Use of hyaluronic acid derivatives in the regeneration of bone and cartilage tissues

Abstract

The present invention relates to the use of derivatives between hyaluronic acid, heterocyclic compounds and naturally occurring amino acids in the form of single, oligomeric or polymeric forms for the treatment of bone diseases, in particular for the regeneration of bone and cartilage tissue.

Description

Technical Field

[0001] The present invention relates to the use of derivatives between hyaluronic acid, heterocyclic compounds and naturally occurring amino acids in the form of single, oligomeric or polymeric forms for the treatment of bone diseases, in particular for the regeneration of bone and cartilage tissues. Background Art

[0002] In the biological field, tissue is defined as a group of cells with similar structure and functionally associated. Thus, it constitutes a higher level of cell organization and plays a specific role in the living organism.

[0003] For the animal kingdom including humans, four basic tissue types can be recognized: epithelial tissue, connective tissue, muscle tissue and nerve tissue, which are further divided into more specific subclasses. In higher animals, different tissue combinations form further organized structures: organs.

[0004] Connective tissue, such as bone, fat, fibrous and nutritive tissue, is a tissue composed of individual cells, interspersed with non-living material called extracellular matrix (ECM). This matrix can be liquid or solid; two extreme examples are blood (where the matrix is plasma) and bone (where the matrix is mineralized tissue and a very rigid matrix). It is due to this special property that bone tissue is sometimes referred to as "hard tissue"; in contrast, "soft tissue" usually refers to other connective tissues.

[0005] Cartilage, the precursor tissue of bone, is also part of connective tissue. It is mainly composed of cells called chondrocytes, which are capable of producing a large amount of extracellular matrix, mainly composed of collagen, elastin and proteoglycans.

[0006] Tissue regeneration must be carried out after injury or disease to ensure complete healing and is a process based on the renewal and differentiation of cells of one or more tissues involved.

[0007] Regenerative medicine is a new research field that has received much attention in recent years; it combines different aspects of medicine, cell biology and bioengineering, with the ultimate goal of regenerating, repairing or replacing damaged or missing tissues. Different research routes involve differentiated cells and stem cells, aiming to optimize the regeneration, healing and / or replacement of damaged tissues.

[0008] In the field of bone and cartilage tissue regeneration, one of the most widely used methods today is the method based on mesenchymal stem cells (MSCs).

[0009] In the case of cartilage, although the differentiation from MSCs to cartilage has been demonstrated, the clinical use of this method remains limited and not entirely successful. Cartilage regeneration can also be achieved by tissue transplantation (autologous or allogeneic) or by techniques known in the literature to stimulate the natural repair process. Thus, in the case of cartilage tissue, the techniques identified so far as the most reliable aim to improve the regenerative properties of the tissue or consist of the transplantation of chondrocytes, with the aim of increasing or healing the residual tissue.

[0010] As for bone tissue, bone marrow remains the selected source for isolating MSCs, from which differentiated bone cells are obtained, although other sources for obtaining MSCs, such as dental tissue, are also known.

[0011] Finally, it is well known that the extracellular matrix plays an important role in the cell differentiation of connective tissues; in particular, the interaction between MSCs and the ECM can improve the osteogenic differentiation of these cells. In fact, the ECM contains various macromolecules, including collagen, adhesive glycoproteins, and glycosaminoglycans (GAGs), which not only have the role of supporting cells and determining tissue structure but also contribute to the dissemination of growth factors and the interaction between cells and the microenvironment, thus affecting cell behavior.

[0012] Despite the large amount of research and progress in the use of stem cells in recent years, the reconstruction of bone and cartilage tissue defects remains a challenge in regenerative medicine because the currently known treatment methods are only partially effective and not always feasible.

[0013] Therefore, new ways and methods are needed to more easily and effectively address the problems of the regeneration and healing of bone and cartilage tissues in organisms. Summary of the Invention

[0014] A class of hyaluronic acid derivatives is described in EP1525244, in which the molecule is associated with at least one heterocyclic compound derived from a purine or pyrimidine base and at least one other organic compound consisting of a naturally occurring amino acid in single, oligomeric, or polymeric form, and the preparation process thereof is also described.

