Amino acid derivative of glucosamine stimulating extracellular matrix synthesis and pharmaceutical composition comprising the same

Abstract

To provide a compound and the use thereof for the treatment for regeneration of connective tissue following the degeneration process of articular cartilage due to osteoarthritis, in the case of humans, the degeneration process of articular cartilage caused by excessive stress, for the treatment of skin aging, and for the prevention of skin cancer.SOLUTION: For example, an amino acid derivative of glucosamine having the following structure is provided.SELECTED DRAWING: None

Description

[Background technology]

[0001] As is well known, the human body and its constituent organs are supported and held together by tissues traditionally referred to by the term "connective tissue", which implies a structural and supportive role. However, connective tissue not only has a supportive function, but also acts as a link to other tissues, participates in the defense mechanisms of the organism, and ensures nutrition for tissues that are not neovascularized.

[0002] All different types of connective tissues consist of two essential components: cell populations and extracellular matrix (ECM). The latter consists of amorphous and fibrous components. The relative proportions of each of these two components vary depending on the type of connective tissue in question.

[0003] The extracellular matrix (ECM) is an organized network of extracellular substances located near the plasma membrane and plays a key role in determining the shape and function of cells. Its components are continuously remodeled to ensure the proper functioning of various physiological processes, but this rhythm slows down with age. Therefore, insufficient remodeling can lead to the development of various diseases, particularly those related to both the locomotor system and the skin (Naba et al., 2015).

[0004] Cartilage is a specialized type of connective tissue. Cartilage is actually defined as a supportive connective tissue composed of a single cell type, the chondrocyte, and an abundant extracellular matrix (ECM). Chondrocytes constitute 2-5% of the tissue and are responsible for producing the components of the extracellular matrix and its continuous remodeling. The matrix is ​​composed of collagen fibers and polymer aggregates, as well as proteoglycans, such as aggrecan. Cartilage is continuously remodeled, a process that requires maintaining a delicate balance between anabolic and catabolic pathways. This also includes:

[0005] The main pathology affecting cartilage tissue is osteoarthritis (OA). It is the most common type of joint disease that causes several musculoskeletal disorders and is characterized by pain, erythema, swelling, effusion around and within the joint, and impaired movement and function at the joint level. In fact, this pathology is caused by friction and defibrillation of the basic substance of articular cartilage. It is characterized by structural changes due to bone loss, which reduces the ability to absorb mechanical stresses under load or movement. As a result, the underlying bone becomes less able to tolerate increased bone formation. In a slow evolutionary form, OA is seen as a paraphysiological condition of joint wear and a manifestation of aging. Although OA can be managed, in its rapidly changing form it actually represents a disease. OA primarily presents degenerative aspects, but also shows more or less intense signs of inflammation. During the progression of OA, an imbalance occurs between the anabolic and catabolic processes involved in the reconstruction of the cartilage matrix. The secretion of proinflammatory cytokines reduces the synthesis of new matrix, while erosion of cartilage occurs as a result of the overactivation of metalloproteases. The final outcome of the osteoarthritic process is a complete blockage of the affected joint and loss of its function.

[0006] A wide variety of approaches are known for treating OA, ranging from preventative measures to pharmacological, physical, and surgical treatments.

[0007] Surgical treatment of OA involves prosthetic replacement procedures. Although it is generally possible to completely eliminate pain symptoms and improve joint function, there are limitations, such as the feasibility of surgery and the lifespan of the implanted prosthesis. Unfortunately, apart from this surgical treatment, the goal of all treatments for OA is only to improve the patient's quality of life by controlling pain, maintaining joint function, and slowing joint deterioration.

[0008] In the field of application of this invention, attention should be paid to the drug therapy of OA. Drugs in this drug therapy can be divided into symptomatic drugs aimed at combating pain and drugs acting on the structure of articular cartilage, called chondroprotectors. Nonsteroidal anti-inflammatory drugs (NSAIDs) are the first-line treatment for pain and the inflammatory phase of the disease. A more effective or more appropriate NSAID for the symptomatic treatment of OA has yet to be identified. The type of NSAID is selected based on several molecular-related properties, such as tolerability, duration of action, and selectivity, as well as clinical response. The most commonly used drugs include ibuprofen, nimesulide, naproxen, ketoprofen, and diclofenac. The limitations of these drugs are related to side effects, particularly gastric toxicity, which preclude their continued use. Paracetamol is NSAIDs are widely used as analgesic agents, especially for patients with gastrointestinal risks or other contraindications to NSAID use. Opioid analgesics have been available for OA in our country for several years, and they are useful for patients who are intolerant to NSAIDs or who respond poorly to non-opiate analgesics. The most commonly used are codeine and tramadol, which can be taken in conjunction with each other or in combination. Other treatments that can sometimes be used in special circumstances include intra-articular infiltration with cortisone preparations or hyaluronic acid-based infiltration, but these treatments have limited long-term efficacy.

[0009] On another level, chondroprotectors are considered a new therapeutic perspective in the treatment of OA because they directly affect cartilage metabolism, inhibit degenerative processes, and promote the synthesis of new extracellular matrix (ECM) by chondrocytes. Studies of these new substances have shown the most promising results, particularly with regard to naturally occurring glucosamine, which is thought to suppress the production of inflammatory factors in cartilage, preventing its deterioration and promoting the formation of new cartilage. Other candidates for this role, such as hyaluronic acid itself, chondroitin sulfate, diacerin extract, soybean extract, and avocado extract, are still under investigation, but their mechanisms of action have not been fully elucidated, and their efficacy is not universally accepted (Cutolo et al., 2015).

[0010] Regarding future therapeutic prospects, great expectations are placed on advances in tissue engineering, and the implantation of autologous chondrocytes, i.e. Transplantation of chondrocytes from the same patient has shown good results, however this treatment remains limited and may only be effective in younger patients, some joints, and in cases of mild damage that does not affect the underlying bone (BL Kidd et al, 2007).

[0011] As far as epidemiology is concerned, osteoarthritis is a disease that affects all vertebrates, while in humans the incidence of this disease is very high, afflicting a large proportion of the world's population, especially those of average age and advanced age. Radiological signs of OA are present in more than 80% of people between the ages of 50 and 65, while actual symptoms in the same age group are present in approximately 20% of women and 15% of men. In Italy, the condition affects approximately 4 million people and is one of the most common reasons for consultation in general medical institutions.

[0012] As mentioned above, in connection with the increase in life expectancy, OA has become a major social problem, not only negatively affecting the quality of life of patients but also inevitably placing a large socio-economic burden on the national healthcare system.

[0013] The present invention therefore generally aims to establish new pharmacological pathways capable of interrupting the evolutionary process of this pathology and preventing the debilitating damage that may result from it, while at the same time restoring a structure that closely resembles that of healthy cartilage and, as far as possible, eliminating the pathological aspects mentioned above.

[0014] Other advantages of the present invention will become apparent from the following detailed description of embodiments of the invention, given as non-limiting examples. Summary of the Invention

[0015] The compounds disclosed herein have been found to exert surprising and unexpected biological effects. In particular, the compounds mentioned, or pharmaceutical compositions containing them, have been shown to moderately stimulate components of the ECM in connective tissues, particularly cartilage tissue, so that these tissues regain a structure closely resembling a previously healthy structure or a cartilage structure free of signs of degeneration due to osteoarthritis, etc.

[0016] The compounds of the present invention described above generally consist of amino acid derivatives of glucosamine, more preferably represented by the N-acetyl-phenylalanine derivative of glucosamine, on which the majority of the following experimental studies have been concentrated.

[0017] The foregoing is generally carried out using in vitro and in vivo methods that allow verification of the mechanism of action and biological activity of innovative amino acid derivatives of glucosamine. This is derived from detailed research by the inventors.

[0018] The above-mentioned class of compounds, specifically the N-acetyl-phenylalanine derivative of glucosamine, has been studied in in vivo models of OA in both rabbits and mice. In this study, intra-articular administration of the compounds into the osteoarthritic knees of rabbits or mice stimulated the synthesis of new ECM, restoring cartilage to a structure closely resembling that of healthy cartilage without osteoarthritis. This indicates that the above-mentioned class of compounds has important regenerative effects.

[0019] To elucidate the molecular mechanism, we investigated glucosamine-derived N-acetyl-phenylalanine in vitro using primary cultured chondrocytes. To study the inflammatory pathway regulated by activator protein (AP)-1, chondrocytes were stimulated with IL-1. Specifically, the above-mentioned N-acetyl-phenylalanine derivative of glucosamine first inhibited the phosphorylation of two kinases, JNK and p38, and as a result, was unexpectedly effective in affecting the activation of c-jun, a component of AP-1. Next, we analyzed its effectiveness in interfering with the inflammatory pathway regulated by the transcription factor nuclear factor κB (NF-κB). This factor is regulated by the IKK complex, which consists of two kinases, IKKα and IKKβ. The above-mentioned N-acetyl-phenylalanine derivative of glucosamine selectively inhibited the kinase activity of IKKα, but not IKKβ, by specifically preventing the nuclear translocation of IKKα kinase, which acts in the cytoplasm.

[0020] Finally, further in vitro studies on human primary chondrocytes were conducted to understand which intracellular pathways are activated by the N-acetyl-phenylalanine derived from glucosamine to promote ECM synthesis. To simulate physiological conditions, human chondrocyte cultures were performed in three dimensions. The cells were cultured in the form of spheroids. This result was unexpected. The compounds of the present invention have been shown to promote the synthesis of type II collagen, a characteristic of cartilage tissue. Furthermore, these experiments demonstrated that simultaneous treatment of cells with glucosamine and the glucosamine-derived N-acetyl-phenylalanine synergistically increased the production of type II collagen and induced the expression of factors that control ECM synthesis, namely, the growth factor IGF-1 (insulin growth factor) and the transcription factor SOX-9 (Sry-related HMGbOX-containing gene), confirming the interesting anabolic effects achieved by the combination of these two molecules. Another important and unexpected discovery was that the amino acid derivatives of glucosamine, more specifically the N-acetyl-phenylalanine derivative of glucosamine, promoted the expression of the hyaluronic acid synthase (HAS-2) enzyme, thereby promoting the production of hyaluronic acid (HA).

[0021] Another encouraging finding for purposes of practicing the present invention is that the compounds of the above-described class of the present invention also prove particularly effective against primary human fibroblasts. It has been found that when these primary human fibroblasts are grown in three dimensions, such as spheroids, they produce relatively large amounts of type I collagen compared to spheroids grown without the compounds of the present invention. Furthermore, when an N-acetyl-phenylalanine derivative of glucosamine is added to the culture medium of fibroblasts stimulated with UVB to a degree that simulates solar UVB irradiation, it is found that the molecule can suppress the inflammatory pathway stimulated by UVB and restore the synthesis of type I collagen inhibited by UVB treatment. Based on this discovery, the inventors of the present application believe that this class of products can inhibit the remodeling of the skin's ECM, resulting in a relative change in turnover, which causes skin atrophy and ultimately He immediately expressed concern that this could potentially lead to skin aging and, in some cases, the initiation of the process of developing skin cancer.

[0022] Therefore, based on the promising results described in both the in vivo and in vitro studies described above, the inventors of the following invention have developed the use of the innovative amino acid derivatives of glucosamine described above, which are generally used for the treatment or prevention of pathologies primarily related to the regulation of the turnover of macromolecules that make up the extracellular matrix (ECM) of connective tissues, particularly cartilage tissue. The following describes this exemplary, non-limiting objective in detail.

[0023] Other features of the present invention are set forth in the following detailed description of one or more specific embodiments, which are protected by various dependent claims. DETAILED DESCRIPTION OF THE INVENTION

[0024] In the following, only some of the possible embodiments of the present invention are given as non-limiting examples, but many other embodiments can also be described based on the specific technical solutions described.

[0025] The present application relates to the use of innovative amino acid derivatives of glucosamine or to the use of pharmaceutical compositions comprising the above-mentioned amino acid derivatives of glucosamine, either as the sole active ingredient or in association with one or more further active ingredients, for preparing medicaments for the treatment or prevention of diseases primarily related to the regulation of the metabolic turnover of macromolecules that make up the ECM of connective tissue, more particularly cartilage tissue.

[0026] The present invention therefore generally aims to provide new pharmacological methods that can interrupt the progression pathway of osteoarthritis and prevent the debilitating damage that can result therefrom.

[0027] The present invention further provides agents capable of promoting the endogenous synthesis of new extracellular matrix. The purpose is to provide.

[0028] The present invention also aims to restore the structure of cartilage to one that closely resembles that of healthy cartilage, i.e., a structure that is not degenerated due to the progression of osteoarthritis or other causes such as excessive stress from strenuous sports activities, by appropriately stimulating cells to produce matrix and components of collagen, proteoglycans, glycosaminoglycans, and aggrecan.

[0029] A further object of the present invention is that, due to the aforementioned ability of this class of products to restore the synthesis of type I and type II collagen through fibroblast activity and to intervene in the remodelling of the dermal ECM, the appropriate use of this class of products in the treatment of skin ageing will reduce or even prevent skin atrophy, and will propose it as a new therapeutic strategy in the treatment of skin cancer.

[0030] The compounds of the present invention described above generally consist of amino acid derivatives of glucosamine having the general structure of Formula 1 below. [ka] Here, the group R represents an amino acid, an N-acyl derivative of an amino acid, or an N-acyl derivative of a peptide, in which the amine group of glucosamine is linked to the carboxyl side chain of the amino acid by a carbamide bond.

[0031] In a particularly preferred embodiment of the present invention, the compound of the present invention described above is preferably represented by N-acetyl-phenylalanine derived from glucosamine, having the structure of the following chemical formula 2: [ka] The compounds of formula 1 or formula 2 according to the present invention may be present as pharmaceutical compositions comprising the compounds alone or in combination with one or more active ingredients, together with one or more excipients or carriers appropriate for the route of administration, selected and prepared in accordance with good manufacturing practice and known standards of pharmaceutical legislation.

