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 monomeric, oligomeric or polymeric form in the treatment of skeletal disorders and in 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 monomeric, oligomeric or polymeric form in the treatment of skeletal disorders, in particular in the regeneration of bone and cartilage tissue. [Background technology]

[0002] In biology, a tissue is defined as a collection of structurally similar cells related by function. Tissues therefore constitute higher levels of cellular life that have specific roles to play within the organism.

[0003] Four basic types of tissue are recognized throughout the animal kingdom, including humans: epithelial, connective, muscular, and nervous tissue (also divided into smaller subtypes). In higher animals, different tissues combine to form more organized structures: organs.

[0004] Connective tissues, such as bone, adipose, fibrous, and trophic tissues, are tissues composed of separate cells intercalated with a non-living substance called the extracellular matrix (ECM). This matrix can be liquid or rigid; two extreme examples are blood (where the matrix is plasma) and bone (where there is mineralized tissue and an extremely rigid matrix). Precisely because of this special property, bone tissue is sometimes referred to as "hard tissue," as opposed to the "soft tissue" commonly referred to as other connective tissues.

[0005] Cartilage (the precursor tissue of bone) is also part of connective tissue. Cartilage is primarily composed of cells called chondrocytes, which are capable of producing large amounts of extracellular matrix, which is primarily composed of collagen, elastin, and proteoglycans.

[0006] Tissue regeneration, which must occur after injury or disease to ensure complete healing, is a process based on the regeneration and differentiation of cells of the involved tissue.

[0007] Regenerative medicine is an emerging field that has attracted interest in recent years, combining different aspects of medicine, cell biology, and bioengineering with the ultimate goal of regenerating, repairing, or replacing damaged or lost tissue. Research into different lineages, both differentiated cells and stem cells, aims to optimize the regeneration, healing, and / or replacement of damaged tissue.

[0008] Today, one of the most widely used approaches in the field of bone and cartilage tissue regeneration is based on the mesenchymal stem cell (MSC) approach.

[0009] Regarding cartilage, although the differentiation of cartilage from MSCs has been demonstrated, the clinical application of this approach is still limited and not always successful. Cartilage regeneration can also be achieved, for example, by tissue transplantation (autograft or allograft) methods or by techniques known in the literature that are adapted to promote the natural healing process. Therefore, the techniques currently identified as the most reliable in the case of cartilage tissue consist of implanting chondrocytes with the aim of improving the regenerative properties of the tissue or augmenting or repairing the remaining tissue.

[0010] With regard to bone tissue, marrow is known to be the source of choice for the isolation of MSCs (to obtain differentiated bone cells from MSCs) and also another source for obtaining MSCs, e.g., dental cells.

[0011] Finally, the extracellular matrix is known to play an important role in connective tissue cell differentiation; in particular, the interaction of MSCs with the ECM improves the osteogenic differentiation of these cells. Indeed, the ECM contains various macromolecules, including collagen, adhesive glycoproteins, and glycosaminoglycans (GAGs), which not only play a role in supporting cells and determining tissue architecture, but also contribute to the propagation of growth factors and cellular interactions with the microenvironment, thus influencing cell behavior.

[0012] Despite the many studies and advances made in recent years in terms of the use of stem cells, the reconstruction of bone and cartilage tissue defects remains a challenge for regenerative medicine, as currently known treatments are only partially effective and not always feasible.

[0013] Therefore, there is a need to find new methods and approaches that easily and effectively solve the problem of regenerating and healing bone and cartilage tissues in the organism.

[0014] EP 1 525 244 describes hyaluronic acid derivatives in which the molecule is associated with at least one heterocyclic compound derived from a purine or pyrimidine base and another organic compound consisting of naturally occurring amino acids in monomeric, oligomeric or polymeric form, together with methods for their preparation.

