method of treating or lessening the severity in a subject of regeneration failure or defect due to IL-36 receptor antagonist deficiency comprising administering a therapeutically effective amount of any of the chitosan-IL-36Ra compounds described herein or a pharmaceutical composition.Compounds and Pharmaceutically Acceptable Compositions for Use

[0079] In some aspects of the disclosure is a chitosan-IL-36Ra compound as described herein or a pharmaceutical composition for use in antagonizing the IL-36R receptor in a subject.

[0080] In some aspects of the disclosure is a chitosan-IL-36Ra compound as described herein or a pharmaceutical composition for use in treating or lessening the severity of a disease or condition in a subject characterized by a deficiency in IL-36 receptor antagonist.

[0081] In some aspects of the disclosure is a chitosan-IL-36Ra compound as described herein or a pharmaceutical composition for use in treating or lessening the severity of osteoarthritis in a subject.

[0082] In some aspects of the disclosure is a chitosan-IL-36Ra compound as described herein or a pharmaceutical composition for use in treating or lessening the severity of myocardial infarction in a subject.

[0083] In some aspects of the disclosure is a chitosan-IL-36Ra compound as described herein or a features a compound of the disclosure or a pharmaceutical composition for use in treating or lessening the severity of fibrotic diseases and disorders in a subject.

[0084] In some aspects of the disclosure is a chitosan-IL-36Ra compound as described herein or a pharmaceutical composition for use in treating or lessening the severity of injury- induced organ damage in a subject.

[0085] In some aspects of the disclosure is a chitosan-IL-36Ra compound as described herein or a pharmaceutical composition for use in treating or lessening the severity of injury- induced tissue damage in a subject.

[0086] In some aspects of the disclosure is a chitosan-IL-36Ra compound as described herein or a pharmaceutical composition for use as a medicament.

[0087] In some aspects of the disclosure is a chitosan-IL-36Ra compound as described herein or a pharmaceutical composition for use in the manufacture of a medicament.

[0088] In some aspects of the disclosure is a chitosan-IL-36Ra compound as described herein or a pharmaceutical composition for use in the manufacture of a medicament for the treatment of disease associated with an IL-36 receptor antagonist deficiency.Administration of Compounds, Pharmaceutically Acceptable Salts, and Compositions

[0089] The compounds and compositions, according to the method of the disclosure, may be administered using any amount and any route of administration effective for treating or lessening the severity of one or more of the disease associated with an IL-36 receptor antagonist deficiency recited herein. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the condition, the particular agent, its mode of administration, and the like. The compounds, salts, and compositions of the invention are preferably formulated in dosage unit form for ease of administration and uniformity of dosage. The expression “dosage unit form” as used herein refers to a physically discrete unit of agent appropriate for the subject to be treated. It will be understood, however, that the total daily usage of the compounds, salts, and compositions of the invention will be decided by the attending physician within the scope of sound medical judgment. The specific effective dose level for any particular subject or organism will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound or salt employed; the specific composition employed; the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound or salt employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound or salt employed, and like factors well known in the medical arts.

[0090] The pharmaceutically acceptable compositions of this invention may be administered to humans and other animals via intra articular injection or the like, depending on the severity of the condition being treated. In certain embodiments, the compound, salts, and compositions of the invention may be administered at dosage levels of about 0.001 mg / kg to about 1000 mg / kg, one or more times a day, effective to obtain the desired therapeutic effect.

[0091] Liquid dosage forms for oral administration include are contemplated herein, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active compound or salt, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 ,3-butylene glycol,dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions may also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

[0092] Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution, suspension or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1 ,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer’s solution, U.S.P. and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables.

[0093] The injectable formulations may be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions that may be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

[0094] In order to prolong the effect of the compounds of the disclosure, it is often desirable to slow the absorption of the compounds from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the compound then depends upon its rate of dissolution that, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered compound form is accomplished by dissolving or suspending the compound in an oil vehicle. Injectable depot forms are made by forming microencapsule matrices of the compound in biodegradable polymers such as polylactidepolyglycolide. Depending upon the ratio of compound to polymer and the nature of the particular polymer employed, the rate of compound release may be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the compound in liposomes or microemulsions that are compatible with body tissues.

