Hyperbranched polyglycerol polyglycidyl ethers and their use as cross-linking materials for polysaccharides

Abstract

The present invention relates to hyperbranched polyglycerol polyglycidyl ethers having a molecular weight of 750 to 15,000 Da and an epoxy equivalent weight of 183 to 7,000 g / eq. The present invention also relates to crosslinked polysaccharides obtained by crosslinking polysaccharides with the hyperbranched polyglycerol polyglycidyl ethers. The present invention also relates to methods for their preparation and their use in therapeutic, cosmetic, agricultural, and food applications.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of European Patent Application No. 19200803.5, filed October 1, 2019.

[0002] The present invention relates to hyperbranched polyglycerol polyglycidyl ethers. In particular, the present invention relates to hyperbranched polyglycerol polyglycidyl ethers having a molecular weight of 750 to 15,000 Da and an epoxy equivalent weight of 183 to 7,000 g / eq. The present invention also relates to crosslinked polysaccharides (crosslinked polysaccharides) obtainable by crosslinking polysaccharides with hyperbranched polyglycerol polyglycidyl ethers, a method for preparing the same, and uses thereof in the food, pharmaceutical, cosmetic, and agricultural industries. [Background technology]

[0003] Carbohydrates, also known as polysaccharides, are the most abundant, readily available, and inexpensive biomolecules and renewable resources on Earth, with annual formation rates exceeding the global production rate of synthetic polymers by many orders of magnitude. They are synthesized by the sun's energy and are fully biodegradable, sustainable materials. Polysaccharides are present in all living organisms and are designed by nature to perform a variety of specific functions.

[0004] In addition to their potential use as important chemical feedstocks and energy producers, polysaccharides are recognized to play important roles in a wide variety of complex biological processes. They are largely involved in mediating cellular recognition processes through interactions with proteins and other biological entities, including cell growth regulation, differentiation, adhesion, cancer cell metastasis, cell trafficking, bacterial and viral inflammation, and the immune response.

[0005] One of the best-known polysaccharides is cellulose. It is a component of at least one-third of higher plants and is therefore undoubtedly the most abundant naturally occurring and reproducible organic compound. In its simplest form, cellulose is composed of β-1,4-linked glucan chains that can be arranged in different ways to give rise to different forms of cellulose. Its chemical structure corresponds to the following formula:

[0006] TIFF0007784138000001.tif34170

[0007] In nature, cellulose is produced in a hierarchical manner, where glucan chains associate with each other to form crystalline and amorphous regions that assemble into higher-order structures such as microfibrils. Cellulose is commonly obtained as the cellulose I crystalline form, in which the glucan chains are aligned parallel to each other. Two forms of the natural crystalline polymer, cellulose, Iα and Iβ, have been shown to exist in different amounts obtained from different sources.

[0008] Specifically, sodium carboxymethylcellulose (NaCMC) is defined as the sodium salt of the carboxymethyl ether of cellulose obtained directly from natural strains of fibrous plant material. The NaCMC molecule is a polymer with a base of β-D-glucose units, the free hydroxyl groups of which are partially replaced by carboxymethyl groups. Its chemical structure corresponds to the following formula, where R is H or CH2CO2Na:

[0009] TIFF0007784138000002.tif32170

[0010] The cellulose derivative NaCMC was initially used as a substitute for starch and natural gums. Large quantities of this cellulose derivative are used in paper, textile processing, detergents, drilling fluids, and protective coatings. Refined grades, known as cellulose gum, are widely used in the food, pharmaceutical, and cosmetic industries.

[0011] Another commonly used polysaccharide is starch, which is abundant and naturally occurring. It is found in the leaves of all green plants, as well as in the seeds, fruits, stems, roots, and tubers of most plants. Most starches are composed of two types of polysaccharides: amylose, a linear α-(1→4)-linked glucan, and amylopectin, an α-(1→4)-linked glucan with 4.2–5.9% α-(1→6) branching bonds. The chemical structures of the amylose and amylopectin units correspond to the following formulas, respectively:

[0012] TIFF0007784138000003.tif39170

[0013] TIFF0007784138000004.tif64170

[0014] Starch occurs as the end product of photosynthesis and serves as the chemical storage form of the sun's energy on Earth. It is estimated that 60-70% of human calorie intake comes from starch. All starch is stored in plants as water-insoluble particles or granules in the chloroplasts of leaves and amyloplasts in other plant tissues. Granules are relatively dense particles of molecules with semi-crystalline properties.

[0015] The major category of enzymes that hydrolyze the α-(1→4) linkages in starch is α-amylase. These enzymes are ubiquitous and are produced by bacteria, fungi, plants, and animals. In mammals, there are two specific sources: the salivary glands, which secrete enzymes into the mouth, and the pancreas, which secretes enzymes into the small intestine. α-amylases are endogenous enzymes that attack the interior of polymeric starch chains, rapidly reducing viscosity. Because of these properties, they are sometimes referred to as liquefying enzymes. When α-amylases encounter starch chains and hydrolyze the α-(1→4) linkages, they also produce low-molecular-weight maltodextrins by a process called multiple attack on one of the two chains initially cleaved. Starch is a common ingredient widely used in many food and non-food applications. However, the application of native starch is often limited due to shortcomings in its physicochemical properties, such as low shear resistance and thermal stability, thermal degradation, and a high tendency toward retrogradation.

[0016] Starch is used as an additive for food processing. Food starch is typically used in foods as a thickener, bulking agent, and emulsion stabilizer, and is an exceptional binder in processed meats. In the pharmaceutical industry, starch is also used as an excipient, tablet disintegrant, binder, and plasma expander. Starch can be chemically modified to function properly under conditions frequently encountered during processing or storage, such as high heat, high shear, low pH, freeze / thaw, and chilling. Resistant starch is a type of starch that escapes digestion in the small intestine of healthy individuals. It is used as an insoluble dietary fiber in processed foods and as a dietary supplement for its health benefits. Resistant starch helps improve insulin sensitivity, increases satiety, and improves markers of colonic function.

[0017] Yet another branched polysaccharide is glycogen, a highly branched polysaccharide of glucose that serves as a form of energy storage in animals, fungi, and bacteria. The polysaccharide structure represents the main storage form of glucose in the body, and glycogen is an analogue of starch with an amylopectin-like structure, but is more extensively branched and compact.

[0018] Finally, hyaluronic acid (HA), also known as hyaluronan or sodium hyaluronate, is an anionic, non-sulfated glycosaminoglycan (GAG). HA is a linear, high molecular weight polysaccharide composed of repeating monomers of D-glucuronic acid and N-acetyl-D-glucosamine linked via alternating β-(1→4) and β-(1→3) glycidic bonds. Its chemical structure corresponds to the following formula:

[0019] TIFF0007784138000005.tif39170

[0020] HA is widely distributed throughout the human body and is a component of connective, epithelial, and neural tissues. It forms a major component of the extracellular matrix (ECM), including the brain ECM, and its deficiency or absence can contribute to epilepsy disorders. It is present in nearly all biological fluids, including synovial fluid and the vitreous humor of the eye. It acts as a lubricant and shock absorber, particularly in joints, and helps prevent cellular damage induced by the constant physical stresses to which cartilage is subjected, thereby maintaining its health. The largest amount of HA is found in the skin, which contains over 50% of the total content in the body. Under normal conditions, HA exists as a free polymer; however, in some tissues, it binds to various proteins to generate proteoglycans or specific cellular receptors.

[0021] The primary functions of HA are to create volume, provide lubrication for tissues, and maintain an open, hydrated, and stable extracellular space in which cells and other ECM components, such as collagen and elastin fibers, are firmly held. HA also plays a role in cellular activity, regulating cell adhesion, migration, and proliferation. In particular, high-molecular-weight HA exhibits anti-angiogenic and anti-inflammatory properties, while low-molecular-weight fragments (<100 kDa) have opposite biological activities: pro-inflammatory, immunostimulatory, and angiogenic. The functions of HA and HA fragments in wound healing and in tumor and cancer growth where the CD44 cell receptor is overexpressed have been widely documented. Due to its unique biological properties, which are highly similar to those of human tissue, HA has attracted considerable attention and interest over the years. These properties have enabled HA to be used in a variety of biomedical applications, such as wound healing, lubrication in osteoarthritis, tissue augmentation, and as a carrier for drug delivery systems. However, native HA has very limited applications due to its poor mechanical properties and rapid in vivo degradation.

[0022] The state of the art has revealed that polysaccharides can be widely degraded. The degradation of polysaccharides, especially HA, can be considered as a depolymerization process mediated by glycosidic bond cleavage, which mainly involves two mechanisms: enzymatic degradation (selective and very rapidly promoted by the specific enzyme hyaluronidase); and oxidative degradation (nonselective and less likely to be promoted by free radicals (ROS) and pro-oxidant enzyme activity that may be present during tissue inflammation). In addition to the above mechanisms, there is always a hydrolytic nonselective degradation mechanism due to water and electrolytes in the tissue.

[0023] In particular, HA degradation is thought to be a highly orchestrated and tightly controlled process that generates HA fragments of precisely defined sizes for desired biological functions. HYAL1 and HYAL2 are the most widely expressed hyaluronidases. HYAL2 (anchored to the cell membrane) cleaves high-molecular-weight HA (>1 MDa) into 20 kDa fragments, and HYAL1 (found in lysosomes) subsequently cleaves these fragments into tetrasaccharides, which are then converted into monosaccharides by several enzymes in the hyaluronidase family (e.g., β-glucuronidase, β-N-acetylglucosaminidase). These degradation products are native to the human body and therefore subject to natural removal processes. As mentioned above, HA can also be naturally degraded in vivo by reactive oxygen species (ROS). The mechanism of HA degradation varies depending on the ROS involved. Disturbances to the tissue environment, regardless of size, can activate the body's immune system, resulting in a transient inflammatory response. Mechanical damage to tissue caused by needle penetration during filler injection can also result in such a response. Due to the aforementioned degradation mechanisms, the in vivo turnover of natural polysaccharides after injection is very short, for example, less than 24 hours in the case of hyaluronic acid, depending on the specific enzymatic activity of the implanted tissue. However, their in vivo duration, and therefore their therapeutic efficacy, may be further reduced after degradation by oxidative stress due to the presence of ROS and pro-inflammatory enzymes.

[0024] To overcome these shortcomings regarding the stability and biocompatibility of polysaccharides, especially HA, several strategies have been disclosed to modulate the physical and mechanical properties of polysaccharides in order to extend the in vivo half-life of HA.

[0025] One of these strategies involves the preparation of cross-linked polysaccharides, such as cross-linked HA, with enhanced stability and longer residence times (i.e., lower degradation rates). Several methods for producing cross-linked polysaccharides, particularly cross-linked HA, have been developed in the state of the art. The resulting cross-linked HA is a cross-linked HA hydrogel with a three-dimensional (3D) network structure formed by covalent cross-linking bonds that retain water within the network but are insoluble in water.

[0026] Chemically crosslinked hydrogels are stable materials and are much more resistant to enzymatic degradation than native HA due to the formation of crosslinks and intermolecular bonds between the HA chains and the crosslinker. Furthermore, this 3D structure may also contribute to their resistance to oxidative degradation due to their anti-inflammatory activity (polyols). The above-mentioned crosslinked HA hydrogels with hydroxyl group modifications allow them to be used in the development of dermal fillers. In this regard, several chemical crosslinkers have been disclosed in the state of the art as suitable crosslinkers for preparing crosslinked polysaccharides, including methacrylamide, hydrazide, carbodiimide (EDC), divinyl sulfone (DVS), epichlorohydrin, and several diepoxy compounds and diglycidyl ethers, such as 1,2,7,8-diepoxyoctane, 1,4-butanediol diglycidyl ether (BDDE), poly(ethylene glycol) diglycidyl ether (PEGDE), and glycerol diglycidyl ether (GDE).

[0027] It is known in the art that cross-linked polysaccharides (such as cross-linked HA hydrogels) with a higher degree of cross-linking are preferred due to their longer lifespan and mechanical strength in vivo. Stronger cross-linked HA hydrogels can also provide sufficient force to elevate tissue and resist subsequent deformation. However, from a health perspective, incorporating a high content of chemical cross-linking agents in HA modification is not feasible. Excessive amounts of cross-linking agents are often toxic to cells and tissues in contact with the hydrogel after implantation. Furthermore, excessive amounts of toxic cross-linking agents may hinder cell differentiation and proliferation and may also cause undesirable reactions with other bioactive components (e.g., free polyols, antioxidants, amino acids, buffers, anesthetics, drugs) incorporated into the hydrogel matrix during its fabrication.

[0028] Divinyl sulfone (DVS) was the crosslinking agent used to crosslink most dermal fillers on the market before the introduction of BDDE, and it is still used in some commercial products today. Its ability as a crosslinker is due to the reactivity of the activated vinyl groups present at the two ends of the molecule. The reaction occurs in an alkaline environment (nucleophilic), by the addition of negatively charged hydroxyl groups present on the polysaccharide chain to the double vinyl bond, resulting in the formation of an ether bond. The reaction is a nucleophilic addition, and no molecules or groups are released after the chemical reaction. However, DVS-crosslinked hyaluronic acid dermal fillers have shown some problems with enzymatic degradation, which may be related to the presence of sulfone groups that act as enzyme inhibitors. Therefore, the filler is difficult to remove by hyaluronidase injection in the event of adverse reactions after implantation. As a result, DVS has gradually fallen out of favor with other crosslinking agents, such as BDDE. Furthermore, DVS is a foreign molecule of synthetic origin, has a degree of toxicity superior to BDDE, and is highly volatile and irritating, which poses problems even for dermal filler manufacturers.

[0029] 1,4-butanediol diglycidyl ether (BDDE) and its reaction with HA have been described in the state of the art. Specifically, HA solution was mixed with BDDE under alkaline conditions to form an ether bond. This reaction involves an epoxy ring-opening process, which takes place in alkaline media, in which the epoxy ring preferentially reacts with the hydroxyl group to form an ether bond. BDDE is commonly used in the preparation of currently commercially available HA-based dermal fillers. Its crosslinking ability is attributed to the reactivity of the epoxy groups present at the two ends of the molecule. Unreacted BDDE has been found to be mutagenic in the model organism Drosophila, but no conclusive carcinogenic effects have been observed in mice. Nevertheless, and due to its mutagenic potential, the amount of unreacted BDDE in dermal fillers must be maintained at trace (i.e., residual) levels. This trace level has been deemed safe after a safety risk assessment by the Food and Drug Administration (FDA). Meanwhile, hydrolyzed BDDE is a diol-ether resulting from the hydrolysis of the epoxide groups in BDDE or the hydrolytic cleavage of crosslinked BDDE. However, the metabolism of hydrolyzed BDDE is not described in the state of the art and is understood to proceed via ether bond cleavage by a family of enzymes called cytochrome P450.In contrast, both natural BDDE and metabolized butanediol are foreign molecules of synthetic origin (petroleum) and possess some degree of toxicity.

[0030] Therefore, due to the potential toxicity of BDDE and its metabolized derivative molecules, as well as the strict regulatory control of the amount of BDDE, BDDE is also gradually being replaced by other safety crosslinking materials.

[0031] To provide a safe cross-linking material for preparing safe cross-linked polysaccharides with improved physical and chemical properties, several studies have disclosed the use of glycerol diglycidyl ether (GDE). GDE is a bis-substituted glycidyl ether of glycerol. In particular, the chemical structure of GDE can correspond to 1,3-GDE and 1,2-GDE, as shown in the following formula:

[0032] TIFF0007784138000006.tif46170

[0033] However, in the state of the art, GDEs are incorrectly referred to as "polyglycerol polyglycidyl ethers" or "polyglycerol diglycidyl ethers" (PPEs) due to the uncertainty of their chemical structure, which are often commercially available as mixtures of bis-substituted glycidyl ethers of glycerol with mono- and even tri-substituted glycidyl ethers of glycerol.

[0034] The use of GDE is particularly advantageous because glycerol is an endogenous molecule naturally present in all human tissues, both in its circulating free form and in the form of triglycerides bound to phospholipids in adipose tissue or cell membranes. Indeed, glycerol is involved in the body's metabolic and catabolic processes and is therefore a safe molecule. Therefore, despite the presence of DVS and BDDE, the presence of GDE and / or its main degradation product (glycerol) does not imply potential toxicity issues. However, the main problem with the use of GDE is the need for a high content of cross-linking material to reduce the degradation rate of the resulting GDE-cross-linked polysaccharides.

[0035] However, cross-linked polysaccharides with a high degree of cross-linking exhibit inadequate flow characteristics due to the contribution of elasticity in addition to viscosity, making them difficult to extrude through thin needles, especially when used in injectable applications (fillers for aesthetics, osteoarthritis treatment, etc.). In fact, the current state of the art has reported various complications and side effects associated with chemical cross-linking agents used for HA stabilization, especially HA fillers treated with excessive amounts of cross-linking agents, such as unwanted pain for patients after application and difficulty for physicians. Any side effects or allergic reactions caused by HA-based fillers are thought to be caused by the cross-linking agents that are not removed during production and are subsequently released in vivo after hydrolysis of the hydrogel.

[0036] Therefore, this research area is currently receiving considerable attention and has become one of the major challenges in hydrogel fabrication (i.e., cross-linking of polysaccharides). However, when the amount of cross-linking agent is reduced, resulting in a decrease in the degree of cross-linking, mechanical properties and / or degradation resistance are compromised. Furthermore, due to issues related to chemical reaction kinetics, low levels of cross-linking agent may not quantitatively complete the chemical reaction in a reasonable time, resulting in only grafting without effective cross-linking of the polymer, or even worse, leaving a large amount of free, unreacted cross-linking agent in the hydrogel, which is difficult to remove and leads to high toxicity, reducing the product's safety for patients.

[0037] Therefore, based on knowledge in the art, there remains a need to provide a safety cross-linked polysaccharide having both a low amount of cross-linking material and a low degree of cross-linking that has sufficient resistance to mechanical and enzymatic / oxidative degradation. Summary of the Invention

[0038] The inventors of the present invention have surprisingly provided a safe cross-linked polysaccharide that has suitable mechanical properties, low enzymatic / oxidative degradation rates, and sufficient biocompatibility for use in in vivo applications.

[0039] In particular, the inventors have found that the hyperbranched polyglycerol diglycidyl ethers (PPEs) of the present invention, having molecular weights of 750 to 15,000 Da and epoxy equivalent weights of 183 to 7,500 g / eq, are useful as crosslinkers that can react with the hydroxyl groups of polysaccharides, allowing for the preparation of crosslinked polysaccharides of the present invention that have a low degree of crosslinking, suitable mechanical properties and degradation rates, while at the same time being safe for application to the skin.

[0040] Without being bound by any theory, the inventors believe that the mechanical properties and degradation resistance of the PPE-crosslinked polysaccharides of the present invention are due to the combined effect of the structure and amount of the hyperbranched PPE of the present invention, as well as the cohesive properties resulting from the covalent and non-covalent interactions between the polysaccharide and the hyperbranched PPE of the present invention. The interaction between the hyperbranched PPE of the present invention and the polysaccharide forms a dense three-dimensional HA network that keeps the hydrogel matrix intact and limits the ability of enzymes to penetrate the implant and degrade the three-dimensional HA network, even at low cross-linking levels.

