Patent Application
20240325435
The present invention relates to a compound consisting of a glycosaminoglycan conjugate with a drug and to a preparation thereof.
The extracellular matrix (ECM) is a dynamic assemblage of interacting molecules that regulate cell functions and interactions in response to stimulation. One class of extracellular matrix macromolecules, the glycosaminoglycans, are molecules known to be involved in a wide array of both normal and abnormal biological processes, including cell migration, differentiation, proliferation, immune response and cytoskeletal organization.
Glycosaminoglycans (GAGs) are unbranched chains composed of repeating disaccharide units. These disaccharide units always contain an amino sugar (N-acetylglucosamine or N-acetylgalactosamine), which in most cases is sulfated, with the second sugar usually being an uronic acid (glucuronic or iduronic). GAGs are highly negatively charged because of the presence of carboxyl or sulfate groups on most of their sugar residues. As such they are strongly hydrophilic. GAGs tend to adopt highly extended conformations and form matrices that are space filling and resistant to compressive forces. Four main groups of GAGs have been distinguished by their sugar residues, the type of linkage between these residues, and the number and location of sulfate groups. They include: (1) hyaluronan, (2) chondroitin sulphate and dermatan sulfate, (3) heparan sulfate and heparin, and (4) keratan sulfate.
Hyaluronan (also called hyaluronic acid or hyaluronate or HA) is the simplest of GAGs. It consists of a regular repeating sequence of non-sulfated disaccharide units, specifically N-acetylglucosamine and glucuronic acid. Its molecular weight can range from 400 daltons (the disaccharide) to over millions of daltons. It is found in variable amounts in all tissues, such as the skin, cartilage, and eye, and in most, if not all, fluids in adult animals. It is especially abundant in early embryos. In articular cartilage, HA can form a large aggregate which is important for the function of cartilage. Furthermore, cell motility and immune cell adhesion is mediated by the cell surface receptor RHAMM (Receptor for Hyaluronan-Mediated Motility) and CD44.
HA is synthesized directly at the inner membrane of the cell surface with the growing polymer extruded through the membrane to the outside of the cell as it is being synthesized. Synthesis is mediated by a single protein enzyme, hyaluronan synthetase (HAS). By contrast, other GAGs are synthesized inside the cell in the Golgi apparatus, possibly in association with some core protein, and then released by exocytosis. HA degradation in vertebrate tissues in vivo is mediated by hyaluronidase, and exoglycosidases that remove sugars sequentially. Mammalian-type hyaluronidases have both hydrolytic and transglycosidase activities and can degrade HA and chondroitin. In connective tissue, the water of hydration associated with HA creates spaces between tissues, thus creating an environment conducive to cell movement and proliferation. HA plays a key role in biological phenomena associated with cell motility including rapid development, regeneration, repair, embryogenesis, embryological development, wound healing, angiogenesis, and tumorigenesis.
CD44 (also known as Pgp-1, Hermes-3, HCAM, ECMR III) is a widely expressed glycoprotein with a molecular weight of 85 to 90 kDa. CD44 is a major cell surface receptor for the glycosaminoglycan, hyaluronic acid (HA). CD44 binds HA specifically, although certain chondroitin-sulfate containing proteoglycans may also be recognized. CD44 plays a role in various cellular and physiological functions, including adhesion to and migration on HA, HA degradation and tumor metastasis. CD44 has also been shown to play a role in extracellular matrix binding, cell migration, lymphocyte activation, lymphocyte homing, and proliferation of bronchial smooth muscle cell (Gunthert et al., 1991, A new variant of glycoprotein CD44 confers metastatic potential to rat carcinoma cells, 5; 65(1):13-24). The CD44 receptor shows a complex pattern of alternative splicing in its variable region of the extracellular domain. CD44 appears to be a particularly important leukocyte receptor for HA and may therefore have a role in the pathogenesis of asthma. In addition, levels of HA, which were increased during experimental asthma in control mice were markedly attenuated in the antibody-treated mice, supporting a role for CD44 in HA metabolism (specifically in the breakdown of high molecular weight HA to pro-inflammatory low molecular weight forms). This may be particularly important because HA-derived oligosaccharides can bind and activate Toll-like receptor. Clearly, the most impressive aspect of the results is the profound magnitude of the beneficial effects of anti-CD44 treatment.
