HK1225657B - Fgf-18 formulation in alginate/collagen hydrogels - Google Patents

Description

FGF-18 formulation in alginate/collagen hydrogel

Technical Field

The present invention relates to the field of pharmaceutical formulations, more particularly to a fibroblast growth factor 18 (FGF-18) protein formulation in an alginate/collagen hydrogel, and to a method for producing the hydrogel/formulation.

Background Art

Fibroblast growth factor 18 (FGF-18) is a member of the fibroblast growth factor (FGF) protein family, closely related to FGF-8 and FGF-17. A characteristic feature of the FGF family is a heparin-binding domain. This putative heparin-binding domain has been identified for FGF-18. Receptor-mediated signaling is hypothesized to initiate after binding to FGF ligands complexed with heparin sulfate proteoglycans on the cell surface.

FGF-18 has been shown to be a proliferative agent for chondrocytes and osteoblasts (Ellsworth et al., 2002; Shimoaka et al., 2002). FGF-18 has been proposed to treat cartilage diseases such as osteoarthritis (OA) and cartilage injury (CI) alone (WO2008/023063) or in combination with hyaluronic acid (WO2004/032849).

Pharmaceutical compositions containing FGF polypeptides are known in the art. WO2012172072 describes a lyophilized formulation containing FGF-18, comprising FGF-18, a buffer, a poloxamer surfactant, and a sugar as a stabilizer. This lyophilized formulation of FGF-18 has shown promising results in treating OA or CI. The current dosing regimen for this lyophilized formulation is a once-weekly injection treatment cycle lasting three weeks. This treatment cycle can be repeated.

In the case of CI, a major drawback of existing formulations is that, upon intra-articular (i.a.) injection, the presence of FGF-18 in the synovial fluid may induce uncontrolled cartilage growth in healthy areas. Of course, this may induce undesirable effects, such as reduced joint mobility. Selectively delivering FGF-18 at the target site level can promote cartilage growth only in damaged areas. Specifically, FGF-18 delivery at the damaged area level would be highly beneficial for CI treatment with microfracture. Microfracture is a surgical technique for articular cartilage repair that works by creating small fractures in the underlying bone. This results in the release of multipotent mesenchymal stem cells from the bone marrow (Ringe J. et al., 2012). Filling the cartilage pores with an injectable gel containing FGF-18 would guide the cells in the gel, which would then serve as both a mechanical support and a drug reservoir for cell growth. For this reason, it is desirable that FGF-18 not be released from the gel, but instead be retained in the matrix.

In tissue engineering, typical scheme is the restriction of growth factor in 3D matrix (i.e. scaffold), and this 3D matrix can be implanted or injected (depending on mechanical properties), to ensure the shape of receiving site. The essential feature of described scaffold is biocompatibility and absorbability. In addition, scaffold must be able to provide the ideal environment that is supplied to growth, propagation and damaged tissue re-formation to cells. Ideally, this matrix should be similar to the identical mechanical properties of original tissue, and should have the available microporosity (interconnected hole with sufficient size) for host cell (Tessmar and 2007).

For example, WO2012113812 describes a nanofibrous scaffold coated with at least one layer of polyanion and one layer of polycation. A therapeutic molecule, such as FGF18, can be incorporated into the scaffold. Specifically, the therapeutic molecule can form a polyanion layer. The scaffold can optionally further comprise osteoblasts in a collagen hydrogel and chondrocytes in an alginate hydrogel, each of which is placed on the coated scaffold. The scaffold is surgically implanted in situ.

Hydrogels are three-dimensional networks of hydrophilic polymer chains capable of absorbing and retaining large amounts of water. Their main characteristic is their ability to swell and shrink, but they are insoluble in aqueous media. Therefore, they can capture active molecules (active pharmaceutical ingredients, or APIs) within their matrix, where they are subsequently slowly released or retained, depending on the presence or absence of specific interactions between the matrix and the API (Lo Presti et al., 2011). The advantage of using injectable hydrogels to treat cartilage diseases is that they can be injected into cartilage defects through arthroscopy, eliminating the need for any invasive surgery that would be required with a solid scaffold.

Among the various known hydrogels, some formulations are based on polymers that undergo a gelation process in response to specific physical or chemical stimuli. These occur as viscous injectable liquids that, upon injection, transform into a visible gel in response to environmental stimuli at the injection site (e.g., changes in temperature, pH, or ionic strength). The composition of such formulations can be adjusted to produce hydrogels with different characteristics, such as viscoelastic properties, microporosity, etc. (WO2008063418; Lo Presti et al., 2011; C. Dispenza et al., 2011). When preparing pharmaceutical compositions containing biologically active proteins, the compositions must be formulated in such a way that the activity of the protein is maintained for an appropriate period of time. Loss of protein activity/stability may be due to chemical or physical instability of the protein, primarily due to denaturation, aggregation, or oxidation. Consequently, the resulting product may be pharmaceutically unacceptable. Although the use of excipients and/or hydrogels is known to increase the stability of a given protein, the stabilizing effect of these excipients is highly dependent on the polymer in the gel, the nature of the excipient, and the biologically active protein itself.

There remains a need for new formulations containing FGF-18 as an active ingredient, wherein the formulations simultaneously maintain the biological activity of the active ingredient and are suitable for injection, preferably for joint injection, allowing for a reduction in the number of injections required for treatment. This feature would reduce the risk of infection and increase patient convenience, as neither surgery nor invasive implantation would be required. Such formulations can be used for administration in the treatment of cartilage disorders (e.g., osteoarthritis or cartilage lesions) in patients.

Summary of the Invention

One object of the present invention is to provide novel formulations comprising FGF-18 protein. More specifically, the formulation is a homogeneous hydrogel comprising FGF-18, wherein the hydrogel is preferably an ion-responsive hydrogel, and more preferably an alginate/collagen gel. The present invention also provides methods for preparing the homogeneous hydrogels of the present invention. The FGF-18-containing hydrogels described herein can be administered for the treatment of cartilage disorders. Of particular interest are alginate/collagen hydrogels that also comprise FGF-18 protein.

In a first aspect, the present invention provides a homogeneous hydrogel (i.e., a gel formulation) comprising or consisting of the following components: alginate, collagen, FGF-18, a sugar as an isotonic/stabilizing agent, and a salt. The formulation is provided as a two-component gelling system, each system being in liquid form when separated. Alternatively, the component gelling system comprising alginate can be in lyophilized form. One of the components comprises or consists of a liquid or lyophilized composition of collagen, FGF-18, a sugar, and alginate (Solution 1). These components form a homogeneous composition. The second component comprises or consists of a liquid salt (Solution 2). Once the two component gelling systems (Solution 1 and Solution 2) are mixed (or combined), a gel is formed. The gel is also homogeneous. In a preferred embodiment, the stabilizer is a sugar or sugar-alcohol, such as sucrose, mannitol, trehalose, or D-sorbitol, and the salt is a divalent cation salt (e.g., a magnesium salt, a copper salt, a zinc salt, or a calcium salt, such as calcium chloride). In a preferred embodiment, in the first component - gelling system (i.e., solution 1), the concentration of alginate is or is about 1-5 weight percent, preferably or is about 2.5-4.5, more preferably or is about 3 or 4 weight percent, the concentration of collagen is or is about 0.1-5 μg/mL, preferably or is about 1 or 2 μg/mL, and the concentration of sucrose is or is about 10-100 mg/mL, preferably or is about 30-70 mg/mL, for example, 30, 40, 50, 60 or 70 mg/mL, more preferably 70 mg/mL; in the second component - gelling system (i.e., solution 2), the concentration of the saline solution is or is about 1-20 mg/mL, preferably 10 mg/mL. When mixed together, the volume ratio of solution 1: solution 2 is 5:1-1:2, more preferably 2:1 (in the case of a lyophilized formulation, the volume of solution 1 is considered before the lyophilization process). Preferably, the FGF-18 is selected from the group consisting of: 1) a polypeptide comprising or consisting of the mature form of human FGF-18 corresponding to a sequence comprising or consisting of residues 28 (Glu) to 207 (Ala) of SEQ ID NO: 1, 2) a polypeptide comprising or consisting of a truncated form of human FGF-18 comprising or consisting of residues 28 (Glu) to 196 (Lys) of SEQ ID NO: 1, and 3) a polypeptide comprising or consisting of SEQ ID NO: 2. More preferably, the FGF-18 is sprifermin, as defined below. The first component gelling system may further comprise a buffer, and/or optionally other excipients.

Solution 1 is preferably co-injected with Solution 2 to form the hydrogel in situ.

In a second aspect, the present invention provides a method for preparing a homogeneous hydrogel of FGF-18, comprising the steps of:

1) preparing a solution 1 comprising or consisting of the following components: FGF-18, alginate, collagen and an isotonic/stabilizing agent,

2) preparing a solution 2 comprising or consisting of a salt, and

3) co-injecting the two solutions to form the gel,

The isotonicity/stabilizing agent is a sugar or sugar alcohol, such as sucrose, mannitol, trehalose, or D-sorbitol, and the salt is a divalent cation salt, such as a magnesium salt, a copper salt, a zinc salt, or a calcium salt (e.g., calcium chloride). Preferably, the pH of the final formulation is maintained at or about 6-8, and more specifically at or about 7. In a preferred embodiment, the FGF-18 is selected from the group consisting of: 1) a polypeptide comprising or consisting of a mature form of human FGF-18 corresponding to a sequence comprising or consisting of residues 28 (Glu) to 207 (Ala) of SEQ ID NO: 1, 2) a polypeptide comprising or consisting of a truncated form of human FGF-18 comprising or consisting of residues 28 (Glu) to 196 (Lys) of SEQ ID NO: 1, and 3) a polypeptide comprising or consisting of SEQ ID NO: 2. More preferably, the FGF-18 is spfmin, as defined below. Solution 1 may further comprise a buffer, and/or optionally other excipients.

In a third aspect, the present invention provides an article of manufacture for pharmaceutical or veterinary use comprising:

1) a first container comprising alginate, collagen, FGF-18 protein, and an isotonic/stabilizing agent (Solution 1) (the composition is homogeneous), and

2) a second container containing salt (Solution 2),

The isotonicity/stabilizing agent is a sugar or sugar alcohol, such as sucrose, mannitol, trehalose, or D-sorbitol, and the salt is a divalent cation salt, such as a magnesium salt, a copper salt, a zinc salt, or a calcium salt (e.g., calcium chloride). Preferably, FGF-18 is selected from the group consisting of: 1) a polypeptide comprising or consisting of a mature form of human FGF-18 corresponding to a sequence comprising or consisting of residues 28 (Glu) to 207 (Ala) of SEQ ID NO: 1, 2) a polypeptide comprising or consisting of a truncated form of human FGF-18 comprising or consisting of residues 28 (Glu) to 196 (Lys) of SEQ ID NO: 1, and 3) a polypeptide comprising or consisting of SEQ ID NO: 2. More preferably, FGF-18 is spfmin, as defined below. Solution 1 may further comprise a buffer and/or optionally other excipients. The contents of the containers are then mixed together in situ and injected simultaneously. Preferably, the first container and the second container of the product are two compartments of a dual chamber or dual injection system.

definition

As used herein, the term "FGF-18 protein" or "FGF-18" refers to a protein that retains at least one biological activity of the human FGF-18 protein. FGF-18 can be native, in its mature form, or in a truncated form. Biological activities of the human FGF-18 protein include significantly increasing osteoblast activity (see WO 98/16644) or chondrogenesis (see WO 2008/023063).