[0041] The hyperbranched PPE of the present invention is prepared by a method including a self-polymerization step of the starting material (i.e., a GDE or a non-hyperbranched PPE). The self-polymerization step means subjecting the GDE or non-hyperbranched PPE to reaction conditions of temperature and time that allow it to have the appropriate molecular weight and epoxy equivalent weight as defined in the present invention.

[0042] Finally, the PPE cross-linked polysaccharides of the present invention exhibit good flow properties and are therefore easy to handle and extrude through thin needles, particularly when used in injection applications. Furthermore, injection of the PPE cross-linked polysaccharides of the present invention results in low or no pain perception by the patient during and after administration.

[0043] As a result, the present invention provides polysaccharide-based hydrogels that are low in cross-linking and chemical modification, and thus molecularly and structurally very similar to natural polysaccharides, with the advantages of high biocompatibility and safety profiles. Surprisingly, even with low cross-linking, polysaccharide-based hydrogels exhibit sufficient mechanical properties and resistance to enzymatic and oxidative degradation due to the high degree of cross-linking, and have the advantage of long life in vivo.

[0044] Thus, a first aspect of the present invention provides a hyperbranched PPE compound of formula (I):

[0045] TIFF0007784138000007.tif81170

[0046] or alternatively A compound of formula (II),

[0047] TIFF0007784138000008.tif54170

[0048] wherein each R1 is independently selected from the group consisting of R2 and R3;

[0049] R2 is

[0050] TIFF0007784138000009.tif21170

[0051] R3 is

[0052] TIFF0007784138000010.tif36170

[0053] b is an integer selected from the group consisting of 1 to 70; c is an integer selected from the group consisting of 1 to 70; d is an integer selected from the group consisting of 1 to 70; and a molecular weight (M) of 750 to 15,000 Da as measured by a liquid chromatography method. w ) and relates to compounds having an epoxy equivalent weight of 183 to 7,000 g / eq as determined by ultrasonic rapid titration with HCl.

[0054] A second aspect of the present invention relates to a cross-linked polysaccharide of formula (III):

[0055] TIFF0007784138000011.tif65170

[0056] or alternatively a cross-linked polysaccharide of formula (IV):

[0057] TIFF0007784138000012.tif51170

[0058] wherein each R1 is independently selected from the group consisting of R2, R3, R4 and R5;

[0059] R2 is

[0060] TIFF0007784138000013.tif17170

[0061] R3 is

[0062] TIFF0007784138000014.tif41170

[0063] R4 is

[0064] TIFF0007784138000015.tif19170

[0065] R5 is

[0066] TIFF0007784138000016.tif25170

[0067] PS is a polysaccharide; it relates to a cross-linked polysaccharide having a cross-linking rate of 0.077 to 0.450%, particularly 0.077 to 0.269%.

[0068] A third aspect of the present invention relates to a method for preparing compound (I) of the first aspect of the present invention, or alternatively a compound of formula (II); where the compound is a compound of formula (I), the method comprises the steps of: a) providing an alkaline solution containing 1,3-glycerol diglycidyl ether; and b) maintaining the solution obtained in step a) at a temperature of 10 to 80°C for 1 minute to 30 days; or alternatively, where the compound is a compound of formula (II), the method comprises the steps of: c) providing an alkaline solution containing 1,2-glycerol diglycidyl ether; and d) maintaining the solution obtained in step c) at a temperature of 10 to 80°C for 1 minute to 30 days.

[0069] A fourth aspect of the present invention relates to a method for preparing a cross-linked polysaccharide of the second aspect of the present invention, comprising the step of cross-linking a polysaccharide with a compound of formula (I) as defined in the first aspect of the present invention, or alternatively with a compound of formula (II).

[0070] A fifth aspect of the invention relates to a composition comprising one or more of the cross-linked polysaccharides of the second aspect of the invention.

[0071] A sixth aspect of the present invention relates to the use of a hyperbranched PPE according to the first aspect of the present invention as a cross-linking agent.

[0072] And finally, the invention relates to the use of the cross-linked polysaccharides of the second aspect of the invention in the fields of pharmacy, cosmetics, food and agriculture.

[0073] In particular, the cross-linked polysaccharide as defined in the second aspect of the invention, wherein the polysaccharide is hyaluronic acid for therapeutic use, is part of the invention. Also relates to the use of the cross-linked polysaccharide as defined in the second aspect of the invention, wherein the polysaccharide is hyaluronic acid, as a dermal filler. DETAILED DESCRIPTION OF THE INVENTION

[0074] All terms used in this application are to be understood in their ordinary meaning as known in the art unless otherwise specified. Other more specific definitions of terms used in this application are as set forth below and are intended to be applied uniformly throughout the specification and claims, unless another expressly defined definition provides a broader definition.

[0075] For purposes of the present invention, any range given includes both the lower and upper endpoints of the range. Given ranges of temperature, time, weight, etc. should be considered approximations unless otherwise specified.

[0076] The term "weight percent (%)" refers to the percentage of each part or component relative to the total weight.

[0077] As disclosed above, a first aspect of the present invention is a compound of formula (I); or alternatively, a compound of formula (II).

[0078] The compounds of formula (I) can be obtained by subjecting a 1,3-GDE or a non-hyperbranched PPE obtained from a 1,3-GDE to the self-polymerization step defined in the method of the present invention. Alternatively, the compounds of formula (II) can be obtained by subjecting a 1,2-GDE or a non-hyperbranched PPE obtained from a 1,2-GDE to the self-polymerization step defined in the method of the present invention.

[0079] For the purposes of the present invention, the PPEs disclosed in the state of the art are branched PPEs but not hyperbranched PPEs. State-of-the-art branched PPEs are prepared by the reaction of glycerol with epichlorohydrin, and their molecular weight can be adjusted by increasing the amount of epichlorohydrin. However, these methods do not involve a self-polymerization step like the method of the present invention. As a result, state-of-the-art branched PPEs (commercially available under the trade name Denacol®) are structurally different from the hyperbranched PPEs of the present invention. In particular, the structure of the branched PPEs disclosed in the state of the art, which are outside the scope of the present invention, is as follows:

[0080] TIFF0007784138000017.tif83170

[0081] The compound of formula (I) or alternatively the compound of formula (II) comprises a molecular weight of 750 to 15,000 Da as determined by liquid chromatography and an epoxy equivalent weight of 183 to 7,500 g / eq as determined by ultrasonic rapid titration with HCl.

[0082] The terms "epoxy equivalent weight," "average EEW," and the abbreviation "EEW" have the same meaning and are used interchangeably. EEW is the weight in grams of a crosslinker containing 1 gram equivalent of epoxy. EEW refers to the content of epoxy groups present in 1 gram of the crosslinker disclosed in the present invention and is expressed in g / eq. EEW was experimentally measured using quantitative ultrasound-assisted titration with HCl for rapid determination of epoxide as disclosed in the state-of-the-art (He, Z., et al., "Ultrasonication-assisted rapid determination of epoxide values ​​in polymer mixtures containing epoxy resins." Analytical Methods, 2014, vol. 6(12), pp. 4257-4261).

[0083] The molecular weight and polydispersity index were determined by liquid chromatography, in particular by using a GPC apparatus equipped with a light scattering detector.

[0084] In one embodiment, the compound of Formula (I) or alternatively the compound of Formula (II) comprises a molecular weight of 800 to 7000 Da by liquid chromatography and an epoxy equivalent weight of 195 to 700 g / eq as determined by ultrasonic rapid titration with HCl. In one embodiment, the compound of Formula (I) or alternatively the compound of Formula (II) comprises a molecular weight of 900 to 4500 Da by liquid chromatography and an epoxy equivalent weight of 200 to 600 g / eq as determined by ultrasonic rapid titration with HCl.

[0085] In one embodiment, the compound of formula (I) or alternatively the compound of formula (II) has a polydispersity index of 1.8 or less as measured by liquid chromatography. In one embodiment, the compound of formula (I) or alternatively the compound of formula (II) has a polydispersity index of 1.6 or less as measured by liquid chromatography, particularly using a GPC instrument equipped with a light scattering detector.

[0086] The terms "polydispersity index," "heterogeneity index," and "dispersity" have the same meaning and are used interchangeably. They refer to a measure of the heterogeneity of the size of molecules or particles in a mixture, specifically the distribution of molecular masses in a given polymer sample. This is represented by the symbol D. For purposes of this invention, polydispersity index refers to molecular mass, which can be calculated using the following formula:

[0087] D M =M w / M n In the formula, M w is the weight-average molar mass, and M n is the number average molar mass.

[0088] The terms "molecular weight", "average molecular weight", "weight average molar mass", "mass average molar mass", "average Mw" and the abbreviation "M W " have the same meaning and are used interchangeably. The mass average molar mass is calculated by the following formula:

[0089] JPEG0007784138000018.jpg22125

[0090] In the formula, N i is the molecular mass M i The mass average molecular mass can be determined by static light scattering, small angle neutron scattering, X-ray scattering, and sedimentation velocity.

[0091] The terms "number average molar mass" and the abbreviation "Mn" have the same meaning and are used interchangeably. Mn is a method for determining the molecular weight of a polymer. Because polymer molecules, such as polysaccharides, are of different sizes (chain lengths for linear polymers), even within the same type, the average molecular weight depends on the method of averaging. The number average molecular weight is the usual arithmetic mean or average of the molecular masses of the individual macromolecules. It is determined by measuring the molecular mass of n polymer molecules, adding up the masses, and dividing by n. Mn is calculated using the following formula:

[0092] JPEG0007784138000019.jpg15125

[0093] In the formula, N i is the molecular mass M i The number average molecular mass of a polymer can be determined by gel permeation chromatography, viscosity measurement (Mark-Houwink equation), colligative methods such as vapor pressure osmometry, end group determination or proton NMR.

[0094] Fragments of the polymer with different molecular weights can be pre-separated by liquid chromatography and revealed or detected by an equipped light scattering detector, before evaluation of the weight-average molar mass "Mw" and number-average molar mass "Mn" of each fragment is carried out by liquid chromatography itself using standards of known Mw.

[0095] In one embodiment, the hyperbranched PPE of the present invention is a compound of formula (I)-16:

[0096] TIFF0007784138000020.tif80170

[0097] The average Mw is 3530 g / mol; the average EEW is 589 g / eq, and the Mw / Mn is 1.53.

[0098] In one embodiment, the hyperbranched PPE of the present invention is a compound of formula (I)-22:

[0099] TIFF0007784138000021.tif79170

[0100] The average Mw is 910 g / mol; the average EEW is 229 g / eq, and the Mw / Mn is 1.36.

[0101] In one embodiment, the hyperbranched PPE of the present invention is a compound of formula (I)-23:

[0102] TIFF0007784138000022.tif79170

[0103] The average Mw is 2400 g / mol; the average EEW is 404 g / eq, and the Mw / Mn is 1.54.

[0104] In one embodiment, the hyperbranched PPE of the present invention is a compound of formula (I)-14:

[0105] TIFF0007784138000023.tif85170

[0106] The average Mw is 940 g / mol; the average EEW is 234 g / eq, and the Mw / Mn is 1.36.

[0107] In one embodiment, the hyperbranched PPE of the present invention is a compound of formula (I)-28:

[0108] TIFF0007784138000024.tif80170

[0109] The average Mw is 990 g / mol; the average EEW is 247 g / eq, and the Mw / Mn is 1.37.

[0110] In one embodiment, the hyperbranched PPE of the present invention is a compound of formula (I)-26:

[0111] TIFF0007784138000025.tif95170

[0112] The average Mw is 2200 g / mol; the average EEW is 365 g / eq, and the Mw / Mn is 1.47.

[0113] In one embodiment, the hyperbranched PPE of the present invention is a compound of formula (I)-21:

[0114] TIFF0007784138000026.tif84170

[0115] The average Mw is 4200 g / mol; the average EEW is 585 g / eq, and the Mw / Mn is 1.55.

[0116] Another aspect of the present invention is a process for preparing a compound of formula (I) or alternatively a compound of formula (II).

[0117] The process for preparing the compound of formula (I) of the present invention comprises the steps of: a) providing an alkaline solution containing 1,3-glycerol diglycidyl ether (1,3-DGE); b) maintaining the solution obtained in step a) at a temperature of 10 to 80°C for 1 minute to 30 days; Includes.

[0118] The method for preparing the compound of formula (II) of the present invention comprises the steps of: c) providing an alkaline solution containing 1,2-glycerol diglycidyl ether (1,2-DGE); d) maintaining the solution obtained in step c) at a temperature of 10 to 80°C for 1 minute to 30 days; Includes.

[0119] 1,3-GDEs and 1,2-GDEs have been previously described in the state of the art and are commercially available (see experimental section).

[0120] In one embodiment, the alkaline solution of step a) or alternatively step c) comprises a base selected from the group consisting of physiologically acceptable alkali metal or ammonium hydroxides (Na, K), alkaline earth metal hydroxides (Mg, Ca, Sr), physiologically acceptable heavy metal hydroxides (Fe, Al, Co, Cu, Se, Zn, Cr, Ni), physiologically acceptable quaternary ammonium cation hydroxides, physiologically acceptable heavy metal or ammonium bicarbonates (NH, Fe, Al, Co, Cu, Se, Zn, Cr, Ni) and mixtures thereof.

[0121] The term "physiologically acceptable" refers to a non-toxic material that is compatible with a biological system, such as a cell, cell culture, tissue, or organism. Preferably, the biological system is a living organism, such as a mammal, particularly a human.

[0122] In one embodiment, the alkaline solution of step a) or alternatively step c) comprises an alkali metal hydroxide or ammonium hydroxide selected from the group consisting of potassium hydroxide (KOH), sodium hydroxide (NaOH), ammonium hydroxide and mixtures thereof, in particular sodium hydroxide.

[0123] In one embodiment, the alkaline solution of step a) or alternatively step c) comprises an alkaline earth metal hydroxide selected from the group consisting of magnesium hydroxide, calcium hydroxide, strontium hydroxide and mixtures thereof.

[0124] In one embodiment, the alkaline solution of step a) or alternatively step c) comprises a heavy metal hydroxide selected from the group consisting of iron hydroxide, aluminum hydroxide, cobalt hydroxide, selenium hydroxide, tin hydroxide, chromium hydroxide, nickel hydroxide, and mixtures thereof.

[0125] In one embodiment, the alkaline solution of step a) or alternatively step c) comprises a quaternary ammonium hydroxide of formula HONR6R7R8R9, wherein each of R6, R7, R8, and R9 is independently selected from the group consisting of (C1-C8) alkyl, or alternatively, two of R5, R6, R7, and R9 form a (C5-C6) cycloalkyl ring.

[0126] In one embodiment, the alkaline solution of step a) or alternatively step c) comprises a heavy metal bicarbonate or ammonium bicarbonate selected from the group consisting of iron bicarbonate, aluminum bicarbonate, cobalt bicarbonate, copper bicarbonate, selenium bicarbonate, zinc bicarbonate, chromium bicarbonate, nickel bicarbonate, ammonium bicarbonate and mixtures thereof, in particular sodium bicarbonate.

[0127] In one embodiment, the alkaline solution of step a) or alternatively step c) comprises 1 to 50 weight percent 1,3-GDE or alternatively 1,2-GDE. In one embodiment, the alkaline solution of step a) or alternatively step c) comprises an alkaline hydroxide and comprises 1 to 50 weight percent 1,3-GDE or alternatively 1,2-GDE. In one embodiment, the alkaline solution of step a) or alternatively step c) comprises an alkaline hydroxide selected from the group consisting of potassium hydroxide (KOH), sodium hydroxide (NaOH), and mixtures thereof, and comprises 1 to 50 weight percent 1,3-GDE or alternatively 1,2-GDE. In one embodiment, the alkaline solution of step a) or alternatively step c) comprises sodium hydroxide (NaOH) and comprises 1 to 50 weight percent 1,3-GDE or alternatively 1,2-GDE.

[0128] In one embodiment, step a) or alternatively step c) of the method of the present invention is carried out at room temperature. The term "room temperature" refers to a temperature of about 25-35°C. In one embodiment, step a) or alternatively step c) of the method of the present invention is carried out in the presence of a solvent. In one embodiment, step a) or alternatively step c) of the method of the present invention is carried out in the presence of a solvent selected from the group consisting of water, (C1-C4) alkyl-OH, (C1-C4) alkyl-CO-(C1-C4) alkyl, (C1-C4) alkyl-CO-O-(C1-C4) alkyl, (C1-C4) alkyl-CN, and mixtures thereof. In one embodiment, step a) or alternatively step c) of the method of the present invention is carried out in the presence of a solvent selected from the group consisting of methanol, ethanol, 1-propanol, 2-propanol, acetone, acetonitrile, ethyl acetate, and mixtures thereof. In particular, the solvent is water.

[0129] The term "alkyl" refers to a saturated straight or branched hydrocarbon chain containing the number of carbon atoms specified in the specification or claims. Examples include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, and tert-butyl groups, among others.

[0130] In one embodiment, step b) or alternatively step d) is carried out by subjecting the 1,3-GDE or alternatively the 1,2-GDE to a temperature of from 10 to 80° C. for a period of from 1 minute to 30 days, during which self-polymerization of the GDE occurs.

[0131] In one embodiment, step b) or alternatively step d) is carried out by subjecting a 1,3-GDE or alternatively a 1,2-GDE to a temperature in the range of 50 to 80° C. for a period of time in the range of 1 minute to 500 minutes. In one embodiment, step b) or alternatively step d) is carried out by subjecting a 1,3-GDE or alternatively a 1,2-GDE to a temperature in the range of 50 to 80° C. for a period of time in the range of 5 minutes to 60 minutes.

[0132] In one embodiment, step b) or alternatively step d) is carried out by subjecting the 1,3-GDE or alternatively the 1,2-GDE to a temperature in the range of 10 to 40° C. for a period of 1 to 30 days. In one embodiment, step b) or alternatively step d) is carried out by subjecting the 1,3-GDE or alternatively the 1,2-GDE to a temperature in the range of 10 to 40° C. for a period of 2 to 7 days.

[0133] Compounds of formula (I) or alternatively compounds of formula (II) obtained by the process defined above, comprising the self-polymerization step b) or alternatively step d) defined above, are also part of the present invention.

[0134] For the purposes of the present invention, the terms "obtainable", "obtained" and equivalent terms are used interchangeably, and in each case the term "obtainable" encompasses the term "obtained".

[0135] All embodiments disclosed above relating to the process for the preparation of compounds of formula (I) or alternatively compounds of formula (II) also apply to compounds of formula (I) or alternatively compounds of formula (II) obtainable by the process of the present invention.

[0136] The use of the hyperbranched PPE of the present invention as a cross-linking agent is also part of the present invention.