HA-CD44 interactions may play an important role in development, inflammation, T cell recruitment and activation, lung inflammation, and tumor growth and metastasis.
Mice with a targeted deletion of standard CD44 and all isoforms develop normally. Studies have suggested an important role for CD44 in inflammatory states such as rheumatoid arthritis and the extravasation of T cells to sites of tissue inflammation. CD44 may have an important role in the recruitment of inflammatory cells in allergen-induced airway inflammation in mice. CD44 has been suggested to play a critical role in regulating chronic inflammation, suggesting that CD44 may have a critical role in regulating macrophage activation independently of interactions with HA (Dianhua Jiang, Hyaluronan in Tissue Injury and Repair, Annu. Rev. Cell Dev. Biol. 2007; 23:435-61).
The ability to control inflammatory and immune responses is central to the therapy of a wide spectrum of diseases. CD44 supports the adhesion of activated lymphocytes to endothelium and smooth muscle cells. Furthermore, ligation of CD44 induces activation of both inflammatory and vascular cells. HA, the principal ligand for CD44, is upregulated in atherosclerotic lesions of apoE-deficient mice and the low-molecular-weight proinflammatory forms of HA stimulate VCAM-1 (vascular cell adhesion molecule-1) expression and proliferation of cultured primary aortic smooth muscle cells, whereas high-molecular-weight forms of HA inhibit smooth muscle cell proliferation. Gal-9 (Galectin-9) can reduce AHR (airway hyperresponsiveness) as well as Th2-associated airway inflammation. Furthermore, administration of Gal-9 as well as anti-CD44 monoclonal antibody inhibited the infiltration of peripheral blood Th2 cells into the airway. Interestingly, Gal-9 directly bound the CD44 adhesion molecule and inhibited interactions with HA. Consistent with the concept that CD44-HA interactions mediate the migration of T cells into the lung, Gal-9 blocked CD44-dependent adhesion of BW5147 mouse T cells to HA. It was concluded that Gal-9 inhibits allergic inflammation of the airway and AHR by modulating CD44-dependent leukocyte recognition of the extracellular matrix (Shigeki Katoh, et al., Galectin-9 Inhibits CD44-Hyaluronan Interaction and Suppresses a Murine Model of Allergic Asthma, American Journal of Respiratory and Critical Care Medicine, 2007 Jul. 1; 176(1):27-35).
The CD44 will be expression at the autoimmune disease likes systemic lupus erythematosus (SLE), Rheumatoid arthritis, Sjögren's syndrome, inflammatory bowel disease (IBD), Ankylosing spondylitis, Psoriatic arthritis, Psoriasis, Dermatomyosistis, Vasculitis, and Behcet's disease. Systemic lupus erythematosus is a prototype autoimmune disease that affects multiorgan systems. Accumulating evidence suggests that hyaluronan and its interaction with its cell surface receptor CD44 plays an important role in mediating pathogenic mechanisms in SLE (Yung S and Chan TM, 2012, The Role of Hyaluronan and CD44 in the Pathogenesis of Lupus Nephritis, Autoimmune Dis. Volume 2012 (2012), Article ID 207190, 9 pages). Rheumatoid Arthritis (RA) is a common autoimmune disorder that results in inflammation of the synovial joints of patients. Though RA affects approximately 1% of the population and is classified as an autoimmune disorder, the molecular event(s) which initiate the evasion of tolerance remain speculative and unconfirmed (Patrick J. Mott, CD44 Antibodies and Immune Thrombocytopenia in the Amelioration of Murine Inflammatory Arthritis, PLoS One, 2013, 8(6): e65805). While Sjögren's syndrome (SS) is more common than related autoimmune disorders, such as systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA), scientific and medical research in SS has lagged behind significantly. This is especially true in the field of SS genetics, where efforts to date have relied heavily on candidate gene approaches (John A. Ice, Genetics of Sjögren's syndrome in the genome-wide association era, J Autoimmun. 2012 August; 39(1-2):57-63). Ankylosing Spondylitis (AS) is a common inflammatory rheumatic disease with a predilection for the axial skeleton, affecting 0.2% of the population. Current diagnostic criteria rely on a composite of clinical and radiological changes, with a mean time to diagnosis of 5 to 10 years (Roman Fischer, 2011, Discovery of Candidate Serum Proteomic and Metabolomic Biomarkers in Ankylosing Spondylitis, Mol Cell Proteomics, 2012 February; 11(2):M111.013904). The results suggest that circulating T lymphocytes bearing activated CD44 are elevated under conditions of chronic inflammation and that these may represent a pathogenically important subpopulation of activated circulating cells that may provide a reliable marker for autoimmune or chronic inflammatory disease activity (Estess P, et al., 1998, Functional activation of lymphocyte CD44 in peripheral blood is a marker of autoimmune disease activity, J Clin Invest. 