Native, or wild-type, human FGF-18 is a protein expressed by chondrocytes of articular cartilage. Human FGF-18 was first designated zFGF-5 and is fully described in WO 98/16644. SEQ ID NO: 1 corresponds to the amino acid sequence of native human FGF-18, which has a signal peptide consisting of amino acid residues 1 (Met) to 27 (Ala). The mature form of human FGF-18 corresponds to the amino acid sequence of SEQ ID NO: 1 from residue 28 (Glu) to residue 207 (Ala) (180 amino acids).

FGF-18 of the present invention can be produced by recombinant methods, for example, as taught in WO2006/063362. Depending on the expression system and conditions, FGF-18 of the present invention is expressed in recombinant host cells with an initial methionine (Met residue) or with a signal sequence for secretion. When expressed in a prokaryotic host (e.g., E. coli), FGF-18 contains an additional Met residue at the N-terminus of its sequence. For example, the amino acid sequence of human FGF-18, when expressed in E. coli, begins with a Met residue at the N-terminus (position 1), followed by residues 28 (Glu) to 207 (Ala) of SEQ ID NO: 1.

As used herein, the term "truncated form" of FGF-18 refers to a protein comprising or consisting of residues 28 (Glu) to 196 (Lys) of SEQ ID NO: 1. Preferably, the truncated form of FGF-18 protein is the polypeptide designated "trFGF-18" (170 amino acids), which begins with a Met residue (at the N-terminus) followed by amino acid residues 28 (Glu) to 196 (Lys) of wild-type human FGF-18. The amino acid sequence of trFGF-18 is shown in SEQ ID NO: 2 (amino acid residues 2-170 of SEQ ID NO: 2 correspond to amino acid residues 28-196 of SEQ ID NO: 1). trFGF-18 is a recombinant, truncated form of human FGF-18 produced in Escherichia coli (see WO 2006/063362). The International Nonproprietary Name (INN) for this specific form of FGF-18 is spformin. Sprefelmin has been shown to have similar activities to mature human FGF-18, for example, it increases chondrocyte proliferation and cartilage deposition, leading to the repair and remodeling of various cartilage tissues (see WO 2008/023063).

- The terms "active molecule" and "active ingredient" refer to the active pharmaceutical ingredient, ie API. The preferred API in the context of the present invention is FGF-18.

- The terms "gel" or "hydrogel" are used interchangeably in this application. They refer to a 3D matrix or scaffold that can be used as a pharmaceutical formulation. They are not solid scaffolds.

- The term "homogeneous" means that the components of the formulation are mixed, blended, stirred or mingled together, ie they do not form separate layers of components.

The term "alginate", also known as alginic acid or algin, refers to any form of alginates. They are well-known gelling agents, especially when used in combination with divalent cation salts. One of the forms that can be used in the context of the present invention is sodium alginate.

The term "collagen" refers to a group of naturally occurring proteins. Although there are nearly 30 different forms of collagen, the main one is type I collagen. Type I collagen and type II collagen are the preferred forms that can be used in the context of the present invention. However, other forms of collagen can be used.

- The term "alginate/collagen" as used in this application refers to a combination of alginate and collagen.

The term "divalent cation salt" refers to, but is not limited to, salts containing magnesium, copper, zinc, or calcium, including magnesium chloride, copper chloride, zinc chloride, or calcium chloride. Preferably, the divalent cation salt of the present invention is not a polycation salt and is not derived from a polycation component (e.g., those disclosed in WO2012/113812).

As used herein, a "stable" solution or formulation is one in which the extent of protein degradation, modification, aggregation, loss of biological activity, etc., is acceptably controlled and does not unacceptably increase over time. Preferably, the formulation retains at least 80% of FGF-18 activity over a period of at least 12 months at room temperature. Stable formulations comprising FGF-18 of the present invention, when stored, for example, at room temperature or 2-8°C, preferably have a shelf life of at least about 12 months, 18 months, more preferably at least 20 months, and even more preferably about 24 months. Methods for monitoring the stability of the FGF-18 formulations of the present invention are available in the art.

As used herein, the term "stabilizer," "stabilizer," or "isotonic agent" is a compound that is physiologically tolerated and imparts suitable stability/tonicity to the formulation. It significantly prevents the net flow of water across cell membranes in contact with the formulation. Stabilizers can also effectively act as cryoprotectants (i.e., lyoprotectants) during freeze drying (lyophilization). Compounds such as glycerol are commonly used for this purpose. Other suitable stabilizers include, but are not limited to, amino acids or proteins (e.g., glycine or albumin), salts (e.g., sodium chloride), and sugars or sugar-alcohols (e.g., dextrose, mannitol, trehalose, sucrose, D-sorbitol, or lactose). According to the present invention, a preferred stabilizer/tonicity agent is a sugar, and even more preferably sucrose.

As used herein, the term "buffer" refers to a solution of a compound that is known to be safe in pharmaceutical or veterinary formulations and has the function of maintaining or controlling the pH of the formulation within the desired pH range for the formulation. Acceptable buffers for controlling pH between moderately acidic and moderately alkaline pH include, but are not limited to, phosphates, acetates, citrates, arginine, TRIS, and histidine buffers. "TRIS" refers to 2-amino-2-hydroxymethyl-1,3-propanediol, and any pharmaceutically acceptable salt thereof. A preferred buffer may be a histidine buffer.

As used herein, the term "solvent" refers to a liquid solvent, whether aqueous or non-aqueous. The choice of solvent depends primarily on the stability of the pharmaceutical compound in the solvent and the mode of administration. Aqueous solvents may consist solely of water or may be composed of water plus one or more miscible solvents and may contain dissolved solutes, such as sugars, buffers, salts, or other excipients. More commonly used non-aqueous solvents are short-chain organic alcohols, such as methanol, ethanol, propanol, short-chain ketones, such as acetone, and polyols, such as glycerol. According to the present invention, preferred solvents are aqueous solvents, such as water or saline solvents.

As used herein, the term "vial" or "container" broadly refers to a reservoir suitable for retaining alginate/collagen in liquid or lyophilized form. Similarly, it will retain a liquid salt mixture. Examples of vials used in the present invention include syringes, ampoules, cartridges, or other such reservoirs suitable for delivering the FGF-18 formulation to a patient by injection (preferably by intra-articular injection). Alternatively, the vial retaining the alginate/collagen solution and the vial retaining the salt are shown as two compartments of a dual-chamber system (syringe or cartridge, e.g., a dual-chamber syringe or dual-needle injection device, etc.). Vials suitable for packaging products for intra-articular administration are well known and recognized in the art.

As used herein, the term "cartilage disease" encompasses diseases resulting from damage caused by traumatic injury or cartilage diseases. Examples of cartilage diseases that can be treated by administering the FGF-18 formulations described herein include, but are not limited to, arthritis, such as osteoarthritis or rheumatoid arthritis, and cartilage damage.

The term "osteoarthritis" is used to refer to the most common form of arthritis. It can be caused by the breakdown of cartilage. Small amounts of cartilage may break off, causing pain and swelling in the joints between bones. Over time, this cartilage may wear away completely, causing bones to rub against each other. Osteoarthritis can affect any joint, but is most common in the hands and weight-bearing joints, such as the hips, knees, feet, and spine. In a preferred example, the osteoarthritis can be knee osteoarthritis or hip osteoarthritis. Those skilled in the art are fully aware of the osteoarthritis classification used in the art, specifically the OARSI assessment system (see, for example, Custers et al., 2007). Osteoarthritis is one of the preferred cartilage diseases that can be treated by administering the FGF-18 formulations of the present invention.

As used herein, the term "cartilage injury" refers to cartilage disease or cartilage damage primarily caused by trauma. Cartilage injury can occur as a result of traumatic mechanical damage, particularly involving accidents or surgery (e.g., microfracture techniques). Sports-related injuries or sports-related wear and tear of joint tissue are also contemplated within this definition.

- The terms "μg" or "mcg" are used interchangeably and refer to a division of the SI unit of mass.

Detailed Description of the Invention

The main purpose of the present invention is an alginate/collagen gel preparation (or hydrogel), which comprises or is composed of the following components: alginate, collagen, FGF-18 protein, sugar as a stabilizer, and salt. The hydrogel is homogeneous. The hydrogel may also include a buffer, and/or other optional excipients. In a preferred embodiment, the stabilizer is a sugar or sugar-alcohol, for example, sucrose, mannitol, trehalose or D-sorbitol, and the salt is a divalent cation salt, for example, a magnesium salt, a copper salt, a zinc salt or a calcium salt (such as calcium chloride). The hydrogel is suitable for injection at the cartilage level. Preferably, the FGF-18 protein is selected from the group consisting of 1) a polypeptide comprising or consisting of the mature form of human FGF-18 corresponding to a sequence comprising or consisting of residues 28 (Glu) to 207 (Ala) of SEQ ID NO: 1, 2) a polypeptide comprising or consisting of a truncated form of human FGF-18 comprising or consisting of residues 28 (Glu) to 196 (Lys) of SEQ ID NO: 1, and 3) a polypeptide comprising or consisting of SEQ ID NO: 2. More preferably, FGF-18 is spfmin.

The advantage of using an injectable homogeneous hydrogel is that the scaffold (or a component of said scaffold) already containing FGF-18 can be injected into the cartilage defect without any invasive surgery using a solid scaffold. Preferably, the injection of said hydrogel is performed by arthroscopy.

Most preferably, the homogeneous hydrogel of the present invention is prepared from two solutions, one comprising a polymer (herein, an alginate system; solution 1) and the other comprising ions (herein, in salt form; solution 2), and is formed in situ after injection by mixing (or merging) the two solutions. The mixing (or merging) of the two solutions is preferably performed by co-injection. Solution 1 may also comprise a buffer, and/or optional other excipients.

In a preferred embodiment, the present invention relates to the use of a homogeneous liquid polymer solution that is capable of undergoing a gelation process upon administration due to changes in ion concentration. In an alternative embodiment, the polymer solution may be in a lyophilized form. When the polymer solution is in such a lyophilized form, the solution may optionally further comprise a lyoprotectant. Known lyoprotectants are, for example, sugars and generally polyols, such as sucrose, mannitol, trehalose or D-sorbitol.

If solution 1 is in lyophilized form, lyophilization is preferably performed using conventional procedures.

The concentration of FGF-18 in Solution 1 is preferably between 0.1 and 300 mcg/mL or about 0.1, 1, 5, 10, 20, 30, 40, 50, 54, 60, 70, 80, 90, 100, 150, 200, 250, or 300 mcg/mL or about 0.1, 1, 5, 10, 20, 30, 40, 50, 54, 60, 70, 80, 90, 100, 150, 200, 250, or 300 mcg/mL or about 0.1 to 100 mcg/mL or about 0.1 to 100 mcg/mL or about 0.1 to 60 mcg/mL or about 0.1 to 60 mcg/mL or about 0.1 to 10 ...

