WO2025136278A1 - A method comprising the layer-to-layer cross-linking processes for obtaining hyaluronic acid compositions - Google Patents

Abstract

The invention relates to a cross-linking method for the improvement of biomechanical strength and rheological properties of hyaluronic acid-containing gels contained in the compositions used in, including but not limited to, intraarticular, intradermal and ophthalmic applications, and a composition containing hyaluronic acid obtained as a result of the aforementioned processes.

Description

A METHOD COMPRISING THE LAYER-TO-LAYER CROSS-LINKING PROCESSES FOR OBTAINING HYALURONIC ACID COMPOSITIONS

TECHNICAL FIELD

The invention relates to a cross-linking method for the improvement of biomechanical strength and rheological properties of hyaluronic acid-containing gels contained in the compositions used in, including but not limited to, intraarticular, intradermal and ophthalmic applications, and a composition containing hyaluronic acid obtained as a result of the aforementioned processes.

PRIOR ART

Hyaluronic acid (abbreviated as HA) is a polyanionic, unsulfated glycosaminoglycan that occurs naturally from N-acetyl-D-glucosamine and beta-glucoronic acid. This substance, with an average molecular weight in the range of 0.1 -10 million Daltons, has a special rheological characteristic due to its high molecular weight, polyanionic character and non-branching chain structure. The volume of the molecule increases about 10000 times when it is hydrated compared to the dry state. HA is the most important basic element of the extracellular matrix in connective tissue. It is present in the vitreous fluid of the eye, hyaline cartilage, joint fluid, dermis and epidermis. It increases volume in the vitreous fluid, acts as a lubricant in tendons and muscles and lubricates the joints, increases the strength of the spine and established the relationship between mother and fetus in the umbilical cord. It also has regulatory roles in cell motility, cellular proliferation, morphogenesis, embryonic development, cancer metastasis, and inflammation.

HA's viscoelastic properties, biocompatibility and non-immunogenicity of HA have enabled it to be used in a number of clinical applications as supplementation of joint fluid in joint arthritis, as viscoelastic auxiliary material in eye surgery, and as facilitator in healing and regeneration of surgical wounds. Recently, HA has also been studied as a drug delivery agent for various routes of administration, including ophthalmic, nasal, pulmonary, parenteral, and topical. The biggest disadvantage of HA, as is known in the art, is that it is rapidly degraded by a family of enzymes called hyaluronidase in the tissue. In order to prevent this, modifications are made with many different chemical compounds that increase the life of HA in the tissue. The most preferred method is to cross-link HA polymer chains to each other with the help of synthetic agents to give more enzymatic resistance. In these methods, at least one of the group of methacrylamide, hydrazide, carbodiimide, divinyl sulfone (abbreviated as DVS), 1 ,4-butanediol diglycidyl ether (abbreviated as BDDE) and polyethylene glycol) diglycidyl ether is used as a cross-linking agent.

BDDE compound is the cross-linking agent used most commonly to eliminate the mentioned technical drawbacks of HA. BDDE is a biodegradable substance with less toxicity than other cross-linking agents. Although BDDE has been proven safe for use over a long period of time, the cross-linking agents used are reactive agents that can be cytotoxic, and in some cases mutagenic. Therefore, the concentration of BDDE that may be present as residue in the final resulting product is limited by the FDA to <2 ppm (two parts per million).

In the patent publication no. JP6479783 B2 document, HA and BDDE, which is used as a cross-linker, were first dissolved in NaOH solution. It was then held at 50 °C for 2 hours for a cross-linking reaction.

In the patent publication no. US10328180 B2 document, HA and BDDE, which is used as a cross-linker, were first dissolved in NaOH solution. It was then held at 50 °C for 2.5 hours for a cross-linking reaction.

In the patent publication no. US20220370573 A1 document, HA and BDDE, which is used as a cross-linker, were first dissolved in NaOH solution. It was then held at 60 °C for 45 minutes for a cross-linking reaction.