[0015] These derivatives of hyaluronic acid, a glycosaminoglycan (GAG) naturally present in the extracellular matrix (ECM), constitute a more stable form than the native form, since hyaluronic acid and related compounds are found at the target sites of the lytic enzyme hyaluronidase, which is usually responsible for its degradation, making its action more difficult. Furthermore, these derivatives exhibit specific three-dimensional structures depending on the type of heterocyclic compound and amino acid chosen, which allow them to modify the microenvironment of the ECM. Summary of the Invention [Problem to be solved by the invention]

[0016] Therefore, the object of the present invention is the use of said hyaluronic acid derivatives in the regeneration of bone and cartilage tissues, based on the high regenerative capacity of said tissues highlighted in the experimental part described herein. [Means for solving the problem]

[0017] According to the present invention, such compounds of hyaluronic acid and at least one heterocyclic compound derived from purines and / or pyrimidines, associated with at least one other organic compound selected from naturally occurring amino acids in monomeric, oligomeric or polymeric form, are used in the treatment of skeletal conditions, in particular in the regeneration of hard tissues.

[0018] According to the present invention, the hyaluronic acid derivative (the use of which is the object of the present invention) is a high molecular weight hyaluronic acid in the range of 400,000 to 4 million Da, preferably 800,000 to 3.5 million Da, more preferably 1.5 to 3 million Da.

[0019] Optionally, the hyaluronic acid derivative, the use of which is the object of the present invention, consists of low molecular weight hyaluronic acid, for example in the range of 80,000 to 400,000 Da.

[0020] The molecular weight of polymers in general and of hyaluronic acid in particular can be determined, for example, by the number-average molecular weight Mn, defined as the average weight of the polymer chain: Mn=Σ (i) NiMi / Σ (i) Ni where Mi is the molecular weight and Ni is the number of chains, or Mw=Σ (i) NiMi 2 / Σ(i)NiMi is the weight average molecular weight Mw, which is defined as

[0021] This amount is influenced more by the fraction with a higher molecular weight, which is greater than the weight average molecular weight.

[0022] The measurement of the average molecular weight of a polymer is important because it indicates a key characteristic of the polymer (to which many of the polymer's properties are related). Molecular weight can be obtained by a variety of techniques, including centrifugal techniques (sedimentation balance), light scattering techniques, and osmometry.

[0023] The rate at which a molecule sediments in an ultracentrifuge is proportional to its molecular weight: indeed, given that molecular weight increases as its volume increases, molecular weight is determined on the basis of sedimentation velocity.

[0024] Light scattering techniques are based on the principle that a light beam does not lose any energy in its path when it traverses a void in a straight line. However, if there are particles of any kind in the space, it is observed that the light beam is scattered or deflected in all directions by the particles present. Thus, the main light beam loses some of its energy and becomes less intense.

[0025] By measuring the intensity of the scattered light, and therefore the optical diffusivity of the polymer itself in a dilute solution in a suitable solvent, a theory can be developed for determining the molecular weight M of a polymer.

[0026] The molecular masses obtained have an average value, which in this case is demonstrated to be the average of the weights.

[0027] Among the above methods, osmometry is the most important and widespread. It involves measuring the osmotic pressure value π for solutions with various polymer concentrations C. Recall that π = CRT.

[0028] If the temperature T at which the measurement is performed is known, the molecular weight can be calculated by remembering that C = mass / molecular weight.

[0029] Instead, the most widely used standard method for the measurement of the physical-chemical properties of polymers is called GPC (Gel Permeation Chromatography).

[0030] According to the invention, the heterocyclic compounds selected are derivatives of purine bases, for example, selected from adenine and guanine, and / or pyrimidine compounds, for example, selected from thymine, cytosine, and uracil. According to the invention, the preferred bases are pyrimidine bases, for example, thymine.

[0031] Other purine or pyrimidine derivatives which may be used to form compounds whose use is the object of the present invention include 5,6-dihydrouracil, 1-methyluracil, 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 -(Δ 2 -isopentyl)adenine, 1-methylguanine, 7-methylguanine, N 2 -methylguanine, N 2 ,N 2 -dimethylguanine.