[0137] A second aspect of the present invention relates to a cross-linked polysaccharide of formula (III) having a cross-linking rate of 0.077 to 0.450%, or alternatively a cross-linked polysaccharide of formula (IV) having a cross-linking rate of 0.077 to 0.450%. In one embodiment, the cross-linked polysaccharide of formula (III) or alternatively the cross-linked polysaccharide of formula (IV) has a cross-linking rate of 0.077 to 0.360%. In one embodiment, the cross-linked polysaccharide of formula (III) or alternatively the cross-linked polysaccharide of formula (IV) has a cross-linking rate of 0.077 to 0.269%.

[0138] In one embodiment, the cross-linked polysaccharide of formula (III) or alternatively the cross-linked polysaccharide of formula (IV) is a polysaccharide comprising hyaluronic acid as defined below, with a cross-linking percentage of 0.077 to 0.450%. In one embodiment, the cross-linked polysaccharide of formula (III) or alternatively the cross-linked polysaccharide of formula (IV) is a polysaccharide comprising hyaluronic acid as defined below, with a cross-linking percentage of 0.077 to 0.360%. In one embodiment, the cross-linked polysaccharide of formula (III) or alternatively the cross-linked polysaccharide of formula (IV) is a polysaccharide comprising hyaluronic acid as defined below, with a cross-linking percentage of 0.077 to 0.269%. In one embodiment, the cross-linked polysaccharide of formula (III) or alternatively the cross-linked polysaccharide of formula (IV) is a polysaccharide comprising hyaluronic acid as defined below, with a cross-linking percentage of 0.077 to 0.140%. In one embodiment, the cross-linked polysaccharide of formula (III) or alternatively the cross-linked polysaccharide of formula (IV) is a polysaccharide comprising hyaluronic acid as defined below, with a cross-linking percentage of 0.140 to 0.450%. In one embodiment, the cross-linked polysaccharide of formula (III) or alternatively the cross-linked polysaccharide of formula (IV) is a polysaccharide comprising hyaluronic acid as defined below, with a cross-linking percentage of 0.140 to 0.360%. In one embodiment, the cross-linked polysaccharide of formula (III) or alternatively the cross-linked polysaccharide of formula (IV) is a polysaccharide comprising hyaluronic acid as defined below, with a cross-linking percentage of 0.140 to 0.269%. In one embodiment, the cross-linked polysaccharide of formula (III) or alternatively the cross-linked polysaccharide of formula (IV) is a polysaccharide comprising hyaluronic acid as defined below, with a cross-linking percentage of 0.270 to 0.450%. In one embodiment, the cross-linked polysaccharide of formula (III) or alternatively the cross-linked polysaccharide of formula (IV) is one in which the polysaccharide is hyaluronic acid as defined below and has a cross-linking rate of 0.270 to 0.360%.

[0139] In one embodiment, the cross-linked polysaccharide of formula (III) or alternatively the cross-linked polysaccharide of formula (IV) is an amylose starch, as defined below, having a cross-linking percentage of 0.124 to 0.204%. In one embodiment, the cross-linked polysaccharide of formula (III) or alternatively the cross-linked polysaccharide of formula (IV) is an amylose, as defined below, having a cross-linking percentage of 0.155 to 0.182%. In one embodiment, the cross-linked polysaccharide of formula (III) or alternatively the cross-linked polysaccharide of formula (IV) is sodium carboxymethylcellulose, as defined below, having a cross-linking percentage of 0.140 to 0.204%.

[0140] In one embodiment, the amount of R2 in the crosslinked polysaccharide of Formula (III) and (IV) is less than R5, based on the total weight of the crosslinked polysaccharide of Formula (III) and (IV). In one embodiment, the amount of R2 in the crosslinked polysaccharide of Formula (III) and (IV) is less than 2 ppm, based on the total weight of the crosslinked polysaccharide of Formula (III) and (IV). The amount of R2 is experimentally determined using quantitative ultrasound-assisted titration with HCl for rapid determination of epoxide as disclosed in He, Z., et al., "Ultrasonication-assisted rapid determination of epoxide values ​​in polymer mixtures containing epoxy resin", Analytical Methods, 2014, vol. 6(12), pp. 4257-4261.

[0141] The polysaccharides of the present invention are "cross-linked" polysaccharides. The term "cross-linked polysaccharide" refers to a polysaccharide having a three-dimensional cross-linked network, the network formed by the starting polysaccharide being broken by one or more cross-linking agents. The term "cross-linking" refers in polymer science to the use of cross-linking to promote differences in the physical properties of polymers (polysaccharides). The term "cross-link" refers to the bond formed by the opening of the epoxide ring of the hyperbranched PPE cross-linker of the present invention by the hydroxyl groups of the polymer (polysaccharide). The terms "cross-linking substance" or "cross-linking agent," used interchangeably herein, refer to a compound capable of cross-linking one or more polymer chains.

[0142] The terms "crosslinking rate", "percentage of crosslinking", "degree of crosslinking" and the abbreviation "CD" have the same meaning and are used interchangeably. They refer to the ratio of PPE-modified polysaccharide monomer units to unmodified polysaccharide monomer units expressed as a percentage. In general, the measurement of the degree of crosslinking (CD) of a material can be assessed using different methods well known in the state of the art. Some methods involve measuring the crosslinking rate of polymer fragments after enzymatic digestion. 1 Direct methods include H NMR or quantitative GPC analysis. Other methods are indirect, using rheological measurements, compression or vibration tests. Usually, all these methods are comparative and can distinguish between different degrees of crosslinking within the same polymer family. In the present invention, the value of the degree of crosslinking is mathematically calculated from the G' value obtained by rheological measurements using the following equation:

[0143] TIFF0007784138000027.tif22170

[0144] During the ceremony, MW rep.unit is the molecular weight of the repeating unit of the polysaccharide monomer, For hyaluronic acid, MW rep.unit = 401 Da; For amylose / glycogen, MW rep.unit = 162 Da; For sodium carboxymethylcellulose, MW rep.unit =242Da.

[0145] Me is calculated mathematically using the inverse formula of Equation 2 below.

[0146] JPEG0007784138000028.jpg19125

[0147] During the ceremony, G' is experimentally obtained by rheological measurements using a rotational rheometer, R·T is the thermal energy, c is the polysaccharide concentration, M n is the number average molecular weight of the polysaccharide.

[0148] In this study, the absolute enzymatic and oxidative degradation rates of each cross-linked polysaccharide hydrogel were mathematically calculated. Three methods were used to monitor the absolute enzymatic and oxidative degradation rates of cross-linked polysaccharide hydrogels depending on the polysaccharide substrate.

[0149] The first method is based on monitoring rheological parameters such as the storage modulus G' during degradation, and the degradation rate rheo is calculated from Equation 3.

[0150] TIFF0007784138000029.tif15170

[0151] Decrease in storage modulus G' during degradation of cross-linked polysaccharide hydrogels caused by disruption of the hydrogel network, G' t <G´ t A negative sign is required to have a positive degradation rate. This first method is suitable for determining the absolute enzymatic and oxidative degradation rates of cross-linked polysaccharide hydrogels containing hyaluronic acid, starch and its derivatives, glycogen, cellulose and its derivatives, pectin, lignin, inulin, guar gum, xanthan gum, alginic acid (alginate), glucomannan, galactomannan, and carrageenan.

[0152] The second method is based on monitoring viscosity parameters such as the dynamic viscosity η during decomposition, which allows the decomposition rate to be monitored. visc is calculated from Equation 4.

[0153] TIFF0007784138000030.tif15170

[0154] The decrease in dynamic viscosity η during the degradation of cross-linked polysaccharide hydrogels caused by the decrease in polymer chain length, η t <η t0 observed, and a negative sign is required to have a positive degradation rate. This second method is suitable for determining the absolute enzymatic and oxidative degradation rates of cross-linked polysaccharide hydrogels containing hyaluronic acid, starch and its derivatives, glycogen, cellulose and its derivatives, pectin, lignin, inulin, guar gum, xanthan gum, alginic acid (alginate), glucomannan, galactomannan, and carrageenan.

[0155] The third method is based on colorimetric monitoring of uronic acid concentration by carbazole assay using a spectrophotometer instrument during degradation, and the degradation rate color is calculated using the following equation 5.

[0156] TIFF0007784138000031.tif15170

[0157] The increase in the concentration of uronic acid during the degradation of cross-linked polysaccharide hydrogels caused by the decrease in polymer chain length and the formation of uronic acid monomer units, [uronic acid] t >[Uronic acid] t0 observed, where a negative sign is required to have a positive degradation rate. This third method is suitable for determining the absolute enzymatic and oxidative degradation rates of cross-linked polysaccharide hydrogels containing hyaluronic acid, pectin, heparin, chondroitin sulfate, dermatan sulfate, xanthan, glucuronan, and alginic acid (alginate).

[0158] In the present invention, the absolute enzymatic and oxidative degradation rates of crosslinked polysaccharide hydrogels are also compared. Over a given period, the relative enzymatic and oxidative degradation rate of each crosslinked polysaccharide hydrogel is calculated as a percentage of the absolute enzymatic and oxidative degradation rate of the crosslinked polysaccharide hydrogel relative to the absolute enzymatic and oxidative degradation rate of a polysaccharide hydrogel crosslinked with a GDE known in the state of the art and used as a reference. Therefore, this is calculated using Equation 6.

[0159] TIFF0007784138000032.tif15170

[0160] During the ceremony, Decomposition rate xl is the absolute degradation rate of polysaccharide hydrogels crosslinked with BDDE, PEGDE and PPE, Decomposition rate GDE is the absolute degradation rate of the cross-linked polysaccharide hydrogel in GDEs known in the state of the art.

[0161] In one embodiment, the cross-linked polysaccharides of the present invention have a relative enzymatic degradation rate of 100% to 37% of the enzymatic degradation rate of a comparative GDE-cross-linked polysaccharide (which is used as a reference value and internal reference). The GDE-cross-linked polysaccharide used as the internal value is obtained by reacting a polysaccharide with a commercially available GDE (as shown in the experimental section). In one embodiment, the cross-linked polysaccharides of the present invention have a relative enzymatic degradation rate of 85 to 40%. In one embodiment, the cross-linked polysaccharides of the present invention have a cross-linking rate of 0.1528 to 0.1877% and a relative enzymatic degradation rate of 84 to 37%.

[0162] In one embodiment, the cross-linked polysaccharides of the present invention have a relative oxidative degradation rate of 100% to 76% of the oxidative degradation rate of a comparative GDE cross-linked polysaccharide (which is used as a reference value and internal reference). The GDE cross-linked polysaccharide used as the internal value is obtained by reacting a polysaccharide with a commercially available GDE (as shown in the experimental section). In one embodiment, the cross-linked polysaccharides of the present invention have a relative oxidative degradation rate of 90 to 75%. In one embodiment, the cross-linked polysaccharides of the present invention have a cross-linking rate of 0.1528 to 0.1877% and an oxidative degradation rate of 76 to 87%.

[0163] As noted above, the absolute enzymatic and oxidative degradation rates of the cross-linked polysaccharides of the present invention are equal to or less than those of the comparative GDE cross-linked polysaccharides, which are used as internal standards and have a lower degree of cross-linking. This is advantageous because it improves both the stability of the hydrogel and the safety and comfort of the patient.

[0164] In one embodiment, the crosslinked polysaccharide of the present invention has an absolute enzymatic degradation rate of 0.8 to 3.5 mmol / h and an absolute oxidative degradation rate of 3.0 to 4.3 mmol / h. In one embodiment, the crosslinked polysaccharide of the present invention has an absolute enzymatic degradation rate of 0.8 to 2.6 mmol / h and an absolute oxidative degradation rate of 3.0 to 3.4 mmol / h. In one embodiment, the crosslinked polysaccharide of the present invention has a crosslinking rate of 0.1528 to 0.1877%, an enzymatic degradation rate of 0.8 to 2.7 mmol / h, and an oxidative degradation rate of 3.0 to 3.4 mmol / h.

[0165] In one embodiment, the cross-linked polysaccharides of the present invention have an average distance between two cross-linking groups (D N In one embodiment, the cross-linked polysaccharide of the present invention has an average distance between two cross-linking groups (D N In one embodiment, the cross-linked polysaccharide of the present invention has an average distance between two cross-linking groups (D N ) is 27 nm or more and 42 nm or less.

[0166] In one embodiment, the cross-linked polysaccharide of the present invention has a polysaccharide concentration of 1 to 50 mg / ml. The polysaccharide concentration can be measured by any suitable method disclosed in the state of the art. In the present invention, the polysaccharide concentration is measured by weighing the dry residue obtained after dehydrating the hydrogel in a ventilated oven at 105°C for an appropriate time.

[0167] In one embodiment, the crosslinked polysaccharide of the present invention has a viscosity of 1 to 200 Pa s as measured by a viscometer. In another embodiment, the crosslinked polysaccharide of the present invention has a viscosity of 10 to 100 Pa s as measured by a viscometer.

[0168] In one embodiment, the cross-linked polysaccharide of the present invention is a cross-linked polysaccharide wherein the polysaccharide is selected from the group consisting of hyaluronic acid, starch and its derivatives, glycogen, cellulose and its derivatives, pectin, lignin, inulin, guar gum, xanthan gum, alginic acid (alginate), heparin, chondroitin sulfate, dermatan sulfate, glucuronan, glucomannan, galactomannan, and carrageenan.

[0169] In one embodiment, the cross-linked polysaccharide of the present invention is that the polysaccharide is hyaluronic acid.For the purpose of the present invention, the terms "hyaluronic acid", "hyaluronan" and the abbreviation "HA" have the same meaning and are used interchangeably.They refer to the natural non-sulfated anionic form of glycosaminoglycan, which is composed of several repeating disaccharide units of N-acetyl-D-glucosamine and D-glucuronic acid and has CAS number 9004-61-9, and also refer to the pharmaceutically or cosmetically acceptable salts of hyaluronic acid.

[0170] For purposes of the present invention, the term "hyaluronic acid" also encompasses pharmaceutically or cosmetically acceptable salts of hyaluronic acid. In one embodiment, the cross-linked polysaccharide of the present invention is one in which the polysaccharide is a pharmaceutically or cosmetically acceptable salt of hyaluronic acid. The type of hyaluronic acid salt that can be used is not limited, as long as it is pharmaceutically or cosmetically acceptable when used for therapeutic purposes. Hyaluronic acid and its salts may differ in some physical properties, but they are equivalent for the purposes of the present invention, and the skin-friendly properties of hyaluronic acid can be extended to pharmaceutically or cosmetically acceptable salts, particularly its sodium salt (i.e., sodium hyaluronate) and potassium salt (potassium hyaluronate). The term "pharmaceutically acceptable salt" refers to a salt suitable for use in pharmaceutical technology to prepare compositions for medical use, and the terms "cosmetically acceptable salt" and "dermatologically acceptable salt," used interchangeably herein, refer to salts that are suitable for use in contact with human skin without toxicity, incompatibility, instability, or undesirable allergic reactions. In particular, the term "pharmaceutically or cosmetically acceptable salts" includes commonly used salts, such as alkali metal salts. Pharmaceutically acceptable salts of hyaluronic acid can be prepared by methods known in the art. Non-limiting examples of pharmaceutically or cosmetically acceptable salts of hyaluronic acid suitable for the present invention include inorganic salts such as sodium hyaluronate, magnesium hyaluronate, potassium hyaluronate, zinc hyaluronate, and cobalt hyaluronate, and organic salts such as tetrabutylammonium hyaluronate. In one embodiment, the pharmaceutically or cosmetically acceptable salt of hyaluronic acid is a selected alkali metal salt of sodium salt (CAS No. 9067-32-7) or potassium salt (CAS No. 31799-91-4).

[0171] In one embodiment, the cross-linked polysaccharide of the present invention is a polysaccharide that is a high molecular weight hyaluronic acid. The term "high molecular weight hyaluronic acid" refers to hyaluronic acid having a molecular weight of 300 kDa or more. In one embodiment, the cross-linked polysaccharide of the present invention is a polysaccharide that is a high molecular weight hyaluronic acid having a molecular weight of 300 kDa to 5000 kDa, particularly 500 kDa to 2000 kDa. In a specific embodiment, the cross-linked polysaccharide of the present invention is a polysaccharide that is a high molecular weight hyaluronic acid having a molecular weight of 1000 kDa to 2000 kDa.

[0172] In one embodiment, the cross-linked polysaccharides of the present invention are Cross-linking degree of 0.2688% Enzymatic degradation rate of 2.8 mmol / h, and Oxidative decomposition rate of 3.3 mmol / h, Cross-linked polysaccharide (III)-4 having the formula: The polysaccharide is hyaluronic acid, The hyperbranched PPE used as the starting material is compound (I)-22.

[0173] In one embodiment, the cross-linked polysaccharides of the present invention are Cross-linking degree of 0.1127% an enzymatic degradation rate of 3.6 mmol / h, and Oxidative decomposition rate of 4.3 mmol / h, Cross-linked polysaccharide (III)-9 having the formula The polysaccharide is hyaluronic acid, The hyperbranched PPE used as the starting material is compound (I)-16.

[0174] In one embodiment, the cross-linked polysaccharides of the present invention are Cross-linking degree of 0.1358% an enzymatic degradation rate of 3.5 mmol / h, and Oxidative decomposition rate of 4.3 mmol / h, Cross-linked polysaccharide (III)-8 having the formula: The polysaccharide is hyaluronic acid, The hyperbranched PPE used as the starting material is compound (I)-22.

[0175] In one embodiment, the cross-linked polysaccharides of the present invention are Cross-linking degree of 0.1877% an enzymatic degradation rate of 0.8 mmol / h, and Oxidative decomposition rate of 3.0 mmol / h, Cross-linked polysaccharide (III)-5 having the formula: The polysaccharide is hyaluronic acid, The hyperbranched PPE used as the starting material is compound (I)-23.

[0176] In one embodiment, the cross-linked polysaccharides of the present invention are Cross-linking degree of 0.1528% an enzymatic degradation rate of 2.7 mmol / h, and Oxidative decomposition rate of 3.4 mmol / h, Cross-linked polysaccharide (III)-6 having the formula: The polysaccharide is hyaluronic acid, The hyperbranched PPE used as the starting material is compound (I)-16.

[0177] In one embodiment, the cross-linked polysaccharides of the present invention are Cross-linking degree of 0.3911% an enzymatic degradation rate of 2.8 mmol / h, and Oxidative decomposition rate of 2.6 mmol / h, and cross-linked polysaccharide (III)-14 having the formula: The polysaccharide is hyaluronic acid, The hyperbranched PPE used as the starting material is compound (I)-22.

[0178] In one embodiment, the cross-linked polysaccharides of the present invention are Cross-linking degree of 0.3049% Enzymatic degradation rate of 1.1 mmol / h, and Oxidative decomposition rate of 2.3 mmol / h, Cross-linked polysaccharide (III)-15 having the formula: The polysaccharide is hyaluronic acid, The hyperbranched PPE used as the starting material is compound (I)-23.

[0179] In one embodiment, the cross-linked polysaccharides of the present invention are Cross-linking degree of 0.2723% Enzymatic degradation rate of 2.9 mmol / h, and Oxidative decomposition rate of 2.6 mmol / h, and cross-linked polysaccharide (III)-16 having the formula: The polysaccharide is hyaluronic acid, The hyperbranched PPE used as the starting material is compound (I)-16.