[0095] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compound or salt is mixed with at leastone inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and / or a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, c) humectants such as glycerol, d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarding agents such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate, h) absorbents such as kaolin and bentonite clay, and i) lubrimayts such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets and pills, the dosage form may also comprise buffering agents.

[0096] Solid compositions of a similar type may also be employed as fillers in soft and hard- filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like. The solid dosage forms of tablets, dragees, capsules, pills, and granules may be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and may also be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions that may be used include polymeric substances and waxes. Solid compositions of a similar type may also be employed as fillers in soft and hard- filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.

[0097] The active compound or salt may also be in microencapsulated form with one or more excipients as noted above. The solid dosage forms of tablets, dragees, capsules, pills, and granules may be prepared with coatings and shells such as enteric coatings, releasecontrolling coatings and other coatings well known in the pharmaceutical formulating art. In such solid dosage forms, the active compound or salt may be admixed with at least one inert diluent such as sucrose, lactose or starch. Such dosage forms may also comprise, as is normal practice, additional substances other than inert diluents, e.g., tableting lubricants and other tableting aids such a magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents. They may optionally contain opacifying agents and may also be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in adelayed manner. Examples of embedding compositions that may be used include polymeric substances and waxes.

[0098] Dosage forms for topical or transdermal administration of a compound or salt of this invention include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants or patches. The active component is admixed under sterile conditions with a pharmaceutically acceptable carrier and any needed preservatives or buffers as may be required. Ophthalmic formulation, eardrops, and eye drops are also contemplated as being within the scope of this invention. Additionally, the invention contemplates the use of transdermal patches, which have the added advantage of providing controlled delivery of a compound to the body. Such dosage forms are prepared by dissolving or dispensing the compound in the proper medium. Absorption enhancers may also be used to increase the flux of the compound across the skin. The rate may be controlled by either providing a rate controlling membrane or by dispersing the compound in a polymer matrix or gel.

[0099] As described generally above, the compounds of the invention are useful as inhibitors of IL-36 receptor signaling. In one embodiment, the compounds are inhibitors of the IL-36 receptor and thus, without wishing to be bound by any particular theory, the compounds and compositions are particularly useful for treating or lessening the severity of a disease, condition, or disorder where activation or hyperactivity of the IL-36 receptor is implicated in the disease, condition, or disorder. When activation or hyperactivity of the IL-36 receptor is implicated in a particular disease, condition, or disorder, the disease, condition, or disorder may also be referred to as a “IL-36 receptor- mediated disease, condition or disorder.” Accordingly, in another aspect, the invention provides a method for treating or lessening the severity of a disease, condition, or disorder where activation or hyperactivity of the IL-36 receptor is implicated in the disease state.

[0100] Although the foregoing disclosure has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to one of ordinary skill in the art in light of the teachings of this disclosure that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims. The following examples are provided by way of illustration only and not by way of limitation. Those skilled in the art will readily recognize a variety of noncritical parameters that could be changed or modified to yield essentially similar results.EXAMPLESGeneral methods.

[0101] In general, the practice of the present disclosure employs, unless otherwise indicated, conventional techniques of chemistry, molecular biology, recombinant DNA technology, and immunology. The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.Abbreviations

[0102] Unless otherwise noted, or where the context dictates otherwise, the following abbreviations shall be understood to have the following meanings:Abbreviation Meaning dH2O Distilled water (H2O) DMM Destabilization of medial meniscus DSL Dynamic Light Scattering EDAC 1 -Ethyl-3-(3-dimethylaminopropyl)carbodiimide GAG Glycosaminoglymays HGC Beta-Cholanic acid-chitosan IHC Immunohistochemistry LCM Laser capturing microdissection TEM Transmission Electron Microscopy Wt Wild typeExample 1Fabrication of Hydrophobic Chitosan (HGC) Nanoparticles