The concentration of the gel component, ie, alginate, in Solution 1 is at or about 1-5 wt %, preferably at or about 2.5-4.5 wt %, and more preferably at or about 3 or 4 wt %.

The stabilizer in the present invention is preferably a sugar or sugar-alcohol, for example, sucrose, mannitol, trehalose, or D-sorbitol. A preferred sugar is sucrose. Preferably, the concentration of the stabilizer in Solution 1 is or is about 10-100 mg/mL, more preferably or is about 30-70 mg/mL, for example, or is about 30, 40, 50, 60, or 70 mg/mL, more preferably or is about 70 mg/mL.

The preferred concentration of the collagen of the present invention in Solution 1 is or is about 0.1-5 mcg/mL, more preferably is or is about 1-2 mcg/mL, and more specifically is or is about 1 or 2 mcg/mL.

The salt in the second component, the gelling system (i.e., solution 2), is preferably a divalent cation salt, such as a magnesium salt, a copper salt, a zinc salt, or a calcium salt (e.g., calcium chloride). The concentration of the salt solution is or is about 1-20 mg/mL, preferably or is about 10 mg/mL.

In a preferred embodiment, solution 1 comprises or consists of the following components: 0.1-100 mcg/mL of FGF-18, 4% by weight of alginate, 70 mg/mL of sucrose, and 2 mcg/mL of collagen; and solution 2 comprises or consists of 10 mg/mL of a divalent cation salt (e.g., calcium chloride). Solution 1 may further comprise a buffer and/or other optional excipients. Solution 1 is homogenous.

When mixed together, the volume ratio of Solution 1:Solution 2 is 5:1-1:2, more preferably 2:1 (in the case of a lyophilized formulation, the volume of Solution 1 is taken into account prior to the lyophilization process).

Once mixed together, the final concentrations of the components are preferably as follows:

- FGF-18: 0.00006-0.2% w/v, e.g., 0.0036% w/v (when the FGF-18 before mixing is 0.1-300 mcg/mL, based on the FGF-18 concentration given in the Examples section)

- Alginate: 0.6-3.33% w/v, e.g., 2.67% w/v (when the alginate before mixing is 4% w/v)

- Collagen: 0.00006-0.003% w/v, e.g., 0.000133% w/v (when collagen before mixing is 2 mcg/mL)

- Stabilizer: 0.6-6% w/v, e.g., 4.67% w/v (when sucrose before mixing is, e.g., 70 mg/mL)

-Divalent cation salt: 0.033-0.66% w/v, e.g., 0.33% w/v (when the salt before mixing is 10 mg/mL)

In a preferred embodiment, the pH of the final formulation is maintained at or about 6-8, more specifically at or about 7.

The present invention also provides a method for preparing a homogeneous hydrogel of FGF-18, comprising the following steps:

1) preparing a first solution (solution 1) comprising or consisting of the following components: FGF-18, alginate, collagen and a stabilizer,

2) preparing a second solution (Solution 2) comprising or consisting of a salt, and

3) co-injecting the two solutions to form the gel,

Wherein, described stabilizer is sugar or sugar-alcohol, for example, sucrose, mannitol, trehalose or D-sorbitol, and described salt is divalent cation salt, for example, magnesium salt, copper salt, zinc salt or calcium salt (for example, calcium chloride).Solution 1 can also comprise buffer, and/or other optional excipient.Solution 1 is homogeneous.In a preferred embodiment, the pH of the final preparation is maintained at 6-8 or is about 6-8, and more specifically, is or is about 7. Preferably, FGF-18 is selected from the group consisting of 1) a polypeptide comprising or consisting of the mature form of human FGF-18 corresponding to a sequence comprising or consisting of residues 28 (Glu) to 207 (Ala) of SEQ ID NO: 1, 2) a polypeptide comprising or consisting of a truncated form of human FGF-18 comprising or consisting of residues 28 (Glu) to 196 (Lys) of SEQ ID NO: 1, and 3) a polypeptide comprising or consisting of SEQ ID NO: 2. More preferably, FGF-18 is a spfmin, as defined herein.

Each compound (i.e., FGF-18, alginate, collagen, stabilizer, and salt) can be used according to any of the above-mentioned concentrations, pH, and/or ratios. Preferably, the ratio of solution 1:solution 2 (volume to volume, i.e., v:v) is or is approximately 1:2-5:1, more preferably is or is approximately 2:1 (in the case of a lyophilized formulation, the volume of solution 1 is taken into account before the lyophilization process).

When the solution 1 of the hydrogel of the present invention is in lyophilized form, it needs to be reconstituted before step 3 (ie before co-injection).

In a third aspect, the present invention provides a pharmaceutical or veterinary preparation comprising:

1) a first container comprising or consisting of alginate, collagen, FGF-18 protein, and a stabilizer (solution 1), and

2) a second container comprising or consisting of a salt (Solution 2),

wherein the stabilizer is a sugar or sugar alcohol, such as sucrose, mannitol, trehalose, or D-sorbitol, and the salt is a divalent cation salt, such as a magnesium salt, a copper salt, a zinc salt, or a calcium salt (e.g., calcium chloride). Solution 1 may further comprise a buffer, and/or optionally other excipients. Solution 1 is homogeneous. Preferably, FGF-18 is selected from the group consisting of: 1) a polypeptide comprising or consisting of the mature form of human FGF-18 corresponding to a sequence comprising or consisting of residues 28 (Glu) to 207 (Ala) of SEQ ID NO: 1; 2) a polypeptide comprising or consisting of a truncated form of human FGF-18 comprising or consisting of residues 28 (Glu) to 196 (Lys) of SEQ ID NO: 1; and 3) a polypeptide comprising or consisting of SEQ ID NO: 2. More preferably, FGF-18 is spfmin, as defined herein.

Each compound (i.e., FGF-18, alginate, collagen, stabilizer, and salt) can be used according to any of the above-mentioned concentrations, pH, and/or ratios. Preferably, the volume ratio (v:v) of solution 1:solution 2 is or is approximately 1:2-5:1, more preferably or is approximately 2:1 (in the case of a lyophilized formulation, the volume of solution 1 is considered before the lyophilization process). The contents of each container are then mixed together in situ and injected simultaneously (e.g., co-injected).

Preferably, the container holding the FGF-18 formulation and the container holding the saline correspond to the two compartments of a dual chamber system or dual injection system (eg, a syringe or cartridge).

When the polymeric solution (ie, Solution 1) is in lyophilized form, the article of manufacture may further comprise a third container comprising or consisting of a solvent required for reconstitution, eg, water or saline solution (eg, 0.9% w/v sodium chloride for injection).

Also described are packaging materials that provide instructions for forming, preferably in situ, the hydrogels of the invention.

Importantly, the inventors surprisingly showed (see Examples) that when the gel was formed using a syringe connector that allowed the simultaneous injection of the two solutions, no residual liquid phase was observed at any polymer concentration. In fact, the contextual injection of the two solutions allowed for faster and more homogeneous mixing of the two liquid streams, which resulted in instantaneous gelation.

The different components of the hydrogel of the present invention can be stored for at least about 12 months to about 24 months. Under preferred storage conditions, the formulation is kept away from bright light (preferably dark) and at refrigerated temperature (at or about 2-8°C) before the first application.

The hydrogel of the present invention needs to be prepared during the injection process.

When the solution 1 of the hydrogel of the present invention is in lyophilized form, it needs to be reconstituted before use. Reconstitution is preferably carried out under sterile conditions using a solvent such as water or saline solution (e.g., 0.9% w/v sodium chloride for injection) before use, i.e., before combining (or mixing) with solution 2, and thus reconstituted before injection. After reconstitution, the volume is preferably the same as before lyophilization, for example, about 0.5 mL to 5 mL, more preferably about 0.5, 1 or 2 mL. The system must be allowed to dissolve and homogenize before it becomes suitable for injection, for example, during 30 minutes. The solution should be used within, preferably within 1 hour of reconstitution.

The present invention provides a homogeneous hydrogel containing FGF-18 suitable for pharmaceutical or veterinary use, especially for single application. The hydrogel containing FGF-18 of the present invention can be administered to improve cartilage repair or treat cartilage diseases, such as osteoarthritis or cartilage damage.

These homogeneous hydrogels are suitable for injection and are alternative delivery systems. In a particularly preferred embodiment, the formulation of the present invention is for intra-articular (i.a.) injection. It can be administered by direct injection into the defect, wherein the gel is preferably formed in situ. In a preferred embodiment of the present invention, i.a. administration is performed in a joint selected from the group consisting of hip, knee, elbow, wrist, ankle, spine, foot, finger, toe, hand, shoulder, rib, scapula, thigh, shin, ankle, and joints along the spinal column. In another preferred embodiment, i.a. administration is performed in a joint of the hip or knee.

The following examples are provided to further illustrate the preparation of the formulations and hydrogels of the present invention. The scope of the present invention should not be limited to the following examples.

Description of the drawings:

Figure 1: Frequency sweep tests for 3 wt% alginate based gels, placebo (w/o FGF), with 54 mcg/mL FGF-18 in alginate solution (w FGF(54)) and 540 mcg/mL FGF-18 in alginate solution (w FGF(540)).

Figure 2: Frequency sweep tests for 4 wt% alginate based gels: placebo (w/o FGF), with 54 mcg/mL FGF-18 in alginate solution (w FGF(54)) and 540 mcg/mL FGF-18 in alginate solution (w FGF(540)).

Figure 3: Frequency sweep tests on 3 wt% pharmaceutical grade (PG) alginate based gels: placebo (w/o FGF), with 54 mcg/mL FGF-18 in alginate solution (w FGF(54)) and 540 mcg/mL FGF-18 in alginate solution (w FGF(540)).

Figure 4: Frequency sweep tests for 4 wt% PG-alginate based gels: placebo (w/o FGF), with 54 mcg/mL FGF-18 in alginate solution (w FGF(54)) and 540 mcg/mL FGF-18 in alginate solution (w FGF(540)).

Figure 5: Swelling ratios of alginate-based gels of candidate placebo (w/o FGF) and active molecule (w FGF), prepared using non-PG sodium alginate.

Figure 6: Swelling ratios of placebo (w/o FGF) and active molecule (w FGF) alginate-based gels without type I collagen, prepared with non-PG sodium alginate.

Figure 7: Swelling of alginate-based gels of candidate placebo (w/o FGF) and active molecule (w FGF), prepared using PG sodium alginate.

Figure 8: SEM images (100x) of alginate-based gels from 3 wt% or 4 wt% polymer liquid solutions containing 0, 1 or 2 mcg/mL type I collagen and 54 mcg/mL FGF-18.

Figure 9: Cross-sectional views of 3D images acquired by CLSM: (a) an alginate-based gel containing 2 μg/mL type I collagen after 72 hours of incubation with HTB94 cells, and (b) an alginate-based gel without collagen after 144 hours of incubation with HTB94 cells. White: HTB94 cells labeled with rhodamine B. Right: top of gel; left: bottom of gel.

