Hydrogel precursor formulation and production process thereof.

(54) Title: FORMULATION OF THE HYDROGEL PRECURSOR AND THE PRODUCTION PROCESS OF THE SAME. (54) Title: HYDROGEL PRECURSOR FORMULATION AND PRODUCTION PROCESS THEREOF.

(57) Summary

The present invention relates to a formulation of the hydrogel precursor, its production process as well as a kit comprising such a formulation and a method of producing a hydrogel using such a formulation. The precursor formulation comprises at least one structural compound, preferably vinyl sulfone (branched acrylated) poly (ethylene glycol), and at least one ligation compound, preferably a peptide with two cisterns, wherein the structural compound and ligation compound are Polymerizable by a selective reaction between a nucleophile and a conjugated unsaturated group or bond. The precursor formulation is in the form of a powder.

(57) Abstract

The present invention relates to a hydrogel precursor formulation, its process of production as well as a kit comprising said formulation and a method of production of a hydrogel using said formulation. The precursor formulation comprises at least one structural compound, preferably vinyl sulfone (acrylated branched) poly (ethylene glycol), and at least one linker compound, preferably a peptide with two cysteines, where said structural compound and said linker compound are polymerizable by a selective reaction between a nucleophile and a conjugated unsaturated bond or group. The precursor formulation is in the form of a powder.

HYDROGEL PRECURSOR FORMULATION AND PRODUCTION PROCESS

OF THE SAME

FIELD OF THE INVENTION

The present invention relates to a formulation of the hydrogel precursor, its production process as well as a kit comprising such formulation and a method of production of a hydrogel using such formulation.

BACKGROUND OF THE INVENTION

The three-dimensional cell culture support structure has been recognized to allow for gene expression patterns and other cell activities that more closely mimic living organisms than conventional two-dimensional cell culture in culture dishes.

This has led to the development of novel families of synthetic polymer hydrogels, which are often called artificial ECMs (aECMs), as they mimic many aspects of the extracellular matrix. A major challenge is to provide a chemistry that allows crosslinking of the matrix in the presence of cells or biomolecules as well as stable binding of biomolecules to the matrix itself.

In recent years, different mechanisms have been developed allowing the formation of gels in the presence of cells or biomolecules. For example, mechanisms based on the self-assembly of low molecular weight building blocks such as peptides (Estroff et al .: Water gelation by small organic molecules; Chem. Rev. 2004;

104 (3); 1201-18) or u'reidopyrimidinone (Zhang S .: Fabrication of novel biomaterials though molecular self-assembly; Nat.

Bio-technol. 2003; 21 (10); 1171-8)) and moderate molecular weight amphiphilic block copolymers were proposed (eg, see Hartgerink et al .: Peptide-amphiphile nonofibers: A versail. E scaffold for the preparation of selfassembling materials; Proc. Nat. Acad. Sci USA 2002;

99(8) ; 5133-8) .

WO 00/454808 describes novel biomaterials, especially for the formation of hydrogels, which have a crosslinking chemistry based on a Michael-type addition reaction between a nucleophile and a conjugated unsaturated bond or group, which allows gel formation in the presence of cells or biomolecules. Furthermore, specific signal molecules can be integrated into the gel matrix by a specific reaction.

A major disadvantage of this system is that it relies on manually mixing at least two precursor components together prior to gelation. In practice, the use of multiple component solutions can be a source of error due to unintended variations in i) the re-suspension conditions of the different components in powder form, ii) the mixing ratios of the solutions precursors, thus leading to more complex use and reproducibility problems of gel compositions.

Additionally, the cost of the process of a manufacturing process of a gel system that requires the mixing of various solutions is more expensive than the cost of the process of a manufacturing process of a gel that requires a simple one.

SUMMARY OF THE INVENTION

It is therefore an object of the present invention to avoid the disadvantages of known hydrogel formulations and specifically to provide a hydrogel precursor formulation that is easy to handle and allows the production of hydrogels with highly reproducible compositions. This objective is met with a hydrogel precursor according to claim 1.

BRIEF DESCRIPTION OF THE FIGURES

Additional details and benefits of the present invention will be apparent from the following figures and examples:

Fig. 1: Schematic representation of one embodiment of a manufacturing process of a hydrogel precursor formulation according to the present invention

Fig. 2: Schematic representation of a second embodiment of a manufacturing process of a hydrogel precursor formulation according to the present invention

Fig. 3: Schematic representation of a third embodiment of a manufacturing process of a hydrogel precursor formulation according to the present invention

DETAILED DESCRIPTION OF THE INVENTION

The hydrogel precursor formulation according to the present invention comprises at least one structural compound and at least one ligation compound. The structural compound and the ligation compound are polymerizable by a selective reaction between a nucleophile and a conjugated unsaturated bond or group. The hydrogel precursor formulation is in the form of an unreacted powder.