[0180] In one embodiment, the cross-linked polysaccharide of the present invention is one in which the polysaccharide is starch or a derivative thereof. For purposes of the present invention, the term "starch" refers to a polysaccharide carbohydrate consisting of multiple glucose units linked by glycosidic bonds. Starch is produced by all green plants as an energy storage material and is a major food source for humans. For purposes of the present invention, the terms "starch derivative" and "modified starch" have the same meaning and are used interchangeably. They refer to starch that has been reacted to modify its properties. Examples of starch derivatives are hydroxyalkyl, carboxyalkylated, alkylcarbonylated, phosphorylated, sulfated, graft-derivatized, and alkylated starches. In one embodiment, the starch derivative is selected from the group consisting of hydroxypropyl starch, sodium starch glycolate, and carboxymethyl starch.

[0181] In one embodiment, the cross-linked polysaccharide of the present invention is one in which the polysaccharide is cellulose or a derivative thereof. For purposes of the present invention, the term "cellulose" refers to a polysaccharide having a backbone formed by D-glucopyranose units linked by β-1,4-glycosidic bonds. The terms "modified cellulose" and "cellulose derivative" have the same meaning and are used interchangeably. They refer to any compound having a cellulose backbone, i.e., D-glucopyranose units linked by β-1,4-glycosidic bonds, that has been chemically modified by the introduction of pendant moieties not found in pure cellulose. Non-limiting examples of cellulose derivatives include methylcellulose, hydroxyethylcellulose, hydroxyethylmethylcellulose, hydroxypropylcellulose, L-hydroxypropylcellulose (low-substituted), hydroxypropylmethylcellulose (HPMC), sodium carboxymethylcellulose, or carboxymethylhydroxyethylcellulose.

[0182] All of the above-described embodiments disclosed for the cross-linked polysaccharides of the present invention also apply individually to each of the polysaccharides specifically listed above, which are hyaluronic acid, starch and its derivatives, glycogen, cellulose and its derivatives, pectin, lignin, inulin, guar gum, xanthan gum, alginic acid (alginate), heparin, chondroitin sulfate, dermatan sulfate, glucuronan, glucomannan, galactomannan, and carrageenan.

[0183] A method for preparing the cross-linked polysaccharides of formula (III) or alternatively formula (IV) of the present invention is also an aspect of the present invention.

[0184] In one embodiment, the process for preparing the cross-linked polysaccharide (III) of the present invention comprises the step of cross-linking a polysaccharide with a compound of formula (I) as defined in the first aspect of the present invention.

[0185] In one embodiment, the method for preparing a cross-linked polysaccharide of formula (III) of the present invention comprises cross-linking a polysaccharide with a compound of formula (I) as defined in the first aspect of the present invention, and the compound of formula (I) can be obtained by a method comprising steps a) and b) as defined above. Thus, the method for preparing a cross-linked polysaccharide of formula (III) of the present invention comprises cross-linking a polysaccharide with a compound of formula (I), and the compound of formula (I) can be obtained by a method comprising the steps of: a) providing an alkaline solution containing 1,3-glycerol diglycidyl ether; and b) maintaining the solution obtained in step a) at a temperature of 10 to 80°C for 1 minute to 30 days.

[0186] In one embodiment, the process for preparing the cross-linked polysaccharide (III) of the present invention comprises the step of cross-linking a polysaccharide with a compound of formula (II) as defined in the first aspect of the present invention.

[0187] In one embodiment, the method for preparing a crosslinked polysaccharide of formula (IV) of the present invention comprises crosslinking a polysaccharide with a compound of formula (II), where the compound of formula (II) can be obtained by carrying out steps c) and d) defined above. Thus, the method for preparing a crosslinked polysaccharide of the present invention comprises crosslinking a polysaccharide with a compound of formula (II), where the compound of formula (II) can be obtained by carrying out steps c) and d) of preparing an alkaline solution containing 1,2-glycerol diglycidyl ether; and d) maintaining the solution obtained in step c) at a temperature of 10 to 80°C for 1 minute to 30 days.

[0188] All embodiments disclosed above for the polysaccharide, the compound of formula (I), the compound of formula (II) and the cross-linked polysaccharide, as well as the reaction conditions of steps a) to d) of the process for preparing the cross-linked polysaccharide of the present invention, also apply to the cross-linked polysaccharide obtainable by this process.

[0189] In one embodiment, the process for preparing a cross-linked polysaccharide of formula (III) comprises: e) cross-linking the polysaccharide with a compound of formula (I) as defined in the first aspect of the present invention; and optionally g) isolating the cross-linked polysaccharide of formula (III) of the present invention; Includes.

[0190] In one embodiment, step e) of the process for preparing a cross-linked polysaccharide of formula (III) comprises: e1) preparing an alkaline solution of 5 to 15 weight percent polysaccharide; e2) providing an alkaline solution containing a compound of formula (I); e3) mixing the solution obtained in step e1) with the solution obtained in step e2) to obtain an alkaline solution containing the polysaccharide and the compound of formula (I); e4) maintaining the alkaline solution obtained in step e3) at a temperature comprised between 20°C and 50°C for 1 hour to 48 hours; Includes.

[0191] In one embodiment, the process for preparing a cross-linked polysaccharide of formula (IV) comprises: f) cross-linking the polysaccharide with a compound of formula (II) as defined in the first aspect of the present invention; and optionally g) isolating the cross-linked polysaccharide of formula (IV) of the present invention; Includes.

[0192] In one embodiment, step f) of the process for preparing a cross-linked polysaccharide of formula (IV) comprises: f1) preparing an alkaline solution of 5 to 15 weight percent polysaccharide; f2) providing an alkaline solution containing a compound of formula (II); f3) mixing the solution obtained in step f1) with the solution obtained in step f2) to obtain an alkaline solution containing the polysaccharide and the compound of formula (II); f4) maintaining the alkaline solution obtained in step f3) at a temperature comprised between 20°C and 50°C for 1 hour to 48 hours; Includes.

[0193] In one embodiment, the alkaline solution of step e3) or alternatively step f3) comprises a polysaccharide at a concentration of 5-15% by weight.

[0194] In one embodiment, the alkaline solution obtained in step e3) or alternatively in step f3) comprises 0.25 to 0.75 M of alkaline base; in particular 0.40 to 0.60 M of alkaline base.

[0195] In one embodiment, the alkaline solution obtained in step e3) or alternatively in step f3) comprises 5 to 15% by weight of polysaccharides; in particular 8 to 10% by weight of polysaccharides.

[0196] In one embodiment, the alkaline solution obtained in step e3) or alternatively in step f3) comprises 0.2 to 1 wt. %; in particular 0.3 to 0.5 wt. % of the compound of formula (I) or alternatively of formula (II).

[0197] In one embodiment, the alkaline solution obtained in step e3) or alternatively in step f3) comprises a weight ratio between the compound of formula (I) and the polysaccharide, or alternatively between the compound of formula (II) and the polysaccharide, of 2 to 40% w / w, expressed as the weight ratio of PPE to dry polysaccharide; in particular, a weight ratio between 2 to 12.5% ​​w / w. The term "weight ratio" refers to the weight relationship between the two components required to enhance the stability and bioavailability of the cross-linked polysaccharide of the present invention after its application. In particular, the relationship between the polysaccharide of the present invention and the hyperbranched PPE was necessary to reduce the enzymatic / oxidative degradation rate and reduce the degree of cross-linking.

[0198] In one embodiment, the alkaline solution obtained in step e4) or alternatively in step f4) contains 1 to 50 mg / mL of polysaccharide.

[0199] The isolation step g) can be carried out according to methods disclosed in the state of the art for isolating compounds and known to those skilled in the art. In one embodiment, the isolation step can include removing the compounds by one or more of the following procedures: evaporation, lyophilization, filtration, decantation and centrifugation, or other suitable techniques known to those skilled in the art. In one embodiment, the isolation step g) includes adjusting the pH of the alkaline solution obtained in step e3) or alternatively f3) to 6.5-7.4. The pH adjustment can be carried out by adding an acid, such as HCl, H3PO3, or a mixture thereof. The pH can be determined by any method known in the state of the art. In the present application, the pH is determined by directly reading the pH value using a pH meter with an electrode immersed in the hydrogel.

[0200] Another aspect of the present invention is a method for producing a semiconductor device comprising: one or more cross-linked polysaccharides of the present invention; one or more suitable excipients or carriers; The appropriate cross-linked polysaccharide and suitable excipients and / or carriers, as well as the amounts thereof, can be readily determined by one skilled in the art depending on the field and type of formulation to be prepared.

[0201] In one embodiment, the composition is a pharmaceutical composition comprising a therapeutically effective amount of one or more pharmaceutically acceptable cross-linked polysaccharides and one or more pharmaceutically acceptable excipients or carriers.

[0202] The term "pharmaceutical composition" refers to a composition suitable for use in pharmaceutical technology for medical applications. As used herein, the term "therapeutically effective amount of a pharmaceutically acceptable cross-linked polysaccharide" refers to an amount of the cross-linked polysaccharide of the present invention that, when administered, is sufficient to prevent the onset of, or alleviate to some extent, one or more symptoms of the disease being addressed. Of course, the dose of a pharmaceutically acceptable cross-linked polysaccharide administered in accordance with the present invention will be determined by the particular circumstances surrounding the case, including the compound being administered, the route of administration, the particular condition being treated, and similar considerations. For purposes of the present invention, the term "pharmaceutically acceptable cross-linked polysaccharide" refers to the active or central component in a composition that directly (therapeutically) effects the diagnosis, prevention, treatment, cure, or alleviation of a disease or condition.

[0203] The pharmaceutical composition of the present invention comprises one or more pharmaceutically acceptable excipients or carriers. The term "pharmaceutically acceptable excipients or carriers" refers to excipients or carriers suitable for use in pharmaceutical technology to prepare compositions for medical use. In one embodiment, the composition of the present invention comprises one or more pharmaceutically acceptable excipients and / or carriers selected from the group consisting of diluents, binders, glidants, disintegrants, lubricants, and mixtures thereof. Furthermore, the pharmaceutical composition of the present invention may contain other ingredients, such as flavorings, coloring agents, and other ingredients known in the art.

[0204] In one embodiment, the pharmaceutical composition comprises one or more pharmaceutically acceptable cross-linked polysaccharides selected from the group consisting of hyaluronic acid, starch and its derivatives, glycogen, cellulose and its derivatives, pectin, lignin, inulin, guar gum, xanthan gum, alginic acid (alginate), heparin, chondroitin sulfate, dermatan sulfate, glucuronan, glucomannan, galactomannan, and carrageenan.

[0205] In one embodiment, the composition is a cosmetic composition comprising a cosmetically effective amount of one or more cosmetically acceptable cross-linked polysaccharides and one or more cosmetically acceptable excipients or carriers.

[0206] The cosmetic composition of the present invention is designed to be applied to the body in an amount ("cosmetically effective amount") appropriate for improving the appearance thereof, or for beautifying, preserving, conditioning, cleaning, coloring or protecting the skin, nails or hair (see Academic Press Dictionary of Science and Technology, 1992, pp. 531; A Terminological Dictionary of the Pharmaceutical Sciences. 2007, pp. 190).Therefore, the cosmetic composition is used adjectively for non-medical use.

[0207] The terms "cosmetically acceptable" or "dermatologically acceptable," as used interchangeably herein, refer to, inter alia, excipients or carriers that are suitable for use in contact with human skin without undue toxicity, incompatibility, instability, or allergic reaction.

[0208] In one embodiment, the pharmaceutical or cosmetic compositions of the present invention are topical compositions that can be formulated in several forms, including, but not limited to, solutions, aerosol and non-aerosol sprays, shaving creams, powders, mousses, lotions, gels, sticks, ointments, pastes, creams, shampoos, shower gels, body washes, or facial cleansers.

[0209] In one embodiment, the pharmaceutical or cosmetic composition is an oral composition, for example, solid compositions such as tablets, granules, and capsules.

[0210] In one embodiment, the pharmaceutical or cosmetic composition is an injectable composition. In one embodiment, the pharmaceutical or cosmetic composition is an injectable composition selected from the group consisting of intramuscular, subcutaneous, or intravenous applications. In one embodiment, the pharmaceutical or cosmetic composition of the present invention is in the form of a parenteral composition suitable for injection, infusion, or implantation into the body. Parenteral compositions as defined above should be sterile and pyrogen-free. They may be in liquid form, such as a solution, emulsion, or suspension, or in solid form packaged in either single-dose or multi-dose containers for appropriate dilution before use. Parenteral compositions may contain excipients or carriers suitable for parenteral administration, which may be pharmaceutical or cosmetic excipients, including, but not limited to, solvents, suspending agents, buffers, substances that render the preparation isotonic with blood, stabilizers, or antimicrobial preservatives. The addition of excipients should be kept to a minimum. If excipients are used, they should not adversely affect the stability, bioavailability, safety, or efficacy of the polymer and / or active agent, or cause toxicity or excessive local irritation. There should be no incompatibility between any of the components of the dosage form.

[0211] The topical composition defined above contains a suitable excipient or carrier for topical administration, which may be a pharmaceutical or cosmetic excipient, including, but not limited to, a skin barrier function repair agent, a hydrating agent, an emollient, an emulsifier, a thickener, a moisturizer, a pH adjuster, an antioxidant, a preservative, a vehicle, or a mixture thereof. The excipient or carrier used is used in an amount sufficient to be compatible with the skin, well tolerated, stable, provide the desired consistency, and facilitate application. Furthermore, the composition of the present invention may contain other ingredients, such as fragrances, colorants, and other ingredients known in the art for use in topical formulations. The topical composition of the present invention may be formulated in several forms, including, but not limited to, solutions, aerosol and non-aerosol sprays, creams, powders, mousses, lotions, gels, sticks, ointments, pastes, and emulsions.

[0212] In one embodiment, the cosmetic composition comprises one or more pharmaceutically acceptable cross-linked polysaccharides selected from the group consisting of hyaluronic acid, starch and its derivatives, glycogen, cellulose and its derivatives, pectin, lignin, inulin, guar gum, xanthan gum, alginic acid (alginate), heparin, chondroitin sulfate, dermatan sulfate, glucuronan, glucomannan, galactomannan, and carrageenan.

[0213] In one embodiment, the composition is an edible composition comprising one or more edible cross-linked polysaccharides and one or more edible excipients or carriers.

[0214] The term "edible" refers to compositions, cross-linked polysaccharides, excipients and carriers that can be consumed by humans or animals without serious adverse health consequences.

[0215] The term "edible acceptable excipient or carrier" refers to an excipient or carrier that is substantially neutral in terms of flavor, so long as it does not significantly alter the sensory characteristics. Furthermore, it is suitable for use in preparing compositions that can be consumed by humans without significant adverse health consequences.

[0216] In one embodiment, the edible composition comprises one or more edible cross-linked polysaccharides selected from the group consisting of hyaluronic acid, starch and its derivatives, glycogen, cellulose and its derivatives, pectin, lignin, inulin, guar gum, xanthan gum, alginic acid (alginate), heparin, chondroitin sulfate, dermatan sulfate, glucuronan, glucomannan, and carrageenan.

[0217] The edible compositions of the present invention can be formulated in several forms, including, but not limited to, solid or liquid forms. Additionally, the edible compositions of the present invention may contain other ingredients, such as colorants and lightfastness agents, and other ingredients known in the state of the art for use in edible compositions.

[0218] In one embodiment, the edible composition is a human food or animal food (feed).

[0219] In one embodiment, the composition is an agricultural composition comprising a biologically effective amount of one or more agriculturally acceptable cross-linked polysaccharides and one or more agriculturally acceptable excipients or carriers.

[0220] The term "agricultural" refers to compositions, cross-linked polysaccharides, excipients and carriers that can be applied to the base and / or foliage of plants to treat plants without serious adverse health consequences.

[0221] In one embodiment, the agricultural composition is selected from the group consisting of fertilizers, macronutrients, micronutrients, pesticides, plant growth regulators, plant hormones and Nod factors.

[0222] As used herein, the term "biologically effective amount" refers to the amount of the cross-linked polysaccharide of the present invention required to produce the desired effect on plants, insects, or plant pests. The effective amount of a substance depends on several factors, including the treatment method, plant species, pest species, type of propagation material, and environmental conditions. For example, a biologically effective amount of an insecticide is the amount of insecticide that protects a plant from damage. This does not mean that the protected plant will not be damaged by pests, but rather that the damage is at a level that allows the plant to produce an acceptable yield of crops.

[0223] The agricultural composition may be formulated in a form selected from the group comprising wettable powders, dusts, water dispersible granules, suspension concentrates, emulsifiable concentrates, tablets, pellets, liquids and oil suspensions.

[0224] In one embodiment, the agricultural composition comprises one or more agriculturally acceptable cross-linked polysaccharides selected from the group consisting of hyaluronic acid, starch and its derivatives, glycogen, cellulose and its derivatives, pectin, lignin, inulin, guar gum, xanthan gum, alginic acid (alginate), heparin, chondroitin sulfate, dermatan sulfate, glucuronan, glucomannan, galactomannan, and carrageenan.

[0225] The use of the cross-linked polysaccharide of the present invention is also part of the present invention. The suitable cross-linked polysaccharide and suitable excipients and / or carriers, as well as the amounts thereof, can be easily determined by those skilled in the art depending on the field and type of formulation to be prepared.

[0226] The pharmaceutical cross-linked polysaccharides of the present invention and pharmaceutical compositions containing them can be used in the pharmaceutical field.Therefore, the pharmaceutical cross-linked polysaccharides of the present invention for use in therapy or alternatively the pharmaceutical compositions of the present invention are also part of the present invention.In one embodiment, the cross-linked HA of the present invention or a composition containing it for use in therapy is provided.

[0227] This aspect can also be described as the use of a pharmaceutical cross-linked polysaccharide for the preparation of a medicament. The present invention also relates to a method for treating a mammal suffering from a disease, comprising administering to the mammal a therapeutically effective amount of a pharmaceutical composition comprising the pharmaceutical cross-linked polysaccharide of the present invention as defined above. In one embodiment, the pharmaceutical composition of the present invention as defined above is a cross-linked polysaccharide for use in the treatment of soft tissue augmentation. This aspect can also be described as the use of a pharmaceutical composition of the present invention comprising cross-linked HA as defined above for the preparation of a medicament for treating a wound. The present invention also relates to a method for treating a mammal suffering from osteoarthritis, comprising administering to the mammal a pharmaceutical composition of the present invention comprising cross-linked HA as defined above.

[0228] All the embodiments defined above for the pharmaceutical cross-linked polysaccharides and pharmaceutical compositions of the invention also apply to their use in therapy.

[0229] The cosmetic cross-linked polysaccharide of the present invention and the cosmetic composition containing it can be used in the cosmetic field.Therefore, the use of the cosmetic cross-linked polysaccharide of the present invention and the cosmetic composition as a skin care agent is also part of the present invention, and skin care includes improving at least one of the symptoms such as roughness, peeling (dry skin), dehydration, stiffness, cracking, and lack of elasticity (resilience).