Figure 10: FGF-18 bioassay in 4 wt% alginate gel with 2 mcg/mL type I collagen.

Figure 11: Sequence of human FGF-18, corresponding to SEQ ID NO: 1 (a), and sequence of spfmin, corresponding to SEQ ID NO: 2 (b).

Figure 12: pH evolution over time of freeze-dried (FD) formulations during 3 months of stabilization under conventional storage conditions (5°C), accelerated conditions (25°C) and stressed conditions (40°C).

Figure 13: Change in % moisture content over time of freeze-dried (FD) formulations during 3 months of stabilization under conventional storage conditions (5°C), accelerated conditions (25°C) and stressed conditions (40°C).

Figure 14: Changes in FGF-18 content over time in freeze-dried (FD) formulations during 3 months of stabilization under conventional storage conditions (5°C), accelerated conditions (25°C) and stressed conditions (40°C).

Figure 15: Changes in alginate content over time in freeze-dried (FD) formulations during 3 months of stabilization under conventional storage conditions (5°C), accelerated conditions (25°C) and stressed conditions (40°C).

Figure 16: a) Changes in intrinsic viscosity over time for lyophilized (FD) formulations during 3 months of stabilization under conventional storage conditions (5°C), accelerated conditions (25°C), and stressed conditions (40°C), calculated as mean ± standard deviation at shear rates of 10-50 s ^-1 . Figure 16b: Effect of shear rate on intrinsic shear viscosity of lyophilized (FD) formulations at time 0 and after 3 months of stabilization under conventional storage conditions (5°C), accelerated conditions (25°C), and stressed conditions (40°C).

Figure 17: Mechanical Property Evaluation. Figure 17a: Storage modulus (G') of the lyophilized (FD) formulation over time at 3 months of stabilization under conventional storage conditions (5°C), accelerated conditions (25°C), and stressed conditions (40°C). Figure 17b: Frequency sweep testing of the lyophilized (FD) formulation at time 0 and after 3 months of stabilization under conventional storage conditions (5°C), accelerated conditions (25°C), and stressed conditions (40°C).

Sequence Description:

SEQ ID NO. 1: Amino acid sequence of original human FGF-18.

SEQ ID NO. 2: Amino acid sequence of recombinant truncated FGF-18 (Spfermin).

Example

Material

The recombinant truncated FGF-18 (trFGF-18 or spfumin) of this example has been produced in-house by expression in E. coli according to the technology described in application WO 2006/063362. In the following examples, spfumin and FGF-18 are used interchangeably.

Other main substances used in the examples are as follows:

-Sodium alginate, Sigma Aldrich A2158,

- Sodium alginate, FMC Biopolymers Keltone LVCR (pharmaceutical grade alginate),

-Sucrose, Merck 1.07653.9029

- Type I collagen from human skin, CalbioChem 234149, and CalbioChem 234138

-Calcium chloride (CaCl2), Merck 1.02382.0250,

-D-(+)-Glucono delta-lactone, Sigma-Aldrich G4750

-Chitosan 75% DD HMW, Sigma-Aldrich 419419,

-Chitosan 95% DD LMW, Faravali 43000,

-Chitosan 95% DD HMW, Heppe Medical 24711,

The following examples report the preparation and characterization of two types of hydrogels: ion-responsive and temperature-responsive hydrogels. Ion-responsive hydrogels have been primarily prepared using sodium alginate, which can form hydrogels in the presence of divalent cations, while temperature-sensitive hydrogels have been primarily prepared using chitosan, a well-known temperature-sensitive polysaccharide (Tomme et al., 2008). The two polymers used in this study are biocompatible natural polysaccharides.

In order to clearly distinguish in this section the preparation before the gelation process and the preparation after gel formation, the former is referred to as "liquid solution" and the latter as "gel", respectively.

method

Gel time and temperature

The gel time of all preparations was evaluated by the "tilt test". Gels were prepared in glass Petri dishes with a diameter of 3 cm and incubated at different temperatures. At predetermined time intervals, the Petri dish was tilted, and the time when the preparation did not show any flow was considered the gel time.

Mechanical properties

The rheological properties of the hydrogels were studied using an AR2000 rheometer (TA Instruments) with a 40 mm diameter and a cone-plate geometry (1° slope). A rough dish was applied to both plates to prevent sample slippage during testing. The thickness of all samples was approximately 1-2 mm, depending on the sample volume. A solvent trap was used to maintain a water-saturated atmosphere to prevent solvent evaporation during testing.

After the hydrogel had fully expanded from its original liquid solution in the Petri dish, the sample was placed on the rheometer and equilibrated at 37°C for 2 minutes. The sample was then tested in a frequency sweep test over a range of 0.1–100 rad/s. The oscillatory stress was set to 10 Pa, with storage as G' and loss as G", and the change in modulus as a function of frequency was recorded.

Expansion performance test

One of the hallmark properties of hydrogels is their ability to swell in the presence of aqueous solutions, attributed to their hydrophilic nature. To evaluate the swelling properties of the gels, they were immersed in simulated synovial fluid (MSF) solutions and weighed over time. The experiments were performed in triplicate in 12-well plates equipped with hanging inserts containing a 0.4 μm porous membrane at the bottom.

Only alginate gels were selected for this test. The gels were prepared in 5 mL vials by mixing 200 μL of calcium chloride solution and 400 μL of a polymer liquid solution, prepared as described in Example 2, and incubated at 37°C for 30 minutes. The gels were then transferred to previously weighed, empty inserts. The inserts containing the gel samples were weighed, and the corresponding weight was considered the reference weight at time zero. The gel-containing inserts were placed in a plate, and 1.5 mL of MSF was added to the wells, ensuring that the porous bottom of the inserts was immersed in the solution. The plates were sealed with parafilm to prevent evaporation and incubated at 37°C on an orbital shaker. At predetermined time points, the gel-containing inserts were removed from the wells. Excess MSF was removed by blotting the porous bottom with filter paper, and the gel-containing inserts were then weighed. The solutions in the wells were collected, stored at -80°C, and replaced with fresh MSF. The composition of MSF is as follows: PBS buffer 1X pH 7.3, F68 (i.e., poloxamer 188) 0.25 g/L, human serum albumin (HSA) 1 wt %, PenStrep 1 wt %. Before use, MSF was filtered through a 0.22 μm filter. Results are reported as swelling ratios, which are the ratio of weight at time t to weight at time t=0.

Scanning electron microscopy (SEM) analysis

Once the gel is formed, scanning electron microscopy is used to analyze the three-dimensional structure of the polymer network. Following the process reported in the paragraph about "gel time," the alginate-based gel of the active molecule was prepared in a glass Petri dish with a diameter of 3 cm, mixing 1 mL of a polymer liquid solution and 0.5 mL of a calcium solution. The resulting gel was then incubated at 37°C for 30 minutes. Six samples were prepared from a polymer liquid solution of 3% by weight or 4% by weight alginate (each containing 0, 1, or 2 μg/mL of type I collagen). The gel was then freeze-dried for 48 hours. The freeze-drying cycle includes a freezing step, a primary drying step, and a secondary drying step. During the freezing step, a transition temperature from room temperature to -45°C is applied, followed by a stage of keeping the gel at -45°C for 4 hours. This was followed by a primary drying step, wherein the temperature was transitioned from -45°C to -10°C at a rate of 1°C/minute under a pressure of 50 mTorr, followed by a stage of keeping the gel at -10°C for 24 hours and 30 minutes under 50 mTorr. The secondary drying step consisted of two stages: a first temperature ramp from -10°C to 21°C at a rate of 1°C/min at 50 mTorr, followed by a stage at 21°C for 12 hours and 30 minutes at 50 mTorr, and then a second temperature ramp from 21°C to 37°C at a rate of 1°C/min at 50 mTorr, followed by a stage at 37°C for 6 hours at 50 mTorr. Finally, room temperature and atmospheric pressure were reached. Each sample was then stained and analyzed by SEM.

In vitro release studies

The same samples used for the swelling test were also analyzed for in vitro release testing. Specifically, the collected phase stored at -80°C (as described above in the "Swelling Performance Test" section) was analyzed by HPLC. Selected samples were also analyzed by Biacore (data not shown).

In vitro testing in animal models

Preliminary tests were conducted in an in vitro animal model to investigate the ability of alginate formulations to adhere to real knee joints. The formulations tested were selected based on the results of the cell invasion assay reported in Example 9. Specifically, the model used consisted of the upper portion of the tibia and the lower portion of the femur, which constitute the knee of a bovine. A piston sampler was used to create holes 1 cm deep and 0.4 cm in diameter in two parts of the knee to simulate the holes surgically created during microfracture surgery. The holes were then filled with a 4% by weight alginate formulation containing type I collagen and 36 μg/mL of FGF-18 at a final concentration of 1.3 μg/mL in the gel. The test was performed using an infusion valve to connect two different syringes, one containing an alginate solution and the other containing a calcium solution. The pistons of the two syringes were pushed simultaneously so that the valves could mix the solutions. The formulations were injected into the holes in both vertical and inverted positions to test the possibility of administration in different possible real-life situations. After incubation at 37°C for approximately 2 hours, the gel was removed from the holes to visually inspect its condition.

In vitro biological tests

A 96-well plate for luminescence testing (assay plate) was coated with 50 μL/well of a 4% sodium alginate solution and 25 μL/well of a 10 mg/mL CaCl2 solution and incubated at 37°C, 5% CO2 for 30 minutes to allow the formation of a basic hydrogel.

A 96-well plate (master plate) was filled with 160 μL/well of assay medium from rows B to H. FGF-18 reference standard and samples were then diluted in assay medium at 10 μg/mL and added in triplicate (100 μL/well) to row A, considering each set of columns as replicates. Using a multichannel pipette, a 1:5 serial dilution was performed from wells in row A to wells in row H by transferring 40 μL from row A to row H.

From the master plate, 25 μL/well of the FGF-18 reference standard dilutions were transferred to a basic hydrogel-coated 96-well plate (assay plate) and incubated at 37° C. and 5% CO 2 for 1 hour.

BaF3/FGFR3c cell cultures seeded at 10,000,000 SM in 75 cm2 culture flasks and starved for 24 hours prior to the assay were diluted at 800,000 cells/mL in assay medium and then added to FGF-18 and basic hydrogel-coated plates at 25 μL/well (20,000 cells/well) and incubated at 37°C and 5% CO2 for 48 hours.

"ATPlite 1Step" reagent was added at 100 μL/well to reveal proliferation after slowly mixing the plate, and the emitted light was read using a luminometer.

Example 1: Pre-formulation

In preliminary formulations, alginate or chitosan was mixed with other excipients to obtain aqueous solutions with acceptable osmolality for i.a. injection (target: 350 mOsm/kg). The solutions were then tested for gelation time and temperature. Further formulations capable of forming hydrogels within 5 minutes and/or at approximately 37°C were prepared using various concentrations of FGF-18 (data not shown).