[0015] The above-mentioned derivatives of hyaluronic acid, a glycosaminoglycan (GAG) naturally present in the extracellular matrix (ECM), constitute a more stable form than the natural one because the compounds associated therewith are found at the target sites of the soluble hyaluronidase that is usually responsible for degrading hyaluronic acid, making its action more difficult. In addition, depending on the type of heterocyclic compound and amino acid selected, these derivatives exhibit a special three-dimensional structure, enabling them to regulate the microenvironment of the ECM.

[0016] Therefore, the aim of the present invention is to use the derivatives of hyaluronic acid for the regeneration of bone and cartilage tissues due to the high regenerative potential for said tissues highlighted in the experimental part attached to this specification.

[0017] Hyaluronic acid and at least one compound derived from a heterocycle of purine and / or pyrimidine, associated with at least one different organic compound selected from naturally occurring amino acids in the form of single, oligomeric or polymeric forms, and thus, according to the present invention, for the treatment of bone disorders, particularly for the regeneration of hard tissues.

[0018] According to the present invention, the hyaluronic acid derivative used for the purposes of the present invention is high molecular weight hyaluronic acid, which ranges between 400,000 and 4,000,000 Da, preferably between 800,000 and 3,500,000 Da, more preferably between 1,500,000 and 3,000,000 Da.

[0019] Optionally, the hyaluronic acid derivative used for the purposes of the present invention consists of low molecular weight hyaluronic acid, for example, having a molecular weight of 80,000 to 400,000 Da.

[0020] Generally, the molecular weight of a polymer, particularly that of hyaluronic acid, for example, the number average molecular weight M n is defined as the average value of the polymer chain weights:

[0021] M n = Σ (i) N i M i / Σ (i) N i

[0022] where M i is the molecular weight and N i is the number of chains, or the weight average molecular weight M w which is defined as:

[0023] M w = Σ (i) N i M i 2 / Σ (i) N i M i

[0024] This quantity is more affected by the higher molecular weight portions and is higher than the weight average molecular weight.

[0025] The determination of the average molecular weight of a polymer is very important because it represents the main characteristic of the polymer and many of its properties are related thereto. The molecular weight can be obtained by a variety of techniques, including centrifugation techniques (sedimentation balance), light scattering techniques, and osmometry.

[0026] The sedimentation velocity of molecules in an ultracentrifuge is proportional to the molecular weight: In fact, assuming that the molecular weight increases with its volume, the molecular weight can be determined based on the sedimentation velocity.

[0027] The principle of light scattering is based on the fact that when a light beam passes straight through empty space, it does not lose energy along its path. On the other hand, if there are any kind of particles in the space, it is possible to observe the scattering of the light beam or deviations in all directions due to the particles present. Thus, the main light beam loses some energy and its intensity decreases.

[0028] By measuring the intensity of the scattered light and thus the photo-diffusivity of the polymer itself in a dilute solution placed in a suitable solvent, it is possible to develop a theory for determining the molecular mass of M polymer.

[0029] The molecular mass obtained has an average value, which can be shown to be the weight-average in this case.

[0030] Among the above methods, the most important and widespread is the osmotic method. Measure the value of the osmotic pressure π of solutions with different polymer concentrations C It should be remembered that

[0031] π = C RT

[0032] When the temperature T at which the measurement is made is known, the molecular weight value will be calculated, remembering that C = mass / molecular weight.

[0033] The most widely used standard technique for determining the chemo-physical properties of polymers is instead called GPC (gel permeation chromatography).

[0034] According to the present invention, the selected heterocyclic compounds are derivatives of purine bases, selected from, for example, adenine and guanine, and / or pyrimidine compounds, selected from, for example, thymine, cytosine and uracil. According to the present invention, the preferred base is a pyrimidine base, such as thymine.

[0035] Other purine or pyrimidine derivatives that can be used to form compounds for the purposes of the present invention can be selected from: 5,6-dihydrouracil, 1-methyluracil (uracyl), 3-methyluracil, 5-hydroxymethyluracil, 2-thiouracil, N 4 -acetylcytosine, 3-methylcytosine, 5-methylcytosine, 5-hydroxymethylcytosine, 1-methyladenine, 2-methyladenine, 7-methyladenine, N 6 -methyladenine, N 6 ,N 6 -dimethyladenine, N 6 -(Δ2 -(Isopentenyl)adenine, 1-methylguanine, 7-methylguanine, N 2 -methylguanine, N 2 ,N 2 -dimethylguanine.