[0103] Glycol chitosan was dissolved in distilled water, followed by dilution with methanol (water / methanol co-solvent (1 :1 v / v) then 5beta-cholanic acid (56 pmol, MW=360.57) was added and stirred until the solutions were optically transparent. To activate carboxylic acid pertaining to 5 -cholanic acid, equal amounts (1 .5 equiv 5[3-cholanic acid]) of EDAC (84 pmol, MW=191 .70) and NHS (84 pmol, MW=115.09) were added into the polymer solution, which allowed formation of the amide linkage by the reaction with primary amino groups in glycol chitosan. The resulting solution was stirred for 24 hours at room temperature, dialyzed (molecular weight cutoff = 10 kDa) for 3 days against the excess amount of water / methanol mixture (1v / 4v), and lyophilized. High, medium, and low MW chitosans as well as lectin and betalain were optionally added to the solution as modulators of the release time of the conjugated proteins depending on the needs.Proposed Generation of Mutated Proteins

[0104] The beta-cholanic, betalain, and lectin provide the chemical charges of chitosan that improve solubility and in part the release time, but a further improvement in release time and binding ability are provided by mutations of IL-36 receptor antagonists. The amine groups of chitosan react with the carboxyl group of amino acid side chains to form a bond that links the chitosan to the protein. To maximize the binding capacity to hydrophobic chitosan, standard methods were used to mutate the IL-36 receptor antagonist protein to have up to 10 aspartic acid residues in tandem that are inserted to either the N- or C-terminal of the protein (Mutants 1 and 4) (FIG. 2 and FIG. 3).

[0105] Glycosaminoglymays (GAGs) are important components of the ECM of articular cartilage. GAGs are essential molecules that regulate diverse biological processes including cell adhesion, differentiation, signaling and growth, by interaction with a wide variety of proteins. With the exception of hyaluronan, GAGs are synthesized covalently bound to core proteins to form proteoglymays such as aggremay that is abundant in articular cartilage. GAGs endow the articular cartilage with resistance to compressive loading and are involved in many biological interactions. Using the heparin binding consensus sequence motifs to bind with GAG prolongs time release and distributes modified proteins across GAG rich tissue, such as cartilage to reach the biological target (i.e. chondrocytes).

[0106] To enable human IL-36 receptor antagonists to bind to GAGs and therefore distribute evenly across tissues such as articular cartilage, standard methods were used to mutate IL- 36RN for insertion of a heparin binding consensus sequence motif. To this purpose, IL-36RN was mutated by inserting the heparin binding consensus sequence motif, namely XBBBXXBX, where B is a basic amino acid exposed on one side and X is a neutral or hydrophobic amino acid, to either the N- or C-terminal of the protein without (Mutant 2) or in tandem with aspartic acids residues (Mutant 3) (FIG. 2).

[0107] The same mutagenesis strategy was used to generate mutated mouse IL-36Ra proteins. Mutant 4 contains up to 10 (1 to 10) aspartic acid residues in tandem that are inserted to either the N- or C-terminal of the protein (FIG. 3). Mutant 5 contains a heparin binding consensus sequence motif without aspartic acid residues while Mutant 6 contains a heparin binding consensus sequence motif in tandem with aspartic acids residues (FIG 3).Proposed Conjugation of HGC Nanoparticles to Modified IL-36 Receptor Antagonist Proteins for Production of ChitoKine36-h1 , ChitoKine36-h2, ChitoKine36-h3, ChitoKine36- m1 , CHitoKine36-m2, and ChitoKine36-m3

[0108] Hydrophobic chitosan nanoparticles will be resuspended in a solution containing distilled water / ethanol (1 :1 , v / v) and then mixed with dH2O containing the mutated protein (any of Mutants 1 , 2, 3, 4, 5, or 6). Additional ethanol will be added to final ratio of distilled water / ethanol (1 :2, v / v) to obtain the respective ChitoKine(s). The solvents will be evaporated using a rotary evaporator to prepare a thin polymer / peptide film. After addition of distilled water, the solution will be gently stirred and then filtered. The resulting filtrates will be collected and lyophilized. Fractions will also be collected at each of the preparation steps to determine protein concentration using either western blot analysis or ELISA for either hlL-36RN and modified hlL- 36RN or mlL-36Ra and modified mlL-36Ra. The schematic in FIG. 4 outlines the conjugate preparation.