[0230] In one embodiment, the cosmetic cross-linked polysaccharide of the present invention and the cosmetic composition containing the same are moisturizers. In one embodiment, the cosmetic cross-linked polysaccharide of the present invention and the cosmetic composition containing the same are skin care agents. In one embodiment, the cosmetic cross-linked polysaccharide of the present invention and the cosmetic composition containing the same are emollients. Emollients are suitable for soothing, calming, soothing, calming, stabilizing, soothing, or calming the skin. In one embodiment, the cosmetic cross-linked polysaccharide of the present invention and the cosmetic composition containing the same are skin barrier restoration agents.

[0231] In one embodiment, the cosmetic cross-linked polysaccharides of the present invention and cosmetic compositions containing same are dermal fillers, in particular the polysaccharide is HA.

[0232] The edible cross-linked polysaccharides of the present invention and edible compositions containing the same can be used in the food industry. In one embodiment, the edible cross-linked polysaccharides of the present invention and edible compositions containing the same are food additives. In one embodiment, the edible cross-linked polysaccharides of the present invention and edible compositions containing the same are texture modifiers such as gums, thickeners, bulking agents, emulsion stabilizers, and binders.

[0233] In one embodiment, for the use of the edible cross-linked polysaccharides of the present invention and edible compositions containing same as food additives, particularly as texture modifiers, the polysaccharides are HA, starch and its derivatives, cellulose and its derivatives, pectin, lignin, inulin, guar gum, xanthan gum, alginic acid (alginate), heparin, chondroitin sulfate, dermatan sulfate, glucuronan, glucomannan, and carrageenan.

[0234] The use of the cross-linked polysaccharides of the invention as carriers for pharmaceutical active ingredients, diagnostic agents, cosmetic compounds, peptides, proteins, antibodies, vaccines and as delivery systems for genes is also part of the present invention.

[0235] Throughout the specification and claims, the word "comprises" and variations of that word are not intended to exclude other technical features, additives, ingredients, or steps. Furthermore, the word "comprises" encompasses the case of "consisting of." Additional objects, advantages, and features of the present invention will become apparent to those skilled in the art upon examination of the specification or may be learned by practice of the present invention. The following examples and drawings are provided by way of illustration and are not intended to limit the invention. Reference signs placed in parentheses in connection with the drawings and in the claims are intended merely to enhance the clarity of the claims and should not be construed as limiting the scope of the claims. Furthermore, the present invention encompasses all possible combinations of the embodiments described herein. [Example]

[0236] List of Abbreviations HA: Hyaluronic acid GAG: glycosaminoglycan CD: degree of crosslinking NaCMC: sodium carboxymethylcellulose HES: Hydroxyethyl starch GlcA: D-glucuronic acid GlcNAc: N-acetyl-D-glucosamine ECM: extracellular matrix ROS: reactive oxygen species HYAL: Hyaluronidase enzyme IA: Inflammatory arthritis EDC: Carbodiimide DVS: Divinyl sulfone BDDE: 1,4-butanediol diglycidyl ether PEGDE: Poly(ethylene glycol) diglycidyl ether GDE: 1,3-glycerol diglycidyl ether PPE: Polyglycerol diglycidyl ether FDA: Food and Drug Administration EEW: Epoxy equivalent weight GPC: Gel Permeation Chromatography SEC: Size Exclusion Chromatography SANS: Small angle neutron scattering 1H NMR: Proton nuclear magnetic resonance PBS: phosphate buffer solution IPN: Interpenetrating Network MMPs: Metalloproteinases Mw: Weight average molecular weight Mn: Number average molecular weight

[0237] material Shyalt ultrapure Sodium hyaluronate (hyaluronic acid), manufactured by Altergon Italy. High amylose starch, from Merck. High amylopectin starch, manufactured by Merck. Sodium carboxymethylcellulose 0.7 MDa, 0.8-0.9 DSAQUALON™, BLANOSE™ and BONDWELL™. Butanediol diglycidyl ether (BDDE), MW=202 Da, from Merck. Polyethylene glycol diglycidyl ether (PEGDE), MW=526 Da, from Merck.

[0238] 1,3-bis(2-oxiranylmethoxy)-2-propanol, also known by the following names, trade names, synonyms, and abbreviations: 1,3-bis(oxiran-2-ylmethoxy)propan-2-ol; 2-propanol, 1,3-bis(oxiranylmethoxy); glycerol 1,3-diglycidyl ether; Denacol EX-313, MW=204 Da, EEW=141 g / eq, manufactured by Nagase ChemteX Corporation; 1,3-glycerol diglycidyl ether, MW=204 Da, manufactured by Merck; 1,3-GDE has the following structure:

[0239] TIFF0007784138000033.tif40170

[0240] 2,3-bis(2-oxiranylmethoxy)-1-propanol, also known by the following names, trade names, synonyms and abbreviations: 2,3-bis(oxiran-2-ylmethoxy)propan-1-ol, 1-propanol, 1,2-bis(oxiranylmethoxy); glycerol 1,2-diglycidyl ether; 1,2-glycerol diglycidyl ether, MW=204 Da, manufactured by Merck; 1,2-GDE has the following structure:

[0241] TIFF0007784138000034.tif44170

[0242] A mixture of 1,3-GDE and 1,2-GDE is also commercially available under the following trade names: Denacol EX-314, MW = 260 Da, EEW = 144 g / eq, manufactured by Nagase ChemteX Corporation; sodium hydroxide pellets (NaOH), from Merck; Hydrochloric acid 37% (HCl), manufactured by Merck; Sodium chloride powder (NaCl), from Merck; potassium chloride powder (KCl), from Merck; Phosphoric acid 85% (H3PO4), from Merck; phosphate-buffered saline (PBS) pellets, from Merck; Distilled water (H2O), from Merck.

[0243] method The measurement methods for average Mw, Mn and Mw / Mn were evaluated by GPC / SEC analysis using a chromatographic instrument equipped with a light scattering detector. The method for determining EEW was evaluated by ultrasound-assisted titration. The pH was calculated by direct reading on a pH meter equipped with an electrode directly immersed in the hydrogel.

[0244] 1. Hyperbranched polyglycerol polyglycidyl ether

[0245] 1.1. Use of 1,3-GDE as a starting material The preparation processes described below can also be carried out by using Denacol® EX-313, commercially available from Nagase, as a source of 1,3-GDE. Nevertheless, these processes can also be carried out by using 1,3-glycerol diglycidyl ether, commercially available from Merck, or by using the trademark Denacol® EX-314, a mixture containing 1,3-DGE, instead of Denacol® EX-313.

[0246] Process 1 Sodium hydroxide pellets (NaOH) were dissolved in water to form an alkaline solution (Solution A) containing 4% NaOH (1 M) based on the total solution weight. 1,3-GDE (Denacol® EX-313) was dissolved in a portion of solution A at room temperature to form an alkaline solution (solution C1) containing 1-50 wt % 1,3-GDE based on the total solution weight. Solution C1 is then added to the 1,3-GDE solution, which is then subjected to self-polymerization of 1,3-GDE. Controlled temperature within the range of 50-80°C for 1-500 minutes The polyglycerol polyglycidyl ether of the present invention was obtained by reacting with the above-mentioned amines (Solution C3 (Examples 1 to 13) disclosed in Table 1).

[0247] Process 2 Sodium hydroxide pellets (NaOH) were dissolved in water to form an alkaline solution (Solution A) containing 4% NaOH (1 M) based on the total solution weight. 1,3-GDE (Denacol® EX-313) was dissolved in a portion of solution A at room temperature to form an alkaline solution (solution C1) containing 1-50 wt % 1,3-GDE based on the total solution weight. Solution C1 is then added to the 1,3-GDE solution, which is then subjected to self-polymerization of 1,3-GDE. Controlled temperature range of 10-40°C for 1-30 days The polyglycerol polyglycidyl ethers (Solution C2) of the present invention disclosed in Table 1 were obtained (Examples 14 to 31).

[0248] 1.1.3. Characterization of the PPE of the Invention The molecular weight and epoxy content of the resulting PPEs after Processes 1 and 2, measured as epoxy equivalent weight expressed as a percentage of the EEW of the starting GDE, were analytically monitored over time using a GPC instrument equipped with a light scattering detector and quantitative titration with HCl, respectively, as previously described. Prior to each analysis, the GDE self-polymerization reaction was stopped by adding 1 M hydrochloric acid solution and PBS to neutralize the alkaline catalyst and return the pH of the polyglycerol polyglycidyl ether solution to 7.0.

[0249] The average molecular weight (MW) and EEW of the resulting polyglycerol polyglycidyl ethers (PPEs) of the present invention for each temperature and time combination are reported in Table 1.

[0250] TIFF0007784138000035.tif187170

[0251] 2.Crosslinked polysaccharide

[0252] 2.1. Cross-linked hyaluronic acid

[0253] 2.1.1. PPE-Crosslinked Hyaluronic Acid of the Present Invention

[0254] 2.1.1.1. Preparation Process

[0255] The PPE-crosslinked hyaluronic acids of Examples 1 to 13 (see Table 2) were prepared according to the process defined below. The PPE-crosslinked hyaluronic acids of Examples 14 to 16 (see Table 2) were prepared according to the same process as in Examples 1 to 13, but using approximately 2.5 times the crosslinker concentration (1.00) of Examples 1 to 13.

[0256] Process 1

[0257] A. Preparation of polyglycerol polyglycidyl ethers of Examples 1-13 following the process disclosed in Section 1.1.2 above.

[0258] B. Preparation of Cross-Linked Polysaccharides

[0259] Alternative B1 Hyaluronic acid (HA) dry powder was gently dissolved in a portion of Solution A at room temperature (25 °C) (or added directly to Solution C3) to form an alkaline solution (Solution B) containing 5–15 wt % hydrated HA based on the total solution weight. Solution C3 is then added to solution B to obtain an alkaline solution with a hydroxide concentration in the final solution ranging from 0.25 to 0.75 M and a HA to PPE ratio of 2 to 12.5% ​​w / w (expressed as the weight ratio of PPE to dry polysaccharide).

[0260] Alternative B2 Solution C3 is then added to solution B to obtain an alkaline solution in which the hydroxide concentration in the final solution is in the range of 0.25-0.75 M, the concentration of HA in the final solution is in the range of 8-10 wt% with respect to the total weight, and the concentration of PPE in the final solution is in the range of 0.2-1 wt% with respect to the total weight. The ratio, expressed as the weight ratio of PPE to dry polysaccharide, is 2-40% w / w.

[0261] C. Reaction and isolation of cross-linked polysaccharides

[0262] The resulting alkaline solution consisting of HA, PPE, and NaOH was then thoroughly mixed at room temperature, and the homogeneous solution was then reacted at a controlled temperature ranging between 20 and 50°C for a period of 1 to 48 hours.

[0263] The PPE-crosslinked hyaluronic acid (hydrogel) was then washed with an acidic aqueous solution (1 M hydrochloric acid and PBS) for 48–120 h to remove unreacted materials and by-products, neutralize the alkaline catalyst, and return the pH of the PPE-crosslinked hyaluronic acid (hydrogel) to 6.5–7.4 by ion exchange to reach the desired final concentration of HA in the PPE-crosslinked hyaluronic acid hydrogel of 1–50 mg / mL, as shown in Table 2.

[0264] PPE cross-linked hyaluronic acid (hydrogel) was filled into syringes and steam sterilized in an autoclave (e.g., 121°C for 15 min) according to the pharmacopoeia-recognized procedure reported in EN ISO 17665-1, Sterilization of health care products—Moist heat Part 1: requirements for the development, validation and routine control of the sterilization process for medical devices.

[0265] The PPE cross-linked hyaluronic acid (hydrogel) can optionally be dehydrated under vacuum at 30° C. or lyophilized (freeze-dried) and stored for subsequent use or analysis.

[0266] Process 2

[0267] A. Preparation of polyglycerol polyglycidyl ethers of Examples 14-31 following the process disclosed in Section 1.1.3 above.

[0268] B. Preparation of Cross-Linked Polysaccharides

[0269] Alternative B1 Hyaluronic acid (HA) was gently dissolved in a portion of Solution A at room temperature (25 °C) (or added directly to Solution C3) to form an alkaline solution (Solution B) containing 5-15 wt% hydrated HA based on the total solution weight. Solution C3 was then added to Solution B to obtain an alkaline solution with a hydroxide concentration in the final solution ranging from 0.25 to 0.75 M and an HA to PPE ratio of 2-12.5% ​​w / w (expressed as the weight ratio of PPE to dry polysaccharide).

[0270] Alternative B2 Solution C3 is then added to solution B to obtain an alkaline solution in which the hydroxide concentration in the final solution is in the range of 0.25-0.75 M, the concentration of HA in the final solution is in the range of 8-10 wt% relative to the total weight, and the concentration of PPE in the final solution is in the range of 0.2-1 wt% relative to the total weight. The ratio, expressed as the weight ratio of PPE to dry polysaccharide, is 2-40% w / w (expressed as the weight ratio of PPE to dry polysaccharide).

[0271] C. Reaction and Isolation of Cross-Linked Polysaccharides

[0272] The resulting alkaline solution consisting of HA, PPE, and NaOH was then thoroughly mixed at room temperature, and the homogeneous solution was then reacted at a controlled temperature ranging between 20 and 50°C for a period of 1 to 48 hours.

[0273] The PPE-crosslinked hyaluronic acid (hydrogel) was then washed with an acidic aqueous solution (1 M hydrochloric acid and PBS) for 48–120 h to remove unreacted materials and by-products, neutralize the alkaline catalyst, and return the pH of the hydrogel to 6.5–7.4 by ion exchange to reach a final concentration of HA in the PPE-crosslinked hyaluronic acid hydrogel of 1–50 mg / mL, as shown in Table 2 .

[0274] The PPE-crosslinked hyaluronic acid (hydrogel) was filled into syringes and steam-sterilized in an autoclave (e.g., 121°C for 15 minutes) according to the pharmacopeia-recognized procedure reported in EN ISO 17665-1, Sterilization of health care products—Moist heat Part 1: requirements for the development, validation, and routine control of the sterilization process for medical devices. The PPE-crosslinked hyaluronic acid (hydrogel) can be optionally dehydrated under vacuum at 30°C or lyophilized (freeze-dried) and stored for further use or analysis.

[0275] 2.1.1.2. Characterization of the PPE-Crosslinked Hyaluronic Acid of the Present Invention (PPE-Crosslinked Hyaluronic Acid Examples 1 to 16)

[0276] The mechanical properties of the PPE-crosslinked hyaluronic acid of the present invention obtained from the PPE of the present invention are disclosed in Table 2. The PPE-crosslinked hyaluronic acid disclosed in Examples 1 to 13 in Table 2 has a hyaluronic acid concentration of 22.0 mg / mL, while Examples 14 to 16 have a crosslinker concentration that is approximately 2.5 times that of Examples 1 to 13.

[0277] TIFF0007784138000036.tif119170

[0278] 2.1.2. Comparative cross-linked hyaluronic acid

[0279] 2.1.2.1. Comparative 1,3-GDE cross-linked hyaluronic acid (Comparative GDE-HA)

[0280] The comparative GDE-HA described below is prepared by using Denacol® EX-313, commercially available from Nagase, as the source of 1,3-GDE. However, these processes can also be carried out by using 1,3-glycerol diglycidyl ether, commercially available from Merck, or by using the trademark Denacol® EX-314, a mixture containing 1,3-DGE, instead of Denacol® EX-313.

[0281] Comparative GDE cross-linked hyaluronic acids of Comparative Examples 1-5 (see Table 4) were prepared according to the process defined below: Comparative GDE cross-linked hyaluronic acid of Comparative Example 6 (see Table 4) was prepared according to the same process as Examples 1-5, but using a cross-linker concentration of 1.00 and having a CD of 0.4037%.

[0282] Preparation Process Sodium hydroxide pellets (NaOH) were dissolved in water to form an alkaline solution (Solution A) containing 4% NaOH (1 M) based on the total solution weight. HA was gently dissolved in a portion of Solution A at room temperature (25 °C) to form an alkaline solution (Solution B) containing 5–15 wt % hydrated HA based on the total solution weight. GDE was dissolved in distilled water at room temperature to form a neutral solution containing 1-10 wt% GDE based on the total solution weight (Solution C1). Optionally, GDE was dissolved in a portion of Solution A at room temperature to form an alkaline solution containing 0.2-2 wt% GDE based on the total solution weight (Solution C2). Solution C (C1 / C2) was then added to Solution B to obtain an alkaline solution with the desired ratio of HA and GDE, with the hydroxide concentration in the final solution ranging from 0.25-0.75 M. The resulting alkaline solution, consisting of HA, GDE, and NaOH, was then thoroughly mixed at room temperature. Next, The homogenous solution is incubated at a controlled temperature ranging between 20 and 50°C for a period of 1 to 48 hours.The GDE cross-linked hyaluronic acid (hydrogel) was then washed with an acidic aqueous solution (1 M hydrochloric acid and PBS) for 48-120 hours to remove unreacted materials and by-products, neutralize the alkaline catalyst, and return the pH of the hydrogel to 6.5-7.4 by ion exchange to reach a concentration of HA in the hydrogel of 1-50 mg / mL, as shown in Table 4 below (GDE Comparative Examples 1-5).

[0283] The hydrogels were filled into syringes and steam sterilized in an autoclave (e.g., 121°C for 15 minutes) according to pharmacopoeia-recognized procedures reported in EN ISO 17665-1. The comparative GDE-crosslinked hyaluronic acid (hydrogel) can optionally be dehydrated under vacuum at 30°C or lyophilized (freeze-dried) and stored for further use or analysis.

[0284] Characterization of comparative GDE cross-linked hyaluronic acid (comparative GDE-HA.1-6)

[0285] The mechanical properties of a comparative GDE-crosslinked hyaluronic acid outside the scope of the present invention obtained from a commercially available 1,3-GDE having a concentration of 22.0 mg / mL of hyaluronic acid are disclosed in Table 4.

[0286] TIFF0007784138000037.tif68170

[0287] 2.1.2.2. Comparative BDDE-crosslinked hyaluronic acid (comparative BDDE-HA)

[0288] Preparation Process Sodium hydroxide pellets (NaOH) were dissolved in water to form an alkaline solution (Solution A) containing 4% NaOH (1 M) based on the total solution weight. Hyaluronic acid was gently dissolved in a portion of Solution A at room temperature (25 °C) to form an alkaline solution (Solution B) containing 5–15 wt % hydrated HA based on the total solution weight. BDDE was dissolved in distilled water at room temperature to form a neutral solution containing 0.2-2 wt% BDDE based on the total solution weight (Solution C1). Optionally, BDDE was dissolved in a portion of Solution A at room temperature to form an alkaline solution containing 1-10 wt% BDDE based on the total solution weight (Solution C2). Solution C (C1 neutral or C2 alkaline) was then added to Solution B to obtain an alkaline solution with the desired ratio of HA and BDDE, with a hydroxide concentration in the final solution ranging from 0.25-0.75 M. The resulting alkaline solution, consisting of HA, BDDE, and NaOH, was then thoroughly mixed at room temperature. The homogeneous solution was then reacted at a controlled temperature ranging from 20 to 50°C for a period of 30 minutes to 48 hours. The BDDE-crosslinked hyaluronic acid (hydrogel) was then washed with an acidic aqueous solution (1 M hydrochloric acid and PBS) for 48–120 h to remove unreacted materials and by-products, neutralize the alkaline catalyst, and return the pH of the hydrogel to 6.5–7.4 by ion exchange to reach a concentration of HA in the comparative BDDE-crosslinked hyaluronic acid (hydrogel) of 1–50 mg / mL as shown in the table below (BDDE-HA Comparative Examples 1–5). The comparative BDDE-crosslinked hyaluronic acid (hydrogel) was filled into syringes and steam sterilized in an autoclave (e.g., 121°C for 15 minutes) according to the pharmacopoeia-recognized procedure reported in EN ISO 17665-1. The comparative BDDE-crosslinked hyaluronic acid (hydrogel) can optionally be dehydrated under vacuum at 30°C and stored for further use or analysis.