Example 2: Preparation of ion-responsive gel (alginate)

Overview

Sodium alginate is used to create ion-responsive gels in the presence of divalent cations (e.g., Ca ²⁺ ) that act as crosslinkers between polymer chains. These gels are capable of forming polymeric networks that create hydrogels. The formulation consists of two solutions, one containing the polymer and the other containing the ions. Once mixed, the solutions form a gel.

For both placebo and active molecule gel, calcium solution is identical, and is made up of calcium chloride (CaCl ₎ solution with a concentration of 10 mg/mL in milliQ water. In polymer liquid solution, in addition to alginate, type I collagen and active molecule (herein, Spfermin) and sucrose are added to adjust the osmotic pressure value. Placebo polymer liquid solution is prepared as follows: a sucrose solution with a concentration of 70 mg/mL in milliQ water is prepared and used to dissolve sodium alginate with a concentration of 3 wt % or 4 wt % under stirring. Once alginate dissolves, a type I collagen solution dissolved in hydrochloric acid and diluted to 30 μg/mL or 60 μg/mL concentration in milliQ water is added under stirring to obtain a collagen concentration of 1 μg/mL or 2 μg/mL in the polymer liquid solution, respectively. For the preparation of active polymer liquid solution, alginate with a concentration of 3 wt % or 4 wt % is dissolved in a sucrose solution with a concentration of 70 mg/mL in milliQ water containing the main body FGF-18 of 54 μg/mL. The same steps as reported for the placebo were then followed for the addition of type I collagen. The gels were prepared by mixing the polymer liquid solution with the calcium solution in a 2:1 (v:v) ratio. The gels were incubated at 37°C for 30 minutes before further characterization.

Initial screening of placebo preparations:

After testing that physiological ions were insufficient to promote the gelation process, the divalent cation Ca2 ⁺ in the form of calcium chloride was selected as the crosslinking agent. In order to avoid the use of calcium, which could cause undesirable calcification phenomena, magnesium chloride was also tested, but the results were less satisfactory.

Possible preparations are then screened, and preliminary placebo preparations are selected. Consider and change different parameters in the screening process, for example, alginate concentration, CaCl _{2concentration} , the volume ratio of polymer and calcium solution, the presence of other excipients, for example, D-(+)-glucono delta-lactone (as gelling retarder), or HSA (being added to PBS to better simulate physiological conditions). Find that the presence of HSA or D-gluconic acid does not have a significant impact for the obtained gel, therefore it is discarded, to avoid increasing useless components.

It is also observed that, among the values tested, the optimum alginate concentration is 2.5% to 4.5% by weight, and in particular 3% or 4% by weight. In fact, lower alginate concentrations (i.e. 1% or 2% by weight) are undesirable because no complete gel formation is recorded. On the other hand, too high alginate concentration (5% by weight) results in extremely viscous polymer liquid solutions that are difficult to control.

The selected preliminary placebo formulation consisted of a 3 wt% alginate liquid solution in water and a 10 mg/mL _CaCl2 solution in PBS in a 2:1 (v:v) ratio.

Addition of FGF-18

To obtain an active formulation, the test added FGF-18 to both solutions. In all of the following tests, the ratio between the two solutions remained constant at 2:1 volume/volume alginate solution: calcium solution. Visual inspection revealed that adding FGF-18 to the alginate solution caused precipitation. As a possible solution, the alginate was dissolved in a PBS buffer containing 0.25g/L of F68 surfactant. However, precipitation still occurred. To overcome this problem, FGF-18 was added to the buffer before the alginate was dissolved. Thus, precipitation in the polymer solution was avoided, and the protein was uniformly distributed, but the physical consistency of the resulting gel appeared to be affected compared to the placebo prepared in water. In addition, analysis of the residual liquid phase after gel formation revealed microscopic precipitation. Further studies confirmed that the precipitation was caused by the interaction between _CaCl2 and PBS, which appeared to lead to the formation of calcium phosphate. Thus, PBS was eliminated from the polymer liquid solution and the calcium solution, and only milliQ water was used as the solvent.

Formulation optimization

In order to obtain a gel with a denser mesh size, an alginate concentration increased to 4% by weight was considered. This should give the material a more durable ability in vivo, so as to have a longer encapsulated protein retention time. Minimization of calcium content was also studied to reduce possible side effects: the minimum amount obtained to obtain a gel, maintaining all other constant parameters, was found to be 4mg/mL. Because the mixing of calcium and polymer solution can cause the immediate formation of gel, it is impossible to detect the osmotic pressure of pH and the final formulation. Therefore, the osmotic pressure of the polymer liquid solution was adjusted to a value of about 300mOsm/kg using 70mg/mL of sucrose. This addition causes changes in gel formation, wherein no residual liquid phase is observed in the absence of sucrose. It is assumed that sucrose interferes with gel formation by interacting with alginate or with calcium. Therefore, the concentration of calcium is again increased to 10mg/mL, resulting in gel formation and no residual liquid and sediment.

The pH of the polymer solution was checked and obtained to be 7.0. The calcium solution showed a pH of 5.3 and an osmolarity of approximately 170 mOsm/kg.

The amount of protein in the polymer liquid solution was set to 54 μg/mL.

Finally, type I collagen was added to the polymer liquid solution to enhance the cell invasion ability of the gel, as reported by Rayatpisheh et al. (2011). According to the literature (Tsai et al., 1998), two different concentrations, 1 and 2 μg/mL, were tested.

At the end of this initial screening, four candidate formulations were selected. For all of them, the calcium solution contained 10 mg/mL calcium chloride in MilliQ water, while the polymer solutions varied in alginate and type I collagen content, as shown in Table 1.

All experiments used readily available sodium alginate, provided by Sigma Aldrich. In the final part of the study, sodium alginate with the same characteristics but certified as pharmaceutical grade (PG) was purchased from FMC BioPolymerKeltone. The four candidate formulations were then re-formulated using PG alginate and compared to non-PG formulations, achieving comparable results upon visual inspection. The swelling and mechanical properties of the gels obtained using PG and non-PG alginate were also compared.

It is expected that although experiments have been performed using calcium chloride, similar results will be obtained using any other divalent cation salt, for example, magnesium, copper or zinc salts.

Example 3: Preparation of temperature-responsive gel (chitosan)

Overview

For screening of chitosan formulations, polymer solutions were prepared using three different chitosans: a high molecular weight (HMW) chitosan with a 95% degree of deacetylation (DD), a low MW chitosan chitosan chitosan with a 95% DD, and a high MW chitosan chitosan chitosan with a 75% DD. The polymer solutions were prepared by gradually adding chitosan to a 0.1N acetic acid solution at 5°C or 25°C with vigorous stirring. The amount of polymer was calculated to achieve a final polymer concentration of 1%, 1.5%, or 2% by weight in the polymer solution. Once the chitosan was completely dissolved, a 10 mM, 100 mM, or 500 mM KH ₂ PO ₄ solution in milliQ water was added with stirring to achieve a final concentration of 1 mM, 10 mM, or 50 mM in the polymer solution. Finally, a 20% by weight solution of β-glycerophosphate (β-GP) in milliQ water was added to adjust the pH of the final solution to 6.0, 6.5, or 7.0. For the formulations that were screened, the final concentration of β-GP in the polymer liquid solution was between 0.5% and 7% by weight. The desired pH was not always achieved because too high a quantity of β-GP was required, the target osmotic pressure of 350 mOsm/Kg was exceeded, or a gel was obtained at room temperature. When available, the polymer liquid solution was incubated at 37°C until gel formation occurred. The osmotic pressure of all formulations screened was determined, and formulations with an osmotic pressure higher than 350 mOsm/Kg, i.e., formulations with a final β-GP concentration higher than 2.5% by weight, were discarded.

Initial screening of placebo preparations:

Temperature-responsive gels are based on polymeric materials that undergo a sol-gel transition in response to changes in the local ambient temperature. Chitosan has been reported to undergo a sol-gel transition in response to temperature changes, but this process is highly influenced by the polymer's molecular weight (MW), its degree of deacetylation (DD), the polymer concentration in the solution, the temperature, time, and mixing speed during polymer dissolution, the final pH of the solution, and the presence of other excipients.

Therefore, a thorough screening of different possible combinations is required. It should be noted that chitosan can only be used in water at an acidic pH. Elevated pH can cause it to aggregate and precipitate. One way to overcome this problem is to use β-GP to increase the pH while maintaining the chitosan in solution.

This study initially focused on HMW chitosan with a 75% DD. Several polymer liquid solutions were prepared in 0.1N hydrochloric acid with final chitosan concentrations ranging from 1% to 2.5% by weight, final β-GP concentrations ranging from 1.6% to 50% by weight (1.6, 5, 5.6, 8, 30, and 50%), various excipients (i.e., pectin, glucosamine, hyaluronic acid, hydroxyethylcellulose, carboxymethylcellulose, and trehalose), and different final pH values ranging from 6.0 to 7.0. These excipients have been reported to play a role in inducing gel formation (Cheng et al., 2010; Schuetz et al., 2008; Yan et al., 2010).

Only one of the formulations screened was able to form a gel after incubation at 37°C for 5 minutes, but the amount of β-GP was above 8% by weight, which is reported in the literature as an upper limit, with concentrations above this being recorded as cytotoxic (Ahmadi et al., 2008). Therefore, the following formulations were all prepared with this limitation in mind. The screening continued to shift towards chitosans with higher DD values. The first trial was based on LMW chitosans with 95% DD. The polymer solutions were all prepared in 0.1N hydrochloric acid, dissolving the polymer at 5°C or 25°C under vigorous stirring.

After the polymer has completely dissolved, the other excipients are added, with β-GP added only at the end. β-GP is responsible for increasing the pH value, which then promotes the gelation process. The first experiments focused on formulations based solely on chitosan and β-GP in different combinations of relative concentrations. It was observed that with high concentrations of chitosan (2% or 3% by weight) and high concentrations of β-GP (8% by weight), gels appeared already at room temperature, and in some cases, also at 5°C.

When the concentrations of the components were reduced, the formulations remained liquid even after prolonged incubation at 37°C. In only one case was gel formation recorded, but after 2 hours of incubation at 37°C, which was too long for the purposes of this study. Therefore, it was necessary to add excipients to improve the formulation. Hydroxyethylcellulose (HEC) was selected as the most suitable excipient and further screening was performed. During this evaluation, the chitosan concentration was 1.5% to 2% by weight, and the initial HEC concentration was 0.5% by weight, but under these conditions, the polymer solution was able to gel even at room temperature during the addition of β-GP if its concentration was above 1.8% by weight. A liquid solution capable of gelling after 13 minutes of incubation at 37°C was obtained using the following composition: 1.5% by weight of chitosan, 0.5% by weight of HEC, and 1.7% by weight of β-GP.

Formulation optimization

In order to improve the formulation, experiments were performed in which the concentration of β-GP was kept almost constant at a value of 1.65-1.7 wt%, the chitosan concentration was varied from 1.5 to 1.8 wt% and the amount of HEC was gradually reduced up to 0.1 wt%.