[0036] Preferably, in the hyaluronic acid derivatives for the use of the purpose of the present invention, the occurrence of the interaction between the hyaluronic acid chain and the purine or pyrimidine base is due to at least one ionic bond between the -COOH residue of the acid and the base center (especially the basic nitrogen) of the heterocyclic base.

[0037] In fact, according to the present invention, hyaluronic acid interacts with at least one purine and / or pyrimidine base selected from the above, and the reaction conditions allow at least one acid center of hyaluronic acid (such as the free carboxyl group in the acid or carboxylate form) and at least one base center of the purine and / or pyrimidine base (also in the free base or ammonium salt form) to form at least one type of ionic bond.

[0038] According to the present invention, the hyaluronic acid derivatives for the use of the purpose of the present invention may contain more than one purine and / or pyrimidine base, and their mutual ratio is variable; thus the derivatives may be represented as "mixed" salts composed of a variable number of purine / pyrimidine bases.

[0039] Due to the presence of -COOH groups in the structure of hyaluronic acid and remaining free, the hyaluronic acid derivatives for the use of the purpose of the present invention also include at least one naturally occurring amino acid or its oligomer or polymer to provide additional salt-forming products. The amino acids that can be used to form these derivatives are selected from, for example: alanine, arginine, asparagine, aspartic acid, glutamic acid, cysteine, phenylalanine, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, proline, serine, tyrosine, threonine, tryptophan and valine. Preferably, the amino acids are selected from lysine and alanine.

[0040] The properties of the derivatives reflect the properties of hyaluronic acid, purine and / or pyrimidine bases and amino acids, which, in turn, are bound by at least one hydrolyzable ionic bond, making the different components more accessible in situ.

[0041] Particularly preferred is the compound currently sold under the name T-LysYal® (T-Lys), which is a derivative of hyaluronic acid, lysine and thymine formed by forming an ionic bond.

[0042] According to the present invention, the compound among hyaluronic acid, at least one heterocyclic compound selected from purine and / or pyrimidine derivatives, and at least one naturally occurring amino acid or its oligomer or polymer can be advantageously used to induce and stimulate cell differentiation in the osteogenic and chondrogenic lineages of MSCs.

[0043] Therefore, according to the present invention, it is possible to use the above-mentioned hyaluronic acid derivatives for the treatment of bone disorders, particularly for the regeneration of bone and cartilage tissues.

[0044] Still according to the present invention, as described above, the hyaluronic acid derivatives are advantageously used to induce and stimulate cell differentiation in the osteogenic and chondrogenic lineages of MSCs.

[0045] According to the present invention, the hyaluronic acid derivatives can also be incorporated into suitable pharmaceutical formulations and / or implantable scaffolds, which can be used to support the tissue regeneration of bone and cartilage.

[0046] The derivatives can be further used for the treatment of the repair and regeneration of bone and cartilage tissues.

[0047] The object of the present invention is to use the derivatives for the treatment related to the tissue regeneration of bone and cartilage tissues. According to the present invention, the derivatives can be advantageously incorporated into an implantable scaffold system or other pharmaceutically suitable formulations.

[0048] As for other "pharmaceutically suitable formulations", it means, but is not limited to, solutions and / or suspensions for parenteral use, solid forms (such as tablets, capsules, granules) or semi-solid forms (such as gels, pastes, creams, ointments) for oral or topical use, intramuscular and / or subcutaneous implants, and other formulations known to experts in the field.

[0049] The potential of the present invention will now be described in the experimental section below, which elaborates on the research conducted by using the hyaluronic acid derivatives and is related to their potential for the cell regeneration of hard tissues in organisms. The following examples are only for illustrative purposes and do not limit in any way. Description of the Drawings

[0050] Figure 1 : The effect of T-Lys on the differentiation of MSCs into the osteoblast lineage. Parts: A) qPCR performed on DBSCs grown in osteogenic medium and stimulated with 0.3% T-Lys and control DBSCs; B) Immunoblotting tests for the expression of Runx-2 and Col 1 proteins; C) Histochemical assay of ALP enzyme (purple staining).