[0289] Comparative characterization of BDDE-crosslinked hyaluronic acid

[0290] The mechanical properties of a comparative BDDE-crosslinked hyaluronic acid outside the scope of the present invention obtained from commercially available BDDE, having a concentration of 22.0 mg / mL of hyaluronic acid, are disclosed in Table 5.

[0291] TIFF0007784138000038.tif60170

[0292] 2.1.2.3. Comparative PEGDE-crosslinked hyaluronic acid (Comparative PEGDE-HA)

[0293] Preparation Process Sodium hydroxide pellets (NaOH) were dissolved in water to form an alkaline solution (Solution A) containing 4% NaOH (1 M) based on the total solution weight. HA was gently dissolved in a portion of Solution A at room temperature (25 °C) to form an alkaline solution (Solution B) containing 5-15 wt% hydrated HA based on the total solution weight. PEGDE was dissolved in distilled water at room temperature to form a neutral solution containing 0.4-4 wt% PEGDE based on the total solution weight (Solution C1). Optionally, PEGDE was dissolved in a portion of Solution A at room temperature to form an alkaline solution containing 1-10 wt% PEGDE based on the total solution weight (Solution C2). Solution C (C1 / C2) was then added to Solution B to obtain an alkaline solution with the desired ratio of HA and PEGDE, with a hydroxide concentration in the final solution ranging from 0.25-0.75 M. The resulting alkaline solution, consisting of HA, PEGDE, and NaOH, was then thoroughly mixed at room temperature. The homogeneous solution was then reacted for a period of 1 to 48 hours at a controlled temperature ranging between 20 and 50°C. The PEGDE-crosslinked hyaluronic acid (hydrogel) was then washed with an acidic aqueous solution (1 M hydrochloric acid and PBS) for 48 to 120 hours to remove unreacted materials and by-products, neutralize the alkaline catalyst, and return the pH of the comparative PEGDE-crosslinked hyaluronic acid (hydrogel) to 6.5 to 7.4 by ion exchange to reach a final concentration of HA in the comparative PEGDE-crosslinked hyaluronic acid (hydrogel) of 1 to 50 mg / mL, as shown in the table below (PEGDE-HA Comparative Examples 1 to 5).

[0294] Comparative PEGDE-crosslinked hyaluronic acid (hydrogel) was filled into syringes and steam sterilized in an autoclave (e.g., 121°C for 15 minutes) according to the pharmacopoeia-recognized procedure reported in EN ISO 17665-1. The hydrogels can be optionally dehydrated under vacuum at 30°C and stored for further use or analysis.

[0295] Comparative characterization of PEGDE-crosslinked hyaluronic acid

[0296] The mechanical properties of a comparative PEGDE-crosslinked hyaluronic acid outside the scope of the present invention, obtained from commercially available PEGDE, having a concentration of 22.0 mg / mL of hyaluronic acid, are disclosed in Table 6.

[0297] TIFF0007784138000039.tif60170

[0298] Assay

[0299] 2.1.3.1. Viability Assay

[0300] The MTT assay was used to assess the viability of grown 3T3 mouse fibroblasts.

[0301] The MTT assay was performed using: Comparative BDDE cross-linked hyaluronic acid (hydrogel) (BDDE-HA Comparative Example 3), Comparative PEGDE-crosslinked hyaluronic acid (hydrogel) (PEGDE-HA Comparative Example 3), PPE-crosslinked hyaluronic acid (hydrogel) of the present invention (PPE-HA Example 3), BDDE, PEGDE, and PPE (PPE Example 22 of the present invention).

[0302] a) MTT assay on cells grown in direct contact with the tested hydrogels. MTT assay was performed on cells grown in direct contact with 100 mg of each test sample.This MTT assay showed a difference in cell viability between all three tested hydrogels compared to the control (represented by culture medium), and this is due to the inhibition of cell proliferation caused mainly by the low available space due to the presence of the hydrogel, rather than toxicity or low biocompatibility.In particular, the cell viability in the presence of the PPE-crosslinked hyaluronic acid of the present invention is significantly higher than that of the control and the comparative crosslinked hyaluronic acid containing BDDE and PEDGE.

[0303] b) MTT assay on cells grown in hydrogel extracts. MTT assay was performed on cells grown in the presence of extracts of the tested hydrogels obtained by immersing the tested crosslinked hydrogels in culture medium for 24 hours. MTT assays performed on cells grown in the presence of extracts from the tested hydrogels showed no difference in cell viability between the hydrogels and the control. This means that all three tested hydrogels did not release toxic substances into the culture medium when immersed in the medium for 24 hours. This is due to the well-controlled manufacturing process, in which all unreacted raw materials and by-products are efficiently removed during crosslinking.

[0304] c) MTT assay on cells grown in direct contact with cross-linking material MTT assays were performed on cells grown in direct contact with a solution containing the crosslinker solution, which contains the same epoxy equivalent weight, after the same steps of the hydrogel manufacturing process, including chemical reaction, washing, and sterilization, but without the addition of polysaccharide. MTT assays show that the viability of cells grown in direct contact with PPE is not significantly different from the control (culture medium in 0.9% NaCl). Microscopic images confirm that cells in direct contact with PPE have good shape and viability when compared to cells grown in direct contact with BDDE and PEGDE, which show a significant decrease in cell viability and signs of disease.

[0305] 2.1.3.2. Enzyme degradation assay

[0306] The enzymatic degradation rate was evaluated using: Comparative Example 3: BDDE-crosslinked hyaluronic acid (hydrogel) BDDE-HA Comparative PEGDE-crosslinked hyaluronic acid (hydrogel) PEGDE-HA Comparative Example 3 Comparative GDE cross-linked hyaluronic acid (hydrogel) GDE-HA Comparative Example 3, and PPE-crosslinked hyaluronic acid (hydrogel) PPE-HA Example 4, PPE-HA Example 5 and PPE-HA Example 6 of the present invention.

[0307] 0.2 mL of each crosslinked hyaluronic acid (hydrogel) tested was treated with 10 μL of 0.1 mg / mL hyaluronidase from bovine samples (type IV-S; 1040 units / mg solid). The tested hydrogels were incubated in PBS at 37°C for 16 hours, and the uronic acids from the degraded hyaluronic acid were evaluated using the carbazole assay. The 22 mg / mL crosslinked hyaluronic acid hydrogel tested responded to hyaluronidase with an enzymatic degradation rate ranging from 74.4% (BDDE crosslinked as a control) to 26.3% of the free uronic acids.

[0308] The comparative BDDE hydrogel (BDDE-HA Comparative Example 3), comparative PEGDE hydrogel (PEGDE-HA Comparative Example 3), and comparative GDE hydrogel (GDE-HA Comparative Example 3) cross-linked hyaluronic acid exhibited similar degrees of cross-linking and therefore comparable amounts of free uronic acid after enzymatic degradation: 74.4, 70.8, and 69.5%, respectively.

[0309] The PPE hydrogels of the present invention (PPE-HA Example 4 and Example 5) exhibit a reduced degree of crosslinking, and therefore a reduced resistance to enzymatic degradation should be expected; however, unexpectedly and counterintuitively, the amount of free uronic acid after enzymatic degradation decreased to 56.7% and 26.3%, respectively, indicating a statistically significant increase in resistance to enzymatic degradation.

[0310] PPE-crosslinked HA PPE-HA Example 6 showed 58.0% of the amount of free uronic acid after enzymatic degradation, which was comparable to the amount of free uronic acid derived from PPE-crosslinked HA from PPE-HA Example 4. This indicates that even though the two hydrogels have two different crosslinking degrees, they have comparable resistance to enzymatic degradation (56.7 and 58.0%, respectively), and in particular, the CD from PPE-HA Example 6 is significantly lower than that of PPE-HA Example 4 (0.2688% and 0.1528%, respectively).

[0311] Therefore, the protective effect of the PPE of the present invention against enzymatic degradation is significantly higher than that of the comparative GDE, BDDE, and PEGDE. In particular, the protective effect of high molecular weight PPE (i.e., 3,000 Da to 15,000 Da) is higher than that of low molecular weight PPE (i.e., 750 Da to less than 3,000 Da) against enzymatic degradation, even though the low molecular weight PPE provides better mechanical properties. The 22 mg / mL HA from the hydrogel in Experiment 51 exhibited greater resistance to in vitro enzymatic degradation by hyaluronidase than the others.

[0312] The results of the enzymatic degradation assays of comparative hyaluronic acid crosslinked with BDDE, PEGDE, GDE of the present invention and hyaluronic acid crosslinked with PPE are summarized in the table below.

[0313] TIFF0007784138000040.tif102170

[0314] Therefore, as shown by the above results, the cross-linked polysaccharides of the present invention prepared by using the hyperbranched PPE of the present invention as a cross-linking material have better resistance to enzymatic degradation (i.e., a lower relative rate of enzymatic degradation) than cross-linked polysaccharides outside the scope of the present invention but having comparable physical properties prepared by using the comparative cross-linking materials disclosed above. In particular, the cross-linked polysaccharides have comparable physical properties (G' values, and therefore D) to the comparative cross-linked polysaccharides outside the scope of the present invention (obtained using commercially available GDE, BDDE, and PEGDE as cross-linking materials). N It has been demonstrated that cross-linked polysaccharides of the present invention (obtained using the hyperbranched PPE of the present invention as a cross-linking agent) having a cross-linking coefficient (C(C)) of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 110, 111, 112, 113, 114, 115, 116, 117,

[0315] Furthermore, the above results also show that the cross-linked polysaccharides of the present invention (obtained by using the hyperbranched PPE of the present invention as a cross-linking material) have a higher enzymatic degradation rate with a smaller amount of cross-linking material, which is the same degradation rate as the comparative cross-linked polysaccharides, but with higher enzymatic degradation rates of G', D', and D'. NNumerically, this is expressed as lower values ​​of C and C. This is advantageous because the amount of cross-linking used is less, resulting in less chemical modification of the polysaccharide, improving biocompatibility and safety after injection into the skin.

[0316] 2.1.3.3. Oxidative Degradation Assay

[0317] sample: The evaluation of the oxidative degradation rate was carried out using: Comparative BDDE cross-linked hyaluronic acid BDDE-HA Comparative Example 3 Comparative PEGDE cross-linked hyaluronic acid PEGDE-HA Comparative Example 3 Comparative GDE cross-linked hyaluronic acid GDE-HA Comparative Example 3, and PPE cross-linked hyaluronic acid PPE-HA Example 4, PPE-HA Example 5 and PPE-HA Example 6 of the present invention.

[0318] method: Resistance to oxidative degradation was evaluated according to the method disclosed in the state of the art (see Bitter, T. and HM Muir, "A modified uronic acid carbazole reaction. Analytical Biochemistry", 1962, vol. 4(4), pp. 330-334): 0.2 mL of each cross-linked hyaluronic acid tested was added to Fenton's reagent (10 µL of 0.1 M Fe in 5 mL of PBS). +2 The samples were treated with PEG / H2O2 and 25 μL of 0.1 M ascorbic acid and incubated at 37°C for 24 hours. Uronic acids from degraded hyaluronic acid were evaluated using a carbazole assay. The 22 mg / mL crosslinked hyaluronic acid tested showed a degradation rate in response to oxidation of 88.0% (PEGDE crosslinked) to 61.6% of free uronic acids by oxidative degradation.

[0319] The comparative BDDE-crosslinked hyaluronic acid (BDDE-HA Comparative Example 3), comparative PEGDE-crosslinked hyaluronic acid (PEGDE-HA Comparative Example 3), and comparative GDE hydrogel-crosslinked hyaluronic acid (GDE-HA Comparative Example 3) exhibited comparable degrees of crosslinking and therefore comparable amounts of free uronic acid after oxidative degradation of 81.2, 88.0, and 80.2, respectively, with the PEGDE-crosslinked hyaluronic acid being the most susceptible to oxidative degradation.

[0320] The PPE-crosslinked hyaluronic acid of the present invention (PPE-HA Example 4 and PPE-HA Example 5) showed a decreased degree of crosslinking, and therefore a decreased resistance to oxidative degradation should be expected. However, unexpectedly and counterintuitively, the amount of free uronic acid after oxidative degradation decreased to 65.9 and 61.6%, respectively, indicating a statistically significant increase in resistance to oxidative degradation. The PPE-crosslinked HA of the present invention from PPE-HA Example 6 showed an amount of free uronic acid after oxidative degradation of 69.6%, which is slightly higher than the free uronic acid derived from the PPE-crosslinked HA from PPE-HA Example 4 and PPE-HA Example 5, but is statistically significantly lower than that of Experiments 3, 8, and 13, indicating that the oxidative degradation resistance of the PPE-crosslinked hyaluronic acid of the present invention is higher than that of the comparative BDDE-, PEGDE-, and GDE-crosslinked polysaccharides, which are outside the scope of the present invention.

[0321] result: The results of the oxidative degradation assays of comparative hyaluronic acid crosslinked with BDDE, PEGDE, GDE of the present invention and hyaluronic acid crosslinked with PPE are summarized in the table below.

[0322] TIFF0007784138000041.tif102170

[0323] Therefore, as shown by the above results and as described above for the enzymatic degradation rate, the cross-linked polysaccharides of the present invention prepared by using the hyperbranched PPE of the present invention as a cross-linking material have better resistance to oxidative degradation (i.e., a lower relative oxidative degradation rate) than cross-linked polysaccharides outside the scope of the present invention but having comparable physical properties prepared by using the comparative cross-linking materials disclosed above.

[0324] Furthermore, the above results also show that the cross-linked polysaccharides of the present invention (obtained by using the hyperbranched PPE of the present invention as a cross-linker) have a higher oxidative degradation rate with a lower amount of cross-linker, which is advantageous because the lower amount of cross-linker used results in less chemical modification of the polysaccharide, improving its biocompatibility and safety after injection into the skin.

[0325] 2.2. Cross-linked amylose starch

[0326] 2.2.1. PPE-Crosslinked Amylose Starch of the Present Invention (PPE-AS)

[0327] The PPE-AS described below is prepared by using Denacol® EX-313, commercially available from Nagase, as a source of 1,3-GDE. However, these processes can also be carried out by using 1,3-glycerol diglycidyl ether, commercially available from Merck, or by using the trademark Denacol® EX-314, a mixture containing 1,3-DGE, instead of Denacol® EX-313.

[0328] Preparation Process Sodium hydroxide pellets (NaOH) were dissolved in water to form an alkaline solution (Solution A) containing 4% NaOH (1 M) based on the total solution weight. 1,3-GDE was dissolved in a portion of Solution A at room temperature to form an alkaline solution (Solution C1) containing 1-50 wt % GDE based on the total solution weight. Solution C1 was then Controlled temperature range of 10-40°C for 1-30 days In, or alternatively, 1 to 48 hours, controlled temperature within the range of 20 to 50°C During this time, self-polymerization of 1,3-GDE occurred to give polyglycerol polyglycidyl ethers having molecular weights and / or specific epoxy contents as disclosed in Table 1 (solution C3).

[0329] High-amylose starch was gently dispersed in a portion of Solution A at room temperature (25°C) to form an alkaline solution (Solution B) containing 5-15 wt% hydrated high-amylose starch based on the total solution weight. Solution C3 was then added to Solution B to obtain an alkaline solution with a hydroxide concentration in the final solution ranging from 0.25 to 0.75 M and a high-amylose starch to PPE ratio. The resulting alkaline solution, consisting of high-amylose starch, PPE, and NaOH, was then thoroughly mixed at room temperature. The homogeneous solution was then reacted at a controlled temperature ranging from 20 to 50°C for a period of 1 to 48 hours.

[0330] The PPE-crosslinked high-amylose starch (hydrogel) was then washed with an acidic aqueous solution (1 M hydrochloric acid and PBS) for 48 to 120 hours to remove unreacted materials and by-products, neutralize the alkaline catalyst, and return the pH of the hydrogel to 6.5 to 7.4 by ion exchange, thereby reaching a concentration of high-amylose starch in the PPE-crosslinked amylose starch (hydrogel) of 1 to 50 mg / mL, as shown in the table below (PPE-AS Examples 1 to 3).

[0331] The PPE cross-linked amylose starch hydrogel was filled into syringes and steam sterilized in an autoclave (e.g., 121°C for 15 minutes) according to the pharmacopoeia-recognized procedure reported in EN ISO 17665-1. The PPE cross-linked amylose starch hydrogel can optionally be dehydrated by freeze-drying and stored for further use or analysis.

[0332] 2.2.2. Comparative GDE Cross-Linked Amylose Starch (Comparative GDE-AS)

[0333] Comparative GDE-crosslinked amylose starch was prepared according to the process disclosed above in Section 2.1.2.1 for comparative 1,3-GDE-crosslinked hyaluronic acid, except that amylose starch was used in place of hyaluronic acid.

[0334] 2.2.3. Characterization of cross-linked amylose starch

[0335] The mechanical properties of the PPE cross-linked amylose starch of the present invention (PPE-AS) and the comparative GDE cross-linked amylose starch (comparative GDE-AS) obtained above are disclosed in the table below.

[0336] TIFF0007784138000042.tif77170

[0337] 2.2.4 Assay

[0338] 2.2.4.1 Enzymatic degradation assay

[0339] sample: The enzymatic degradation rate was evaluated using: Comparative GDE cross-linked amylose starch (hydrogel) GDE-AS Comparative Example 1, and PPE cross-linked amylose starch (hydrogel) PPE-AS Example 1, PPE-AS Example 2 and PPE-AS Example 3 of the present invention.

[0340] method: 0.2 mL of each tested cross-linked amylose starch hydrogel was treated with 10 μL of 0.1 mg / mL α-amylase from Bacillus licheniformis (lyophilized powder, 500–1,500 units / mg protein). The tested hydrogels were incubated in PBS at 37°C for 16 hours, and the hexose monomers from the degraded amylose starch hydrogels were evaluated using the carbazole assay. 22 mg / mL of the tested cross-linked amylose starch hydrogels showed relative enzymatic degradation rates of 80.3%–41.7% in response to α-amylase.