In the patent publication no. EP3218023 B1 document, HA and BDDE, which is used as a cross-linker, were first dissolved in NaOH solution. It was then held at 33.33 °C for 4 hours for a cross-linking reaction. In the relevant technical field, research and development activities are carried out to make improvements in the biomechanical strength and rheological properties of HA used in many different functions.

BRIEF DESCRIPTION OF THE INVENTION

In order to improve the biomechanical strength and rheological properties of HA, crosslinking processes are carried out using various cross-linking agents in the art. The most commonly used agent for cross-linking HA in the art is BDDE.

BDDE, defined as diepoxide cross-linker, reacts with the 6-hydroxyl group of HA to form ether bonds under alkaline conditions. This reaction is mostly carried out by temperature catalysis. Cross-link modification is achieved by forming an ether bond between the 6- hydroxyl group on the linear HA molecules and the epoxy rings on the BDDE molecule. Thus, it forms two new ether bonds between the HA molecules of linear structure and the BDDE molecule. The concentration of HA and BDDE used in the cross-linking reaction, temperature, reaction time, and the molarity of the sodium hydroxide (abbreviated as NaOH) solution used to dissolve HA affect the specifications of the final finished product, especially Theologically. By changing these parameters, cross-linked HA hydrogels with desired rheological properties can be obtained.

Cross-linking processes of HA with the present methods and compounds in the relevant technical field are considered insufficient for the current technology. In particular, it has been determined that the HA obtained as a result of cross-linking processes does not have sufficient biomechanical properties.

Another problem is that HA does not have sufficient enzymatic and chemical stability as a result of cross-linking processes. This situation causes HA to show insufficient resistance against such effects.

Furthermore, as a result of cross-linking processes, HA has structural inconsistency and an inhomogeneous structure.

In order to eliminate all the technical drawbacks mentioned here, the present inventors aim to introduce innovations in cross-linking processes for HA. In this respect, the present invention reveals a method for carrying out high-efficiency cross-linking processes of HA in compositions used in various applications.

The primary object of the present invention is to introduce a cross-linking process for HA in which high biomechanical strength and desired rheological properties can be achieved for the relevant technical field.

Another object of the present invention is to introduce a cross-linking process for HA in which enzymatic and chemical stability can be achieved for the relevant technical field.

DETAILED DESCRIPTION OF THE INVENTION

In this detailed description, the subject of the invention relates to a cross-linking method for HA contained in components suitable for use in many different technical fields, and is described by way of non-limiting examples only for a better understanding of the subject matter.

HA, as is known, is rapidly degraded by a family of enzymes called “hyaluronidase” in the tissue. In order to prevent this, cross-linking processes of HA are carried out with cross-linking agents.

In this invention, the field of use for the compositions containing HA is not specified and is not limited to the field of use for the scope of protection of the invention. Essentially, compositions containing HA have been discussed in intraarticular applications, intradermal applications, and ophthalmic applications, however, any technical field in which HA subjected to cross-linking processes as characterized by the invention in accordance with the state of the art can be used is considered to be a suitable technical field for the subject of the present invention.

In this invention, the cross-linking process is carried out in at least two stages. The first of these, the first cross-linking processes, is carried out with the cross-linking agent(s) commonly used in the art.

Since it is mentioned to be in at least two stages, as can be expected, more than one cross-linking process will be carried out for HA. In order to make the process steps more clearly understandable, the cross-linking processes applied in the art will be referred to as the first cross-linking process and the additional cross-linking process as characterized by this invention will be referred to as the second cross-linking process.

Accordingly, in the present invention, the process of cross-linking is carried out in stages. Firstly, the first cross-linking agent and HA are added in certain ratios (by weight) at a certain temperature control and the first cross-linking processes are carried out for certain durations. Subsequently, a second cross-linking process will be carried out at another stage. Similarly, the second cross-linking processes are also carried out for certain durations with the addition of certain ratios (by weight) at a certain temperature control according to the cross-linker to be used. On the other hand, the subject of the present invention is also referred to as layer-to-layer cross-linking technology.

In the invention, for the first cross-linking process, at least one of the group of methacrylamide, hydrazide, carbodiimide, divinyl sulfone, 1 ,4-butanediol diglycidyl ether, and polyethylene glycol) diglycidyl ether is used as the cross-linking agent. If preferred, it may be the case that the cross-linking agent is a mixture containing more than one component to be selected from this group.