Several candidate formulations were selected using this strategy. However, the study was not continued in this direction because it was found that the HEC excipient may contain contaminants (reportedly cytotoxic) and, on the other hand, cause a modulation of the gelation process in the presence of chitosan (Hoemann et al., 2007). Other excipients tested in previous experiments, such as pectin or glucosamine, did not produce positive results.

Final screening efforts were then initiated using three chitosan polymers that differed in molecular weight and DD: HMW chitosan with a 75% DD, HMW chitosan with a 95% DD, and LMW chitosan with a 95% DD. The study was planned to use LMW chitosan with an 85% DD, but this material was not available before the study was completed. In this study, each chitosan was tested at three fixed concentrations of 1, 1.5, and 2% by weight, and polymer solutions were prepared to have final pH values of 6.0, 6.5, and 7.0. To reduce the amount of β-GP used and thereby increase the pH of the solution, the polymers were dissolved in 0.1N acetic acid rather than the 0.1N HCl used in previous experiments. The osmolality of the final solution was also monitored and maintained below approximately 350 mOsm/kg. Therefore, formulations that required excessive amounts of β-GP to reach the desired pH and also resulted in excessively high osmolality values were discarded. In these screening tests, the contribution of ionic strength was also investigated, since it has been reported that the presence of salts may contribute positively to the gelation process (Filion et al., 2007).

Since sodium salts must be avoided to prevent possible interactions with proteins, _KH₂PO₄ was _selected and added to the polymer solutions to final concentrations of 1 mM, 10 mM, or 50 mM. Chitosan with a 75% DD did not yield positive results and was therefore discarded entirely. HMW chitosan with a 95% DD yielded positive results: chitosan concentrations above 1 wt% required excessive amounts of β-GP to achieve a fixed pH, exceeding the target osmolality value, and the 1 wt% formulation failed to form a gel at 37°C. Two candidate formulations were selected using LMW chitosan with a 95% DD because they underwent a solution-gel transition at 37°C, but the preparation of these polymer solutions was completely unreproducible. Indeed, significant variations in the time required to obtain a gel and the physical macroscopic characteristics of the polymer liquid solutions were observed, depending on the time taken to dissolve the polymer, the speed of mixing during polymer dissolution and excipient mixing, and, ultimately, the temperature and volume of the prepared solution.

This high variability in the results led to the decision to discontinue the studies on this polymer.

Example 4: Gelation time and temperature

All formulations studied in this work were intended to be used as in situ forming gels. Therefore, an important requirement for ion- and temperature-responsive gels is a fast gel time. In fact, it is important to identify formulations that have good gelling properties but whose gelling process is neither too fast (to allow injection) nor too slow (to avoid the risk of obtaining loose material). The gel time is determined using a tilt test: the solution is turned upside down, and a gel is considered to have formed if no liquid flow is observed.

Ion-based formulations

In the case of alginate-based preparations, the gelation process is affected by the concentration of the calcium solution and the relative ratio between the calcium and alginate solutions. The gel is prepared in a glass Petri dish, first adding the alginate solution, then adding the calcium solution, and attempting to homogenize the two solutions as much as possible. If the amount of calcium chloride is not enough, no gel formation is observed, even at 37°C for a long time. Increasing the amount of cross-linking agent (calcium) and adjusting the polymer solution: solution ratio, a gel is formed almost instantly after the two solutions are mixed together. In some cases, a residual liquid phase is observed. After incubation at 37°C, the time required for the complete disappearance of the liquid is considered the gelation time.

The gelation time varies from 5 minutes to 2 hours, and in some cases, does not realize the complete disappearance of liquid. The gelation of the polymer liquid solution adopting 4 % by weight alginate is faster than that adopting 3 % by weight. Adding type I collagen does not show any influence for gelation time. The results are summarized in Table 2. It is 5 minutes that two candidate preparations of the polymer solution of 4 % by weight alginate concentration show gelation time, while two candidate materials of 3 % by weight polymer solution show complete gelation in 15 minutes.

Importantly, when gel formation was performed using a syringe connector that allowed simultaneous injection of two solutions, no residual liquid phase was observed at any polymer concentration. In fact, the coupled injection of the two solutions allowed for faster and more homogeneous mixing of the two streams, which resulted in instantaneous gelation.

It is expected that although experiments have been performed using calcium chloride, similar results will be obtained using any other divalent cation salt, for example, magnesium, copper or zinc salts.

Temperature-based formulations

The temperature-responsive gels were designed to be liquid at room temperature during preparation and handling, but to gel after injection (under physiological conditions of 37°C). Testing was performed by incubating different chitosan-based formulations at 5°C, 25°C, and 37°C to monitor gelation times as a function of temperature using a tilt test. The results are summarized in Table 3.

Depending on the concentration of chitosan and β-GP, the gelling properties vary significantly. With over-concentrated solutions of β-GP, gelling already occurs at room temperature during the preparation of the liquid solution. Therefore, it is important not only to optimize the formulation by varying the concentrations of the components, but also to dissolve and mix all the ingredients at 5°C and then incubate the liquid solution at 37°C. After appropriate optimization, as described in Example 3, gel formation was obtained after 15 minutes at 37°C, but the results were not found to be reproducible. In particular, it was observed that the gelling time was influenced by the speed and time required to dissolve the chitosan and mix it with the other components.

Example 5: Mechanical properties

Hydrogels are a group of semisolid materials composed of hydrophilic polymer networks with high water retention capacity. Generally speaking, hydrogels have mechanical characteristics (viscoelastic properties) between viscous liquids and elastic solids (Anseth et al., 1996). It is important that their mechanical properties are determined and tested under conditions similar to the target in vivo environment.

Dynamic mechanical analysis characterizes the viscoelastic properties of hydrogels, reflecting a combined viscous and elastic response, following application of an oscillatory stress (typically shear). Viscoelastic materials are characterized by a storage modulus, G’, and a loss or viscous modulus, G”, which respectively describe the elastic and viscous contributions to the dynamic stress-strain behavior.

From a rheological point of view, hydrogels are defined as viscoelastic materials with G’>G”, where both G’ and G” are independent of the oscillation frequency (Peppas et al., 2000). Specifically, the higher the G’, the stronger the hydrogel, and the greater the difference between G’ and G”, the lower the liquid-like versus solid-like performance. In this section, we report on the investigation of the viscoelastic properties of alginate hydrogels by performing frequency sweep tests using a rheometer equipped with a cone-plate geometry (see Experimental Section). In the frequency sweep test, an oscillatory shear stress was applied over a range of frequencies (0.1-100 rad/s). G’ and G” were then plotted against frequency.

Figure 1 shows the results of a frequency sweep for 3 wt% alginate gels with different collagen contents (0, 1, 2 μg/mL) and different FGF-18 contents (0, 54, 540 μg/mL) in alginate solution. All samples were analyzed after incubation at 37°C for 30 minutes after addition of calcium solution. An assay using 540 μg/mL of FGF-18 in alginate solution was performed to investigate whether a tenfold higher protein concentration would affect the internal structure of the material.

As can be seen, all samples exhibit typical properties of gels, showing G'>>G", which are independent of angular frequency. The G' values for all samples are between approximately 300 and 400 Pa. These differences in G' do not represent substantial differences in the elastic response of these materials. This suggests that the low concentration of collagen in the formulation (1 or 2 μg/mL) does not affect the mechanical properties of the gel at time point 0. However, it may promote a higher stability of the gel after incubation, as will be shown in the next section.

Importantly, it was also noted that the presence of 54 or 540 μg/mL of FGF-18 in the alginate solution did not affect the mechanical properties of the material. In fact, no significant differences or trends were identified between the placebo and active molecule gels at the two different protein concentrations. This is an important parameter because it allows the formulation of FGF-18 in alginate solution over a wide range of protein concentrations (0-540 μg/mL) without affecting the properties of the matrix.

Figure 2 shows the results obtained for the 4 wt% alginate candidate, the corresponding placebo, and the placebo without collagen. Again, in this case, all samples showed typical properties of gels, G'>>G", independent of angular frequency. Similarly, no effect was observed for the two collagens and FGF-18 (54 or 540 μg/mL in the alginate solution), with all G' values being around 380 and 500 Pa. It can be expected that higher concentrations of alginate would lead to higher crosslink densities, and thus elastic moduli, G'. However, the differences recorded were not significant.

Regardless, it is important to emphasize that the calcium solution added to the 3 wt% and 4 wt% alginate solutions was identical. This means that exposing the 3 wt% alginate solution to a higher relative concentration of crosslinker may even result in a denser network, depending on the arrangement of the polymer chains in the solution.

At the end of the study, pharmaceutical grade (PG) alginate was available. To ensure that the purity level of the alginate did not affect the gel structure, a frequency sweep test was also performed on a PG alginate-based gel. The results are shown in Figures 3 and 4.

The results obtained with PG alginate were very comparable to those obtained with non-PG alginate. The only difference was a slightly wider spread in the results for the 3 wt% and 4 wt% gels. This can likely be attributed to slight differences in the molecular weight distribution of the raw materials, even though the technical parameters for PG and non-PG alginates are reported to the same specifications.

However, again, no particular trends or significant differences were observed between the 3 wt% and 4 wt% systems, and the slightly higher G' values observed in the 4 wt% system were also confirmed in the PG feedstock.

Example 6: Expansion Performance

A typical parameter that hydrogel materials have been evaluated for is their ability to swell or shrink in the presence of liquid. This test is used to predict the performance of a material when injected under in vivo conditions where it would come into contact with physiological synovial fluid.

This study was performed only on the alginate-based gels prepared according to Example 2, since the chitosan formulation was discarded (as explained in Example 3). Therefore, these four candidate alginate-based gels were incubated in contact with MSF at 37°C and their performance was monitored for 30 days (see "Swelling Performance Test" in the Methods section).

Figure 5 reports the one-month swelling performance of four candidate alginate-based gels, compared to placebo and active molecule gels. As a reference, the same gels were also tested without type I collagen to investigate its effect on the gel's structural characteristics. These results are shown in Figure 6. Figures 5 and 6 refer to non-pharmaceutical grade alginate (non-PG).

In both cases, the higher the alginate concentration, the higher the expansion ratio. This performance seems to be different from the result obtained by rheological measurement. Specifically, higher polymer concentration should form denser network, which is then associated with the ability of lower absorption aqueous solution. And, it is necessary to consider making Ca2 ⁺ concentration remain constant, so that the ratio between polymer and cross-linking agent is changed by making polymer concentration change. Lower cross-linking agent accessibility (availability) can cause forming some regions, and the polymer in these regions is not cross-linked, thus allowing water to easily see through. This can explain the difference in expansion ratio between the candidate of 3 % by weight and 4 % by weight alginate, and is confirmed by the SEM photo shown in the next part.