1998 Sep. 15; 102(6):1173-82). Inflammatory bowel disease (IBD), including Crohn's disease (CD) and ulcerative colitis (UC), shares clinical and immunological features with psoriasis. Genome-wide association studies have found common susceptibility genes. However, epidemiologic data evaluating the association between psoriasis, psoriatic arthritis and risk of IBD are sparse. This research aimed to evaluate the association between psoriasis, psoriatic arthritis and incident CD and UC among women in the USA. Psoriasis with concomitant psoriatic arthritis is associated with an increased risk of incident CD (Wen-Qing Li, 2013, Psoriasis, psoriatic arthritis and increased risk of incident Crohn's disease in US women, Ann Rheum Dis. 2013 July; 72(7):1200-5). Dermatomyositis (DM) is a connective-tissue disease related to polymyositis (PM) that is characterized by inflammation of the muscles and the skin. While DM most frequently affects the skin and muscles, it is a systemic disorder that may also affect the joints, the esophagus, the lungs, and, less commonly, the heart. Vasculitis is a group of disorders that destroy blood vessels by inflammation. Both arteries and veins are affected. The pathophysiology of vasculitis is not well understood, but the ensuing inflammatory response has been generally well described (Henry S. Su, 2012, Vasculitis: Molecular Imaging by Targeting the Inflammatory Enzyme Myeloperoxidase, Radiology, 2012 January; 262(1):181-90). Behcet's disease (BD) is the only systemic vasculitis involving both arteries and vein in any sizes. It is frequently encountered in rheumatology clinics. It has some major morbidities and even fatal outcomes in some cases (M. B. Owlia, 2012, Behcet's Disease: New Concepts in Cardiovascular Involvements and Future Direction for Treatment, ISRN Pharmacol, 2012: 760484).
Interferon alpha (IFNα) conjugated with polyethylene glycol (PEG) has been widely used for the treatment of hepatitis C virus (HCV) infection as a once-a-week injection formulation. However, the PEGylated IFNα has a low efficacy of 39% and a side effect after repeated injections possibly due to the nonspecific delivery with PEGylation. Therefore, target specific long-acting hyaluronic acid-interferon alpha (HA-IFNα) conjugate was developed for the treatment of HCV infection. HA-IFNα conjugate was synthesized by coupling reaction between aldehyde modified HA and the N-terminal group of IFNα. The IFNα content could be controlled in the range of 2-9 molecules per single HA chain with a bioconjugation efficiency higher than 95%.
Several studies have implicated CD44 in direct interactions between bacteria and host cells, as well as in signaling events that alter host cells and make them more susceptible to infection. Streptococcus pyogenes, for example, attaches to cells through its hyaluronan-rich polysaccharide capsule (or cell wall). HA binds to CD44, which in turn triggers tyrosine phosphorylation of several host-cell proteins, as well as cytoskeletal rearrangements that cause ruffles and the extension of lamellipodia. As a result, the intercellular adhesion is loosened owing to a reduced number of tight junctions and of E-cadherin, a process that allows the entry of bacteria into subepithelial tissue.
WO94/09811 describes the use of CD44 in treating inflammation or detecting cancer metastasis. The authors show that CD44 is upregulated in inflammatory conditions and CD44 peptides are capable of inhibiting T-cell activation. No data or claims are presented on inhibition of metastasis by CD44 and no claims are made towards use of CD44 for inhibiting tumor growth or angiogenesis. WO 99/45942 discloses the use of HA-binding proteins and peptides including CD44 to inhibit cancer and angiogenesis-dependent diseases. This publication uses metastatin, a 38 kDa fragment of the cartilage link protein, as well as a HA-binding peptide derived from this fragment to inhibit pulmonary metastasis of B16 mouse melanoma and Lewis lung carcinoma. In the case of the HA-binding peptide, growth of B16 melanoma on chicken CAM and endothelial cell migration on HA have been inhibited. In both publications the use of HA-binding peptides is directly related to their ability to bind hyaluronic acid.