In a preferred embodiment, in the first cross-linking process, the cross-linking agent is dissolved in a solution. Among said solution components, at least one of the components of sodium hydroxide, potassium hydroxide, ammonium hydroxide, triethyl amine, pyridine is used. In a preferred embodiment sodium hydroxide (abbreviated as NaOH) is present as a basic component. The cross-linking agent is present to provide properties such as homogeneous distribution in solution, carrying out a controlled reaction, and increasing the interaction between chains.

One of the important parameters for the first cross-linking process is the amount of crosslinking agent(s) to be used. Accordingly, the ratio of the amount of cross-linking agent(s) to the amount of HA by weight (m_Cross-iinker/mhyaiuronicacid) is at a value in the range of 0.01 and 0.20.

As is known in the art, the first cross-linking process is temperature catalyzed. Accordingly, it is critical to carry out the processes at appropriate temperature values. In this invention, the ambient temperature for the first cross-linking processes is a value between 35 to 50 °C.

Another important parameter is the cross-linking duration. The cross-linking duration for the first cross-linking is between 75 to 100 minutes. The reason why these durations are preferred is that the concentration of the cross-linking agent(s) present in the medium decreases in the medium and the agent(s) do not affect the reaction rate.

The first cross-linking process carried out with BDDE cross-linking agent was carried out under the same conditions (NaOH concentration, temperature and reaction time) to fulfill the above mentioned conditions for Sample-1 , Sample-2 and Sample-3 studies. In these studies, the only variable parameter is the ratio of the amount of cross-linking agent(s) to the amount of HA by weight (m_Cross-iinker/mhyaiuronic acid). For Sample-1 , Sample-2 and Sample-3 studies, the varying m_Cross-iinker/m_hyaiuronic acid ratio and the rheological values obtained are given in Table 1 .

Table 1. m_Cross-iinker/m_hyaiuronic acid ratios and rheological values according to first crosslinking model

As mentioned earlier, the cross-linking processes of the present invention are carried out in stages. The innovative aspect of the invention is that after the first cross-linking process has been carried out, at least one additional cross-linking process is also carried out.

As is known, a cross network is formed by the reaction between the cross-linking agent(s) used for the first cross-linking processes and HA. During these cross-linking reactions, some reactions occur that do not take place between HA and the first crosslinking agent(s). Due to the reactions that do not take place, some hydroxyl groups remain free after the first cross-linking process of HA. The reason for this is that a modification occurs as a result of the first cross-linking agent(s) reacting with HA at one end. This modification is also referred to as "pendant".

The object of the present invention is to create a secondary cross-link network by interacting with the free hydroxyl groups remaining from the cross-linking agent(s) that react with HA at one end as a result of the first cross-linking processes. In the present invention for this purpose, the second cross-linking processes are carried out as a stagewise process step.

In the invention, for the second cross-linking processes, at least one cross-linking agent which can interact with HA and has two functional groups is used as the cross-linking agent.

In this invention, at least one of the compounds of methacrylamide, hydrazide, carbodiimide, divinyl sulfone, 1 ,4-butanediol diglycidyl ether, and polyethylene glycol) diglycidyl ether is used as the second cross-linking agent. In this way, the second crosslinking processes can achieve their goal, and both functional ends of the cross-linking agent can interact with the free HA hydroxyl groups.

In a preferred embodiment, in the second cross-linking process, the cross-linking agent is dissolved in a solution. Among said solution components, at least one of the components such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, triethyl amine, pyridine is used. In a preferred embodiment sodium hydroxide (abbreviated as NaOH) is present as a basic component. The cross-linking agent is present to provide properties such as homogeneous distribution in solution, carrying out a controlled reaction, and increasing the interaction between chains.

It is critical that the ratio of the amount of cross-linking agent which will be used in the second cross-linking processes to the amount of HA by weight (m_Cross-iinker/mhyaiuronic acid) is at a value in the range of 0.01 and 0.15. In this way, it is possible to carry out the second cross-linking processes of the cross-linking agent(s) by interacting with HA without leaving any free hydroxyl groups on the HA molecules.