The formulation has the advantage that the powder can simply be resuspended, preferably in a buffer solution, to start the gelling reaction. Mixing of different components is not required, therefore greatly reducing the probability of erroneous relationships between at least one structural compound and at least one compu bond computation. Thus, this increases the reproducibility of the hydrogels produced from these hydrogel precursors. . Furthermore, the hydrogel precursor of the present invention provides ease of use.

The hydrogel precursor formulation of the present invention is in the form of a powder. The powder can comprise particles that have any size or shape.

Alternatively, the powder can also be provided as a pill or pressed tablet. More preferably, the powder is provided in the form of a stable compact cake, for example, at the bottom - of a container.

The powder is unreacted, which means that almost none of the at least one structural compound has reacted with at least one ligation compound by the selective reaction. Preferably more than 70%, more preferably more than 85%, and more preferably more than

95% of the compounds have not undergone the selective reaction.

Selective reaction is a reaction between a nucleophile and a nucleophilic addition conjugated unsaturated group or bond. Such reactions are also known as Michael-type addition reactions.

The structural compound has a functionality of at least three, but more preferably the structural compound has a functionality of four or more. By functionality is meant the number of reactive sites in a molecule.

The structural compound is preferably selected from the group consisting of oligomers, polymers, natural or biosynthetic proteins or peptides and polysaccharides.

Preferably, the structural compound is a polymer selected from the group consisting of poly (ethylene glycol), poly (ethylene oxide), poly (vinyl alcohol), poly (ethylene-co-vinyl alcohol), poly (acrylic acid), poly (ethylene-co-acrylic acid), poly (ethyloxazoline), poly (vinyl pyrrolidone), poly (ethyl-co-vinyl pyrrolidone), poly (maleic acid), poly (ethylene-co-maleic acid), poly (acrylamide), or blocks of poly (ethylene oxide) -co-poly (propylene oxide) copolymers or mixtures thereof. More preferably, the structural compound is a poly (ethylene glycol), more preferably a poly (ethylene glycol) branched with three, at four or more branches.

The ligation compound has a functionality of at least two and is selected from the group consisting of oligomers, polymers, natural or bio-synthetic proteins or peptides and polysaccharides. or mixtures thereof.

Preferably, the ligation compound is a peptide sequence, more preferably containing an adhesion site, a growth factor binding site, or a protease binding site.

The nucleophile is preferably a strong nucleophile, such as a thiol or a thiol-containing group. The nucleophile could also be any other type of nucleophile known in the art, as long as it is strong enough to undergo the selective reaction, for example, such as an amine. Furthermore, the conjugated unsaturated group is preferably an acrylate, an acrylamide, a quinone, or a vinylpyridinium. More preferably, the unsaturated group is a vinyl sulfone.

Additionally, the hydrogel precursor formulation of the present invention may comprise at least one bioactive compound, preferably comprising a RGD peptide sequence, which is 'conjugatable to the structural compound through a selective reaction between a nucleophile and a bond' unsaturated conjugate or group.

The bioactive compound can comprise an adhesion site, such as the fibronectin RGD sequence or the YISG sequence of. l.aminine; a growth factor binding site, such as a heparin binding site;

a protease binding site or a therapeutically active compound. Preferably, the bioactive compound comprises. a cell adhesion site, more preferably an RGD sequence.

The bioactive compound comprises at least one active group capable of undergoing the self-selective reaction. More preferably, the bioactive compound comprises at least one nucleophilic group, and more preferably a thiol group.

The bioactive compound is conjugatable to the structural compound through a self-selective reaction between a nucleophile and a conjugated unsaturated group or bond. More preferably, this self-selective reaction is the same reaction as the self-selective reaction between the structural compound and the compound of. ligation, especially between the same type of nucleophile and conjugated unsaturated group or bond. Alternatively, the bioactive compound can be conjugated to the structural compound through a self-selective reaction before the polymerization of the ligation compound with the structural compound.

The structural compound is preferably a multi-branched poly (ethylene glycol) (PEG) with functionalized end groups. More preferably, the end groups are functionalized with vinyl sulfone. More preferably, · the structural compound is a PEG-tri (vinyl sulfone) or PEG-tetra (vinyl sulfone). The vinyl sulfone functionalization of the PEG alcohol groups can be carried out with any suitable reaction known in the art. By using a branched PEG with three, four or more branches it is possible to produce structural compounds with a functionality of three, four or more.