U.S. Pat. No. 8,192,744 shows that soluble recombinant CD44 hyaluronic acid binding domain (CD44HABD) inhibits angiogenesis in vivo in chick and mouse and thereby inhibits human tumor growth of various origins. The invention discloses soluble non glycosylated CD44 recombinant proteins as a novel class of angiogenesis inhibitors based on targeting of vascular cell surface receptor.
Thus, the prior art discloses the potential use of CD44 to specify that any effects are dependent on HA-CD44-interaction. Consequently, all utility ascribed this far to CD44-HA conjugate is directly dependent on their ability to bind hyaluronic acid.
However, some drugs are still not successfully conjugated onto HA and further experiments should be carried out to confirm the potential usefulness of HA as site-delivery carrier of active compound. In particular, the prior art has not shown that the interactions between the surface cell receptor CD44 and a conjugate of HA with an active compound can be profitably exploited for a target delivery of such active compound in diseases characterized by an overexpression of CD44 obtaining an effective therapeutic improvement of the same.
The purpose of the present invention is to provide new compound based on the conjugation of HA with active compound suitable for a site delivery of such active compound in diseases overexpressing the surface cell receptor CD44.
The present invention, therefore, provides a compound conjugating glycosaminoglycan with a drug, wherein the drug is used for treating diseases of inflammation that are highly related with the expression of CD44.
In a first aspect, it is an object of the invention a compound consisting of a conjugate from a glycosaminoglycan and an active compound, wherein the active compound is conjugated by means of a functional group to a carboxylic group of the glycosaminoglycan, its derivative, or a salt thereof to form a covalent conjugation, and wherein the active compound includes anti-inflammatory drug, anti-allergy drug and steroid.
The glycosaminoglycan of the conjugate according of the present invention is preferably hyaluronic acid.
Furthermore, the glycosaminoglycan conjugate according of the present invention is preferably for use for treating inflammation diseases.
Therefore, in a second aspect it is a further object of the invention the use of a compound consisting of a conjugate from a glycosaminoglycan and an active compound, wherein the active compound is conjugated by means of a functional group to a carboxylic group of the glycosaminoglycan, its derivative, or a salt thereof to form a covalent conjugation, and wherein the active compound consists of Celecoxib, Fexofenadine, Budesonide, and Prednisolone for the treatment of inflammation and for the preparation of pharmaceutical compositions for said therapeutic treatment.
Yet in a further aspect, it is an object of the present invention the method for preparing a compound consisting of a conjugate from a glycosaminoglycan and an active compound, wherein the active compound is conjugated by means of a functional group to a carboxylic group of the glycosaminoglycan, its derivative, or a salt thereof to form a covalent conjugation, and wherein the active compound consists of Celecoxib, Fexofenadine, Budesonide, and Prednisolone.
To adequately describe the present invention, references to embodiments thereof are illustrated in the appended drawings. These drawings herewith form a part of the specification. However, the appended drawings are not to be considered limiting in their scope.
Other objectives, advantages and novel features of the invention will become more apparent from the following detailed description when taken in conjunction with the accompanying drawings.
In general, a drug orally administered or injected into circulation system must directly arrive at its targeted treatment area, and therefore the drug effect on targeted disease and normal organ is very similar because the concentration and specificity are not so high on the targeted site, limited by safety profile.
In order to improve the therapy efficacy combining the same with good safety profile, one strategy is to modify the drug to be more target-selective to the disease area through a covalent binding the drug with a carrier. This is a need particularly felt in the field of anti-inflammatory therapy as previously anticipated.
At this purpose, the inventor has conceived the idea to exploit the interactions between the hyaluronic acid (HA) and its receptor CD44 for a target delivering active substance.
The idea to maintain the relative higher concentration of the drug on the targeted site versus normal tissue or organ has been established by the inventor following long-term study and experiment on HA.