A preferred embodiment of the invention is the use of cross-linker amounts in crosslinking prOCeSSeS SUCh that the ratiO Of m_Second cross-linking agent^first cross-linking agent iS 1 .1. The second cross-linking processes are also temperature-catalyzed. Accordingly, it is critical that the reaction medium is at a value in the range of 35 to 50 °C for the interaction of HA with the second cross-linking agents targeted in the present invention.

The reaction times determined for carrying out the second cross-linking processes are also critical. Certain durations must elapse for the second cross-linking agent(s) to interact with HA. This determined duration is a value in the range of 75 to 100 minutes.

The rheological results of the samples obtained in the first cross-linking study are given in Table 1 . The samples obtained after the first cross-linking process were subjected to a second cross-linking process. Herein, samples were created by applying the same parameters as in the first cross-linking process, and the second cross-linking process was performed on these samples with the same reaction parameters (NaOH concentration, temperature, and reaction time). In the second cross-linking process Sample-4, Sample-5, and Sample-6 were prepared such that the ratio of m_second cross-linking agent ■ ITIfirst cross-linking agent (1 :1 ) is the same. For these studies, m cross-linker/lTlhyaluronic acid ratios and rheological values of gels obtained as a result of cross-linking are given in Table 2.

Table 2. m_Cross-iinker/mhyaiuronicacid ratios and rheological values according to second crosslinking model

Taking all these studies into account, the traditional HA cross-linking method (addition of cross-linking agent in one step) studies were carried out. In this study, Sample-7, Sample-8 and Sample-9 gels were created in such that all parameters (NaOH concentration, temperature, reaction time and m_Cross-iinker/mhyaiuronicacid ratio) are the same in the cross-linking process. As a result of this study, m cross-linker/lTlhyaluronic acid ratiOS and rheological values of the gels are given in Table-3.

Table 3. m_Cross-iinker/mhyaiuronicacid ratios and rheological values according to traditional HA cross-linking model

As seen in the results of the studies, when the layer-to-layer cross-linking technology and traditional HA cross-linking (one-step cross-linking agent addition) were compared under the same conditions, an increase in the biomechanical strength and rheological values of HA was observed with the new method.

All samples obtained in the invention (Sample -1 to Sample-9) are formulated as crosslinked HA in phosphate buffer solution at a value in the range of 10 to 30 mg/mL and Lidocaine.HCI at a value in the range of 1 to 5 mg/mL.

In another aspect, the invention relates to the provision of HA-containing composition obtained by applying layer-by-layer cross-linking processes characterized by the invention.

There are no major changes in the biomechanical strength values of HA subject to and obtained by the technical teachings characterized by the invention compared to the first cross-linking processes. Accordingly, it can be contemplated that the biomechanical strength values of the obtained HA will remain the same after the second cross-linking process. However, it has been determined that there is an increase of up to 60% in the elastic module values of the HA obtained. The reason for the increase in these values is that there is cross-linking between unreacted linear HA molecules in the solution medium, linear HA molecules react with partially cross-linked HAs (cross-linked HA hydrogel formed as a result of the reaction in the first stage) in the medium, and there is cross-linking between partially cross-linked HAs in the medium again. Considering the results of the elastic module of HA obtained, it can reach 500 Pa values at 1 Hz. In the same way, it was determined that increases of up to 35% were observed in the complex viscosity values of the HA obtained. The complex viscosity value of the obtained HA can reach up to 250 Pa.s.

Accordingly, it has been determined that HA, which has been subjected to layer-to-layer cross-linking processes as characterized in the invention, reaches higher elastic values and high complex viscosity values without sacrificing biomechanical strength values.

It has been determined that HA, which has been subjected to layer-to-layer cross-linking processes as characterized in the invention, has achieved improvements in its enzymatic and chemical stability properties.