[0032] Preferably, in the derivatives of hyaluronic acid whose use is the subject of the present invention, the interaction between the hyaluronic acid chain and the purine or pyrimidine base occurs by means of at least one ionic bond between the -COOH residue of said acid and the basic center, in particular the basic nitrogen, of said heterocyclic base.

[0033] In particular, according to the present invention, hyaluronic acid is reacted with at least one purine and / or pyrimidine base selected from the group described above under conditions allowing the formation of at least one ion between at least one acid center of the hyaluronic acid, e.g., a free carboxyl group in the form of an acid or carboxylate salt, and at least one basic center of the purine and / or pyrimidine base in the form of a free base or ammonium salt.

[0034] According to the present invention, the derivatives of hyaluronic acid, the uses of which are the subject of the present invention, can contain one or more types of purine and / or pyrimidine bases in variable mutual ratios, and therefore, said derivatives are represented by "mixed" bases consisting of a variable number of purine / pyrimidine bases.

[0035] The hyaluronic acid derivatives, the use of which is the subject of the present invention, also contain at least one naturally occurring amino acid, or its polymer, to provide additional chloride products, thanks to the -COOH groups present in the structure of hyaluronic acid and remaining free.The amino acids 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.

[0036] The characteristics of the derivatives reflect those of hyaluronic acid, purine and / or pyrimidine bases, and amino acids, which are then linked by at least one easily hydrolyzable ionic type bond to form another compound that is readily available "in situ."

[0037] Particularly preferred is the compound currently available under the name T-LysYal® (T-Lys), a derivative of hyaluronic acid, lysine and thymine by ionic type bonding.

[0038] According to the present invention, said compounds between hyaluronic acid, a heterocyclic compound selected from purine and / or pyrimidine derivatives, and at least one naturally occurring amino acid, or an oligomer or polymer thereof, are advantageously used to initiate and stimulate cell differentiation of MSCs along the osteogenic and chondrogenic lineages.

[0039] According to the invention, the above-mentioned derivatives of hyaluronic acid can therefore be used for the treatment of skeletal conditions, in particular in the regeneration of bone and cartilage tissue.

[0040] Furthermore, according to the present invention, said derivatives of hyaluronic acid are advantageously used to initiate and stimulate cell differentiation of MSCs along the osteogenic and chondrogenic lineages, as already mentioned.

[0041] According to the present invention, the hyaluronic acid derivatives can also be incorporated into suitable pharmaceutical preparations and / or implantable scaffolds used to support bone and cartilage tissue regeneration.

[0042] The derivatives are further used in therapy for the repair and regeneration of bone and cartilage tissue.

[0043] The object of the present invention is the use of said derivatives in the treatment of tissue regeneration of bone and cartilage tissue. According to the invention, said derivatives are advantageously formulated in an implantable scaffold system or other suitable pharmaceutical preparation.

[0044] Other "suitable pharmaceutical formulations" means, but are not limited to, solutions and / or suspensions for parenteral use, solids (e.g., tablets, capsules, granules) or semi-solids (e.g., gels, pastes, creams, ointments) for oral or topical use, intramuscular and / or subcutaneous implantation, and other formulations known to those skilled in the art.

[0045] The feasibility of the present invention is described in the "Experimental Section" below, which discloses studies carried out using said hyaluronic acid derivatives in relation to their ability for cellular regeneration of hard tissues in the body. The following examples are for illustrative purposes only and are not limiting. [Brief explanation of the drawings]