A comparison between Figures 5 and 6 shows that the presence of type I collagen affects swelling properties, particularly in terms of kinetics. While in the absence of collagen, the entire gel increased in weight (monitored for approximately 15 days) before reaching a plateau. The presence of collagen, independent of concentration, resulted in a sinusoidal trend. In the presence of collagen, the first plateau was reached after two days, indicating equilibrium swelling corresponding to the maximum amount of water the gel could retain. This value was lower than the corresponding swelling ratio in the absence of collagen, indicating a tighter structure of the gel, as expected after the addition of collagen. The subsequent swelling ratio increased after 15 days of incubation, indicating the onset of network erosion, as the lattice loosened, leading to absorption of the MSF solution. A second plateau was then reached after 24 days and maintained until the end of the one-month observation period. Further erosion of the network would be expected to lead to further weight gain, resulting in an initial increase and then a decrease in the swelling ratio until complete erosion occurred. In the absence of collagen (Figure 6), a continuous increase in the swelling ratio was observed after a period of slow erosion. This behavior is likely related to the simultaneous erosion and water uptake (attributable to the looser structure of the gel).

As mentioned above, no differences were noted between the gels obtained using 1 or 2 μg/mL type I collagen in the polymer solution.

Interestingly, no differences were observed between the placebo and active molecule gels. This suggests that the 36 μg/mL final concentration of FGF-18 in the gel did not affect the internal structure of the material. These experiments were repeated using the same formulation, this time using PG alginate, and the results are shown in Figure 7.

As can be seen, the results are perfectly comparable to those obtained with non-PG alginates, with the only difference being a minor deviation in the swelling ratios of the 3 wt% and 4 wt% alginate gels compared to the non-PG material. The experiment was run for up to 50 days for the 4 wt% gels, which showed the expected performance, i.e. a decrease in weight after a second plateau, indicating slow, continuous erosion.

Example 7: Scanning Electron Microscope (SEM) Analysis

Once the gel was formed, SEM images were taken to study the microstructure of the material. The analysis was performed on freshly prepared, then freeze-dried, gels.

A comparison of samples with and without type I collagen, performed at two polymer concentrations, is shown in FIG8 , showing that collagen has no significant effect on the microporosity of the 3 wt % alginate gel, and only a slight effect for only 4 wt %, which exhibits a slightly tighter structure when collagen (1 or 2 μg/mL) is added.

From the micrographs, a difference in porosity between the two materials was observed, with the 4 wt% alginate gel showing more voids than the 3 wt%. This can also be explained by the relative concentration of crosslinker in the 3%.

The weight (wt) of the alginate preparation was higher than that of the 4 wt% case. This could induce a denser structure in the 3 wt% gel.

These images are consistent with the swelling results and appear to contradict the mechanical property analysis. Indeed, the larger voids present in the 4 wt% gel explain the higher swelling observed compared to the 3 wt% gel. On the other hand, the slightly higher elastic modulus G' observed for the 4 wt% gel can be explained by the presence of non-crosslinked or loosely crosslinked alginate chains in the macropores, which nonetheless contribute to G'.

Example 8: In vitro release studies

In vitro release studies were performed in conjunction with swelling tests, with the absorption phase from each well collected at each time point considered. All samples from the active alginate-based gels were analyzed by RP-HPLC. In addition, selected samples were analyzed by Biacore, a more specific and sensitive analytical method, to confirm the results obtained by RP-HPLC.

Before initiating this experiment, the feasibility of the test was evaluated. Specifically, the recovery of FGF-18 in a well plate system was investigated. Indeed, based on previous studies, it is known that proteins tend to adhere to various plastic materials, such as polystyrene, and membranes. The recovery of known amounts of free FGF-18 was evaluated under different experimental conditions. The possible positive effects of the presence of F68 surfactant and/or pre-treatment of the wells with concentrated solutions of HSA or FGF-18 were tested. The experiment was performed as follows: a solution of FGF-18 at a concentration of 500 μg/mL was added to the insert and to each well:

-a) a solution of 1 wt% HSA in PBS,

-b) 1 wt% HSA solution in PBS+F68,

-c) and d) are the same as a) and b), but the wells are pretreated with a concentrated HSA solution,

-e) and f) Same as a) and b), but the wells were pretreated with the FGF-18 concentrated fraction.

After incubation at 37°C for 16 hours, the well and basket phases were analyzed for all samples, and the recovery reached approximately 75%. Therefore, none of the strategies used to improve the assay had a significant positive effect, as the recovery was comparable. In vitro release was then performed using a 1 wt% HSA solution in PBS, using F68 0.25 g/L as the absorption phase.

In the experiment, the gel was kept under incubation conditions for 30 days. FGF-18 was not detected in any of the collected fractions by HPLC or Biacore. This suggests that the protein was trapped in the gel matrix during the observation period.

These results are expected given the strong interaction between FGF-18 and alginate. Indeed, given the structures of these two molecules, both ionic and hydrophobic interactions are likely to occur. Furthermore, FGF-18 is a heparin-binding protein, which exhibits high-energy secondary interactions with heparin. Alginate is a naturally occurring carbohydrate with a molecular structure similar to heparin, making it highly likely that these two macromolecules will experience extremely strong secondary interactions.

To confirm this, separation experiments were performed, showing that only HIC- or heparin-coated columns were able to separate FGF-18 and alginate.

Example 9: In vitro cell invasion assay

Administration of FGF-18 in a formulation can produce a 3D structure after injection, as in the case of alginate-based hydrogels, which can localize the active molecule (API) at the injection site. Simultaneously, the formulation can generate a scaffold in which the growing cartilage can be anchored. Thus, for selected alginate-based gels, important requirements are the compatibility of the material with chondrocyte cells and the ability to be invaded by the cells. Therefore, based on the in situ formation of FGF-18-loaded hydrogels, the cell invasion, cytotoxicity, and chemotaxis properties of alternative formulations were investigated.

In vitro cell invasion assays were performed using two chondrosarcoma cell lines: the ATDC5 murine chondrogenic cell line and the CRL-7891 human chondrosarcoma cell line, which are commonly used to study chondrocyte performance. The experiments were performed on gels formed from 4% or 3% by weight alginate liquid solutions containing 0, 1, or 2 μg/mL type I collagen and 54 μg/mL FGF-18.

Cell invasion was monitored after 24, 48, 72 and 144 hours of incubation. The gel was removed from the wells and thin slices were cut, placed on glass and observed on an Axiovert 200 microscope with a Zeiss A-Plan 10x/0.25 eyepiece glass. In this way, gel sections can be analyzed and cells can be positioned at different levels of penetration depth. Photos were collected and processed by AxioVision 4.2 software. Some samples were also analyzed by confocal laser scanning microscopy (CLSM) using an FV10 Olympus for limited time demonstration applications. For these experiments, the gels were treated with a 0.2 μg/mL rhodamine B solution in PBS, which was added to the culture medium in the wells 1 hour before analysis. Rhodamine B was used to stain the cells, and the samples were then excited with a 553 nm laser for visual inspection.

Observation in 24,48,72 and 144 hours has 3 % by weight or 4 % by weight polymer liquid solution, contains or does not contain the cell invasion of the gel based on alginate of collagen.At each time point, cut gel thin slice and observe gel cross section (data not shown).First observe the good compatibility of preparation and cell, because the gel based on alginate seems all nontoxic for two kinds of cell lines, it even shows the compatible properties with different species (mouse and people) origin.

In the formulation with 4 wt% alginate gel, more cells and cell clusters were found inside the gel.

Can observe different performance for the alginate gel that comprises 2 μ g/mL type I collagen of 4 weight %.Compared to other preparations, more cells invade this gel.In addition, cell does not produce cluster, but seems to be well distributed as the cell that single and hole separates along gel.This one side seems to disclose that it is in healthy state.Finally, observe that it passes through and enters gel matrix over time.Comparatively contain 2 μ g/mL collagen and do not contain the gel of 4 weight % of collagen, observe that the invasion in the presence of collagen is faster and more homogeneous (referring to Fig. 9).This analysis is carried out by CLSM, has confirmed the ability of cell invasion gel matrix.

Example 10: Ex vivo testing in animal models

Therapies based on these alginate formulations could be combined with microfracture strategies. Therefore, in order to demonstrate the compatibility of the gels with this technique, preliminary studies were performed in an ex vivo animal model, and thus the bovine knee was used.

Experiments were performed on a candidate gel of 4 wt% alginate containing 2 μg/mL collagen type I. This gel was chosen from other candidates because it showed the most promising results in terms of cell invasion as shown above.

Cavities are made on the upper tibia and lower femur, which have the same size (1cm deep × 4mm diameter) expected for microfracture surgery. Then, the cavity is filled with candidate gel, and the filling adopts a syringe connector to mix alginate solution and calcium solution respectively when injecting. This allows instantaneous homogeneous gel to be obtained without any water drop phenomenon. Injection is carried out in vertical and inverted positions, confirming that in both cases, the gel remains in the cavity, and no material is observed to drip. Joints are also subjected to vibration and rapid movement, proving that the gel is well fixed in the cavity, and no material loss is observed. Then, after incubation for 2 hours at 37°C, the joints with the injected gel are evaluated: the gel still fills the entire cavity, and, once taken out, it appears slightly reddish, indicating that the blood overflowing from the cleft is absorbed by the gel.

These preliminary results demonstrate the feasibility of alginate formulations in microfracture strategies.

Example 11: In vitro biological test

Cell proliferation assays performed as described in the Methods section showed that FGF-18 entrapped in the gel was biologically active with a clear dose-response curve, as shown in FIG10 .

in conclusion

Ion-responsive gels were obtained from sodium alginate after addition of calcium chloride as a cross-linking agent, whereas thermo-responsive gels were obtained from chitosan with addition of β-GP to adjust the pH, without any precipitation of the polymers.

Alginate- or chitosan-based formulations were tested at different polymer concentrations, and the addition of other excipients was considered to adjust the pH and osmolarity of the formulation and, in some cases, facilitate the gelation process.

The gelation time results showed that the alginate-based gels exhibited extremely fast and reproducible gelation compared to chitosan. Therefore, the alginate gels were selected for further development.

Four candidate gel formulations were selected for characterization of mechanical properties, swelling, in vitro release, and cell invasion. They were formed by adding 10 mg/mL calcium chloride solution to an alginate solution containing 3% or 4% alginate by weight, 1 or 2 μg/mL type I collagen, 70 mg/mL sucrose, and 54 μg/mL FGF-18. The alginate solution volume to calcium volume ratio was 2:1.

The mechanical properties of the candidate gels were unaffected by either collagen concentration or FGF-18 concentration (at least up to 540 μg/mL). This is an important goal, as the target concentration of FGF-18 in the hydrogels has not yet been determined, allowing for the formulation of FGF-18 in these matrices at a wide range of concentrations without affecting the characteristics of the final gel. No major differences in elastic modulus G' were observed between the 3 wt % and 4 wt % alginate gels, with all displaying values between approximately 300 and 500 Pa, with the 4 wt % gels displaying higher values.

All candidate gels show the same swelling properties, indicating similar hydration and degradation processes. Only in absolute values, there is a difference, with 4 % by weight alginate gel being slightly higher. This result is consistent with the SEM micrograph, which demonstrates the higher dimensions of the pores of these gels.

After 15 days of incubation at 37°C in simulated synovial fluid, all candidates showed a first degradation process, indicated by weight gain. Initial degradation of the network typically results in a looser structure, which is thus able to accommodate a larger amount of water. After one month, continued network degradation led to the onset of visible gel erosion, indicated by a slow weight loss. Meanwhile, after 50 days of observation, the gel was still not completely eroded, as indicated by the presence of some gel in the porous basket (50-day swelling ratio) (Lo Presti C. et al., 2011; Dang et al., 2011).

Carry out cell invasion test. The gel formulation showing the most promising cell invasion results comprises 4 wt % alginate and 2 μg/mL collagen. Then, this formulation was selected to carry out in vitro experiments to study the adhesion properties of this gel. These in vitro experiments carried out on cattle knees show that the candidate alginate gel can be injected using a dual syringe for alginate and calcium solution, and injection in either vertical or reversed directions does not show any dripping. In addition, the gel shows perfect filling and adhesion to the cartilage cavity simulating microfracture conditions (data not shown).

Cell proliferation assays revealed that FGF-18 trapped in the gel exhibited biological activity with a clear dose-dependent curve. Even after 50 days of observation, the gel had not completely degraded, and in vitro release experiments showed no evidence of FGF-18 release in simulated synovial fluid. This suggests that FGF-18 remains within the gel and is therefore likely to be more effective in the long-term effects on cells that have migrated into the gel. Therefore, these results are promising.

Example 12: Freeze-dried formulation

After preparing a polymer liquid solution (see Example 2) comprising FGF18, sugar (sucrose) and type I collagen, the solution was dispensed into vials. Each vial (10 ml Fiolax Clear 45x24, glass form SCHOTT) was filled with 2 mL of the polymer liquid solution. All filled vials were subjected to a freeze-drying process. Specifically, the process comprises the following steps:

- Cooling period, from 25°C to -40°C, lasting 1 hour, where product freezing begins

- Freezing period, -40°C for 4 hours, during which the product remains frozen

- Vacuum period, where the pressure drops suddenly to a vacuum

- First drying period, the temperature is increased from -40℃ to -10℃ for 30 minutes, and then at -10℃ for 10 hours

- Second drying period, in which the temperature is increased from -10°C to 21°C for 30 minutes and then at 21°C for 34 hours

- A third drying period in which the temperature was increased from 21°C to 37°C for 16 minutes and then maintained at 37°C for 20 hours.

After the lyophilization process is complete, the vials are sealed with stoppers and stored at 2-8°C.

The lyophilized product needs to be reconstituted before injection. It needs to be equilibrated at room temperature, then 2 mL of WFI (water for injection) is injected into the vial, and the vial is then vortexed and shaken to facilitate the reconstitution of the lumps. The system is allowed to dissolve and homogenize for 30 minutes, after which it becomes suitable for injection.

The lyophilized formulations were then subjected to a 3-month stability study at 3 different temperatures. Conventional storage conditions at 5°C, 25°C to implement accelerated stability, and under stress conditions at 40°C. After reconstitution of the samples with 2 ml of water, analyses were performed at 0, 2, 4, 8, and 12 weeks. The subsequent parameters were as follows:

pH

Water content

FGF18 content

Alginate content

Alginate MW distribution

Viscosity

Mechanical properties

Cell invasion ability

Below are the key results up to 12 weeks.

pH : No significant changes in the pH of the formulations were noted over incubation time at all three temperatures. The pH of the FD formulation was higher than that of the liquid form (most likely due to the filtration and lyophilization processes it underwent), but it did not change over time. The data shown in Figure 12 indicate that the formulations were stable for up to 12 weeks under typical storage conditions.

Moisture content : Residual moisture content was within the acceptance criteria (3%). This value remained constant for formulations stored at 2-8°C, but increased slightly over time after 12 weeks at 25°C and increased even more after 2 weeks at 40°C, as expected. This data (see Figure 13) indicates that the formulations are stable for up to 12 weeks under conventional storage conditions.

FGF18 content: No significant changes in the FGF18 content of the formulations were observed over time at all three different temperatures, with the total content being slightly below the target concentration of 54 μg/ml, which is most likely due to material losses during the different processing steps and in particular during filtration. The differences observed at different time points are most likely due to variations in the method, which is still under development, considering both the amount of FGF18 close to the detection limit of the method and the variation in the vial contents, which is also evidenced by the significant STD DEV of the results: in fact, the high viscosity of the solution does not allow for accurate fill volumes to be manipulated at the laboratory level. In summary, the data indicate that the formulation is stable under conventional storage conditions for up to 12 weeks (see Figure 14).

Alginate Content: No significant changes in alginate content were observed over time at all three temperatures. The observed changes are most likely due to variations in the method, which is still under development, and to variations in vial contents, as the solution viscosity does not allow for a very accurate filling process at the laboratory level. The data shown in Figure 15 demonstrate that the formulation is stable for up to 12 weeks under typical storage conditions.

Alginate MW distribution: under all three kinds of different temperatures, over time, do not observe the change of the chromatographic characteristics of preparation, thereby do not have the change on overall polymer molecular weight distribution: peak indication broad MW distribution, from greater than 870kDa to about 40kDa, wherein maximum value is at about 520kDa.MW distribution is assessed by comparing with the standard reference of the MW of good determination.The small platform detected by the left part of peak is attributed to collagen.Data show that preparation is stable under conventional storage conditions and reaches 12 weeks (data not shown).

Viscosity: A slight decrease in formulation viscosity was observed at time 0 for samples stored at 25°C and 40°C, which was attributed to accelerated degradation conditions, while no significant difference was observed for samples stored under conventional conditions (2-8°C): the observed changes were most likely due to process variations and the filling process. The data indicate that the formulation is stable for up to 12 weeks under conventional storage conditions (see Figure 16).

Mechanical Properties: Alginate FD formulations reconstituted with water at each time point in the stability study were mixed with calcium chloride solution to form hydrogels. The mechanical properties of the resulting hydrogels were studied. No change in the storage modulus G' was observed over time at all three different temperatures, resulting in no change in mechanical properties. The results shown in Figure 17 demonstrate that the formulations were stable for up to 12 weeks under standard storage conditions.

Cell invasion ability: The alginate FD formulation reconstituted with water at each time point of the stability study was mixed with a calcium chloride solution to form a hydrogel. The cell invasion ability of the resulting hydrogel was studied. The hydrogel was incubated with cells for 72 hours and then analyzed by confocal laser scanning microscopy. Over time, considering samples stored under the same temperature conditions, no significant changes in the number of viable cells capable of penetrating into the hydrogel matrix were observed (data not shown). At each time point, the number of penetrating cells shown by the sample stored at 40°C was higher than that of the sample stored at 2-8°C, indicating that incubation at higher temperatures caused partial changes in the polymer matrix. The data show that the formulation is stable for up to 12 weeks under conventional storage conditions.

Conclusion: The lyophilized alginate formulations showed good stability for up to 12 weeks under conventional storage conditions (2-8°C) and appeared to be at least as stable as the corresponding liquid formulations.

sheet

Table 1: Combinations of calcium and polymer liquid solutions for four candidate alginate-based formulations

Table 2: Gel time of active alginate-based formulations

Table 3: Gel time of selected chitosan-based formulations at 37°C

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Claims (16)

1. A two-component gelling system, wherein the gelling system is formed by the following: Solution 1: The first component, which contains FGF-18, alginate, collagen, and sugars as stabilizers, or is composed of them, and Solution 2: The second component, which contains or is composed of divalent cation salts; FGF-18 is selected from the following group: a. A polypeptide containing amino acid residues 28-207 of SEQ ID NO:1 or composed thereof, b. A polypeptide comprising amino acid residues 28-196 of SEQ ID NO:1 or composed thereof, and c. Contains SEQ ID NO:2 or a polypeptide thereof. 2. The two-component gelling system of claim 1, wherein the stabilizer is sucrose. 3. The two-component gelling system as described in claim 1 or 2, wherein the divalent cation salt is a calcium salt, magnesium salt, copper salt, or zinc salt. 4. The two-component gelling system of claim 1 or 2, wherein, in the respective solutions, the concentration of the alginate is 3-4% by weight, the concentration of the collagen is 1-2 mcg/mL, the concentration of the stabilizer is 10-100 mg/mL, and the concentration of the salt is 1-20 mg/mL. 5. The two-component gelling system as described in claim 1 or 2, wherein the ratio of solution 1 to solution 2 is 2:1 (volume to volume). 6. A method for generating a homogeneous hydrogel, comprising the following steps: Step a. Prepare a first homogenized solution containing, or consisting of, FGF-18, alginate, collagen, and sugars as stabilizers. Step b. Prepare a second solution containing, or consisting of, a divalent cation salt. Step c. Mix the two solutions and then inject to form a hydrogel, or inject the two solutions together to form a hydrogel; FGF-18 is selected from the following group: a. A polypeptide containing amino acid residues 28-207 of SEQ ID NO:1 or composed thereof, b. A polypeptide comprising amino acid residues 28-196 of SEQ ID NO:1 or thereof, and c. Contains SEQ ID NO:2 or a polypeptide thereof. 7. The method of claim 6, wherein the stabilizer is sucrose. 8. The method of claim 6 or 7, wherein the divalent cation salt is a calcium salt, magnesium salt, copper salt, or zinc salt. 9. The method of claim 6 or 7, wherein, in the respective solution, the concentration of the alginate is 3-4% by weight, the concentration of the collagen is 1-2 mcg/mL, the concentration of the stabilizer is 10-100 mg/mL, and the concentration of the salt is 1-20 mg/mL. 10. The method of claim 6 or 7, wherein the ratio of the first homogeneous solution to the second solution is 2:1 (volume to volume). 11. A manufactured article comprising two containers, wherein: 1) The first container contains or is composed of a first homogeneous solution, wherein the first homogeneous solution contains, or is composed of, FGF-18, alginate, collagen, and sugar as a stabilizer, and 2) The second container contains or is composed of a second solution, the second solution containing or being composed of a divalent cation salt; FGF-18 is selected from the following group: a. A polypeptide containing amino acid residues 28-207 of SEQ ID NO:1 or composed thereof, b. A polypeptide comprising amino acid residues 28-196 of SEQ ID NO:1 or thereof, and c. Contains SEQ ID NO:2 or a polypeptide thereof. 12. The manufactured article as claimed in claim 11, wherein the stabilizer is sucrose. 13. The manufactured article as claimed in claim 11 or 12, wherein the divalent cation salt is a calcium salt, magnesium salt, copper salt, or zinc salt. 14. The manufactured article as claimed in claim 11 or 12, wherein, in the respective solution, the concentration of the alginate is 3-4% by weight, the concentration of the collagen is 1-2 mcg/mL, the concentration of the stabilizer is 10-100 mg/mL, and the concentration of the salt is 1-20 mg/mL. 15. The manufactured article as claimed in claim 11 or 12, wherein the ratio of the first homogeneous solution to the second solution is 2:1 (volume to volume). 16. The manufactured article as claimed in claim 11 or 12, wherein the first container and the second container are two compartments of a dual-chamber system or two compartments of a dual-needle injection device.