Within the scope of the present invention, the HA-containing composition obtained by layer-to-layer cross-linking technology is a viscoelastic gel that can be used in the field of orthopedics for synovial fluid support, reduction of inflammation due to osteoarthritis, reduction of cartilage surface destruction, and regeneration of cartilage surface.

Within the scope of the present invention, the HA-containing composition obtained by layer-to-layer cross-linking technology is a viscoelastic gel to be used in the field of dermatology to protect the natural balance of the dermis in the process of photo-aging, to maintain natural remodeling by increasing collagen synthesis and reducing its destruction, to minimize oxidative stress due to photoaging and inflammation, and to correct cosmetic defects.

Within the scope of the present invention, the HA-containing composition obtained by layer-to-layer cross-linking technology can be used in drug delivery systems in addition to dermatology and orthopedic applications. Layer-to-layer cross-linking technology can facilitate the controlled release of the drug active ingredient.

The scope of protection of the invention is specified in the appended claims and cannot be limited to what is described for illustrative purposes in this detailed description. It is clear that a person skilled in the art can produce similar embodiments in the light of what is explained above, without deviating from the main theme of the invention.

Claims

1 . A cross-linking method for improving the biomechanical and rheological properties of HA contained in the components suitable for use in many different technical fields, characterized in that it is carried out in at least two stages and comprises the following process steps,

- subjecting HA to the first cross-linking processes, wherein for said first cross-linking processes the ratio of m_Cross-iinker/mhyaiuronicacid by weight is at a value between 0.01 to 0.2,

- subjecting HA to second cross-linking processes in order to ensure interaction with hydroxyl groups on the linear structured HA in that have not reacted as a result of first cross-linking processes and/or the free hydroxyl groups remaining from the cross-linking agent(s) reacting with HA at one end wherein for said second cross-linking processes, the ratio of m_Cross-iinker/mhyaiuronic acid by weight is at a value between 0.01 to 0.15.

2. A method according to Claim 1 , characterized in that, in the first cross-linking process, at least one of the group of methacrylamide, hydrazide, carbodiimide, divinyl sulfone, 1 ,4-butanediol diglycidyl ether, and polyethylene glycol) diglycidyl ether having at least two functional groups is used as the cross-linker.

3. A method according to one of the preceding claims, characterized in that the crosslinker to be used in the first cross-linking process is contained in a solution.

4. A method according to Claim 3, characterized in that said solution is at least one of the group of sodium hydroxide, potassium hydroxide, ammonium hydroxide, triethyl amine, and pyridine.

5. A method according to one of the preceding claims, characterized in that the first cross-linking processes are carried out at a value between 35 to 50 °C.

6. A method according to one of the preceding claims, characterized in that the first cross-linking processes are carried out for a duration between 75 to 100 minutes.

7. A method according to one of the preceding claims, characterized in that at least one of the group of methacrylamide, hydrazide, carbodiimide, divinyl sulfone, 1 ,4- butanediol diglycidyl ether, and polyethylene glycol) diglycidyl ether having at least two functional groups is used as the cross-linker to be used in the second crosslinking process.

8. A method according to one of the preceding claims, characterized in that the crosslinker to be used in the second cross-linking process is contained in a solution.

9. A method according to Claim 8, characterized in that said solution is at least one of the group of sodium hydroxide, potassium hydroxide, ammonium hydroxide, triethyl amine, and pyridine.

10. A method according to one of the preceding claims, characterized in that in the crosslinking processes, the processes are carried out such that IT1 second cross-linking agent-^first cross-linking agent ratio is 1 :1.

11 . A method according to one of the preceding claims, characterized in that the second cross-linking processes are carried out at a value between 35 to 50 °C.

12. A method according to one of the preceding claims, characterized in that the second cross-linking processes are carried out for a duration between 75 to 100 minutes.

13. A method according to one of the preceding claims, characterized in that the HA obtained as a result of cross-linking processes is allowed to interact with Lidocaine.HCI in a phosphate-containing solution.

14. A method according to Claim 13, characterized in that HA is present at a value in the range of 10 to 30 mg/ml in phosphate-containing solution and Lidocaine.HCI at a value in the range of 1 to 5 mg/ml in phosphate-containing solution.