[0109] The morphology and size distribution of nanoparticles loaded or unloaded with proteins will be characterized using dynamic light scattering and transmission electron microscopy. Biological assays will be performed to evaluate biological activity in vivo and in vitro as well as binding receptor affinity. Biodistribution will be performed using Cibacron Brilliant Red-3BA staining for chitosan nanoparticles.

[0110] The conjugation with hydrophobic chitosan nanoparticles to mutated proteins generated:ChitoKine36-h1 in which Mutant 1 is conjugated with hydrophobic chitosan nanoparticles ChitoKine36-h2, in which Mutant 2 is conjugated with hydrophobic chitosan nanoparticlesChitoKine36-h3, in which Mutant 3 is conjugated with hydrophobic chitosan nanoparticlesChitoKine36-m1 , in which Mutant 4 is conjugated with hydrophobic chitosan nanoparticlesChitoKine36-m2, in which Mutant 5 is conjugated with hydrophobic chitosan nanoparticlesChitoKine36-m3 in which Mutant 6 is conjugated with hydrophobic chitosan nanoparticlesExample 2Determination of Protein Loading Efficiency Using HGC-BSA and HGC-mlL-36Ra (wt)

[0111] HGC protein loading efficiency was evaluated via Western blot using the fractions collected (fractions designated as F1 to F4) at each of the preparation and purification steps. The loading efficiency of BSA to HGC, expressed as the percentage of the starting concentration (F1 ) to the concentration detected in the final fraction (F4), was determined to be 84±3% and the loading ability of mlL-36Ra (wt) was determined to be been 82±4%. Increasing the loading efficiency of IL-36 receptor antagonists motivated the mutational studies (Mutants 1 - 6).

[0112] FIG. 5 shows the size and morphology of HGC after protein loading by DSL and TEM analyses. The mean diameter of HGC nanoparticles is 196 nm (FIG. 5A). HGC-BSA (FIG. 5B) and HGC-mlL-36Ra (wt) (FIG. 5C) had a larger diameter respectively of 361 nm (FIG. 5B) and 350nm (Figure 5C). TEM analyses indicate that HGC-BSA and HGC-mlL-36Ra (wt) loaded nanoparticles display a spherical shape similar to unloaded HGC but larger. To determine HGC- protein stability, DSL analyses were performed daily for 5 days in HGC and HGC-BSA preparations maintained at 4 °C. No signifimayt changes in the average size were observed in the protein loaded and unloaded preparations.

[0113] Similar studies will be conducted to determine the loading efficiency of IL-36 receptor antagonist mutants 1 through 6.Determination of Protein Release Efficiency using HGC-BSA and HGC-mlL-36Ra (wt)

[0114] To determine the release of BSA and IL-36Ra (wt) from HGC-BSA and HGC-mlL- 36Ra (wt) respectively, a time-course release profile was performed. The protein release profile from HGC nanoparticles was determined in PBS (pH 7.4) at 37°C, maintaining the protein gradient by regularly replacing the PBS dialysate solution at various time points. Each solution was mixed with the previous and subjected to Western Blot analysis. Protein was also quantified by densitometric analyses. Serial dilutions of recombinant protein solution were used to generate an internal standard curve to evaluate the concentration. The BSA release profile showed that 84±3% of the protein was released, from the HGC nanoparticles within 48 hours following linear kinetics; a minimal additional release was observed after that time. These results demonstrated the sustained release properties of HGC nanoparticles at a physiological pH.