[0341] result: While the comparative GDE cross-linked amylose starch (GDE-AS Comparative Example 1) and the PPE cross-linked amylose starch of the present invention (PPE-AS Example 1) exhibited comparable degrees of cross-linking, the PPE hydrogels of the present invention (PPE-AS Examples 2 and 3) exhibited reduced degrees of cross-linking, and therefore reduced resistance to enzymatic degradation should be expected; however, unexpectedly and counterintuitively, the amount of free hexose monomers after enzymatic degradation decreased to 41.7% and 80.0%, respectively, indicating a statistically significant increase in resistance to enzymatic degradation.

[0342] After enzymatic degradation, the PPE-crosslinked HA PPE-AS Example 3 showed a comparable amount of free hexose monomer to that derived from the PPE-crosslinked starch from PPE-AS Example 1, indicating that even though the two hydrogels have two different crosslinking degrees, their resistance to enzymatic degradation is comparable (80.0% and 80.3%, respectively), and in particular, the CD from PPE-AS Example 3 is significantly lower than that of PPE-AS Example 1 (0.1548% and 0.1981%, respectively).

[0343] Thus, the protective effect of the PPEs of the present invention against enzymatic degradation is significantly higher than that of the comparative GDE. In particular, the protective effect of high molecular weight PPEs (i.e., 3,000 Da to 15,000 Da) is higher than that of low molecular weight PPEs (i.e., 750 Da to less than 3,000 Da) against enzymatic degradation, even though the low molecular weight PPEs provide better mechanical properties. 22 mg / mL AS from PPE-AS Example 2 showed greater resistance to in vitro enzymatic degradation by hyaluronidase than the others.

[0344] The results of the enzymatic degradation assays of comparative amylose starches cross-linked with GDE and amylose starches cross-linked with PPEs of the present invention are summarized in the table below.

[0345] TIFF0007784138000043.tif60170

[0346] Therefore, as shown by the above results, the cross-linked polysaccharides of the present invention prepared by using the hyperbranched PPE of the present invention as a cross-linking material have better enzymatic degradation resistance (i.e., a lower relative enzymatic degradation rate) than cross-linked polysaccharides outside the scope of the present invention but having comparable physical properties prepared by using the comparative cross-linking materials disclosed above. In particular, the cross-linked polysaccharides of the present invention (obtained using the hyperbranched PPE of the present invention as a cross-linking material) have been shown to have higher enzymatic resistance (i.e., a lower enzymatic degradation rate). Furthermore, the above results also show that the cross-linked polysaccharides of the present invention (obtained by using the hyperbranched PPE of the present invention as a cross-linking material) have a higher enzymatic degradation rate with a smaller amount of cross-linking material.

[0347] 2.2.4.2 Oxidative degradation assay

[0348] sample: The oxidative degradation rate was evaluated using: Comparative GDE cross-linked amylose starch GDE-AS Comparative Example 1, and PPE cross-linked amylose starches of the present invention PPE-AS Example 1, PPE-AS Example 2 and PPE-AS Example 3.

[0349] method: Resistance to oxidative degradation was evaluated according to the method disclosed in the state of the art (see Bitter, T. and HM Muir, "A modified uronic acid carbazole reaction. Analytical Biochemistry", 1962, vol. 4(4), pp. 330-334): 0.2 mL of each cross-linked amylose starch tested was added to Fenton's reagent (10 µL of 0.1 M Fe in 5 mL of PBS). +2The hydrogels were treated with 25 μL of 0.1 M ascorbic acid (H2O2 / HO and 25 μL of 0.1 M ascorbic acid) and incubated at 37 °C for 24 h. The liberation of hexose monomers from the degraded amylose starch hydrogels was assessed by the carbazole assay. The crosslinked amylose starch tested at 22 mg / mL showed degradation rates ranging from 83.8% to 77.4% in response to oxidation.

[0350] result: While the comparative GDE-crosslinked amylose starch (GDE-AS Comparative Example 1) and the PPE-crosslinked amylose starch of the present invention (PPE-AS Example 1) exhibited comparable degrees of crosslinking, the PPE-crosslinked amylose starches of the present invention (PPE-AS Examples 2 and 3) exhibited reduced degrees of crosslinking, and therefore reduced resistance to oxidative degradation should be expected; however, unexpectedly and counterintuitively, the amount of free hexose monomer after oxidative degradation decreased to 83.8, 77.4, and 81.0 for PPE-AS Example 1, PPE-AS Example 2, and PPE-AS Example 3, respectively, indicating a statistically significant increase in resistance to oxidative degradation.

[0351] Therefore, as shown by the above results and as described above for the oxidative degradation rate, the cross-linked polysaccharides of the present invention prepared by using the hyperbranched PPE of the present invention as a cross-linking material have better resistance to oxidative degradation (i.e., a lower relative oxidative degradation rate) than cross-linked polysaccharides outside the scope of the present invention but having comparable physical properties prepared by using the comparative cross-linking materials disclosed above.

[0352] The results of the oxidative degradation assays of comparative amylose starches cross-linked with GDE and amylose starches cross-linked with PPEs of the present invention are summarized in the table below.

[0353] TIFF0007784138000044.tif60170

[0354] Therefore, as shown by the above results and as described above for the enzymatic degradation rate, the cross-linked polysaccharides of the present invention prepared by using the hyperbranched PPE of the present invention as a cross-linking material have better resistance to oxidative degradation (i.e., a lower relative oxidative degradation rate) than cross-linked polysaccharides outside the scope of the present invention but having comparable physical properties prepared by using the comparative cross-linking materials disclosed above. Furthermore, the above results also show that the cross-linked polysaccharides of the present invention (obtained by using the hyperbranched PPE of the present invention as a cross-linking material) have a higher oxidative degradation rate with a smaller amount of cross-linking material.

[0355] 2.3. Cross-linked amylopectin starch / glycogen

[0356] 2.3.1. PPE-Crosslinked Amylopectin Starch / Glycogen (PPE-ASG) of the Invention

[0357] The PPE-ASG described below is prepared by using Denacol® EX-313, commercially available from Nagase, as a source of 1,3-GDE. However, these processes can also be carried out by using 1,3-glycerol diglycidyl ether, commercially available from Merck, or by using the trademark Denacol® EX-314, a mixture containing 1,3-DGE, instead of Denacol® EX-313.

[0358] Preparation Process Sodium hydroxide pellets (NaOH) were dissolved in water to form an alkaline solution (Solution A) containing 4% NaOH (1 M) based on the total solution weight. 1,3-GDE was dissolved in a portion of Solution A at room temperature to form an alkaline solution (Solution C1) containing 1-50 wt % GDE based on the total solution weight. Solution C1 was then 1 to 30 days at temperatures between 10 and 40°C , or alternatively, 1 to 48 hours at a controlled temperature between 20 and 50°CThe reaction was allowed to proceed, during which self-polymerization of 1,3-GDE occurred, to give the polyglycerol polyglycidyl ether of the present invention having the molecular weight and / or specific epoxy content disclosed in Table 1 (Solution C3).

[0359] The high-content amylopectin starch / glycogen was gently dispersed in a portion of Solution A at room temperature (25°C) to form an alkaline solution (Solution B) containing 5-15 wt% hydrated high-content amylopectin starch / glycogen based on the total solution weight. Solution C3 was then added to Solution B to obtain an alkaline solution with a hydroxide concentration in the final solution ranging from 0.25 to 0.75 M and a high-content amylopectin starch / glycogen to PPE ratio. The resulting alkaline solution, consisting of high-content amylopectin starch / glycogen, PPE, and NaOH, was then thoroughly mixed at room temperature. The homogeneous solution was then reacted at a controlled temperature ranging from 20 to 50°C for a period of 1 to 48 hours.

[0360] The PPE-crosslinked high-content amylopectin starch / glycogen (hydrogel) of the present invention (PPE-ASG Examples 1-3) was then washed with an acidic aqueous solution (1 M hydrochloric acid and PBS) for 48-120 hours to remove unreacted materials and by-products, neutralize the alkaline catalyst, and return the pH of the hydrogel to 6.5-7.4 by ion exchange to reach a concentration of 1-50 mg / mL of the PPE-crosslinked high-content amylopectin starch / glycogen (hydrogel) of the present invention as shown in the table below (PPE-ASG Examples 1-3).

[0361] The PPE cross-linked amylopectin starch / glycogen (hydrogel) was filled into syringes and steam sterilized in an autoclave (e.g., 121°C for 15 minutes) according to the pharmacopoeia reported in EN ISO 17665-1. The PPE cross-linked amylopectin starch / glycogen (hydrogel) of the present invention could optionally be dehydrated in a ventilated oven at 50°C and stored for further use or analysis.

[0362] 2.3.2. Comparative GDE Cross-Linked Amylopectin Starch / Glycogen (Comparative GDE-ASG)

[0363] Comparative GDE cross-linked amylopectin starch / glycogen (Comparative GDE-ASG) was prepared according to the process disclosed above in Section 2.1.2.1 for Comparative GDE cross-linked hyaluronic acid, except that amylopectin starch / glycogen was used in place of hyaluronic acid.

[0364] 2.3.3. Characterization of Cross-Linked Amylopectin Starch / Glycogen

[0365] The mechanical properties of the PPE cross-linked amylopectin starch / glycogen of the present invention (PPE-ASG) and the comparative GDE cross-linked amylopectin starch / glycogen (comparative GDE-ASG) obtained above, having a concentration of 22.0 mg / mL, are disclosed in the table below.

[0366] TIFF0007784138000045.tif102170

[0367] 2.3.4 Assay

[0368] 2.3.4.1 Enzymatic degradation assay

[0369] sample: The enzymatic degradation rate was evaluated using: Comparative GDE cross-linked amylopectin starch / glycogen (hydrogel) GDE-ASG Comparative Example 1, and PPE cross-linked amylopectin starch / glycogen (hydrogels) PPE-ASG Example 1, PPE-ASG Example 2, and PPE-ASG Example 3 of the present invention.

[0370] method: Resistance to enzymatic degradation was assessed according to the method disclosed in the enzymatic degradation assay in Section 2.2.4.1 above.

[0371] result: The PPE hydrogels of the present invention (PPE ASG Example 2 and PPE ASG Example 3) exhibit a reduced degree of crosslinking, and therefore a reduced resistance to enzymatic degradation should be expected; however, unexpectedly and counterintuitively, the amount of free hexose monomer after enzymatic degradation decreased to 76.0% and 89.4%, respectively, indicating a statistically significant increase in resistance to enzymatic degradation.

[0372] While the comparative GDE-crosslinked amylopectin starch / glycogen (GDE-ASG Comparative Example 1) and the PPE-crosslinked amylopectin starch / glycogen of the present invention (PPE-ASG Example 1) exhibited comparable degrees of crosslinking, the PPE-crosslinked amylopectin starch / glycogen of the present invention (PPE-ASG Examples 2 and 3) exhibited reduced degrees of crosslinking, and therefore, reduced resistance to oxidative degradation should be expected. However, unexpectedly and counterintuitively, the PPE-crosslinked HA PPE-ASG Example 3 exhibited an amount of free hexose monomer after enzymatic degradation equivalent to the amount of free hexose monomer derived from the PPE-crosslinked starch from PPE-ASG Example 1. This indicates that even though the two hydrogels have two different degrees of crosslinking, their resistance to enzymatic degradation is comparable (89.4% and 85.7%, respectively). In particular, the CD from PPE-ASG Example 3 is significantly lower than that of PPE-ASG Example 1 (0.1241% and 0.1708%, respectively).

[0373] Thus, the protective effect of the PPEs of the present invention against enzymatic degradation is significantly higher than that of the comparative GDE. In particular, the protective effect of high molecular weight PPEs (i.e., 3,000 Da to 15,000 Da) is higher than that of low molecular weight PPEs (i.e., 750 Da to less than 3,000 Da) against enzymatic degradation, even though the low molecular weight PPEs provide better mechanical properties. 22 mg / mL AS from PPE-AS Example 2 showed greater resistance to in vitro enzymatic degradation by hyaluronidase than the others.

[0374] The results of the enzymatic degradation assays of comparative amylopectin starch / glycogen cross-linked with GDE and amylopectin starch / glycogen cross-linked with PPEs of the present invention are summarized in the table below.

[0375] TIFF0007784138000046.tif51170

[0376] Therefore, as shown by the above results, the cross-linked polysaccharides of the present invention prepared by using the hyperbranched PPE of the present invention as a cross-linking material have better enzymatic degradation resistance (i.e., a lower relative enzymatic degradation rate) than cross-linked polysaccharides outside the scope of the present invention but having comparable physical properties prepared by using the comparative cross-linking materials disclosed above. In particular, the cross-linked polysaccharides of the present invention (obtained using the hyperbranched PPE of the present invention as a cross-linking material) have been shown to have higher enzymatic resistance (i.e., a lower enzymatic degradation rate). Furthermore, the above results also show that the cross-linked polysaccharides of the present invention (obtained by using the hyperbranched PPE of the present invention as a cross-linking material) have a higher enzymatic degradation rate with a smaller amount of cross-linking material.

[0377] 2.3.4.2 Oxidative Degradation Assay

[0378] sample: The oxidative degradation rate was evaluated using: Comparative GDE cross-linked amylopectin starch / glycogen GDE-ASG Comparative Example 1, and PPE cross-linked amylopectin starch / glycogen PPE-ASG Example 1, PPE-ASG Example 2 and PPE-ASG Example 3 of the present invention.

[0379] method: Resistance to oxidative degradation was assessed according to the method disclosed in the oxidative degradation assay in Section 2.2.4.2 above.

[0380] result: While the comparative GDE-crosslinked amylopectin starch / glycogen (GDE-ASG Comparative Example 1) and the PPE-crosslinked amylopectin starch / glycogen of the present invention (PPE-ASG Example 1) exhibited comparable degrees of crosslinking, the PPE-crosslinked amylose starches of the present invention (PPE-ASG Examples 2 and 3) exhibited reduced degrees of crosslinking, and therefore reduced resistance to oxidative degradation should be expected. However, unexpectedly and counterintuitively, the amount of free hexose monomers after oxidative degradation decreased to 81.2, 74.5, and 87.0 for PPE-ASG Example 1, PPE-ASG Example 2, and PPE-ASG Example 3, respectively, indicating a statistically significant increase in resistance to oxidative degradation.

[0381] Therefore, as shown by the above results and as described above for the oxidative degradation rate, the cross-linked polysaccharides of the present invention prepared by using the hyperbranched PPE of the present invention as a cross-linking material have better resistance to oxidative degradation (i.e., a lower relative oxidative degradation rate) than cross-linked polysaccharides outside the scope of the present invention but having comparable physical properties prepared by using the comparative cross-linking materials disclosed above.

[0382] The results of the oxidative degradation assays of comparative amylopectin starch / glycogen cross-linked with GDE and amylopectin starch / glycogen cross-linked with PPEs of the present invention are summarized in the table below.

[0383] TIFF0007784138000047.tif56170

[0384] Therefore, as shown by the above results and as described above for the enzymatic degradation rate, the cross-linked polysaccharides of the present invention prepared by using the hyperbranched PPE of the present invention as a cross-linking material have better resistance to oxidative degradation (i.e., a lower relative oxidative degradation rate) than cross-linked polysaccharides outside the scope of the present invention but having comparable physical properties prepared by using the comparative cross-linking materials disclosed above. Furthermore, the above results also show that the cross-linked polysaccharides of the present invention (obtained by using the hyperbranched PPE of the present invention as a cross-linking material) have a higher oxidative degradation rate with a smaller amount of cross-linking material.

[0385] 2.4. Cross-linked sodium carboxymethylcellulose

[0386] 2.4.1. PPE-Cross-Linked Sodium Carboxymethylcellulose (PPE-CMC) of the Present Invention

[0387] The PPE-CMC described below is prepared by using Denacol® EX-313, commercially available from Nagase, as a source of 1,3-GDE. However, these processes can also be carried out by using 1,3-glycerol diglycidyl ether, commercially available from Merck, or by using the trademark Denacol® EX-314, a mixture containing 1,3-DGE, instead of Denacol® EX-313.

[0388] Preparation Process Sodium hydroxide pellets (NaOH) were dissolved in water to form an alkaline solution (Solution A) containing 4% NaOH (1 M) based on the total solution weight. 1,3-GDE was dissolved in a portion of Solution A at room temperature to form an alkaline solution (Solution C1) containing 1-50 wt % GDE based on the total solution weight. Solution C1 was then 1 to 30 days at temperatures between 10 and 40°C , or alternatively, 1 to 48 hours at a controlled temperature between 20 and 50°CThe reaction was allowed to proceed, during which self-polymerization of 1,3-GDE occurred, to give the polyglycerol polyglycidyl ether of the present invention having a molecular weight and / or a specific epoxy content as disclosed in Table 1 (Solution C3).

[0389] Sodium carboxymethylcellulose (NaCMC) was gently dispersed in a portion of Solution A at room temperature (25°C) to form an alkaline solution (Solution B) containing 5-15 wt% hydrated NaCMC based on the total solution weight. Solution C3 was then added to Solution B to obtain an alkaline solution with the desired ratio of NaCMC and PPE, with the hydroxide concentration in the final solution ranging from 0.25 to 0.75 M. The resulting alkaline solution, consisting of NaCMC, PPE, and NaOH, was then thoroughly mixed at room temperature. The homogeneous solution was then allowed to react at a controlled temperature ranging between 20 and 50°C for a period of 1 to 48 hours. The PPE-crosslinked NaCMC (hydrogel) of the present invention (PPE-CMC) was then washed with an acidic aqueous solution (1 M hydrochloric acid and PBS) for 48-120 hours to remove unreacted materials and by-products, neutralize the alkaline catalyst, and return the pH of the hydrogel to 6.5-7.4 by ion exchange to reach a concentration of NaCMC in the PPE-crosslinked NaCMC (hydrogel) of the present invention of 1-50 mg / mL as shown in the table below (PPE-CMC). The PPE-crosslinked NaCMC (hydrogel) of the present invention was filled into syringes and steam sterilized in an autoclave (e.g., 121°C for 15 minutes) according to the pharmacopoeia reported in EN ISO 17665-1. The hydrogel can optionally be dehydrated by freeze-drying and stored for further use or analysis.

[0390] 2.4.2. Comparative GDE Cross-Linked Sodium Carboxymethylcellulose (Comparative GDE-CMC)

[0391] Comparative GDE-crosslinked NaCMC was prepared according to the process disclosed above in Section 2.1.2.1 for comparative GDE-crosslinked hyaluronic acid, except that NaCMC was used in place of hyaluronic acid.

[0392] 2.4.3. Characterization of Cross-Linked NaCMC

[0393] The mechanical properties of the PPE-crosslinked NaCMC of the present invention (PPE-CMC Examples 1 to 3) and comparative GDE-crosslinked NaCMC (GDE Comparative Example 1) obtained above, each having a concentration of 22.0 mg / mL, are disclosed in the table below.

[0394] TIFF0007784138000048.tif85170

[0395] 2.4.4 Assay

[0396] 2.4.4.1 Enzymatic degradation assay

[0397] sample: The enzymatic degradation rate was evaluated using: Comparative GDE cross-linked sodium carboxymethylcellulose (hydrogel) GDE-CMC Comparative Example 1, and PPE cross-linked sodium carboxymethylcellulose (hydrogels) of the present invention PPE-CMC Example 1, PPE-CMC Example 2, and PPE-CMC Example 3.

[0398] method: 0.2 mL of each cross-linked sodium carboxymethylcellulose (hydrogel) tested was treated with 10 μL of 0.1 mg / mL pectinase from Aspergillus niger. The hydrogels were incubated in PBS at 37 °C for 16 hours, and the hexose monomers from the degraded sodium carboxymethylcellulose hydrogels were evaluated using the carbazole assay.

[0399] result: The comparative GDE-crosslinked sodium carboxymethylcellulose (GDE-CMC Comparative Example 1) and the PPE-crosslinked sodium carboxymethylcellulose of the present invention (PPE-CMC Example 1) exhibit comparable degrees of crosslinking. The PPE hydrogels of the present invention (PPE-CMC Example 2 and PPE-CMC Example 3) exhibit a reduced degree of crosslinking, and therefore, a reduced resistance to enzymatic degradation should be expected. However, unexpectedly and counterintuitively, the amount of free hexose monomer after enzymatic degradation decreased to 64.6% and 85.4% for PPE-CMC Example 2 and PPE-CMC Example 3, respectively, indicating a statistically significant increase in resistance to enzymatic degradation.

[0400] Thus, the protective effect of the PPEs of the present invention against enzymatic degradation is significantly higher than that of the comparative GDE. In particular, the protective effect of high molecular weight PPEs (i.e., 3,000 Da to 15,000 Da) is higher than that of low molecular weight PPEs (i.e., 750 Da to less than 3,000 Da) against enzymatic degradation, even though the low molecular weight PPEs provide better mechanical properties. 22 mg / mL AS from PPE-AS Example 2 showed greater resistance to in vitro enzymatic degradation by hyaluronidase than the others. The results of the enzymatic degradation assays of comparative sodium carboxymethylcellulose cross-linked with GDE and sodium carboxymethylcellulose cross-linked with PPE of the present invention are summarized in the table below.

[0401] TIFF0007784138000049.tif60170

[0402] Thus, the protective effect of the PPEs of the present invention against enzymatic degradation is significantly greater than that of the comparative GDEs. In particular, the protective effect of high molecular weight PPEs (i.e., 3000 Da to 15000 Da) is greater than that of low molecular weight PPEs (i.e., 750 Da to less than 3000 Da) against enzymatic degradation, even though the lower molecular weight PPEs provide better mechanical properties.

[0403] 2.4.4.2 Oxidative Degradation Assay

[0404] sample: The oxidative degradation rate was evaluated using: Comparative GDE cross-linked sodium carboxymethylcellulose GDE-CMC Comparative Example 1, and PPE cross-linked sodium carboxymethylcellulose of the present invention PPE-CMC Example 1, PPE-CMC Example 2 and PPE-CMC Example 3.

[0405] method: Resistance to oxidative degradation was assessed according to the method disclosed in the oxidative degradation assay in Section 2.2.4.2 above.

[0406] result: While the comparative GDE-crosslinked sodium carboxymethylcellulose (GDE-CMC Comparative Example 1) and the PPE-crosslinked sodium carboxymethylcellulose of the present invention (PPE-CMC Example 1) exhibited comparable degrees of crosslinking, the PPE-crosslinked sodium carboxymethylcellulose of the present invention (PPE-CMC Example 2 and PPE-CMC Example 3) exhibited a reduced degree of crosslinking; therefore, a decrease in resistance to oxidative degradation should be expected. However, unexpectedly and counterintuitively, the amount of free hexose monomer after oxidative degradation decreased to 92.6, 83.7, and 82.5 for PPE-CMC Example 1, PPE-CMC Example 2, and PPE-CMC Example 3, respectively, indicating a statistically significant increase in resistance to oxidative degradation.

[0407] Therefore, as shown by the above results and as described above for the oxidative degradation rate, the cross-linked polysaccharides of the present invention prepared by using the hyperbranched PPE of the present invention as a cross-linking material have better resistance to oxidative degradation (i.e., a lower relative oxidative degradation rate) than cross-linked polysaccharides outside the scope of the present invention but having comparable physical properties prepared by using the comparative cross-linking materials disclosed above. The results of the oxidative degradation assays of comparative sodium carboxymethylcellulose cross-linked with GDE and sodium carboxymethylcellulose cross-linked with PPE of the present invention are summarized in the table below.

[0408] TIFF0007784138000050.tif60170

[0409] 3. Determination of injection force through needle from pre-filled syringe of PPE cross-linked polysaccharide of the present invention

[0410] This test can determine the injection force through the needle. In particular, the peak injection force refers to the force required by the compound or composition to overcome static friction. For cross-linked polysaccharides, this value also depends on their uniformity, the quality of the syringe, and the lubricity of the barrel. Usually, this value represents the quality of the entire system (compound, syringe, and needle). Therefore, a lower injection force value results in a lower pain perception without increasing the injection rate.

[0411] Test sample: Comparative Example 5: BDDE-crosslinked hyaluronic acid (hydrogel) BDDE-HA Comparative PEGDE-crosslinked hyaluronic acid (hydrogel) PEGDE-HA Comparative Example 5 Comparative GDE cross-linked hyaluronic acid (hydrogel) GDE-HA Comparative Example 5, and PPE-crosslinked hyaluronic acid (hydrogel) PPE-HA Example 10, PPE-HA Example 11 and PPE-HA Example 12 of the present invention.

[0412] The effectiveness of needle injection was evaluated by performing tests on the injected samples. The selection of the 27G and 30G gauge lengths, and in general the correct needle, is strongly influenced by the physicochemical and mechanical properties of the hydrogel. The injection force was measured with a texture analyzer (Stable Micro Systems, TA.XT Plus) as the force (N) required to inject the hydrogel through the 27G and 30G needles over a distance of 50 mm at a rate of 10 mm / min (0.2 mL / min) using a 1 mL syringe.

[0413] A comparison of the peak and average injection forces through a 27G needle for comparative hyaluronic acid (hydrogels) crosslinked with BDDE, PEGDE and GDE using standard methods, and hyaluronic acid (hydrogels) crosslinked with the PPE of the present invention (all crosslinkers having the same equivalent epoxy content when added to the polysaccharide) is disclosed in the table below.

[0414] TIFF0007784138000051.tif68170

[0415] As shown by the above results, the cross-linked polysaccharides of the present invention have lower peak injection force values ​​than comparative cross-linked polysaccharides outside the scope of the present invention. The cross-linked polysaccharides of the present invention are advantageous because they are easier to inject and reduce pain perception and skin trauma without compromising (increasing) injection speed. Furthermore, the cross-linked polysaccharides of the present invention are advantageous because they are more convenient for practitioners.

[0416] Citation List He, Z., et al.,´´Ultrasonication-assisted rapid determination of epoxide values ​​in polymer mixtures containing epoxy resin´´.Analytical Methods,2014,vol.6(12),pp.4257-4261.

[0417] For reasons of completeness, the various aspects of the invention are set out in the following numbered paragraphs.

[0418] Item 1. A compound of formula (I), TIFF0007784138000052.tif82170 or alternatively, A compound of formula (II), TIFF0007784138000053.tif54170 wherein each R1 is independently selected from the group consisting of R2 and R3; R2 is TIFF0007784138000054.tif26170 and R3 is TIFF0007784138000055.tif40170 and b is an integer selected from the group consisting of 1 to 70; c is an integer selected from the group consisting of 1 to 70; d is an integer selected from the group consisting of 1 to 70; The compound is Molecular weights (M) of 750 to 15,000 Da determined by liquid chromatography w ) have; With an epoxy equivalent weight of 183 to 7,000 g / eq, as determined by ultrasonic rapid titration with HCl. compound.

[0419] Item 2. Molecular weight (M w ) is 800-7000 Da as determined by liquid chromatography; Epoxy equivalent weight is 195-700 g / eq as determined by ultrasonic rapid titration with HCl; The compound according to item 1.

[0420] Item 3. Molecular weight (M w ) is 900-4500 Da as determined by liquid chromatography; The epoxy equivalent weight is 200 to 600 g / eq as measured by ultrasonic rapid titration using HCl. The compound according to item 1 or 2.

[0421] Item 4. A polydispersity index (M) of less than 1.8 as determined by liquid chromatography W / M n ) 4. The compound according to any one of items 1 to 3.

[0422] Item 5. A cross-linked polysaccharide of formula (III): TIFF0007784138000056.tif61170 or alternatively, A cross-linked polysaccharide of formula (IV): TIFF0007784138000057.tif51170 wherein each R1 is independently selected from the group consisting of R2, R3, R4 and R5; R2 is TIFF0007784138000058.tif20170 and R3 is TIFF0007784138000059.tif41170 and R4 is TIFF0007784138000060.tif23170 and R5 is TIFF0007784138000061.tif24170 and PS is a polysaccharide; The cross-linked polysaccharide has a cross-linking rate of 0.077 to 0.269%. Cross-linked polysaccharide.

[0423] Item 6. The relative enzymatic degradation rate is 100% to 37.9%; The relative oxidative decomposition rate is 100% to 76.8%. Item 5. The cross-linked polysaccharide according to item 5.

[0424] Item 7. The polysaccharide is selected from the group consisting of hyaluronic acid, starch and its derivatives, glycogen, cellulose and its derivatives, pectin, lignin, inulin, guar gum, xanthan gum, alginic acid (alginate), glucomannan, galactomannan and carrageenan; preferably hyaluronic acid. 7. The cross-linked polysaccharide according to item 5 or 6.

[0425] Item 8. The average distance between two crosslinking groups (D N ) is 27nm to 42nm, 8. The cross-linked polysaccharide according to any one of items 5 to 7.

[0426] Item 9. The concentration of the polysaccharide is 1 to 50 mg / ml. 9. The cross-linked polysaccharide according to any one of items 5 to 8.

[0427] Item 10. The polysaccharide can be obtained by a method comprising a step of crosslinking with a compound selected from the group consisting of compounds of formula (I), compounds of formula (II) and mixtures thereof, as described in any one of items 1 to 4, 10. The cross-linked polysaccharide according to any one of items 5 to 9.

[0428] Item 11. 11. A cross-linked polysaccharide according to item 10, obtainable by a process comprising a step of cross-linking a polysaccharide with a compound of formula (I), wherein the compound of formula (I) is a) providing an alkaline solution containing 1,3-glycerol diglycidyl ether; b) maintaining the solution obtained in step a) at a temperature of 10 to 80°C for 1 minute to 30 days; 11. The cross-linked polysaccharide according to item 10, which is obtained by a method comprising the steps of: or alternatively, 11. A cross-linked polysaccharide according to item 10, obtainable by a process comprising a step of cross-linking a polysaccharide with a compound of formula (II), wherein the compound of formula (II) is c) providing an alkaline solution containing 1,2-glycerol diglycidyl ether; d) maintaining the solution obtained in step c) at a temperature of 10 to 80°C for 1 minute to 30 days; 11. The cross-linked polysaccharide according to item 10, which is obtained by a method comprising the steps of: or alternatively, 11. Cross-linked polysaccharides according to item 10, obtainable by a process comprising a step of cross-linking a polysaccharide with a mixture of a compound of formula (I) and a compound of formula (II), The compound of formula (I) a) providing an alkaline solution containing 1,3-glycerol diglycidyl ether; b) maintaining the solution obtained in step a) at a temperature of 10 to 80°C for 1 minute to 30 days; obtained by a process comprising: The compound of formula (II) c) providing an alkaline solution containing 1,2-glycerol diglycidyl ether; d) maintaining the solution obtained in step c) at a temperature of 10 to 80°C for 1 minute to 30 days; 11. The cross-linked polysaccharide according to item 10, obtained by a method comprising the steps of:

[0429] Item 12. In step a), the alkaline solution comprises 1 to 50 weight percent of 1,3-glycerol diglycidyl ether; In step c), the alkaline solution contains 1 to 50 weight percent of 1,2-glycerol diglycidyl ether; Item 12. The cross-linked polysaccharide according to item 11.

[0430] Item 13. A process for preparing compound (I) or alternatively compound of formula (II) according to any one of items 1 to 4, comprising: When the compound is a compound of formula (I), the method comprises: a) providing an alkaline solution containing 1,3-glycerol diglycidyl ether; b) maintaining the solution obtained in step a) at a temperature of 10 to 80°C for 1 minute to 30 days; Includes; or alternatively, When the compound is a compound of formula (II), the method comprises: c) providing an alkaline solution containing 1,2-glycerol diglycidyl ether; d) maintaining the solution obtained in step c) at a temperature of 10 to 80°C for 1 minute to 30 days; A preparation method comprising:

[0431] Item 14. One or more cross-linked polysaccharides according to any one of items 5 to 12, one or more suitable excipients or carriers; A composition comprising:

[0432] Item 15. 13. A pharmaceutical composition comprising a therapeutically effective amount of one or more pharmaceutically acceptable cross-linked polysaccharides according to any one of items 5 to 12, and one or more pharmaceutically acceptable excipients or carriers for use in therapy. Item 15. The composition according to item 14.

[0433] Item 16. 13. A cosmetic composition comprising a cosmetically effective amount of one or more cosmetically acceptable cross-linked polysaccharides according to any one of items 5 to 12 and one or more cosmetically acceptable excipients or carriers as skin care agents, in particular as dermal fillers. Use of the composition according to item 14.

Claims

1. A cross-linked polysaccharide of formula (III): or alternatively, A cross-linked polysaccharide of formula (IV): In the formula, each R １ is R ２ , R ４ and R ５ are independently selected from the group consisting of: R ２ teeth, and R ４ teeth, and R ５ teeth, and PS is a polysaccharide; The cross-linked polysaccharide has a cross-linking rate of 0.077 to 0.450%. Cross-linked polysaccharide.

2. PS is a polysaccharide; and the cross-linked polysaccharide has a cross-linking rate of 0.077 to 0.269%. The cross-linked polysaccharide of claim 1.

3. R ２ is less than 2 ppm based on the total weight of the cross-linked polysaccharides of formulas (III) and (IV); The cross-linked polysaccharide of claim 1 or 2.

4. The relative enzymatic degradation rate of the cross-linked polysaccharide is 100% to 37.9% of the enzymatic degradation rate of a comparative glycerol diglycidyl ether (GDE) cross-linked polysaccharide; the relative oxidative degradation rate of the cross-linked polysaccharide is 100% to 76.8% of the oxidative degradation rate of a comparative glycerol diglycidyl ether (GDE) cross-linked polysaccharide; The cross-linked polysaccharide of claim 1.

5. The polysaccharide is selected from the group consisting of hyaluronic acid, starch and its derivatives, glycogen, cellulose and its derivatives, pectin, inulin, guar gum, xanthan gum, alginic acid (alginate), heparin, chondroitin sulfate, dermatan sulfate, glucuronan, glucomannan, galactomannan and carrageenan; preferably hyaluronic acid. The cross-linked polysaccharide of any one of claims 1 to 4.

6. The average distance between two crosslinking groups (D Ｎ ) is 27 nm to 42 nm; The cross-linked polysaccharide of any one of claims 1 to 5.

7. the polysaccharide PS is present in the cross-linked polysaccharide at a concentration of 1 to 50 mg / ml; The polysaccharide concentration is determined by weighing the dry residue obtained after dehydrating the hydrogel for an appropriate time in a ventilated oven at 105°C. The cross-linked polysaccharide of claim 1.

8. A compound of formula (I), A compound of formula (II), and mixtures thereof, Each R １ is R ２ and R ２ teeth, and b is an integer selected from the group consisting of 1 to 70; c is an integer selected from the group consisting of 1 to 70; d is an integer selected from the group consisting of 1 to 70; The formula (I) or the formula (II) has a molecular weight (Mw) of 750 to 15,000 Da as measured by a liquid chromatography method; Epoxy equivalent weight of 183 to 7,000 g / eq as determined by ultrasonic rapid titration with HCl cross-linking the compound; A process for preparing a cross-linked polysaccharide as defined in any one of claims 1 to 7.

9. the molecular weight (MW) of the compound of formula (I) or the compound of formula (II) is 800 to 7000 Da as measured by a liquid chromatography method; The molecular weight (MW) of the compound of formula (I) or the compound of formula (II) is 195 to 700 g / eq, as measured by ultrasonic rapid titration with HCl; The process of claim 8.

10. the molecular weight (MW) of the compound of formula (I) or the compound of formula (II) is 900 to 4500 Da as measured by a liquid chromatography method; The compound of formula (I) or the compound of formula (II) has an epoxy equivalent molecular weight (MW) of 200 to 600 g / eq, as measured by ultrasonic rapid titration with HCl; 10. The process according to claim 8 or 9.

11. The compound of formula (I) or the compound of formula (II) has a polydispersity index (M) of 1.8 or less as measured by liquid chromatography. Ｗ / M Ｎ ) 11. A process according to any one of claims 8 to 10.

12. The polysaccharide is crosslinked with a compound of formula (I), and the compound of formula (I) is a) providing an alkaline solution containing 1,3-glycerol diglycidyl ether; b) maintaining the solution obtained in step a) at a temperature of 10 to 80°C for 1 minute to 30 days; obtained by a process comprising: or alternatively, The polysaccharide is crosslinked with a compound of formula (II), the compound of formula (II) being c) providing an alkaline solution containing 1,2-glycerol diglycidyl ether; d) maintaining the solution obtained in step c) at a temperature of 10-80°C for 1 minute to 30 days; obtained by a process comprising: or alternatively, the polysaccharide is crosslinked with a mixture of a compound of formula (I) and a compound of formula (II), The compound of formula (I) a) providing an alkaline solution containing 1,3-glycerol diglycidyl ether; b) maintaining the solution obtained in step a) at a temperature of 10 to 80°C for 1 minute to 30 days; obtained by a process comprising: The compound of formula (II) c) providing an alkaline solution containing 1,2-glycerol diglycidyl ether; d) maintaining the solution obtained in step c) at a temperature of 10-80°C for 1 minute to 30 days; 12. The process according to any one of claims 8 to 11, comprising:

13. In step a), the alkaline solution comprises 1 to 50 weight percent 1,3-glycerol diglycidyl ether; In step c), the alkaline solution comprises 1 to 50 weight percent 1,2-glycerol diglycidyl ether. The process of claim 12.

14. One or more cross-linked polysaccharides according to any one of claims 1 to 7; one or more suitable excipients or carriers; A composition comprising:

15. 10. A pharmaceutical composition comprising a therapeutically effective amount of one or more pharmaceutically acceptable cross-linked polysaccharides according to any one of claims 1 to 7, and one or more pharmaceutically acceptable excipients or carriers for use in therapy.

15. The composition of claim 14.

16. 10. A cosmetic composition comprising a cosmetically effective amount of one or more cosmetically acceptable cross-linked polysaccharides according to any one of claims 1 to 7 and one or more cosmetically acceptable excipients or carriers as skin care agents, in particular as dermal fillers. Use of the composition according to claim 15.

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