[0051] Figure 2: Role of T-Lys in the deposition of mineral matrix during osteogenic differentiation of MSCs. Deposition of mineral matrix was tested by ARS (red staining) in cells treated with T-Lys, hyaluronic acid, and control cells.

[0052] Figure 3 : Effect of T-Lys on the expression of typical markers in chondrocyte cultures. qPCR was performed on chondrocyte pellet cultures grown in chondrogenic medium and stimulated with 0.3% T-Lys and a negative control group (Ctr).

[0053] Figure 4 : Effect of T-Lys on chondrocyte proliferation and tissue growth. Part: A) Images and measurements of cultures of control and T-Lys-treated dissected chondrocytes; B) Images and measurements of deposition of cartilage matrix in control and T-Lys-treated dissected chondrocytes; C) Theoretical reconstruction of the thickness of cultures of control and T-Lys-treated chondrocytes. Detailed implementation

[0054] Experimental part Example

[0055] Effect of T-Lys on the differentiation of MSCs into the osteoblast lineage.

[0056] Dental bud stem cells (DBSCs) were used as a source of MSCs and differentiated in osteogenic medium for 12 days. Part of the test cells were treated with medium supplemented with 0.3% T-Lys (T-Lys - treated group) at each change. Part of the cells were not treated with T-Lys and served as the control group (Ctr). The mRNA levels of the early markers of typical osteoblasts, Runx-2 and type I collagen (Col 1), were determined by real-time PCR in Ctr and T-Lys samples.

[0057] Figure 1 (A) shows how the expression of both markers was significantly increased in T-Lys-treated cells compared to Ctr cells, indicating that T-Lys treatment improved the ability of MCSs to differentiate into the osteoblast lineage.

[0058] The protein expression levels of these osteoblast markers were further evaluated in T-Lys and Ctr cells by Western blot analysis.

[0059] Figure 1 (B) highlights how the levels of Runx-2 and Col 1 proteins were increased in T-Lys-treated cells compared to Ctr cells, thus validating the mRNA expression trend.

[0060] Then, histochemical tests were performed to explore the expression of another osteoblast marker, alkaline phosphatase (ALP) enzyme, in response to treatment with T-Lys. The results of this experiment, as Figure 1 (C) shows, showed that during osteogenic differentiation, stimulation of MSCs with T-Lys significantly increased the purple staining identifying ALP expression.

[0061] All of the above results indicate that T-Lys is capable of increasing the ability of MSCs to differentiate into osteoblast-like cells. Example

[0062] Role of T-Lys in the deposition of mineral matrix during osteogenic differentiation of MSCs

[0063] To thoroughly investigate the role of this new molecule in the osteogenic differentiation of MSCs, DBSC cultures under mineralization conditions were performed on different samples for 21 days: Ctr (without any addition, as a negative control group); HA (addition of unmodified hyaluronic acid as a positive control group) and T-Lys (where cells were treated with 0.3% T-Lys, as a treatment group).

[0064] The role of T-Lys in the deposition of the mineral matrix of DBSC was quantitatively analyzed by histochemical tests using alizarin red staining (ARS) by a colorimetric technique. The mineralization ability of cells treated with 0.3% T-Lys was shown to be significantly higher than both Ctr and HA.

[0065] These data show how T-Lys is capable of increasing the osteogenic ability of MSCs, also by stimulating its ability to produce a mineralized matrix. Example

[0066] Role of T-Lys in the effect on the subcellular distribution of ανβ3 integrin

[0067] Integrins are receptors for ECM molecules, important in cell adhesion but also in the mediation of proliferation and differentiation signals. In particular, ανβ3 integrin is the receptor for an osteoprotein called osteopontin, which is fundamentally important in determining the differentiation of MSCs into the osteogenic lineage. Therefore, it was evaluated whether treatment with T-Lys could affect the subcellular distribution of ανβ3 integrin.