Preferably, the ligation comprises at least two nucleophilic groups, preferably thiol groups. Thiols are strong nucleophiles that readily undergo Micháel-like addition reactions with unsaturated bonds or groups at a physiological pH. In addition, thiols are commonly found in biological systems, so their use has no problems regarding toxicity.

The ligation compound is preferably a peptide comprising at least two cysteines, preferably located near the -N- and C-termini of the peptide.

Synthesizing peptides with two or more cysteine ​​residues is straightforward. Furthermore, it is possible to introduce specific protease sites into the peptide in order to produce degradable hydrogels, for example for in-vivo use. Additionally, by varying the amino acids close to cysteines, it is possible to change the p'Ka value of the thiol group.

Preferably, the cysteines are located in the N- and C-termini of the peptide, resulting in peptides with the

NOT:

1), structure H₂N-CXXXXXXXXC-COOH (SEQ ID preferably with an N-acetylated term, Ac; and a term

C amidated, NH2; where C is the letter representation of cysteine ​​and X represents any amino acid except cysteine. The peptide can be of any length, so the number of X (X_n) can be any number.

Preferably, the peptide · is 16 amino acids in length. Alternatively, the cysteines can be located in one or more amino acids far from the N- terminus or

C-, for example, resulting in peptides with the general structure H₂NX_mCX_nCX_p-COOH (SEQ ID NO: 2), where m, n and p can be any integer, including zero.

More preferably, the ligation compound is a peptide with the sequence NH₂-GCRE-XXXXXXXX-ERCG-COOH (SEQ ID

NO: 3). Glycine (G) serves as a spacer, Arginine (R) increases the reactivity of the thiol group of nearby cysteine, and glutamic acid (E) improves the solubility of the peptide in aqueous solutions.

More preferably, the sequence of the ligation compound is NH₂-GCRE-GPQGIWGQERCG-COOH [SEQ ID NO: 4] or NH₂GCREGDQGIAGFERCG-COOH [SEQ 'ID NO: 5], again preferably with an acetylated N-terminus and an amidated C-terminus.

The peptides for the ligation and bioactive compounds should be synthesized and processed in acidic solvents, more preferably in solutions containing trifluoroacetate (TFA). The residual TFA bound to the peptide powder after the peptide synthesis has the effect of lowering the pH of a suspension of water containing the respective peptide below 4.

The structural compound and / or the ligation compound are selected in such a way that the reaction rate of the selective reaction between the structural compound and the ligation compound is hampered or highly reduced under mixing conditions. Preferably, the reaction rate is highly reduced to a pH of 4 or below compared to a pH of 7 or above.

A selection of the compounds in this manner enables a precursor formulation to be provided which will readily undergo a gelation reaction under physiological conditions, but which will allow their preparation under conditions such that no or almost no selective reaction occurs.

Preferably, the structural and / or ligation compound are selected such that the reaction rate of the self-selective reaction is at least twice as fast at a pH of 7.5 compared to a pH of 7.0.

Another object of the present invention is to provide a production process for a hydrogel precursor formulation. This problem is solved by a conformity process as claimed in claim 8.

. 12

The process includes the stages of:

- providing a first solution Ά comprising at least one structural compound;

- providing a second solution B comprising at least one ligation compound;

- mix solutions A and B; and

- lyophilization of the resulting precursor solution.

At least one structural compound and at least one ligation compound are polymerizable by a selective reaction between a nucleophile and a conjugated unsaturated bond or group. Both solutions A and B are mixed under conditions that stop the selective reaction.

This process enables the production of a hydrogel precursor formulation in the form of a powder comprising both a structural compound and the ligation compound. The mixing conditions have to be selected in such a way that the auto-selective reaction is stopped. This means that the reaction rate is low enough that a very large fraction of the compounds in both solutions A and B does not react through the self-selective reaction prior to lyophilization. Preferably more than 70%, more preferably more than 85%, more preferable more than 90% of the molecules in both solutions have not undergone the selective reaction prior to the lyophilization step.

Mixing conditions should be selected by adjusting the pH, concentration of different compounds, process time, temperature, or solvent conditions.

Most preferably, mixing is carried out at a pH of or less. Especially when a thiol is used as a nucleophile, a pH of 4 or less sufficiently stops the self-selective reaction. Mixing is preferably done at room temperature.

It is important that solution A is added to the solution

B, since the pH of solution A is around 7, while the pH of solution B is below 4. If solution B was added to solution A, the self-selective polymerization reaction would start during the step mixing. When Solution A is added to Solution B, the pH of the resulting solution will always be less than 4, thus stopping the reaction. .