[0115] Similar studies will be conducted to determine the release efficiency of IL-36 receptor antagonist mutants 1 through 6 at time points including 24, 40, 48, 64, and 72 hours.Determination of Tissue Distribution using HGC-BSA

[0116] Interphalangeal joints were implanted with HGC, HGC-BSA or left non-injected and adjacent frozen sections were either subjected to CBR-3BA staining or BSA immunohistochemistry (IHC) (FIG. 6). HGC and HGC-BSA nanoparticles implanted into joints stained uniquely red with CBR-3BA, while no staining was detectable in joints that were noninjected (FIG. 6A-C). To determine whether BSA was detectable within the joints implanted with HGC-BSA IHC was performed for BSA. BSA is not produced in rodents meaning BSA-IHC detects only the BSA that is delivered by HGC. As shown in FIG. 6D, BSA was detected within the joints injected with HGC-BSA (4 hours after injection), while no signal was detectable in joints injected only with HGC (FIG. 6E) or left non-injected (FIG. 6F). However, the BSA was not evenly distributed across the articular cartilage surface, leaving several regions without BSA. This motivated the mutation of IL-36 receptor antagonist (human or mouse) to include a heparin binding consensus sequence motif for binding to GAGs that are uniformly distributed in the articular cartilage. This provides a lodging for an even distribution of mutated proteins released from ChitoKine36-h2, ChitoKine36-h3, ChitoKine36-m2, and ChitoKine36-m3 across the articular cartilage and any other tissue reach in GAGs.

[0117] Similar studies will be conducted to determine the tissue distribution of IL-36 receptor antagonist mutants 1 through 6.Example 5Evaluation of Chrondroprotective Role of IL-36 Receptor Antagonist

[0118] Morphologically, the position of each future joint is first delimited by the interzone development, consisting of a region of compact mesenchymal cells between two adjacent growth plates. The distinct gene expression profile where interzone cells emerge between the forming growth plates, was studied using Spatial Transcriptomics gene expression profile screening. RNA samples from E14.5 interzone cells and adjacent growth plate chondrocytes were obtained using laser capturing microdissection (LCM). After verifying RNA quality, specimens were subjected to microarray analysis. Normalized data were expressed as fold changes of a maydidate gene in the interzone sample compared to the expression found in the sample from adjacent growth plate chondrocyte. hlL-36RN was increased by 4.34 folds in the interzone compared to adjacent growth plate chondrocytes, indicating that hlL-3RN is a joint morphogenic articular cartilage chondroinductive factor.

[0119] h II-36RN was evaluated for its chondroprotective role in maintaining the homeostasis of the articular cartilage and its role related to OA degeneration. In human and mouse OA, adeficiency in IL-36 receptor antagonist correlates with the severity of cartilage degeneration. FIG 7, consistently indicate a decrease in hlL-36RN in human cartilage and in several models for OA in experimental animals. In human knee cartilage with a range of histologically graded OA severity (from intact, grade 0 to end stage), (FIG. 7) the decrease in expressions of hlL-36RN correlated with OA severity. Using an ex-vivo model for injury-induced OA (FIG. 7B), in which Grade 0 human articular cartilage was mechanically injured (impacted), hlL-36RRN’s expression decreased after impact and remained decreased with the progression of degeneration (2 days and 14 days after impact).Evaluation of mlL-36Ra Replacement in mlL-36Ra Deficient Cells

[0120] To test if the mlL-36Ra deficiency had a functional role in OA, a mouse model of injury-induced OA (destabilization of medial meniscus -DMM- model) was used for mlL-36Ra (daily i.a. injections for 5 days) or PBS injections after DMM / sham procedure. To perform volumetric analyses of all joint tissues, a CT-phosphotungstic acid imaging system was used that visualizes soft tissues (articular cartilage, synovium) and allows Co-registration, 3D Reconstruction, volumetric quantitative analyses of five joint tissues. In DMM-mlL-36Ra mice compared to DMM-PBS mice, it was noted an upright trend in the subchondral bone and synovium thickness, and an increase in cartilage thickness (FIG. 8).

[0121] Similar studies will be conducted to determine the treatment efficacy of replacing IL- 36 receptor antagonist deficient tissues with IL-36 receptor antagonist mutants 1 through 6. Many modifications and variations of the embodiments described herein may be made without departing from the scope, as is apparent to those skilled in the art. The specific embodiments described herein are offered by way of example only.