[0068] The subcellular distribution of the integrin was analyzed by confocal microscopy in DBSCs and Ctrs treated with T-Lys. This analysis was performed only 4 days after osteogenic differentiation to compensate for the tendency of the cells to rapidly form multiple layers, which would impede their microscopic observation. In Ctr cells, ανβ3 integrin was shown to be distributed at several sites, while T-Lys treatment induced a different organization of this receptor, with more localization at focal adhesion sites. Thus, 4 days after differentiation, the receptor was still distributed throughout the cell under control conditions, while in T-Lys cells it was present in focal adhesions. "Strings" (a typical pattern of ανβ3 integrin involved in focal adhesions) were detected in T-Lys cells but not in Ctr cells. These results suggest that the effect of T-Lys on DBSC differentiation may be mediated by ανβ3 rearrangement. Example

[0069] Effect of T-Lys on the expression of typical markers in chondrocyte cultures

[0070] To mimic the microstructure of three-dimensional tissues and avoid the inappropriate dedifferentiation that chondrocytes are prone to during two-dimensional growth, human articular chondrocytes collected from patients undergoing orthopedic surgery were grown in pellet cultures. The cell pellets were grown for 28 days under chondrogenic conditions. The control group (Ctr) was treated according to a conventional protocol, while the T-Lys group received 0.3% T-Lys added at each carrier change. At the end of the culture period, the chondrocyte culture pellets were lysed and subjected to gene expression analysis for evaluation. The mRNA levels of typical chondrogenic markers: Sox-9, collagen type II (Col II), collagen type X (Col X), and aggrecan were determined in both groups of real-time PCR samples.

[0071] Figure 3 The results of these tests are shown, which demonstrate that, considering three of these four markers, their expression was positively increased in the T-Lys group. In particular, the expression of Sox-9, which is the main transcription factor involved in chondrogenic differentiation, was significantly increased in T-Lys-treated cells compared to Ctr cells. Based on this result, Col II and Col X, typical proteins of the extracellular matrix of cartilage, were also increased by T-Lys treatment, again indicating that this molecule supports and improves chondrocyte differentiation. On the other hand, aggrecan, a proteoglycan of cartilage, had its expression unaffected. Example

[0072] Effect of T-Lys on chondrocyte proliferation and tissue growth

[0073] After 28 days of differentiation under the conditions described in Example 4, the chondrocyte aggregates were fixed with 4% paraformaldehyde and embedded, sectioned and subjected to histological staining and examination. Morphological examination of the cut chondrocyte culture aggregates by light microscopy showed that the T-Lys was larger than that of the control group. This result is visible in Figure 4 (A).

[0074] To quantify the aggregate size, the samples were sectioned (5 μm thick) and the area of each section obtained from the two groups of cells was measured by ImageJ software. Figure 4 The graph in (A) shows that the average surface area in the T-Lys treatment group was significantly larger than that of the control group.

[0075] Then the aggregates were stained with safranin O to highlight the chondrocytes ( Figure 4 (B)). The staining showed the presence of the cartilage matrix (orange staining), demonstrating that the cells were able to differentiate and produce CME components under these culture conditions; the nuclei were counterstained with hematoxylin ( Figure 4 (B)). To check whether the T-Lys treatment also had an effect on the cell number, the cells (100 x 100 μm) in the selected fields of view of each section were counted. Figure 4 The graph in (B) depicts the cell numbers of the two cultures. It can be seen that the cell number in the treatment group was significantly increased compared with the control group. Interestingly, more sections (slices) with a consistent thickness (5 μm), referred to as "slices", could be obtained in the case of the T-Lys samples compared with the control group.

[0076] In Figure 4 (C), this difference is depicted in the graph, where the number of "slices" has been multiplied by the slice thickness (5 μm) to reconstruct the theoretical thickness of the entire culture aggregate. These results demonstrated that T-Lys stimulated the proliferation, differentiation and matrix secretion of chondrocytes.

[0077] DETAILED DESCRIPTION OF THE DRAWINGS

[0078] Figure 1 : Effect of T-Lys in the differentiation of MSCs into the osteoblast lineage

[0079] A) qPCR was performed on DBSC and DBSC Ctr grown in osteogenic medium and stimulated with 0.3% T-Lys. Each graph depicts the mean ± standard error of 3 independent experiments performed in triplicate. ∗P < 0.02 compared with the control group. The expression has been normalized to β2-microglobulin (B2M). The graph shows that treatment with T-Lys significantly increased the expression of the two osteoblast markers Runx-2 and Col1.