Solution A preferably comprises 5-10% w / v of at least one structural compound. More preferably, Solution A comprises 7.5% w / v of at least one structural compound.

Additionally, solution B preferably comprises

0.1-2% w / v of at least one ligation compound. More preferably, solution B comprises 1% w / v of at least one ligation compound. · This concentration of the ligation compound provides good solubility of the compound in the solution.

Using the concentrations of the structural and ligation compounds as mentioned above for both solutions A and B leads to the formation of a compact powder after the lyophilization step. This compact powder will form a cake-like layer at the bottom of a container, which is favorable.

Additionally, using these relatively low concentrations of both compounds during the production process additionally reduces the likelihood of unwanted premature reactions between structural compounds and ligation. Additionally, material losses in subsequent production stages are reduced with these concentrations compared to high concentrations.

Furthermore, solution A and / or solution B are preferably solutions of at least one structural compound or at least one ligation compound, respectively, in distilled water.

Thus, both compounds are presented in an undamped solution. Due to the adhesion of trifluoroacetic acid to the peptide ligation compound of solution B, the pH of this solution will decrease. This leads to a pH that is preferably less than 4 for the resulting mixture of solutions A and B. More preferably, the pH of the resulting solution is around 3.5.

Alternatively, before mixing with solution B, solution A can be mixed with additional solution C comprising a biologically active compound that is dimerizable with the structural compound by a selective reaction between a nucleophile and a conjugated unsaturated bond or group. This bioactive compound can comprise an 'adhesion site, such as the RGD sequence of fibronectin or the YISG sequence of laminin; a growth factor binding site, such as a heparin binding site; a protease binding site or a therapeutically active compound. Preferably, the bioactive compound comprises a cell adhesion site, more preferably an RGD sequence.

Preferably solution C comprises 0.1 to

10% w / v of the biologically active compound. More preferably, the. solution C comprises between 0.1 and a

5%, more preferably 0.1 to 2% of the biologically active compound.

Variation of the respective amounts of the structural compound in solution A and the biologically active compound in optional solution C as well as the concentration and nature (eg, amino acid sequence) of the ligation compound in solution B allows the production of the hydrogel precursor formulations with different characteristics.

Although the possibilities of varying the concentration of the compounds in each of solution A and B are many, it is preferred that the concentration of the compounds be selected such that the molar ratio of the nucleophile to the conjugated unsaturated group or bond results in optimal physicochemical properties, such as maximum shear modulus and minimum thickening characteristics of. the final gels. Normally the optimal ratio of the nucleophile to the conjugated unsaturated group or bond is within the range of 0.8: 1 to 1.3: 1. This ensures the formation of a hydrogel where almost all the active groups have undergone the selective reaction, so that the side reactions of any of the reactive groups are greatly reduced.

Furthermore, the precursor solution can be filtered before the lyophilization step. The filtration is preferably a sterile filtration. Any of the undissolved compounds as well as bacterial contamination can be removed from the mixture prior to the lyophilization step.

Preferably, the pre-mixed precursor solution is aliquoted and filled into containers, preferably sterile containers, prior to the lyophilization step. This allows the · production of simple containers containing. a defined amount of a hydrogel precursor formulation. The containers can be of any suitable material, such as plastic or glass. The containers are preferably vials.

The containers are preferably filled with sterile nitrogen gas and capped immediately after the lyophilization step. This protects the hydrogel precursor powder from contact with moisture and / or oxygen, which can lead to premature polymerization or oxidation of nucleophiles.

Another object of the present invention is the use of a hydrogel precursor formulation as described herein for the manufacture of a hydrogel.

A further object of the present invention is to provide a simple, to use system for the production of hydrogels with highly reproducible results. This problem is solved with a kit according to claim 20.

The kit of the present invention comprises at least one container filled with a hydrogel precursor formulation as described herein and one container with a reaction buffer. The container preferably contains an amount of precursor formulation powder, which will result in a gel with predefined characteristics when re-suspended with a defined amount of reaction buffer.