The results, from which the present invention originates, are fully described in the examples and are hereinafter briefly summarized.
In fact, the present invention finds support on the result showing that hyaluronic acids having different average molecular weights (MW) have an adhesion index higher in injured tissue than in normal tissue and that the HA with low average molecular weight performs better than the HAs with high average molecular weights. In particular, as shown in
The attachment condition of free dye and HA-dye on cell lines of HT29 and HCT15 of mice for 4 weeks was conducted. The free dye was injected into the tail vein of the mice. The result showed that the two different CD44 expression cancer cells without any difference in attachment result. The ratio of attachment area of HT29 is 50.15%, whereas HCT15 is 49.86%. However, when the HA conjugated dye was injected into the mice tail vein, the more CD44 expression cancer cell HT 29 showed significant concentration of HA conjugated dye, but the less CD44 expression HCT15 showed very limited result. The ratio of attachment area of HT29 is 74.15%, whereas HCT15 is 25.85%. The result can show that when dye conjugated with HA, the concentration of dye was increased owing to HA attached on CD44 abundant site.
CD44 highly related diseases include cancer, infection and inflammation. Infectious pathogens include, but not limited to, some viruses, bacteria, fungi, protozoa, multicellular parasites, and aberrant proteins known as prions. These pathogens are the cause of disease epidemics, in the sense that without the pathogen, no infectious epidemic occurs. In a preferred embodiment, infection disease includes lower respiratory infections, HIV/AIDS, diarrheal diseases, tuberculosis, malaria, measles, pertussis, tetanus, meningitis, syphilis, hepatitis B, sepsis, and tropical diseases. Inflammatory abnormalities are a large group of disorders which underlie a vast variety of human diseases. In a preferred embodiment, inflammation disease includes acne vulgaris, allergic diseases, asthma, autoimmune diseases, celiac disease, chronic prostatitis, glomerulonephritis, hypersensitivities, inflammatory bowel diseases, pelvic, inflammatory disease, reperfusion injury, rheumatoid arthritis, sarcoidosis, transplant rejection, vasculitis, and interstitial cystitis.
In the specification and in the claims the term “drug” or “compound” or “agent” for use in the present invention may comprise anti-asthma drug, anti-histamine drug, anti-infection drug, anti-inflammatory drug, anti-allergy drug, anti-virus drug, immunosuppressant, NSAID (Non-Steroidal Anti-Inflammatory Drug), and steroid. Among them, anti-allergy drug, anti-infection drug, anti-inflammatory drug, and steroid are preferred. The majority of anti-allergy drugs can be divided into anti-histamines, anti-inflammatory agents, decongestants and steroid such as Fexofenadine, cetirizine, chlorpheniramine maleate, pseudoephedrine, and Budesonide. The majority of anti-infection drugs can be divided into local anti-infection means that are used externally on skin or mucous membranes without entering the blood stream and metabolizing in the liver; and systemic anti-infection means which are used to treat internal organ infections through oral or injected administration. The majority of anti-inflammatory drugs can be divided into non-steroidal anti-inflammatories or NSAIDs, COX-2 antagonist and steroid.
In another embodiment, examples of appropriate drugs are anti-asthma drug, anti-fungal drug, anti-histamine drug, anti-inflammatory drug, anti-virus drug, NSAID and steroid thereof. In a preferred embodiment, the conjugation of anti-asthma drug including Salbutamol; NSAID including Nimesulide, Celecoxib, Meloxicam, Diclofenac and Piroxicam; anti-allergy drug including Fexofenadine; anti-fungal drug including Amphotericin B; anti-virus drug including Ribavarin; and steroid including Budesonide and Prednisolone to HA were preferred. Further, COX-2 antagonist includes Celecoxib.
The aim of the present invention is binding or conjugating HA with a drug aforesaid, with or without a linker or spacer, by carboxyl group, hydroxyl group, or amino group of HA to accomplish working effect on specific location and specific time. Therefore, HA as a target delivery vehicle to carry the drug to the specific site that has abundant CD44 can produce better treatment efficacy and safety.