[0032] In light of the above experimental evidence, a preferred embodiment of the present invention is to provide a concentration of 0.5 wt.% to 4. It is envisioned that by providing an intra-articular or intradermal injection solution comprising a compound having formula 1, or a pharmaceutically acceptable salt, solvate, hydrate, or stereoisomer thereof, at a concentration of 5% by weight, more preferably 1% to 3% by weight, together with a known carrier suitable for said administration route, a pharmaceutical composition suitable for parenteral administration, more specifically for intra-articular or transdermal administration, can be provided.

[0033] In a further preferred embodiment, the pharmaceutical composition described above comprises an active ingredient combination consisting of an amino acid derivative of glucosamine having the present invention's chemical formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, or stereoisomer thereof at a concentration of 0.5% to 4.5% by weight, more preferably 1% to 3% by weight, and even more preferably 2.5% by weight, and glucosamine and / or hyaluronic acid at a concentration of 0.1% to 0.4% by weight, more preferably 0.25% by weight. In particular, the above-described pharmacological combination is based on experimental evidence demonstrating that the addition of glucosamine has a synergistic effect on the pharmacological activity of the drug, and that the addition of hyaluronic acid is clearly indicated by the properties of this molecule, imparting a higher viscosity to the formulation, which provides a lubricating effect when administered to a joint. It should be noted that while all human skeletal joints can be targeted and benefit from the above-described administration, administration to the knee is particularly preferred.

[0034] A further embodiment of the present invention aimed at the above-mentioned therapeutic and prophylactic effects aims to provide a pharmaceutical composition suitable for the topical administration route by formulating a cream consisting of a compound having chemical formula 1, or a pharmaceutically acceptable salt, solvate, hydrate or stereoisomer thereof, in a base cream at a concentration of 0.1% to 2% by weight, more preferably at a concentration equal to 1% by weight.

[0035] Although the present invention has been described according to preferred embodiments for illustrative and non-limiting purposes, those skilled in the art can make variations and / or modifications without departing from the relevant scope of protection as defined in the following dependent claims.

Claims

1. The general structure of Formula 1 below: 【Chemical 1】 Or an amino acid derivative of glucosamine having a pharmaceutically acceptable salt, solvate, hydrate, or stereoisomer thereof, wherein the group R represents an amino acid, an N-acyl derivative of an amino acid, or an N-acyl derivative of a peptide, and the amine group of the glucosamine is linked to the carboxyl side chain of the amino acid by a carbamide bond.

2. Preferably, the N-acetyl-phenylalanine derivative of glucosamine has the following structural formula 2: 【Chemistry 2】 Or, the amino acid derivative of glucosamine described in claim 1, which is composed of a pharmaceutically acceptable salt, solvate, hydrate, or stereoisomer thereof.

3. 3. The amino acid derivative of glucosamine according to claims 1 and 2, characterized in that it is in the form of a pharmaceutical composition suitable for parenteral administration.

4. The amino acid derivative of glucosamine according to claim 3, characterized in that the parenteral administration route includes the intra-articular route and the transdermal route.

5. The amino acid derivative of glucosamine described in claim 3, characterized in that it is contained in the pharmaceutical composition at a concentration of 0.5% to 4.5% by weight, more preferably 1% to 3% by weight, together with a carrier suitable for the administration route.

6. The pharmaceutical composition of claim 3, wherein the amino acid derivative of glucosamine is combined with an active ingredient consisting of glucosamine and / or hyaluronic acid, the concentration of the amino acid derivative of glucosamine being 0.5% to 4.5% by weight, more preferably 1% to 3% by weight, and the concentration of each of glucosamine and / or hyaluronic acid being 0.1% to 0.4% by weight, more preferably 0.25% by weight, and the composition is present together with a carrier suitable for the administration route.

7. 3. The amino acid derivative of glucosamine according to claims 1 and 2, characterized in that it is in the form of a pharmaceutical composition suitable for topical administration.

8. The amino acid derivative of glucosamine according to claim 7, which is contained in the pharmaceutical composition as a base cream at a concentration of 0.1% to 2% by weight, more preferably at a concentration equal to 1% by weight.

9. 9. The amino acid derivative of glucosamine according to any one of claims 1 to 8, which is used in regenerative therapy of connective tissue.

10. Use of an amino acid derivative of glucosamine described in any one of claims 1 to 9 in the treatment of connective tissue regeneration following the degenerative process of articular cartilage due to osteoarthritis, in humans, the degenerative process of articular cartilage due to excessive stress, as well as in the treatment of skin aging and in the prevention of skin cancer.

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