The reaction buffer is preferably at a pH above 7. More preferably the reaction buffer is at a pH between 7 and 8. The buffer is preferably HEPES,

<td>preferably</td><td>in concentration 0.3M with pH</td><td>adjusted to</td><td>a</td>

<td>value between 7</td><td>and 8. This allows a</td><td>reaction</td><td>of</td>

<td colspan="2">sufficiently rapid polymerization. An additional object of this</td><td>invention</td><td>is</td>

<td>provide a</td><td>easy to use method for</td><td>produce</td><td>a</td>

<td>hydrogel. East</td><td colspan="2">goal is achieved with a method</td><td>of</td>

<td>compliance with The method</td><td>claim 22. of production of a hydrogel</td><td>understands</td><td>the</td>

stages of:

- Re-suspend a formulation of the hydrogel precursor as described herein in a buffer solution having pH 7, more preferably with a buffer solution having a pH between 7 and 8;

Optionally add a cell culture suspension to the precursor suspension;

Melt a gel precursor with the precursor suspension; and

- Polymerization of the gel precursor for at least 30 minutes, preferably for 30 to 45 minutes, preferably in an incubator at 37 ° C

The hydrogel precursor formulation of the present invention is polymerizable under physiological conditions, which allow the addition, of a cell culture to the precursor suspension so that the cells can be uniformly distributed in the suspension prior to gelation. This would not be easily possible with any other precursor system.

Figure 1 shows a schematic representation of one embodiment of a manufacturing process of a hydrogel precursor formulation according to the present invention. Solution A comprising 7.5% w / v branched PEG with 4 vinyl sulfone functionalized branches is added to solution B comprising 1% w / v of a

<td>peptide sequence</td><td>with</td><td>two</td><td>cysteines,</td><td>. a close to the</td>

<td>terminal C and the other one</td><td>near</td><td>of</td><td>the N, in the</td><td>mixing stage</td>

<td>one · For example, 275</td><td>mL of</td><td>the</td><td>solution to</td><td>comprising 7.5%</td>

w / v functionalized PEG is added to 425 mL of solution B comprising 1% w / v of a ligation peptide.

Solutions A and B are prepared by suspending the respective compounds in distilled water. For solution B, the peptide ligation compound is preferably added to the water in small portions. Mixing is carried out under continuous stirring with a magnetic stirrer at 400 RPM.

The thus obtained precursor solution 4 is subsequently subjected to a sterile filtration step 5, for example, using a Mini Kleenpak filter (PALL Corp.) with a PTFE membrane with an absolute rating of 0.2pm, providing the filtered precursor solution 6. This The solution is then subjected to lyophilization step 7, resulting in the formulation of the hydrogel precursor 8 as a powder. The resulting powder is in the form of a stable compact cake.

Lyophilization step 7 can be carried out by first freezing the solution on a shelf at -50 ° C for 150 min, followed by a first drying step at -10 ° C for 570 min at a pressure of 0.26 mbar. A second drying stage follows at a temperature of 20 ° C for 180 min at a pressure of

0.02 mbar.

In Figure 2 a second embodiment of a manufacturing process of a hydrogel precursor formulation according to the present invention is schematically depicted. In this embodiment, Solution A comprising 7.5% w / v of a branched PEG with 4 vinyl sulfone functionalized branches is mixed with Solution C comprising 2% w / v of a peptide comprising a RGD sequence in the mixing stage. two.

For example, 275mL of Solution A comprising 7.5% w / v of a branched four functionalized PEG is mixed with 5mL of Solution C comprising 2% w / v of a bioactive compound. This solution is then subsequently mixed with Solution B comprising 1% w / v of a peptide ligation comprising two cysteines, one near the terminal

C and the other near terminal N, in the mixing stage

1.

The precursor solution thus obtained 4 is subsequently subjected to a sterile filtration step 5, providing the filtered precursor solution 6. This solution is then subjected to the lyophilization step 7, resulting in the formulation of the hydrogel precursor 8 in the form of a dust.

Figure 3 shows a third embodiment of a manufacturing process of a hydrogel precursor formulation according to the present invention. Solution A comprising 7.5% w / v branched PEG with 4 vinyl sulfone functionalized branches is mixed with Solution B comprising 1% w / v. of a cysteine ​​ligation peptide sequence close to the C and N terminal in mixing step 1. Solutions A and B are prepared by suspending the respective compounds in distilled water. Mixing is carried out under continuous stirring with a magnetic stirrer, preferably at 400 RPM. The precursor solution thus obtained 4 is subsequently subjected to a sterile filtration step 5, providing the filtered precursor solution 6.

This solution is then aliquoted into containers aliquot-processing step 9. Each container may contain only a small amount, preferably

0.3-0.4 mL. The containers are sealable and are preferably made of glass. The precursor solution processed by aliquots 10 is lyophilized in lyophilization step 7 to produce the precursor formulation powder 8.

NOVELTY OF THE INVENTION

Having described the present invention, it is considered as a novelty, and therefore the content of the following is claimed as property: