HK1205750B - Method of preparing a composition based on hyaluronic acid - Google Patents

Description

Method for preparing hyaluronic acid-based compositions

Technical Field

The present invention relates to a method for preparing a composition, such as a gel, based on a polysaccharide, such as hyaluronic acid, to a composition as such, to a kit comprising a syringe and said composition, and to the use of said composition as a dermatological filler.

Background Art

Gels, such as hydrogels based on polysaccharides and water, are known to be used as dermal fillers. Such gels are typically prepared by chemical crosslinking of various polysaccharides in an aqueous medium. Suitable polysaccharides are, for example, based on hyaluronic acid, as it is present in the same or similar compositions in various organisms. Hyaluronic acid, for example, is a major component of the skin, where it participates in tissue repair. Therefore, it offers minimal side effects and allows for safe application.

EP 1818344 relates to a method for preparing a cross-linked hyaluronic acid gel, comprising stirring and mixing a mixture containing 10 w/v% or more of hyaluronic acid, a cross-linking agent and water under acidic or alkaline conditions.

EP 2 054 039 (WO 2008/018796) relates to a viscoelastic hydrogel composition comprising first and second microparticles capable of interacting with each other via stereocomplex interactions, wherein the first microparticles comprise a cross-linked first hydrophilic polymer, the first microparticles comprise an external graft of a first oligomer or co-oligomer containing a first chiral site, the first chiral site comprising a first chiral monomer, and wherein the second microparticles comprise a cross-linked second hydrophilic polymer, the second microparticles comprise a second oligomer or co-oligomer containing a second chiral site, the second chiral site comprising a second chiral monomer having a chirality opposite to that of the first chiral monomer, wherein the first chiral site and the second chiral site interact non-covalently with each other. The hydrophilic polymer may be hyaluronic acid.

EP 2 178 923 (WO 2009/018076) relates to a process for preparing cross-linked hyaluronic acid, which process comprises contacting hyaluronic acid with a polyethylene glycol-based cross-linking agent.

WO 2011/119468 relates to a hydrogel for soft tissue augmentation comprising a cross-linked biocompatible polymer having zero-length cross-links and optionally at least one further active ingredient embedded in the cross-linked biocompatible polymer.

EP 2 152 329 (WO 2008/068297) relates to a subcutaneously or intradermally injectable implant in the form of a single-phase hydrogel comprising a gel consisting of cross-linked hyaluronic acid and one of its physiologically acceptable salts.

EP 2 170 961 (WO 2009/021526) relates to hyaluronic acid dispersions for use in aesthetic medicine and plastic surgery, wherein the dispersed phase comprises particles made of cross-linked hyaluronic acid and the continuous phase comprises substantially linear hyaluronic acid.

EP 1 699 500 (WO 2005/067994) relates to a hyaluronic acid composition comprising cross-linked, water-insoluble, hydrated hyaluronic acid gel particles. The composition is useful for augmenting tissue in a subject in need of tissue augmentation, and relates to a method for stabilizing cross-linked HA, the method comprising hydrating the water-insoluble, dehydrated cross-linked HA with a physiologically compatible aqueous solution containing a local anesthetic, wherein the storage modulus value G' of the stabilized composition is at least about 110% of the G' value of the unstabilized composition, and to the stabilized HA composition.

WO 2010/015900 relates to soft tissue fillers, such as dermal and subdermal fillers, based on hyaluronic acid and pharmaceutically acceptable salts thereof, wherein the hyaluronic acid-based composition may include a therapeutically effective amount of at least one anesthetic, such as lidocaine. The hyaluronic acid-based composition comprising lidocaine has enhanced stability relative to conventional compositions comprising lidocaine, for example, when subjected to sterilization techniques or stored for extended periods. Methods and processes for preparing such hyaluronic acid-based compositions are also provided.

FR 2 919 999 relates to a cosmetic or pharmaceutical composition comprising hyaluronic acid and divalent cations. Said composition can be used for treating wrinkles.

EP 2 254 584 (WO 2009/098127) relates to biocompatible injectable products capable of releasing zinc and/or at least one zinc-in-form sugar salt, to compositions containing said products, and to their use, in particular for filling or increasing the volume of biological tissue or for replacing or supplementing biological fluids.

EP 2 155 212 (WO 2008/139122) relates to a combination of hyaluronic acid and at least one hyaluronic acid degradation inhibitor, which is intended in particular for use in dermatology and plastic surgery in humans.

EP 0 839 159 B1 discloses a method for preparing a cross-linked biocompatible polysaccharide gel composition. The method comprises cross-linking a polysaccharide in the presence of a multifunctional cross-linking agent, wherein a viscoelastic gel is formed.

EP 1 711 552 B1 relates to a method for producing a biocompatible cross-linked gel comprising the steps of cross-linking a biocompatible polymer, diluting the cross-linked polymer with a non-cross-linked polymer, and terminating the cross-linking reaction.

EP 0 466 300 B1 relates to a method for obtaining a biocompatible viscoelastic gel suspension, said method comprising mixing a biocompatible gel comprising cross-linked hyaluronic acid and a second polymer which may be, for example, hyaluronic acid, to form a two-phase mixture, and to a gel as such.

Purpose of the Invention

One object of the present invention is to provide a method for preparing a polysaccharide-based composition, such as a gel, which can be used as a dermal filler. The composition should have excellent stability, i.e., it should not change its properties, in particular its viscoelastic properties, after application, and it should be tailored to dermatological requirements. In addition, the gel should have excellent skin tissue compatibility.

Summary of the Invention

This object is achieved with a method for preparing a composition, such as a gel, comprising a crosslinked first polymer, optionally a second polymer which may be crosslinked or non-crosslinked, and water, wherein the first and second polymers are selected from polysaccharides; and with a composition prepared according to the above method.

In particular, according to a first aspect, the present invention relates to a method for preparing a composition, such as a gel, comprising a cross-linked first polymer, optionally a second polymer, which may be cross-linked or non-cross-linked, and water, wherein the first and second polymers are selected from polysaccharides, the method comprising at least steps (i) to (iv):

(i) cross-linking a mixture comprising the first polymer and water;

(ii) after the cross-linking in step (i), terminating the cross-linking;

(iii) optionally mixing the product obtained in step (ii) with the second polymer;

(iv) dialyzing the product obtained in step (ii) or step (iii).

In one embodiment, the presence of the second polymer is mandatory. In this embodiment, the process requires mixing the product obtained in step (ii) with the second polymer according to step (iii).

In one embodiment, the presence of the second polymer is not essential. In this embodiment, the process does not require mixing the product obtained in step (ii) with the second polymer according to step (iii).

In one embodiment, the first and second polymers may be the same.

In one embodiment, the first and second polymers may be different from each other.

In one embodiment, the first polymer is cross-linked and the second polymer is non-cross-linked.

In one embodiment, the first and second polymers are selected from hyaluronic acid and salts thereof.

In one embodiment, the first and second polymers are selected from hyaluronic acid or a salt thereof.

In one embodiment, the hyaluronate is a sodium salt.

In one embodiment, the first polymer used in step (i) has a molecular weight Mw of from 1.5 MDa to less than 3.5 MDa, or from 2.0 MDa to less than 3.5 MDa, or from 2.5 MDa to less than 3.0 MDa.

In one embodiment, the second polymer used in step (iii) has a molecular weight of at least 3.0 MDa or at least 3.5 MDa.

In one embodiment, the weight of the second composition is less than 5%, or less than 4%, based on the weight of the first composition, for example in the range of 0.01-5%, or in the range of 0.1-4%, or in the range of 0.1-2.5%, or 0.2-2.0%, or 0.5-1.5%.

In one embodiment, the method comprises, after step (iv), a further step (v):

(v) admixing an anesthetic or antiarrhythmic agent, such as lidocaine, lidocaine hydrochloride, lidocaine hydrochloride monohydrate, or tetracaine, to the product obtained in step (iv).

In one embodiment, the method comprises, after step (iv), or after step (v), a further step (vi):

(vi) filling the product obtained in step (iv) or step (v) into a syringe and sterilizing it.

In one embodiment, the mixture in step (i) further comprises an alkaline phosphate buffer.

In one embodiment, the second polymer is provided in step (iii) in a mixture with a phosphate buffer.

In one embodiment, the anesthetic or antiarrhythmic agent, such as lidocaine or tetracaine, provided in step (v) is provided in a mixture with a phosphate buffer.

In one embodiment, the reaction temperature of step (i) is 0-40°C, such as 15-40°C; or 25-35°C, or 25-30°C, or 30 to 35°C.

In one embodiment, the reaction temperature of step (ii) is 0-30°C, such as 0-10°C; or 3-7°C.

In one embodiment, the reaction temperature of step (iii) is 0-30°C, such as 0-10°C; or 3-7°C.

In one embodiment, the reaction temperature of step (iv) is 0-30°C, such as 0-10°C; or 3-7°C.

In one embodiment, in step (i), diglycidyl ether is used as a cross-linking agent.

In one embodiment, in step (i), 1,4-butanediol diglycidyl ether (BDDE) is used as a cross-linking agent.

In one embodiment, step (ii) comprises step (ii.1):

(ii.1) The product obtained in step (i) is subjected to acid treatment.

In one embodiment, step (ii) comprises steps (ii.1) and (ii.2):

(ii.1) subjecting the product obtained in step (i) to acid treatment;

(ii.2) extruding the product obtained in step (ii.1); or

Extruding the product obtained in step (ii.1) through a sieve; or

The product obtained in step (ii.1) is pressed through a sieve having a mesh size in the range of 500-600 μm.

In one embodiment, the dialysis step according to step (iv) further comprises steps (iv.1) to (iv.3): (iv.1) extruding the product obtained in step (iii) through a first sieve, and subsequently extruding the extruded product of the first sieve through a second sieve, wherein the mesh size of the second sieve is smaller than the mesh size of the first sieve; or

extruding the product obtained in step (iii) through a first sieve, and then extruding the extruded product of the first sieve through a second sieve, and then extruding the extruded product of the second sieve through a third sieve, wherein the mesh size of the second sieve is smaller than the mesh size of the first sieve, and the mesh size of the third sieve is smaller than the mesh size of the second sieve;

(iv.2) loading the product obtained in step (iv.1) into a dialysis membrane;

(iv.3) The filled membrane obtained in step (iv.2) is placed in the dialysis solution.

In one embodiment, the dialysis step according to step (iv) further comprises steps (iv.1) to (iv.3):

(iv.1) extruding the product obtained in step (iii) through a first sieve having a mesh size of 325-425 μm; and then extruding the extruded product of the first sieve through a second sieve having a mesh size of 175-225 μm; and then extruding the extruded product of the second sieve through a third sieve having a mesh size of 110-170 μm;

(iv.2) loading the product obtained in step (iv.1) into a dialysis membrane having a molecular weight cut-off of 12,000-14,000 Da;

(iv.3) The filled membrane obtained in step (iv.2) is placed in the dialysis solution.

Any one of the above-described embodiments may be combined with at least one other embodiment selected from the above-described embodiments.

According to a second aspect of the present invention, the present invention relates to a composition, such as a gel, comprising a cross-linked first polymer, optionally a second polymer, which may be cross-linked or non-cross-linked, and water, wherein the first and second polymers are selected from polysaccharides, and the composition is obtainable by the method according to the first aspect or by the method according to the first aspect and any embodiment or any combination of at least two embodiments defined therein.

According to a third aspect of the present invention, the present invention relates to a kit comprising a syringe and a composition according to the second aspect, or a composition prepared according to the method of the first aspect.

According to a fourth aspect of the present invention, the present invention relates to the use of the composition according to the second aspect in cosmetic applications, or to the use of the composition prepared by the method according to the first aspect in cosmetic applications.

In one embodiment, the composition is used as a dermal filler.

According to a fifth aspect of the present invention, the present invention relates to a composition, such as a gel, according to the second aspect for use as a medicament.

Detailed Description of the Invention

According to said first aspect, the present invention relates to a process for preparing a composition comprising a crosslinked first polymer, an optionally second polymer which may be crosslinked or non-crosslinked, and water, wherein said first and second polymers are selected from polysaccharides, said process comprising at least steps (i) to (iv):

(i) cross-linking a mixture comprising the first polymer and water;

(ii) after the cross-linking in step (i), terminating the cross-linking;

(iii) optionally mixing the product obtained in step (ii) with the second polymer;

(iv) dialyzing the product obtained in step (ii) or step (iii).

As used in this disclosure, the term "composition" encompasses a product comprising a crosslinked first polymer, optionally a second polymer, which may be crosslinked or non-crosslinked, and water.

In one embodiment, the composition is a gel, such as a hydrogel. As used herein, the term "gel" encompasses products that have both viscous and elastic properties. Thus, the term encompasses viscoelastic products. In popular science, gels are sometimes characterized as jelly-like substances. The viscoelastic properties of a gel can be determined by measuring the loss modulus and storage modulus of the gel.

The ratio of the loss modulus G" to the storage modulus G' can be expressed by the loss factor tanδ = G"/G'. The higher the loss factor, the closer the product's properties are to a Newtonian flow. The viscosity of the product can be expressed by the term η*. Suitable methods for determining tanδ and η* are known in the art.

In the process of the invention, the composition or the composition according to the invention, such as a gel, obligatorily requires the use of a polysaccharide as the first polymer and, if a second polymer is used, also as the second polymer.

As used herein, the term "polysaccharide" includes carbohydrate molecules composed of or comprising repeating monomer units linked together by glycosidic bonds. Generally, the polysaccharide comprises more than 10 monosaccharide units. Examples of polysaccharides are polysaccharides such as starch, glycogen, cellulose, chitin or hyaluronic acid, or mixtures thereof.

In a preferred embodiment, the polysaccharide is hyaluronic acid. The hyaluronic acid may be provided in the form of a salt thereof, such as a sodium salt. A mixture of the acid and a salt thereof, such as a sodium salt, may also be provided.

Therefore, the term "hyaluronic acid" as used herein is a synonym for terms such as "hyaluronan" or "hyaluronate." Hereinafter, hyaluronic acid may be abbreviated to the term "HA."

HA is a non-crosslinked polymer of disaccharides. It can have a length of up to 25,000 disaccharide units. The molecular weight of HA can range from 5,000 to 20,000,000 Da.

HA has a widely recognized meaning in the art. HA is commercially available in grades having different molecular weights (Mw) and/or different molecular weight distributions. It is obtained in a non-crosslinked form and serves as the starting material in step (i) of the process according to the invention.

Step (i)

Step (i) requires crosslinking a mixture comprising the first polymer and water.

The term "crosslinking" as used herein comprises crosslinking at least two different polymer chains of the polysaccharide by a chemical bond or multiple chemical bonds. As a result, the molecular weight of the first polymer increases and thus the viscosity and/or elasticity increases.

In one embodiment, cross-linking is performed by a cross-linking agent.

Suitable cross-linking agents for cross-linking polysaccharides such as hyaluronic acid are known in the art.

In one embodiment, crosslinking agents based on epoxide structures can be used in the process according to the invention.

In one embodiment, diglycidyether is used for cross-linking.

In one embodiment, 1,4-butanediol diglycidyl ether (BDDE) is used for cross-linking. This compound is commercially available.

In one embodiment, the cross-linking agent is used in an amount of 5-15% (volume of cross-linking agent/weight of hyaluronic acid), such as 6-14% (v/w), or 7-12% (v/w).

Advantageously, the temperature of the cross-linking reaction according to step (i) can be controlled.

In one embodiment, the cross-linking according to step (i) is effected at a temperature in the range of 0-40°C.

In another embodiment, the temperature of step (i) is controlled so as to be carried out in the temperature range of 15-40°C.

In one embodiment, the temperature of step (i) is 25-35°C.

In one embodiment, the temperature of step (i) is controlled so that the crosslinking is performed in the temperature range of 25-30°C.

In another embodiment, the temperature of step (i) is controlled so that the cross-linking is carried out at a temperature range of 30-35°C, or from above 30°C to 35°C.

Such a temperature or temperature range can ensure a very homogeneous crosslinking avoiding as far as possible inhomogeneous particles. Furthermore, in one embodiment, the control of the temperature allows a tailored adjustment of the viscoelastic properties of the composition according to the invention.

In one embodiment, if the crosslinking according to step (i) is carried out at a higher temperature, such as in a temperature range from 30°C to 35°C, the resulting composition, such as a gel, has stronger viscoelastic properties than a composition in which the crosslinking is carried out at a lower temperature, such as at a temperature of 25-30°C. This difference can be characterized by measuring the storage modulus and loss modulus of the composition according to methods known in the art. Accordingly, in one embodiment, the appropriate choice of the reaction temperature in step (i) allows the preparation of compositions, such as gels, having different viscoelastic properties.

As used herein, the term "mixture" encompasses a combination of two or more substances that are mixed but not chemically bound to each other. Thus, the term "mixture" refers to a physical combination of the first polymer (which is a polysaccharide) and water. The mixture may be provided in the form of a solution, a suspension, or a colloid.

The first polymer, ie the first polysaccharide, ie HA, typically has a molecular weight Mw in the range of 1.0-4.0 MDa, or from 1.5 MDa to less than 3.5 MDa, or from 2.0 MDa to less than 3.5 MDa. These molecular weight Mw ranges refer to the ranges before cross-linking.

In one embodiment, the first polysaccharide has a molecular weight Mw of from 2.5 MDa to less than 3.0 MDa, also prior to cross-linking.

Water may be provided as tap water, distilled water, or deionized water.

In one embodiment, the mixture used in step (i) additionally comprises a buffer solution.

In one embodiment, the buffer solution is a phosphate buffer solution.

In one embodiment, the phosphate buffer solution is made of sodium chloride, anhydrous disodium hydrogen phosphate, sodium dihydrogen phosphate dihydrate and water.

In one embodiment, the buffer is an alkaline buffer.

In one embodiment, the pH of the buffer is 6.8-7.6, or 7.0-7.4, or 7.1-7.3.

In one embodiment, the pH of the final composition prepared according to the method of the present invention is adjusted to a range of 6.5-7.5, such as 6.7-7.2, or 6.8-7.1 or 6.8-6.9. Such a pH can support the compatibility of the composition with skin tissue. In one embodiment, the buffer is used to adjust the pH.

In one embodiment, step (i) may be achieved by mixing the first polymer, water, the crosslinking agent, and optionally a buffer solution, and stirring the mixture for a predetermined time, wherein the temperature is controlled not to exceed a predetermined set point.

Step (ii)

Step (ii) requires termination of the cross-linking performed in step (i).

Termination of the cross-linking reaction according to step (ii) is imperative since otherwise it is possible to obtain a composition or gel that is too viscous or viscous and elastic to allow a reasonable use as a dermal filler, as long as the gel contains a compound capable of producing cross-linking, such as the cross-linking agent used in step (i), the respective viscous or viscoelastic properties of the composition or gel will not be constant.

Basically, compounds which react with the cross-linking agent and thus inactivate it can be used to terminate the cross-linking reaction.

In one embodiment, since an epoxide-type crosslinking agent is used in step (i), termination of crosslinking may be achieved by adding a compound that cleaves the epoxide moiety so that no further crosslinking with suitable groups in the polysaccharide can occur.

In one embodiment, cleavage of the epoxide and thus termination of the cross-linking reaction in step (i) can be achieved by an acid.Both organic and inorganic acids can be used for termination of cross-linking.

In one embodiment, a mineral acid such as hydrochloric acid may be used.

The compound for terminating the cross-linking reaction may be applied in a buffer solution, such as the buffer solution used in step (i).

Such a solution may be referred to as a “quenching solution.” Accordingly, the termination according to step (ii) may be achieved by quenching the cross-linked mixture obtained according to step (i).

In one embodiment, termination of the cross-linking according to step (ii) is effected at a temperature in the range of 0-30°C.

In another embodiment, the temperature of step (ii) is controlled so as not to exceed 20°C, or 15°C.

In one embodiment, the temperature in step (ii) is 0-10°C, or 3-7°C, such as 5°C.

The termination according to (ii) can be achieved by adding a compound for termination to the mixture according to step (i), and stirring for a predetermined time, for example.

Termination of the crosslinking reaction is important for the process according to the invention since the composition or gel thus obtained has excellent stability, i.e. its properties, in particular its viscosity or its viscoelastic properties, do not change after application, for example as a dermal filler in skin tissue.

In one embodiment, termination is further supported by extruding the cross-linked product obtained in step (i) in step (ii), such as by extruding the product through a sieve. Without being bound by theory, it is believed that the high shear forces applied during extrusion provide complete mixing of the cross-linking agent and the compound for terminating the cross-linking reaction in the composition or gel. As a result, the cross-linking agent is completely or almost completely inactivated, thereby preventing further cross-linking and thus preventing further undesirable increases in molecular weight and viscosity and/or elasticity.

The term "extruding through a sieve" encompasses terms such as "passed through a sieve," or "pressed through a sieve," or "directed through a sieve," or "filtered."

Thus, in one embodiment, step (ii) comprises step (ii.1):

(ii.1) Extruding the cross-linked product obtained in step (i).

In another embodiment, step (ii) comprises steps (ii.1) and (ii.2):

(ii.1) subjecting the product obtained in step (i) to acid treatment;

(ii.2) Extruding the product obtained in step (ii.1); or extruding the product obtained in step (ii.1) through a sieve.

In one embodiment, step (ii) comprises steps (ii.1) and (ii.2):

(ii.1) subjecting the product obtained in step (i) to acid treatment;

(ii.2) extruding the product obtained in step (ii.1); or

Extruding the product obtained in step (ii.1) through a sieve; or

The product obtained in step (ii.1) is pressed through a sieve having a mesh size in the range of 500-600 nm.

In one embodiment, the mesh size of the sieve used in step (ii.2) is about 560 μm, such as 558.8 μm (0.022")

Step (iii)

Optional step (iii) entails mixing the product obtained in step (ii) with the second polymer.

In one embodiment, the presence of the second polymer is not necessary and therefore step (iii) is omitted in the reaction sequence of steps (i) to (iv).

In another embodiment, the presence of the second polymer is essential, and therefore step (iii) must be performed in the reaction sequence of steps (i) to (iv).

The term "mixing" as used herein encompasses mixing of the cross-linked polymer obtained in step (ii) with the second polymer used in step (iii), wherein the resulting mixture comprises the cross-linked first polymer and the second polymer, which may also be cross-linked, however, the second polymer may also be provided in a non-cross-linked form, and the resulting mixture has different physical properties relative to the first and second polymers.

The second polymer may also be a polysaccharide. This polymer may be the same as the first polymer, or may be different therefrom.

Thus, in one embodiment, the second polymer is the same HA as the first polymer used in step (i), or is a different HA than the HA used in step (i).

In one embodiment, the first polymer has the same molecular weight as the second polymer. In another embodiment, the molecular weights are different from each other. The term "molecular weight" of the second polymer refers to the corresponding molecular weight of the polymer before mixing and optionally cross-linking the second polymer.

In one embodiment, the second polymer has a molecular weight of at least 3.0 MDa, or at least 3.5 MDa, or at least 4.0 MDa.

In one embodiment, the second polymer has a molecular weight of at least 3.0 MDa, with an upper limit of 20 MDa, or 10 MDa, or 8 MDa, or 6 MDa, or 4 MDa, respectively.

In one embodiment, the second polymer has a molecular weight of at least 3.5 MDa, with an upper limit of 20 MDa, or 10 MDa, or 8 MDa, or 6 MDa, or 4 MDa, respectively.

In one embodiment, the second polymer has a molecular weight of at least 4.5 MDa, with an upper limit of 20 MDa, or 10 MDa, or 8 MDa, or 6 MDa, or 4 MDa, respectively.

In one embodiment, the second polymer is provided in a buffer, such as a buffer that may be used in step (i) or step (ii).

In one embodiment, the mixing according to step (iii) is carried out at a temperature in the range of 0-40°C, or 0-30°C.

In another embodiment, the temperature of step (iii) is controlled so as not to exceed 20°C, or 15°C.

In one embodiment, the temperature of step (iii) is 0-10°C, or 3-7°C, such as 5°C.

In one embodiment, the mixing according to step (iii) is performed by stirring the cross-linked product and the second polymer, wherein the cross-linking has been terminated according to step (ii).

In one embodiment, the weight of the second composition is less than 5%, or less than 4%, based on the weight of the first composition, for example in the range of from 0.01-5%, or in the range of 0.1-4%, or in the range of 0.1-2.5%, or 0.2-2.0%, or 0.5-1.5%.

In one embodiment, step (iii) allows for tailored adjustment of the viscosity and elasticity, or the viscosity or elastic properties, of the target composition or gel, which are essential for use as a dermal filler. Such properties can be achieved by selecting the following parameters: a suitable temperature range in step (i) and/or the weight ratio of the first and second polymers in step (iii) and/or the amount of crosslinker used in step (i) and the amount of the second polymer optionally used, assuming that the second polymer is used in step (iii) and is used in crosslinked form.

Furthermore, the use of a second polymer in step (iii) can improve the flowability of the composition through a sieve, if a sieving step is performed. However, generally, a lower extrusion force is required to extrude the composition through the sieve or sieves compared to a composition without the second polymer. Furthermore, the use of the second polymer in a non-crosslinked form can advantageously improve the flowability of the composition according to the present invention through the needle of a syringe used to apply the composition in cosmetic applications. Consequently, less force can be used to extrude the composition through the needle.

Step (iv)

Step (iv) requires dialysis of the product obtained in step (ii) or (iii).

In one embodiment, this step is used to remove foreign compounds or particles from the gel obtained in step (iii). Foreign compounds or particles may adversely affect the physical properties of the composition, such as the gel, and/or may adversely affect the compatibility of the composition or gel with skin tissue. Therefore, in one embodiment, step (iv) is used to reduce or avoid an inflammatory response that may occur when the composition according to the present invention is injected into skin tissue.

In another embodiment, this step is used to adjust the swelling of the gel obtained in step (ii) or step (iii).

As used herein, the term "swelling" or "swellability" encompasses the ability of a gel to absorb water.

In one embodiment, when subjected to step (iv), the swelling of the gel obtained in step (iii), i.e., the amount of water absorbed during dialysis, is 5-25%, such as 6-23%, or 7-22%, or 9-21%, based on the total weight of the gel.

In another embodiment, the swelling or swellability is 7-18%, or 8-15%.

In one embodiment, the swelling or swellability creates a swelling pressure that enables the HA matrix to withstand compressive forces (eg, when injected into skin tissue and the skin tissue is subjected to compressive forces).

In one embodiment, step (iv) serves to remove foreign compounds or particles from the gel obtained in step (ii) or step (iii) and to regulate the swelling of the gel obtained in step (ii) or step (iii).

Thus, step (iv) serves, inter alia, to provide a further improved HA composition than products known in the art.

Therefore, in addition to step (ii) (termination of the crosslinking), step (iv) (dialysis) is another important reaction step in the sequence of steps necessary for preparing the composition according to the invention, such as a gel. In particular, the combination of steps (ii) and (iv) in the reaction sequence according to the invention allows the provision of a composition, such as a gel, having the properties described, which should be achieved in accordance with the problem posed.

In one embodiment, dialysis is performed using a dialysis membrane having a predetermined molecular weight cut-off. Such dialysis membranes are commercially available.

As used herein, the term "molecular weight cutoff (MWCO)" refers to the lowest molecular weight solute (in Daltons) retained by a membrane used for dialysis, or the molecular weight at which a defined percentage of analyte is prohibited from diffusion through the membrane.

Commercially available dialysis membranes typically have a MWCO of 1,000-100,000 Da.

In one embodiment, the dialysis membrane used has a MWCO of 12,000-14,000 Da.

In one embodiment, dialysis is performed with a dialysis solution containing a buffer.

In one embodiment, the buffer is the buffer used in step (i), or the buffer used in step (ii), or the buffer used in step (iii).

In one embodiment, the dialysis according to step (iv) is performed at a temperature in the range of 0-30°C.

In another embodiment, the temperature in step (iv) is controlled so as not to exceed 20°C, or 15°C.

In one embodiment, the temperature in step (iv) is 0-10°C, or 3-7°C, such as 5°C.

In one embodiment, the method according to the invention is carried out so that the temperature in step (i) is 25-35°C, and the temperature in steps (ii) to (iv) is 0-10°C, respectively; or the method according to the invention is carried out so that the temperature in step (i) is 25-35°C, and the temperature in steps (ii) to (iv) is 3-7°C, such as 5°C, respectively.

In one embodiment, before dialyzing the product obtained in step (iii), the product is subjected to a sieving step, or multiple sieving steps, to further homogenize the product, respectively to remove heterogeneous particles or any other particles that may adversely affect its use as a dermal filler.

In one embodiment, the dialysis step (iv) comprises steps (iv.1) to (iv.3):

(iv.1) extruding the product obtained in step (ii) or step (iii) through a first sieve, and subsequently extruding the extruded product of the first sieve through a second sieve, wherein the mesh size of the second sieve is smaller than the mesh size of the first sieve; or

extruding the product obtained in step (ii) or (iii) through a first sieve, and then extruding the extruded product of the first sieve through a second sieve, and then extruding the extruded product of the second sieve through a third sieve, wherein the mesh size of the second sieve is smaller than the mesh size of the first sieve, and the mesh size of the third sieve is smaller than the mesh size of the second sieve;

(iv.2) loading the product obtained in step (iv.1) into a dialysis membrane;

(iv.3) The filled membrane obtained in step (iv.2) is placed in the dialysis solution.

Such use of a sieve prior to dialysis can further enhance the effectiveness of the dialysis step. Suitable selection of the mesh size of the sieve further enhances the removal of foreign compounds and particles, such as gelled particles, that could negatively impact the desired homogeneity of the product. Thus, in one embodiment, the sieving step or steps employed in the method according to the invention allow for the preparation of a particularly homogeneous composition, i.e., a particularly homogeneous gel comprising the first polymer, optionally the second polymer, and water. Homogeneity is a desirable property of the composition, which is achieved according to the method of the invention and supports and improves the intended application, such as cosmetic or pharmaceutical use, of the composition.

In one embodiment, the dialysis step (iv) comprises steps (iv.1) to (iv.3):

(iv.1) extruding the product obtained in step (ii) or step (iii) through a first sieve having a mesh size of 325-425 μm; and then extruding the extruded product of the first sieve through a second sieve having a mesh size of 175-225 μm; and then extruding the extruded product of the second sieve through a third sieve having a mesh size of 110-170 μm;

(iv.2) loading the product obtained in step (iv.1) into a dialysis membrane having a molecular weight cut-off of 12,000-14,000 Da;

(iv.3) The filled membrane obtained in step (iv.2) is placed in the dialysis solution.

In one embodiment, the gel obtained in step (ii) or step (iii) is squeezed into the dialysis membrane through a first sieve or screen having a mesh size of about 380 μm, such as 381 μm (0.015"), then through a second sieve or screen having a mesh size of about 200 μm, such as 203.2 μm (0.008"), and then through a third sieve or screen having a mesh size of about 140 μm, such as 139.7 μm (0.0055"). The filled dialysis membrane or filled membranes are then placed in a container containing a suitable dialysis solution, such as the buffer solution used for step (i) according to the first aspect of the invention.

In one embodiment, if a mixing step (iii) is performed after step (ii) and before the dialysis step (iv), step (iii) facilitates a sieving step performed after step (iv) according to step (iv.1).

In one embodiment, the dialysis is performed by stirring the contents of the container. In one embodiment, the dialysis solution can be replaced once or at least twice with fresh dialysis solution. In one embodiment, the replacement interval is 8-18h, or 10-14h, such as 12±2h. In one embodiment, the dialysis according to step (iv) is allowed to proceed for 30-45h, or 35-39h, such as 37±2h.

In one embodiment, the composition obtained in step (iv) has a tan δ measured at a frequency of 0.7 Hz and 30° C. between 0.1 and 0.9, such as 0.1 to 0.5, or 0.2 to 0.4.

In another embodiment, the composition obtained in step (iv) has a tan δ measured at a frequency of 0.7 Hz and 30° C. between 0.1 and 3.5.

In one embodiment, at a frequency of 0.7 Hz and 30° C., η* is in the range of 2,000 mPa*s to 200,000 mPa*s, wherein tan δ is between 0.1-0.9, such as 0.1 to 0.5, or 0.2 to 0.4.

In one embodiment, the composition obtained in step (iv) has a tan δ measured at a frequency of 0.7 Hz and 30° C. of between 0.10 and 3.5. In one embodiment, the corresponding viscosity η* is in the range of 2,500 mPa*s to 145,000 mPa*s, or 4,000 to 145,000 mPa*s.

In one embodiment, the composition obtained in step (iv) has a tan δ measured at a frequency of 0.7 Hz and 30° C. of between 0.10 and 3.5. In one embodiment, the composition obtained in step (iv) has a tan δ measured at a frequency of 0.7 Hz and 30° C. of between 0.10 and 0.25.

Step (v)

Although the composition according to the present invention allows for the application of the composition with minimal adverse skin reactions, in one embodiment, a local anesthetic and/or antiarrhythmic drug may be added to the composition or gel according to the present invention obtained in step (iv), if necessary or desired. Such drugs can alleviate itching, burning, and pain that may be caused by skin inflammation when the composition or gel according to the present invention is injected into skin tissue.

Suitable drugs are known in the art.

In one embodiment, lidocaine is used as a local anesthetic and/or antiarrhythmic drug. This drug is known as a dental anesthetic or a local anesthetic for minor surgery, for example, for injection.

In one embodiment, lidocaine is used in the form of a salt, such as the hydrochloride salt, and/or in the form of a hydrate, such as the monohydrate.

Accordingly, the term "lidocaine" as used herein includes its salts and hydrates.

In one embodiment, lidocaine is used in an amount of 0-1 wt% or 0-0.5 wt% based on the weight of the composition or gel.

In one embodiment, the weight is from 0.3% to 0.35%.

In one embodiment, the weight is 0.3% or 0.35%.

In another embodiment, tetracaine is used. The term "tetracaine" as used herein includes its salts and hydrates. Tetracaine can be used in the same amount as lidocaine.

In another embodiment, a mixture of lidocaine and tetracaine may be used.

Accordingly, in one embodiment, the method according to the present invention further comprises step (v) after step (iv):

(v) incorporating an anesthetic or an antiarrhythmic agent or both into the product obtained in step (iv).

In one embodiment, the method according to the present invention further comprises step (v) after step (iv):

(v) adding lidocaine, lidocaine hydrochloride, or lidocaine hydrochloride monohydrate to the product obtained in step (iv); or adding tetracaine to the product obtained in step (iv); or adding lidocaine and tetracaine to the product obtained in step (iv).

In one embodiment, the composition obtained in step (v) has a tan δ measured at a frequency of 0.7 Hz and 30° C. of between 0.1 and 0.9, such as between 0.1 and 0.5, or between 0.2 and 0.4.

In one embodiment, the composition obtained in step (v) has a tan δ measured at a frequency of 0.7 Hz and 30° C. of between 0.1 and 3.5 or between 0.15 and 3.4.

In one embodiment, at a frequency of 0.7 Hz and 30° C., η* is between 2,000 mPa*s and 200,000 mPa*s, wherein tan δ is between 0.1 and 0.9, such as 0.1 to 0.5, or 0.2 to 0.4.

In one embodiment, the composition obtained in step (v) has a tan δ of 0.10 to 3.5 measured at a frequency of 0.7 Hz and 30° C. In one embodiment, the corresponding viscosity η* is in the range of 2,500 mPa*s to 145,000 mPa*s or 4,000 to 145,000 mPa*s.

In one embodiment, the composition obtained in step (v) has a tan δ measured at a frequency of 0.7 Hz and 30° C. of between 0.10 and 3.5. In one embodiment, the composition obtained in step (v) has a tan δ measured at a frequency of 0.7 Hz and 30° C. of between 0.10 and 0.25.

In one embodiment, the composition obtained in step (v) has a tan δ measured at a frequency of 0.7 Hz and 30° C. of between 0.1 and 3.5. In one embodiment, the corresponding viscosity η* is in the range of 2,000 mPa*s to 150,000 mPa*s. In one embodiment, the composition obtained in step (v) has a tan δ measured at a frequency of 0.7 Hz and 30° C. of between 0.15 and 3.5. In one embodiment, the corresponding viscosity η* is in the range of 2,500 mPa*s to 145,000 mPa*s or 4,000 to 145,000 mPa*s.

In one embodiment, the composition obtained in step (v) has a tan δ of 0.15 to 0.25 measured at a frequency of 0.7 Hz and 30° C. In one embodiment, the corresponding viscosity η* is in the range of 15,000 mPa*s to 28,000 mPa*s.

Step (vi)

Finally, in one embodiment, the product obtained in step (iv) or step (v) can be filled into a syringe. This is because the product obtained according to the method of the present invention is intended to be used by injection.

In one embodiment, the product obtained in step (iv) or step (v) is filled into syringes and sterilized.

Accordingly, in one embodiment, the method according to the present invention further comprises step (vi) after step (iv) or step (v):

(vi) filling the product obtained in step (iv) or step (v) into a syringe and sterilizing it.

In one embodiment, the filling is performed by extruding the product obtained in step (iv) or step (v) into a syringe.

Sterilization can be performed by methods known in the art. As used herein, the term "sterilization" includes any process that eliminates or removes or kills various forms of microbial life, including transmissible agents (such as fungi, bacteria, viruses, spore forms, etc.), present on the surface of the syringe and/or in the composition or gel prepared according to the method of the present invention. Sterilization can be achieved by methods known in the art, such as the application of heat, chemicals, radiation, high pressure, or filtration, or suitable combinations thereof.

In one embodiment, sterilization is performed before filling according to step (vi), ie the composition or gel obtained in step (iv) or step (v) and the syringe are sterilized independently of each other.

In another embodiment, sterilization is performed during the filling process according to step (vi).

In another embodiment, sterilization is performed after filling according to step (vi).

In one embodiment, the total content of HA in the final composition, such as a gel, is in the range of 1-5 wt % based on the total weight of the composition. In another embodiment, the total content is in the range of 1.5-4 wt %, or 2-2.5 wt %.

In one embodiment, the product obtained according to step (iv) or according to step (v) or according to step (vi) is an isotonic sterile viscoelastic composition such as a gel. The composition or gel is injectable and can be used as a graft to increase the volume of skin tissue, i.e. to augment it.

In one embodiment, the product obtained according to step (iv) or according to step (v) or according to step (vi) is an isotonic sterile viscoelastic injectable gel or implant for increasing the volume of, for example, facial skin tissue, or for correcting moderate or deep wrinkles.

In one embodiment, the skin tissue comprises or is tissue of the lips.

In another embodiment, the skin tissue comprises or is skin tissue with moderate to severe facial wrinkles or folds such as nasolabial folds.

In one embodiment, the composition prepared according to the first aspect of the present invention provides a safe, effective, biocompatible, non-immunogenic composition that is easy to distribute and store and does not require allergy testing. Furthermore, when applied to skin tissue, the composition exhibits acceptable long-term durability. In one embodiment, when applied to skin tissue, the composition prepared according to the present method is stable for a substantial period of time.

Accordingly, in one embodiment, the present invention relates to a method for preparing a composition according to the first aspect or according to the method of the first aspect and any embodiment or any combination of at least two embodiments defined therein, wherein the composition obtained in step (iii) is dialyzed in step (iv) to have a pH of 6.5-7.5, such as 6.7-7.2, or 6.8-7.1, or 6.8-6.9.

Accordingly, in one embodiment, the present invention relates to a method for preparing a composition according to the first aspect or according to the method of the first aspect and any embodiment or any combination of at least two embodiments defined therein, wherein the gel obtained in step (iii) is dialyzed in step (iv) to have a swelling capacity of 5-25%, such as 6-23% or 7-22% or 9-21%, based on the total weight of the gel.

In one embodiment, the present invention relates to a method for preparing a composition according to the first aspect or according to the method of the first aspect and any embodiment or any combination of at least two embodiments defined therein, wherein the composition obtained in step (iv) or step (v) has a tan δ measured at a frequency of 0.7 Hz and 30° C. of between 0.1 and 0.9 and/or a viscosity η* in the range of 2,000 mPa*s to 200,000 mPa*s; or a tan δ measured at a frequency of 0.7 Hz and 30° C. of between 0.1 and 3.5 and/or a viscosity η* in the range of 2,000 mPa*s to 150,000 mPa*s; or the composition obtained in step (iv) or step (v) has a tan δ measured at a frequency of 0.7 Hz and 30° C. of between 0.15 and 3.5 and/or a viscosity η* in the range of 0.7 Hz to 30° C. The viscosity η* measured is in the range of 2,100 mPa*s to 145,000 mPa*s, or from 2,500 to 145,000 mPa*s, or from 4,000 to 145,000 mPa*s; or the composition obtained in step (iv) or step (v) has a tan δ measured at a frequency of 0.7 Hz and 30°C of between 0.10 and 0.25 and/or has a viscosity η* measured at a frequency of 0.7 Hz and 30°C of between 15,000 mPa*s and 28,000 mPa*s; or the composition obtained in step (iv) or step (v) has a tan δ measured at a frequency of 0.7 Hz and 30°C of between 0.10 and 0.25 and/or has a viscosity η* measured at a frequency of 0.7 Hz and 30°C of between 22,000 mPa*s and 28,000 mPa*s.

In one embodiment, the invention relates to a method for preparing a composition according to the first aspect or according to the method according to the first aspect and any embodiment or any combination of at least two embodiments defined therein, wherein the composition is stable when injected into skin tissue for at least three months, such as at least four months, or five months, or six months.

In one embodiment, the invention relates to a method for preparing a composition according to the first aspect or according to the method according to the first aspect and any embodiment or any combination of at least two embodiments defined therein, wherein upon injection into skin tissue, the anesthetic and/or antiarrhythmic agent of step (v), such as lidocaine or tetracaine, or lidocaine and tetracaine, is released; and wherein the composition is sterile.

In one embodiment, the present invention relates to a method for preparing a composition according to the first aspect or according to the method according to the first aspect and any embodiment or any combination of at least two embodiments defined therein, wherein the total content of HA in the final composition, such as a gel, is 1-5 wt%, such as 1.5-4 wt%, or 2-2.5 wt%, based on the total weight of the composition.

In one embodiment, the invention relates to a method for preparing a composition according to the first aspect or according to the method according to the first aspect and any embodiment or any combination of at least two embodiments defined therein, wherein the composition does not irritate skin tissue when injected into skin tissue.

In one embodiment, the present invention relates to a method for preparing a composition according to the first aspect or according to the method according to the first aspect and any embodiment or any combination of at least two embodiments defined therein, wherein when the composition is in the form of a gel, the composition is used as an injectable tissue filler.

In one embodiment, the present invention relates to a method for preparing a composition according to the first aspect or according to the method according to the first aspect and any embodiment or any combination of at least two embodiments defined therein, wherein the hyaluronic acid, or the hyaluronic acid and the anesthetic and/or antiarrhythmic agent are the only active ingredients of the composition.

According to a second aspect, the present invention relates to a composition, such as a gel, comprising a crosslinked first polymer, optionally a second polymer, which may be crosslinked or non-crosslinked, and water, wherein the first and second polymers are selected from polysaccharides such as HA.

In one embodiment, the composition, such as a gel, is obtainable by the method according to the first aspect of the invention, or the method according to the first aspect of the invention and any embodiment or any combination of at least two embodiments defined therein, comprising a crosslinked first polymer, optionally a second polymer which may be crosslinked or non-crosslinked, and water, wherein the first and second polymers are selected from polysaccharides.

In one embodiment, the composition, such as a gel, is obtainable by the method according to the first aspect of the invention, or the method according to the first aspect of the invention and any embodiment or any combination of at least two embodiments defined therein, and consists of a cross-linked first polymer, optionally a second polymer, which may be cross-linked or non-cross-linked, and water, and optionally an anesthetic and/or antiarrhythmic agent, wherein the first and second polymers are selected from polysaccharides.

According to a third aspect, the present invention relates to a kit comprising a syringe and the composition, such as a gel, according to the second aspect, or a syringe and a composition, such as a gel, prepared according to the method of the first aspect.

According to a fourth aspect, the present invention relates to the use of the composition, such as a gel, according to the second aspect, or to the use of the composition, such as a gel, prepared according to the method of the first aspect, in cosmetic applications.

In one embodiment, the composition, such as a gel, is used as a dermal filler.

As used herein, the term "dermal filler" refers to a composition such as a gel prepared according to the present invention that is suitable for increasing the volume of skin tissue, ie, filling skin tissue.

In one embodiment, the composition, such as a gel, is used to fill skin tissue, such as facial skin tissue, and/or to correct moderate or severe wrinkles.

In one embodiment, the composition is an injectable composition, ie, when applied, it is injected into the skin tissue.

According to a further aspect, the composition according to the second aspect may be used as or in a pharmaceutical composition.

In one embodiment, the composition is for use in medical applications that require the use of a hyaluronic acid based composition such as a gel.

Thus, according to a fifth aspect, the present invention relates to a composition, such as a gel, according to the second aspect for use as a medicament.

According to a sixth aspect, the present invention relates to a method for preparing a composition, such as a gel, comprising a cross-linked first polymer, optionally a second polymer, which may be cross-linked or non-cross-linked, and water, wherein the first and second polymers are selected from polysaccharides, the method comprising at least a first sieving step (a).

The term "sieving step" includes the step of squeezing the cross-linked polymer, such as cross-linked hyaluronic acid, and water through a sieve.

In one embodiment, the term "screening step" refers to the process of squeezing the product obtained in any of steps (i) to (vi) defined in the first aspect of the present invention through a sieve. The term "squeezing through a sieve" (e.g., as mentioned in the first method of the present invention) encompasses terms such as "through a sieve," "pressing through a sieve," "controlling through a sieve," or "filtering."

The term "sieve" encompasses the term "filter".

The term "sieve" or "filter" encompasses any device having pores or holes through which a liquid can penetrate, wherein particles that may be contained in the liquid can be removed or trimmed to fit through the pores of the sieve or filter. Thus, in one embodiment, the size of the particles is adjusted by screening or filtering. For example, the sieve can be provided in the form of a mesh of wire or fibers, such as plastic fibers. Suitable sieves are known in the art of screening and filtering.

In one embodiment, the process comprises at least a second screening step (b).

In another embodiment, the method comprises at least a third sieving step (c) in addition to the first sieving step (a) and the second sieving step (b).

In another embodiment, the method comprises, in addition to the first screening step (a) and the second screening step (b) and the third screening step (c), at least one further screening step, or at least two further screening steps or at least three further screening steps or at least four further screening steps.

In one embodiment, the second sieving step (b) is performed with a second sieve having a mesh size smaller than the mesh size of the first sieve used in the first sieving step (a).

In another embodiment, the third sieving step (c) is performed with a third sieve having a mesh size smaller than the mesh size of the second sieve used in the second sieving step (b).

In another embodiment, the third sieving step (c) is carried out using a third sieve having a mesh size smaller than the mesh size of the second sieve used in the second sieving step (b), and the third sieve correspondingly has a mesh size smaller than the mesh size of the first sieve used in the first sieving step (a).

In another embodiment, each subsequently used sieve has a mesh size that is smaller than the mesh size of the sieve used in the preceding sieving step.

In one embodiment, the first sieve has a mesh size in the range of 200-600 μm.

In another embodiment, the first sieve has a mesh size in the range of 200-600 μm; and the second sieve has a mesh size in the range of 100-400 μm.

In another embodiment, the first sieve has a mesh size in the range of 200-600 μm; and the second sieve has a mesh size in the range of 100-400 μm; and the third sieve has a mesh size in the range of 50-300 μm.

In one embodiment, the first sieve has a mesh size in the range of 300-500 μm.

In another embodiment, the first sieve has a mesh size in the range of 300-500 μm; and the second sieve has a mesh size in the range of 100-300 μm.

In another embodiment, the first sieve has a mesh size in the range of 300-500 μm; and the second sieve has a mesh size in the range of 100-300 μm; and the third sieve has a mesh size in the range of 50-200 μm.

In one embodiment, the first sieve has a mesh size in the range of 325-425 μm.

In another embodiment, the first sieve has a mesh size in the range of 325-425 μm; and the second sieve has a mesh size in the range of 175-225 μm.

In another embodiment, the first sieve has a mesh size in the range of 325-425 μm; and the second sieve has a mesh size in the range of 175-225 μm; and the third sieve has a mesh size in the range of 110-170 μm.

In one embodiment, said sieving according to at least one of said sieving steps (a), (b) or (c) is carried out at a temperature of 5-30°C, such as 5-25°C or 5-20°C or 5-15°C or 5-10°C.

In one embodiment, before or after the first screening step (a), or before or after the second screening step (b), or before or after the third screening step (c), the method comprises at least one of the following steps (i) to (vi) as defined in the first aspect of the invention.

Accordingly, in one embodiment of the sixth aspect, before or after the first screening step (a), or before or after the second screening step (b), or before or after the third screening step (c), the method comprises at least one of the following steps (i) to (vi):

(i) cross-linking a mixture comprising a first polymer and water;

(ii) following the cross-linking in step (i), terminating the cross-linking;

(iii) optionally mixing the product obtained in step (ii) with a second polymer;

(iv) dialyzing the product obtained in step (ii) or step (iii);

(v) incorporating an anesthetic or antiarrhythmic agent, such as lidocaine, lidocaine hydrochloride, lidocaine hydrochloride monohydrate, tetracaine, or lidocaine and tetracaine, into the product obtained in step (iv);

The product obtained in step (v) is filled into syringes and sterilized.

In another embodiment, before or after the first screening step (a), or before or after the second screening step (b), or before or after the third screening step (c), the method comprises at least one of the following steps (i) to (vi):

(i) cross-linking a mixture comprising a first polymer and water;

(ii) following the cross-linking in step (i), terminating the cross-linking;

(iii) optionally mixing the product obtained in step (ii) with a second polymer;

(iv) dialyzing the product obtained in step (i), or step (ii), or step (iii);

(v) incorporating an anesthetic or antiarrhythmic agent, such as lidocaine, lidocaine hydrochloride, lidocaine hydrochloride monohydrate, tetracaine, or lidocaine and tetracaine, into the product obtained in step (i), step (ii), or step (iii);

(vi) filling the product obtained in step (i), or step (ii), or step (iii) into a syringe and sterilizing it.

In one embodiment, the first screening step (a) is performed subsequent to step (i).

In another embodiment, the first sieving step (a) and the second sieving step (b) are performed subsequent to step (i).

In another embodiment, the first sieving step (a), the second sieving step (b), and the third sieving step (c) are performed subsequent to step (i).

In one embodiment, the first screening step (a) is performed subsequent to step (ii).

In another embodiment, the first sieving step (a) and the second sieving step (b) are performed subsequent to step (ii).

In another embodiment, the first sieving step (a) and the second sieving step (b) and the third sieving step (c) are performed subsequent to step (ii).

In one embodiment, the first screening step (a) is performed subsequent to step (iii).

In another embodiment, the first sieving step (a) and the second sieving step (b) are performed subsequent to step (iii).

In another embodiment, the first sieving step (a) and the second sieving step (b) and the third sieving step (c) are performed subsequent to step (iii).

In one embodiment, the first screening step (a) is performed subsequent to step (iv).

In another embodiment, the first sieving step (a) and the second sieving step (b) are performed subsequent to step (iv).

In another embodiment, the first sieving step (a) and the second sieving step (b) and the third sieving step (c) are performed subsequent to step (iv).

In one embodiment, the first screening step (a) is performed subsequent to step (v).

In another embodiment, the first sieving step (a) and the second sieving step (b) are performed subsequent to step (v).

In another embodiment, the first sieving step (a) and the second sieving step (b) and the third sieving step (c) are performed subsequent to step (v).

The use of a sieve facilitates the removal of foreign compounds and particles, such as gelled particles, that could adversely affect the desired homogeneity of the product, or helps adjust the size of the particles to fit through the pores of the sieve by applying shear forces. Thus, in one embodiment, the sieving step or steps used in the method according to the invention allow the preparation of a particularly homogeneous composition, i.e., a particularly homogeneous gel comprising the first polymer, optionally the second polymer, and water. This homogeneity supports and enhances the intended application, such as cosmetic or pharmaceutical use, of the composition.

According to a seventh aspect, the present invention relates to a composition, such as a gel, comprising a cross-linked first polymer, and optionally a second polymer, which may be cross-linked or non-cross-linked, and water, wherein the first and second polymers are selected from polysaccharides, and the composition, such as a gel, can be prepared or obtained by the method defined in the sixth aspect of the present invention or the method defined in any embodiment or any combination of at least two embodiments defined in the sixth aspect.

According to an eighth aspect, the present invention relates to a kit comprising a syringe and a composition prepared or obtainable by the method defined in the sixth aspect of the present invention or any embodiment of the sixth aspect; or to a kit comprising a syringe and a composition defined in the seventh aspect of the present invention.

According to a ninth aspect, the present invention relates to the use of a composition prepared or obtainable by a method as defined in the sixth aspect of the invention or any embodiment or any combination of at least two embodiments as defined in the sixth aspect of the invention; or to the use of said composition as defined in the seventh aspect of the invention, in cosmetic applications; or as a dermal filler.

According to the tenth aspect of the present invention, the present invention relates to a composition, such as a gel, comprising a cross-linked first polymer, and optionally a second polymer, which may be cross-linked or non-cross-linked, and water, wherein the first and second polymers are selected from polysaccharides, and the composition, such as a gel, can be prepared or obtained by the method defined in the sixth aspect of the present invention or the method defined in any embodiment or any combination of at least two embodiments defined in the sixth aspect, or relates to the composition defined in the seventh aspect of the present invention, which is used as a medicine.

According to an eleventh aspect, the present invention relates to the use of at least one sieve or at least one sieving step in the preparation of a composition comprising cross-linked hyaluronic acid and water.

In one embodiment of this application, at least two sieves or two sieving steps are used.

In another embodiment, at least three sieves or at least three sieving steps are used.

DETAILED DESCRIPTION

Example 1

Preparation of buffer solution

A buffer solution is prepared from sodium chloride, anhydrous disodium hydrogen phosphate, sodium dihydrogen phosphate dihydrate and water by dissolving these salts in water.

Preparation of HA in buffer

Following the preparation of the buffer solution, sodium hyaluronate having a molecular weight of from 2.5 MDa to less than 3.0 MDa was added to a quart mixing bowl and a portion of the buffer solution was added. The materials were mixed at 250 rpm for 2.0-2.5 hours with the bowl jacket set at 50°C. Following the mixing, the materials were cooled to 5-7°C.

Addition of alkaline solution

A first alkaline solution was prepared by dissolving sodium hydroxide in the above buffer solution. Next, a second alkaline solution was prepared by dissolving sodium hydroxide in the above buffer solution. The first alkaline solution was then added to the contents of the mixing bowl, and the contents were mixed at 250 rpm, 5°C jacket setting for 30 to 40 minutes.

Cross-linking reaction [step (i)]

The crosslinking solution was prepared by adding BDDE to a portion of the second alkaline buffer solution. This alkaline solution containing the crosslinker was added to the contents of the mixing bowl and mixed at 500 rpm, 5°C jacket setting for 10 to 15 minutes. The mixing speed was then reduced to 100 rpm and the temperature setting was changed to 30°C. Once the temperature reached 28°C, the mixing was turned off and the contents were allowed to stand for approximately 3 hours.

Preparation of quenching solution for terminating the cross-linking reaction

1 M HCl solution was added to a portion of the buffer solution to form a quenching solution.

Quenching the reaction [step (ii)]

The temperature setting of the jacket was set to 5°C and the quench solution was added to the contents of the bowl. The contents were then mixed at 500 rpm for 10 to 15 minutes.

Processing of cross-linked HA

The polymer obtained in step (ii) is then cut into segments, possibly into blocks or strips. The blocks or strips may be 1.27 cm x 1.27 cm x 1.27 cm (0.5 x 0.5 x 0.5 inches) or smaller in size. The blocks or strips are then mixed at 150 rpm and a jacket setting of 5°C for about 2.5 to 3.0 hours. Following the mixing, the mixed product is extruded through a 558.8 μm (0.022") screen and returned to the mixing bowl and further mixed at 150 rpm and a jacket setting of 5°C for 2.0 to 2.5 hours.

The second polymer is provided

HA (sodium salt) with Mw ≥ 3.0 MDa was added to a portion of the buffer solution. The materials were mixed for a short period of time using an overhead mixer.

Preparation of gel [step (iii)]

A portion of the second polymer in buffer (1% w/w) was added to the contents of the one quart mixing bowl. The contents were mixed at 250 rpm for 1 to 5 minutes.

Dialysis reaction [step (iv)]

A dialysis membrane with a MWCO of 12,000 to 14,000 Da is hydrated in sterile water. The gel obtained in step (iii) is then squeezed into the dialysis membrane through a sieve with a mesh size of about 380 μm, such as 381 μm (0.015"), then through a sieve with a mesh size of about 200 μm, such as 203.2 μm (0.008"), and then through a sieve with a mesh size of about 140 μm, such as 139.7 μm (0.0055"). The dialysis membrane is filled and has an effective length of about 20.3 cm (8 inches) and a total length of about 25.4 cm (10 inches). The membrane is then placed in a container containing the buffer solution. The container is brought to a set point of 5°C and the substance is stirred. The dialysis solution is changed twice at intervals of 12±2 h. The dialysis is allowed to proceed for 37±2 h.

Extrusion of the product obtained in step (iv) [step (vi)]

After dialysis, the membranes are combined, mixed and extruded twice through a sieve having a mesh size of about 140 μm, such as 139.7 μm (0.0055”), and then further mixed under vacuum for 30-40 minutes. The resulting material is extruded into syringes and steam sterilized.

The product obtained after step (iv) and contained in the syringe after step (vi) is an isotonic, sterile, viscoelastic, injectable gel. This gel can be used as an implant suitable for increasing the volume of, for example, facial skin tissue, i.e., for filling said skin tissue, and/or for correcting moderate or deep skin wrinkles.

Example 2

The reaction was carried out according to Example 1, but with the difference in the cross-linking step (i):

The crosslinking solution was prepared by adding BDDE to a portion of the second alkaline buffer solution. This alkaline solution containing the crosslinker BDDE was added to the contents of the mixing bowl and mixed at 500 rpm and a 5°C jacket setting for 10 to 15 minutes. The mixing speed was then reduced to 100 rpm and the temperature setting was changed to 33.33°C. Once the temperature reached 31.33°C, the mixing was turned off and the contents were allowed to stand for approximately 3 hours.

The product obtained after step (iv) and contained in the syringe after step (vi) is an isotonic, sterile, viscoelastic, injectable gel. This gel can be used as an implant suitable for increasing the volume of, for example, facial skin tissue, i.e., for filling said skin tissue, and/or for correcting moderate or deep skin wrinkles. This gel has higher elasticity and viscosity than the gel according to Example 1.

Example 3

The reaction is carried out according to Example 1, except that an anesthetic and an antiarrhythmic agent such as lidocaine are added to the gel obtained after step (iii):

Addition of Lidocaine [Step (v)]

The lidocaine hydrochloride monohydrate solution is dissolved in the buffer solution and added to the dialysis membrane containing the gel prepared according to step (iii) in an amount of 0.35 wt.-%, based on the gel. The substance can be squeezed twice through a sieve with a mesh size of about 140 μm, such as 139.7 μm (0.0055") and further mixed under vacuum for 30 to 40 minutes.

The product obtained after step (v) and contained in the syringe after step (vi) is an isotonic, sterile, viscoelastic, injectable gel. This gel can be used as an implant suitable for increasing the volume of, for example, facial skin tissue, i.e., for filling said skin tissue, and/or for correcting moderate or deep skin wrinkles.

Example 4

The reaction was carried out according to Example 1, but with the difference in the cross-linking step (i):

The crosslinking solution was prepared by adding BDDE to a portion of the second alkaline buffer solution. This alkaline solution containing the crosslinker BDDE was added to the contents of the mixing bowl and mixed at 500 rpm and a 5°C jacket setting for 10 to 15 minutes. The mixing speed was then reduced to 100 rpm and the temperature setting was changed to 27°C. Once the temperature reached 25°C, the mixing was turned off and the contents were allowed to stand for approximately 3 hours.

The product obtained after step (iv) and contained in the syringe after step (vi) is an isotonic, sterile, viscoelastic, injectable gel. This gel can be used as an implant suitable for increasing the volume of, for example, facial skin tissue, i.e., for filling said skin tissue, and/or for correcting moderate or deep skin wrinkles. This gel has lower elasticity and viscosity than the gel according to Example 1.

Example 5

The reaction was carried out according to Example 1, but with the differences in the cross-linking step (i) and the mixing step (iii):

The crosslinking solution was prepared by adding BDDE to a portion of the second alkaline buffer solution. This alkaline solution containing the crosslinker BDDE was added to the contents of the mixing bowl and mixed at 500 rpm and a 5°C jacket setting for 10 to 15 minutes. The mixing speed was then reduced to 100 rpm and the temperature setting was changed to 30°C. Once the temperature reached 27°C, the mixing was turned off and the contents were allowed to stand for approximately 3 hours.

The second polymer is provided

HA (sodium salt) with Mw ≥ 3.0 MDa was added to a portion of the buffer solution. The materials were mixed for a short period of time using an overhead mixer.

Preparation of gel [step (iii)]

A portion of the second polymer in buffer (3% w/w) was added to the contents of the one quart mixing bowl. The contents were mixed at 250 rpm for 1 to 5 minutes.

The product obtained after step (iv) and contained in the syringe after step (vi) is an isotonic, sterile, viscoelastic, injectable gel. This gel can be used as an implant suitable for increasing the volume of, for example, facial skin tissue, i.e., for filling said skin tissue, and/or for correcting moderate or deep skin wrinkles.

Example 6

The reaction was carried out according to Example 1, but with the differences in the cross-linking step (i) and the mixing step (iii):

The crosslinking solution was prepared by adding BDDE to a portion of the second alkaline buffer solution. This alkaline solution containing the crosslinker BDDE was added to the contents of the mixing bowl and mixed at 500 rpm and a 5°C jacket setting for 10 to 15 minutes. The mixing speed was then reduced to 100 rpm and the temperature setting was changed to 25°C. Once the temperature reached 22°C, the mixing was turned off and the contents were allowed to stand for approximately 3 hours.

The second polymer is provided

HA (sodium salt) with Mw ≥ 3.0 MDa was added to a portion of the buffer solution. The materials were mixed for a short period of time using an overhead mixer. The portion of the crosslinking solution used in step (i) was then added to the second polymer in the buffer solution.

Preparation of gel [step (iii)]

A portion of the cross-linked second polymer in buffer (1% w/w) was added to the contents of the one quart mixing bowl. The contents were mixed at 250 rpm for 1 to 5 minutes.

The product obtained after step (iv) and contained in the syringe after step (vi) is an isotonic, sterile, viscoelastic, injectable gel. This gel can be used as an implant suitable for increasing the volume of, for example, facial skin tissue, i.e., for filling said skin tissue, and/or for correcting moderate or deep skin wrinkles.

Example 7

The reaction is carried out according to Example 2, except that an anesthetic and an antiarrhythmic agent such as lidocaine are added to the gel obtained after step (iii):

Addition of Lidocaine [Step (v)]

The lidocaine hydrochloride monohydrate solution is dissolved in the buffer solution and added to the dialysis membrane containing the gel prepared according to step (iii) in an amount of 0.35 wt.-%, based on the gel. The substance can be squeezed twice through a sieve with a mesh size of about 140 μm, such as 139.7 μm (0.0055") and further mixed under vacuum for 30 to 40 minutes.

The product obtained after step (v) and contained in the syringe after step (vi) is an isotonic, sterile, viscoelastic, injectable gel. This gel can be used as an implant suitable for increasing the volume of, for example, facial skin tissue, i.e., for filling said skin tissue, and/or for correcting moderate or deep skin wrinkles.

Example 8

The reaction was carried out according to Example 1, except that the mixing step (iii) after step (ii) was omitted.

The product obtained after step (iv) and contained in the syringe after step (vi) is an isotonic, sterile, viscoelastic, injectable gel. This gel can be used as an implant suitable for increasing the volume of, for example, facial skin tissue, i.e., for filling said skin tissue, and/or for correcting moderate or deep skin wrinkles.

Examples 9-14

The reaction was carried out according to Example 2-7, except that the mixing step (iii) after step (ii) was omitted.

The products obtained after step (iv) and contained in the syringe after step (vi), respectively, are isotonic, sterile, viscoelastic, injectable gels. Such gels can be used as implants suitable for increasing the volume of, for example, facial skin tissue, i.e., for filling said skin tissue, and/or for correcting moderate or deep skin wrinkles.

Claims (23)

1. A method for preparing a composition comprising a crosslinked first polymer, optionally a second polymer, the second polymer being crosslinked or non-crosslinked, and water, wherein the first and second polymers are selected from polysaccharides, the method comprising at least steps (i), (ii), and (iv) and optionally step (iii): (i) Crosslinking a mixture containing the first polymer and water; (ii) After the crosslinking in step (i), the crosslinking is terminated; (iii) Optionally, the product obtained in step (ii) is mixed with the second polymer; (iv) The product obtained in step (ii) or step (iii) by dialysis; The dialysis step (iv) includes steps (iv.1) to (iv.3): (iv.1) The product obtained in step (ii) or (iii) of extruding through a first sieve, and subsequently extruding the extruded product of the first sieve through a second sieve, wherein the sieve aperture size of the second sieve is smaller than that of the first sieve; or The product obtained in the first sieve extrusion step (ii) or (iii), and subsequently extruded through a second sieve, and then extruded through a third sieve, wherein the aperture size of the second sieve is smaller than that of the first sieve, and the aperture size of the third sieve is smaller than that of the second sieve; or The product obtained by extruding through a first sieve with a sieve aperture size of 325-425 μm in step (ii) or (iii); and then extruding the extruded product of the first sieve through a second sieve with a sieve aperture size of 175-225 μm; and then extruding the extruded product of the second sieve through a third sieve with a sieve aperture size of 110-170 μm. (iv.2) The product obtained in step (iv.1) is packed into a dialysis membrane; or the product obtained in step (iv.1) is packed into a dialysis membrane having a molecular weight cutoff of 12,000-14,000 Da. (iv.3) Place the packed membrane obtained in step (iv.2) into the dialysis solution. 2. The method of claim 1, wherein the first and second polymers may be the same or different from each other. 3. The method according to claim 1 or 2, wherein the second polymer is non-crosslinked. 4. The method according to claim 1 or 2, wherein the first and second polymers are selected from hyaluronic acid and/or its sodium salt. 5. The method according to claim 1 or 2, wherein the first polymer used in step (i) has a molecular weight Mw ranging from 2.5 MDa to 3.0 MDa but not 3.0 MDa. 6. The method according to claim 1 or 2, wherein the second polymer used in step (iii) has a molecular weight of at least 3.0 MDa. 7. The method according to claim 1 or 2, wherein the weight of the second polymer is less than 5% based on the weight of the first polymer. 8. The method according to claim 1 or 2, further comprising step (v): (v) Incorporate an anesthetic and/or an antiarrhythmic agent into the product obtained in step (iv). 9. The method according to claim 1 or 2 further comprises step (vi): (vi) The product obtained in step (iv) is loaded into a syringe and sterilized. 10. The method according to claim 8 further comprises step (vi): (vi) The product obtained in step (v) is loaded into a syringe and sterilized. 11. The method according to claim 1 or 2, wherein step (ii) comprises step (ii.1), or steps (ii.1) and (ii.2): (ii.1) subject the product obtained in step (i) to acid treatment; (ii.2) The product obtained in the extrusion step (ii.1); or The product obtained by sieving and extrusion step (ii.1); or The product obtained in the sieve extrusion step (ii.1) using a sieve aperture size in the range of 500-600 μm. 12. The method according to claim 1, wherein the composition is a gel. 13. The method according to claim 8, wherein the anesthetic and/or antiarrhythmic agent is lidocaine, or lidocaine hydrochloride, or lidocaine hydrochloride monohydrate, or tetracaine, or lidocaine and tetracaine. 14. The method according to claim 10, wherein the sieve aperture size is 558.8 μm. 15. The method according to claim 1 or 2, wherein the weight of the second polymer is less than 4% based on the weight of the first polymer. 16. The method according to claim 1 or 2, wherein the weight of the second polymer is in the range of 0.01-5% based on the weight of the first polymer. 17. The method according to claim 1 or 2, wherein the weight of the second polymer is in the range of 0.1-4% based on the weight of the first polymer. 18. The method according to claim 1 or 2, wherein the weight of the second polymer is in the range of 0.1-2.5% based on the weight of the first polymer. 19. The method according to claim 1 or 2, wherein the weight of the second polymer is in the range of 0.2-2.0% based on the weight of the first polymer. 20. The method according to claim 1 or 2, wherein the weight of the second polymer is in the range of 0.5-1.5% based on the weight of the first polymer. 21. A composition obtained by the method according to any one of claims 1-20. 22. A kit comprising a syringe and the composition of claim 21. 23. Use of the composition of claim 21 in the preparation of a medicament.