[0046] [Figure 1] Figure 1 shows the effect of T-Lys on the differentiation of MSCs into osteoblast lineage. A): Quantitative PCR performed on DBSCs grown in osteogenic medium and stimulated with 0.3% T-Lys and control DBSCs; B): Immunoblot test for the expression of Runx-2 and Col 1 proteins; C): Histochemical analysis for ALP enzyme (purple staining). [Figure 2]Figure 1 shows the effect of T-Lys on mineral matrix deposition during osteogenic differentiation of MSCs. Mineral matrix deposition was tested by ARS (red staining) in cells treated with T-Lys, hyaluronic acid, and control. [Figure 3] Figure 1 shows the effect of T-Lys on the expression of representative markers in cartilage tissue cultures. Quantitative PCR performed on chondrogenic pellet cultures grown in chondrogenic medium and stimulated with 0.3% T-Lys and a negative control group (Ctr) is shown. [Figure 4] The effect of T-Lys on chondrocyte differentiation and tissue proliferation is shown. A): Images and measurements of culture pellets of dissected chondrocytes treated with T-Lys and the control group; B): Images and measurements of cartilage matrix deposition of dissected chondrocytes treated with T-Lys and the control group; C): Theoretical reconstruction of the thickness of culture pellets of chondrocytes treated with T-Lys and the control group. DETAILED DESCRIPTION OF THE INVENTION

[0047] "Experiment section" [Example]

[0048] Effect of T-Lys on osteogenic differentiation of MSCs towards osteoblast lineage Dental germ stem cells (DBSCs) were used as a source of MSCs and differentiated in osteogenic medium for 12 days. A portion of the test cells was treated with 0.3% T-Lys added to the culture medium at each change (T-Lys-treated group). A fraction of cells not treated with T-Lys was used as the control group (Ctr). Real-time PCR was used to measure the mRNA levels of representative early markers of osteoblasts, Runx-2, and Collagen I (Col 1). Figure 1A) shows how the expression of both markers was significantly increased in T-Lys-treated cells compared to Ctr cells, suggesting that T-Lys treatment improves the potential of MSCs to differentiate into osteoblastic lineages. The protein expression levels of these osteoblast markers were further assessed in T-Lys and Ctr cells by Western blot analysis. Figure 1B) highlights how Runx-2 and Col 1 protein levels are increased in T-Lys-treated cells compared to Ctr cells, thus confirming the trend in mRNA expression. Next, histochemical tests were performed to examine the expression of another osteoblast marker, alkaline phosphatase (ALP) enzyme, in response to T-Lys treatment. The results of this experiment (shown in Figure 1C) showed that stimulation of MSCs with T-Lys during osteoblast differentiation significantly increased purple staining, which evidenced the expression of ALP. All the above results demonstrate the increased potential of MSCs to differentiate into osteoblast-like cells. [Example]

[0049] Effect of T-Lys on mineral matrix deposition during osteogenic differentiation of MSCs To thoroughly investigate the effect of this novel molecule on osteogenic differentiation, DBSCs were cultured under mineralization conditions for 21 days in different samples: Ctr (as a negative control, no additives); HA (as a positive control, unmodified hyaluronic acid was added); and T-Lys (as a treatment group, cells were treated with 0.3% T-Lys). The effect of T-Lys on the deposition of mineral matrix in DBSCs was analyzed using Alizarin Red Staining (ARS) histochemical test and quantified using colorimetric method. The mineralization ability of DBSCs treated with 0.3% T-Lys was demonstrated to be significantly higher than that of both Ctr and HA. These data demonstrate how T-Lys can increase the osteogenic potential of MSCs by stimulating their ability to generate more mineralized matrix. [Example]

[0050] Effect of T-Lys on the intracellular distribution of alpha V beta 3 integrin Integrins are receptors for extracellular matrix molecules that are important not only for cell adhesion but also for mediating proliferation and differentiation signals. In particular, alphaVbeta3 integrin is a receptor for a bone protein called osteopontin, which is fundamentally important for determining differentiation toward the osteogenic lineage. Therefore, we evaluated whether treatment with T-Lys affects the intracellular distribution of alphaVbeta3 integrin. The molecular distribution of this integrin was analyzed by confocal microscopy in DBSCs treated with T-Lys and Ctr. To compensate for the fact that cells rapidly tend to form multiple layers that interfere with microscopic observation, analysis was performed after only 4 days of osteogenic differentiation. In Ctr cells, alphaVbeta3 integrin was found to be distributed at several sites, whereas T-Lys treatment induced a distinct organization of this receptor (more localized at focal adhesion sites). Thus, after 4 days of differentiation, control receptors were still found to be distributed throughout the cells, whereas in T-Lys cells, control receptors were present at focal adhesions. "Strings" (a typical pattern of alphaVbeta3 integrin associated with focal adhesions) were detectable in T-Lys but not in Ctr cells. These results suggest that the effect of T-Lys on DBSC differentiation is mediated by alphaVbeta3 integrin. [Example]