As used herein, in general, the term “linker” or “spacer” means an organic moiety that connects two parts of a compound. Linkers typically comprise a direct bond or an atom such as oxygen or sulfur, a unit such as SS, NH, C(O), C(O)NH, SO, SO2, SO2NH or a chain of atoms, such as substituted or unsubstituted alkyl where one or more methylenes can be interrupted or terminated by O, S, S(O), SO2, NH, NH2, C(O). The term “linker” or “spacer” of the present invention may be absent and denotes any chemical compound present between the drug and the HA which may be removed chemically, enzymatically or may decompose spontaneously; it also contains at least one other group useful for linking the drug, e.g. amino, thiol, further carboxyl groups, etc. The linker or spacer may be a polypeptide, a peptide, or a lipid.
Suitable linkers or spacers are e.g. adipic dihydrazide (ADH), linear or branched, aliphatic, aromatic or araliphatic C2-C20 dicarboxylic acids, amino acids, peptides.
The role of the linker, whenever present, consists in creating an arm or a spacer between the hyaluronic acid and the drug. The linker engages, on one side, the HA via the amide, carboxyl group, hydroxyl group, or amino group linkage and, on the other side, the drug via any possible covalent-type bond.
When the linker or spacer is a dicarboxylic acid, the carboxylic group forming the ester bond with the drug may be the hydroxyl group of the compound. When the linker or spacer is a dihydrazide, the amino group forming the amide bond with HA may be the free carboxylic group of the HA. Preferred linkers or spacers are: succinic acid to drug, adipic dihydrazide to HA.
In the preferred embodiment, the present invention provides a compound consisting of a conjugate from a glycosaminoglycan, preferably hyaluronic acid, and an active compound, wherein the active compound is conjugated by means of a functional group to a carboxylic group of the glycosaminoglycan, its derivative, or a salt thereof to form a covalent conjugation, and wherein the active compound consists of anti-inflammatory drug, anti-allergy drug and steroid.
The active compounds anti-inflammatory drug including Celecoxib which is a COX-2 antagonist, anti-allergy drug including Fexofenadine, and the steroid including Budesonide and Prednisolone can be bound preferably directly or indirectly through a linker by means the functional carboxylic group of the HA and the —NH2 group of the active compounds.
In a preferred embodiment of the present invention, the covalent conjugation, either direct or indirect through a linker, between one of the functional carboxyl groups of HA and of the active compound can be either an amidic bond or an ester bond.
In case of indirect conjugation by means of a linker, said linkers are selected from a dihydrazide, adipic dihydrazide, polypeptide, a peptide, a lipid, an aminoacid or a linear or branched, aliphatic, aromatic or araliphatic C2-C20 dicarboxylic acids.
The preferred HA for conjugation has an average molecular weight in the range comprised from 5 kDa to 2000 kDa and the conjugation involves at least 40% of the carboxyl group of HA.
In order to treat the acute or chronic inflammation, where the acute or chronic inflammation is induced or caused by infection, injury, autoimmune disease, or allergy and the autoimmune disease including Rheumatoid arthritis, the preferred embodiment of the formulation or dosage form of the present invention including an excipient and/or diluent to formulate an administrating dosage form for oral or circulation system use. The more preferred embodiment of the oral dosage form is selected from the group consisting of solid dosage form, solution including, but not limited to suspension, tablet including, but not limited to controlled-release tablet, and capsule including, but not limited to enteric-coated capsule. The more preferred embodiment of the circulation system or systemic administration form is selected from the group consisting of intro-venous (IV), intra-muscle (IM) and subcutaneous (SC).
The following examples are given for the purpose of illustrating various embodiments of the invention and are not meant to limit the present invention in any fashion.
The following whole process of HA-dye conjugation must be kept in dark. The synthesis of HA-ADH
The synthesis of HA-ADH-FITC
Result: The fluorescent view can show the attachment site and amount of dye on HCT15 (
Result: As shown in
This application is a divisional application of U.S. Non-Provisional patent application Ser. No. 14/469,028, filed on Aug. 26, 2014, which claims priority to U.S. Provisional Patent Application Ser. No. 61/871,352, filed on Aug. 29, 2013, the content of which is hereby incorporated by reference in their entireties.
| Number | Date | Country | |
|---|---|---|---|
| 61871352 | Aug 2013 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 14469028 | Aug 2014 | US |
| Child | 18740235 | US |