Claims

CLAIMS1 . A compound for the treatment of diseases characterized by an IL-36 receptor antagonist deficiency comprising: a polypeptide comprising the amino acid sequence of SEQ ID No: 1 or a sequence which is at least 70% homologous thereto; and a chitosan nanoparticle.

2. The compound of claim 1 , wherein the chitosan nanoparticle is covalently linked by an amine group to a carboxyl group of the polypeptide.

3. The compound of claim 1 , wherein the polypeptide comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7.

4. The compound of claims 1 - 3, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 1 or a sequence which is at least 80% homologous thereto, or an analogue, derivative, fragment, variant or peptidomimetic thereof.

5. The compound of claims 1 - 3, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 2 or a sequence which is at least 80% homologous thereto, or an analogue, derivative, fragment, variant or peptidomimetic thereof.

6. The compound of claims 1 - 3, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 3 or a sequence which is at least 80% homologous thereto, or an analogue, derivative, fragment, variant or peptidomimetic thereof.

7. The compound of claims 1 - 3, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 4 or a sequence which is at least 80% homologous thereto, or an analogue, derivative, fragment, variant or peptidomimetic thereof.

8. The compound of claims 1 -3, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 5 or a sequence which is at least 80% homologous thereto, or an analogue, derivative, fragment, variant or peptidomimetic thereof.

9. The compound of claims 1 - 3, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 6 or a sequence which is at least 80% homologous thereto, or an analogue, derivative, fragment, variant or peptidomimetic thereof.

10. The compound of claims 1 - 3, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 7 or a sequence which is at least 80% homologous thereto, or an analogue, derivative, fragment, variant or peptidomimetic thereof.11 . The compound according to any of the claims 1 - 10, wherein the nanoparticle comprises beta-cholanic acid-ch itosan.

12. The compound of according to any of the claims 1 - 11 , where in the nanoparticle further comprises a cholanic acid core.

13. The compound according to any of the claims 1 to 12, wherein the polypeptide is covalently coupled to the surface of the nanoparticle.

14. The compound according to any of the claims 1 to 12, wherein the nanoparticle has amino or amine functionality for attachment of the polypeptide to the surface of the nanoparticle.

15. A pharmaceutical composition comprising the compound of any one of claims 1-13 and a pharmaceutically acceptable carrier.

16. A pharmaceutical composition comprising a therapeutically effective amount of the compound of any one of claims 1 to 15 or the compound of claim 1 and one or more pharmaceutically acceptable carriers or vehicles.

17. A method of treating or lessening the severity in a subject of a disease characterized by an IL-36 receptor antagonist deficiency comprising administering a therapeutically effective amount of the compound according to any of the claims 1 to 14 or a pharmaceutical composition according to any one of the claims 15 to 16.

18. The method of claim 17, wherein the disease comprises osteo-arthritis, myocardial infarction, fibrotic diseases and disorders, injury-induced organ damage, or injury-induced tissue damage.

19. The method of claim 17, wherein the administration comprises administration to a tissue.

20. The method of claim 19, wherein the tissue is cartilage.21 . The method of claim 17, wherein the administration comprising administration to a knee joint, shoulder joint, or hip joint.

22. The method of claim 17, wherein the subject is a mammal.

23. The compound according to any one of claims 1 to 14 or a pharmaceutical composition according to any one of claims 15 to 16 for use in a method of treating osteo-arthritis in a subject.

24. The compound according to any one of claims 1 to 14 or a pharmaceutical composition according to any one of claims 15 to 16 for use in treating or lessening the severity of a disease or condition in a subject characterized by a deficiency in IL-36 receptor antagonist.

25. Use of the compound of any one of claims 1 to 14, or the pharmaceutical composition of any one of claims 15 to 16, as a medicament.

26. Use of the compound of any one of 1 to 14, or the pharmaceutical composition of any one of claims 15 to 16, in the manufacture of a medicament for the treatment of disease associated with an IL-36 receptor antagonist deficiency.

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