[0051] Effect of T-Lys on the expression of representative markers in chondrocyte cultures Human articular cartilage collected from patients undergoing orthopedic surgery was grown in pellet culture to mimic the microarchitecture of three-dimensional tissues and avoid the inappropriate dedifferentiation of cartilage that can easily occur when grown in two dimensions. Cell pellets were grown under chondrogenic conditions for 28 days. The control group (Ctr) was treated according to the general protocol, while the T-Lys group received 0.3% T-Lys supplemented with each change of vehicle. At the end of the culture period, cartilage culture pellets were solubilized and evaluated for gene expression analysis. mRNA levels of representative chondrogenic markers: Sox-9, Collagen II (Col II), Collagen X (Col X), and aggrecan were measured in both groups of real-time PCR samples. Figure 3 shows the results of these tests, demonstrating that the expression of three of the four markers considered was significantly increased in the T-Lys group. In particular, the expression of Sox-9 (a key transcription factor involved in chondrogenic differentiation) was significantly increased in T-Lys-treated cells compared to Ctr. The results also showed that Col II and Col X (representative proteins of the extracellular matrix of cartilage) were increased by T-Lys treatment, indicating that these molecules support and improve chondrocyte differentiation. On the other hand, the expression of aggrecan (a cartilage proteoglycan) was not affected. [Example]

[0052] Effect of T-Lys on chondrocyte proliferation and tissue growth After 28 days of differentiation under the conditions described in Example 4, chondrocyte pellets were fixed with 4% paraformaldehyde, incorporated, fractionated, histochemically stained, and examined. Morphometric examination of dissected chondrocyte culture pellets by light microscopy revealed that those in the T-Lys group were larger than those in the control group. This result is shown in Figure 4A. To measure the pellet size, the samples were sectioned (5 μm thick), and the area of each section obtained for the two cell groups was measured using the software Image-J. The graph in Figure 4A shows that the average surface area was significantly greater in the T-Lys-treated group than in the control group. The pellets were then stained with Safranin O to highlight chondrocytes (Figure 4B). The staining indicated the presence of cartilage matrix (orange staining), demonstrating that the cells were able to differentiate and generate CME components under these culture conditions (nuclei were counterstained with hematoxylin) (Figure 4B). To check whether T-Lys also had an effect on cell number, cells in selected fields (100 × 100 μm) were counted for each section. The graph in Figure 4B displays the cell number for the two cultures, showing a significant increase in the treated group compared to the control group. Interestingly, more sections with a uniform thickness (5 μm), called "slices," were obtained in the T-Lys sample compared to the control group. This differentiation is represented graphically in Figure 4C, where the number of "slices" is multiplied by the section thickness (5 µm), thus reconstructing the theoretical thickness of the culture pellet as a whole. These results demonstrate that T-Lys stimulates chondrocyte proliferation, their differentiation, and matrix secretion.

