TW202513100A - Cross-linked material comprising polysaccharide moieties interconnected by ester bonds - Google Patents

Abstract

The present invention relates to a method for preparing a cross-linked material, 5 comprising reaction of at least some carboxylic acid residues of polysaccharide moieties with at least some hydroxy residues of polysaccharide moieties to form ester bonds cross-linking polysaccharide moieties covalently with each other, wherein the reaction is triggered by one or more triazine-based activating agents. Furthermore, the invention refers to a cross-linked material obtainable from such 10 method, compositions comprising such, and uses thereof.

Description

Translated fromChinese

包含以酯鍵互連的多醣部分之交聯材料Cross-linked material comprising polysaccharide moieties interconnected by ester bonds

本發明關於用於製備交聯材料之方法，其包括多醣部分之至少一些羧酸殘基與多醣部分之至少一些羥基殘基反應以形成酯鍵而使多醣部分彼此共價交聯，其中該反應係藉由一或多種基於三𠯤之活化劑觸發。此外，本發明意指可由此種方法獲得之交聯材料、包含此等物之組成物及其用途。The present invention relates to a method for preparing a crosslinking material, which comprises reacting at least some of the carboxylic acid residues of a polysaccharide moiety with at least some of the hydroxyl residues of a polysaccharide moiety to form ester bonds to covalently crosslink the polysaccharide moieties to each other, wherein the reaction is triggered by one or more trihydrium-based activators. Furthermore, the present invention relates to crosslinking materials obtainable by such a method, compositions comprising the same and their use.

顏面和身體重塑係逐漸受到關注。例如，填平顏面及/或身體的皺紋、皮膚回春、乳房重建或隆胸、其他類型之軟組織增補係經常令人感興趣的。為了避免外科介入的需求，已開發出或正在開發一些可經皮下注射或注射在皮膚深層內之軟組織填充劑。Facial and body remodeling is gaining interest. For example, filling of facial and/or body wrinkles, skin rejuvenation, breast reconstruction or augmentation, and other types of soft tissue augmentation are often of interest. To avoid the need for surgical intervention, some soft tissue fillers have been or are being developed that can be injected subcutaneously or deep into the skin.

軟組織填充劑典型地為凝膠，諸如水凝膠。使用此類軟組織填充劑，特別是真皮填充劑的開業者典型地期望此類填充劑在感興趣的條件下進行投予時不會導致有毒或免疫副作用，顯示良好的生物相容性，可無負擔地注射並基於天然材料。同時，填充劑應餘留在空間限定區中，並在生物系統中，諸如在注射時具有足夠穩定性。Soft tissue fillers are typically gels, such as hydrogels. Practitioners using such soft tissue fillers, particularly dermal fillers, typically desire that such fillers do not cause toxic or immunological side effects when administered under the conditions of interest, exhibit good biocompatibility, be injectable without burden, and be based on natural materials. At the same time, the filler should remain in a spatially defined area and be sufficiently stable in a biological system, such as when injected.

在該技術領域中適用於軟組織填充之材料為多醣，例如玻尿酸(HA)。許多多醣，諸如玻尿酸具有良好的生物可接受性。然而，一項重大缺點為此類基於多醣，諸如玻尿酸之材料的生物降解相對較快速，並且此填充材料不適合用於長期解決方法中。其在經其投予之受試者中的壽命有限，經常小於數個月之期望最小範圍。當此種多醣在體內快速降解時，黏度不合意地快速降低，並且填充效果不足夠持久。此外，基於玻尿酸之填充劑的儲存性和保存期限經常有限。包含未修飾多醣之儲存產物經常易於部分降解。然後，黏度降低，並且隨後投予之此類儲存並因此部分降解的產物在投予的受試者中具有甚至更短的壽命。Materials suitable for soft tissue filling in this technical field are polysaccharides, such as hyaluronic acid (HA). Many polysaccharides, such as HA, have good bioacceptability. However, a major disadvantage is that such materials based on polysaccharides, such as HA, biodegrade relatively quickly and such filling materials are not suitable for use in long-term solutions. Their lifespan in subjects to whom they are administered is limited, often less than the expected minimum range of several months. When such polysaccharides are rapidly degraded in the body, the viscosity decreases undesirably quickly and the filling effect is not sufficiently long-lasting. In addition, the storability and shelf life of fillers based on hyaluronic acid are often limited. Storage products containing unmodified polysaccharides are often susceptible to partial degradation. The viscosity then decreases and subsequently administered such stored and thus partially degraded products have an even shorter lifetime in the administered subject.