[0080] B) Immunoblotting tests for the expression of proteins Runx-2 and Col 1; each graph depicts the mean optical density calculated relative to the constitutive protein (β-actin Housekeeping gene ) + standard error in 3 independent experiments performed in triplicate. ∗P < 0.001 compared to the control group. Representative immunoblot images are also depicted on the left side of the figure. The graphs show how the measured parameters in cells treated with T-Lys are higher than those in the control group.

[0081] C) Histochemical tests for ALP enzyme (purple staining) were performed on DBSCs stimulated with T-Lys and cultured under osteogenic conditions for 7 days, compared to the control group. The graph depicts the quantified percentage of positive staining (*P < 0.01) compared to the control group, analyzed from 3 independent experiments performed in quadruplicate. Data are shown as mean ± standard error. Representative images of the cultures are also depicted on the left side of the figure. The graphs show how the T-Lys samples have higher alkaline phosphatase expression.

[0082] Figure 2 : Role of T-Lys in the deposition of mineral matrix during osteogenic differentiation of MSCs

[0083] The deposition of mineral matrix was tested by ARS (red staining) in cells treated with T-Lys, hyaluronic acid, and Ctr, under osteogenic conditions for 21 days. The graphs show the quantification of the optical density of the dye, extracted from the colored cell layer, as mean percentage ± standard error, and represent 3 independent experiments performed in quadruplicate. ∗P < 0.01, #P < 0.001 compared to the negative control group (Ctr); @P < 0.01 compared to the positive control group (HA). Representative images of the cultures are also depicted on the left side of the figure. The graphs show how the T-Lys samples have higher mineral matrix deposition than the untreated samples and the samples treated with native hyaluronic acid.

[0084] Figure 3 : Effect of T-Lys on the expression of typical markers in chondrocyte cultures

[0085] qPCR was performed on pellet cultures of chondrocytes grown in chondrogenic medium and stimulated with 0.3% T-Lys and a negative control group (Ctr). Each graph depicts the mean ± standard error of 3 independent experiments performed in triplicate. Compared to the control group, for Sox-9, *P<0.04, for Col II, *P<0.001, and for Col X, *P<0.01. Expression was normalized to beta-2 microglobulin (B2M). The graphs show that treatment with T-Lys significantly increased the expression of the chondrocyte markers Sox-9, Col II, and Col X, with no effect on aggrecan expression.

[0086] Figure 4 : Effects of T-Lys on chondrocyte proliferation and tissue growth

[0087] A) Cultured pellet of chondrocytes in sections were photographed using a 20x objective lens under an optical microscope and analyzed by morphological examination of each region using Image-J software. The selected images are representative images of three different experiments, with the scale bar described in the lower right corner of the figure: 75 μm. Each graph depicts the mean ± standard error of 3 independent experiments performed in triplicate, *P<0.0003. Pellets treated with T-Lys appeared larger than those of the control group.

[0088] B) Measurement of cartilage matrix deposition using safranin O staining, with hematoxylin counterstaining of chondrocyte nuclei. Images were taken with a 40x lens, and the scale bar is shown in the upper left corner of the control figure: 25 μm. Each graph depicts the mean ± standard error of 3 independent experiments performed in triplicate, *P<0.04. The number of cells in the T-Lys samples was greater than that in the control group.

[0089] C) The graph shows the theoretical reconstructed thickness of the chondrocyte culture pellet, where the number of obtained sections is multiplied by the section thickness, expressed in μm. The group treated with T-Lys showed a greater thickness.

Claims

1. Use of a compound in the preparation of a pharmaceutical preparation for the regeneration of bone and cartilage tissues, said compound being a derivative formed by an ionic bond of hyaluronic acid, lysine and thymine, wherein the regeneration of the bone and cartilage tissues is based on increasing the osteogenic capacity of MSCs and improving the differentiation of chondrocytes.

2. Use of a compound in the preparation of an implantable scaffold for the regeneration of bone and cartilage tissues, said compound being a derivative formed by an ionic bond of hyaluronic acid, lysine and thymine, wherein the regeneration of the bone and cartilage tissues is based on increasing the osteogenic capacity of MSCs and improving the differentiation of chondrocytes.

Images