[0053] [Detailed explanation of the figure] Figure 1: Effect of T-Lys on differentiation of MSCs towards osteoblast lineage. A) Quantitative PCR performed on DBSCs and DBSC Ctr grown in osteogenic medium and stimulated with 0.3% T-Lys. Each graph represents the mean ± standard error of three independent experiments performed in triplicate. *P<0.02 (compared to the control group). Expression was normalized to microglobulin 2 (B2M). The graph shows that treatment with T-Lys significantly increased the expression of two osteoblast markers, Runx-2 and Col-1. B) Immunoblot test for the expression of proteins Runx-2 and Col 1: Each graph represents the mean optical density calculated for the constitutive protein (β-actin housekeeping gene) plus the standard error of three 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 factors are greater in T-Lys-treated cells than in the control group. C) Histochemical assay for ALP enzyme (purple staining) performed on DBSCs maintained under osteogenic conditions for 7 days and stimulated with T-Lys compared to the control group. The graph represents the quantification (%) of positive staining with respect to the control group (*P<0.01) and is derived from the analysis of three independent experiments performed in quadruplicate. Data are shown as mean ± standard error. Representative images of culture wells are also depicted on the left of the figure. The graph shows how T-Lys samples have greater expression of alkaline phosphatase enzyme.

[0054] Figure 2: Effect of T-Lys on mineral matrix deposition during osteogenic differentiation of MSCs. Mineral matrix deposition, as tested by ARS (red staining), in cells treated with T-Lys, hyaluronic acid, and Ctr over 21 days under osteogenic conditions. The graph shows the quantification (as mean % ± standard error) of the optical density of the dye extracted from the pigmented cell layer and is representative of three independent experiments performed in quadruplicate. *P<0.01, P<0.001 for the negative control group (Ctr), P<0.01 for the positive control group (HA). Representative images of culture wells are also depicted on the left of the figure. The data show how T-Lys samples had greater mineral matrix deposition than both untreated samples and samples treated with native hyaluronic acid.

[0055] Figure 3: Effect of T-Lys on the expression of representative markers in the chondrocyte lineage. Quantitative PCR was performed on chondrocyte pellet cultures grown in chondrogenic medium and stimulated with 0.3% T-Lys and the negative control group (Ctr). Each graph represents the mean ± standard error of three independent experiments performed in triplicate. *P<0.04 for Sox-9, *P<0.001 for Col II, and *P<0.01 for Col X (compared to the control group). Expression was normalized to microglobulin 2 (B2M). The graph shows that treatment with T-Lys increases the expression of chondrocyte markers Sox-9, Col II, and Col X, but has no effect on aggrecan expression.

[0056] Figure 4: Effect of T-Lys on chondrocyte proliferation and tissue growth. A) Compartmentalized chondrocyte culture pellets were photographed under a light microscope using a 20x objective lens and analyzed by using the software Image-J for area morphometric examination. The selected image is representative of three different experiments, and the scale bar is shown in the lower right corner of the figure: 75 μm. The graph shows the mean ± standard error of three independent experiments performed in triplicate: *P<0.0003. The pellets treated with T-Lys were significantly larger than those of the control group. B) Cartilage matrix deposition was measured using Safranin O staining, and chondrocyte nuclei were counterstained with hematoxylin. Images were taken with a 40x lens, and the scale bar is shown in the upper left corner of the control image: 25 μm. The graph represents the mean ± standard error of three independent experiments performed in triplicate: *P<0.04. The number of cells in the T-Lys sample is higher than that in the control group. C) The graph shows the theoretical reconstruction of the thickness of the chondrocyte cultures (number of sections obtained multiplied by the thickness of the obtained slices (expressed in μm)). The group treated with T-Lys shows a greater thickness.

Claims

1. A pharmaceutical preparation for bone and cartilage tissue regeneration, comprising derivatives of hyaluronic acid, thymine and lysine by ionic type bonding.

2. The pharmaceutical preparation according to claim 1, which is used for inducing and stimulating cell differentiation in the osteogenic and chondrogenic lineages of mesenchymal stem cells.

3. A pharmaceutical preparation as described in claim 2, wherein the mesenchymal stem cells are tooth germ stem cells.

4. An implantable scaffold for bone and cartilage tissue regeneration, comprising a derivative of hyaluronic acid, thymine and lysine by ionic type bonding.

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