因此，需要提供其他具有適合的生物穩定性之填充材料。Therefore, there is a need to provide other filling materials with suitable biostability.

已考慮使多醣，諸如玻尿酸與合成的異生素(xenobiotic)交聯劑，例如基於環氧化物連接子丁二醇二環氧丙基醚(BDDE)交聯，如(例如)US-A 2012/0190644、WO 2015/149941或WO 2020/030629中所描述般，或與二-或多親核官能交聯劑交聯，諸如US-A 2020/0140626中所描述般。此種材料具有數項缺點。例如，此類未反應或半反應之二價連接子的殘餘物可能有害並限制此類材料的可使用性。此類反應性連接子具有最大可投予含量，因此基於安全理由，對於使用此物製備得到的填充材料亦然。當投予有其需要之受試者並在該受試者中降解時，會產生異生素和不可降解或難降解的代謝物，此狀況特別是在美容和藥物用途一般係不樂見的。因此，需要避免此類異生素連接子部分。It has been considered to crosslink polysaccharides, such as hyaluronic acid, with synthetic xenobiotic crosslinkers, for example based on epoxide linkers butanediol diglycidyl ether (BDDE), as described, for example, in US-A 2012/0190644, WO 2015/149941 or WO 2020/030629, or with di- or polynucleophilic crosslinkers, as described in US-A 2020/0140626. Such materials have several disadvantages. For example, the residues of such unreacted or half-reacted divalent linkers may be harmful and limit the usability of such materials. Such reactive linkers have a maximum administrable content, and therefore for safety reasons, the same applies to the filler materials prepared using them. When administered to a subject in need thereof and degraded in the subject, xenobiotics and non-degradable or poorly degradable metabolites are produced, which is generally undesirable, particularly in cosmetic and pharmaceutical applications. Therefore, it is desirable to avoid such xenobiotic linker moieties.

為了克服此等缺點，已考慮與蛋白質、胜肽和胺基酸交聯。在此背景下，已考慮使多醣，諸如玻尿酸與交聯蛋白質交聯。例如，如WO 2011/119468中所描述般，玻尿酸與彈性蛋白交聯。此外，多醣亦與絲纖維蛋白交聯，諸如WO 2022/268871中所描述般。申請案CN-A 105713211描述玻尿酸與高含量之包含兩個胺基的胺基酸離胺酸連接。在無胺基酸存在下，不清楚是否可完成交聯，若可完成，則不清楚交聯程度和化學鍵。In order to overcome these drawbacks, crosslinking with proteins, peptides and amino acids has been considered. In this context, crosslinking polysaccharides, such as hyaluronic acid, with crosslinking proteins has been considered. For example, hyaluronic acid is crosslinked with elastin, as described in WO 2011/119468. In addition, polysaccharides are also crosslinked with fibrous proteins, as described in WO 2022/268871. Application CN-A 105713211 describes hyaluronic acid linked to a high content of the amino acid lysine containing two amine groups. In the absence of amino acids, it is unclear whether crosslinking can be achieved, and if so, the degree of crosslinking and the chemical bonds are unclear.

此類方法的缺點為蛋白質、胜肽或胺基酸的存在性並非總是理想的。過程相當複雜，因為其包含數種成分，並且視所使用的蛋白質而定，蛋白質序列內的醯胺鍵可能斷裂。在某些情況下，蛋白質和胜肽可具有不期望的免疫性反應。因此，無此類互連的蛋白質、胜肽和胺基酸地交聯多醣，諸如玻尿酸是理想的。The disadvantage of this method is that the presence of proteins, peptides or amino acids is not always ideal. The process is quite complex, since it involves several components and, depending on the protein used, amide bonds within the protein sequence may be broken. In some cases, proteins and peptides can have an undesirable immunological reaction. Therefore, cross-linked polysaccharides, such as hyaluronic acid, without such interconnected proteins, peptides and amino acids are ideal.

在此背景下，認為藉由具有羧酸殘基以及胺基之多醣彼此交聯可形成醯胺鍵。為達此目的，修飾多醣，諸如醣胺聚醣以包含與游離羧基共軛之游離胺基，諸如WO 2019/002369中所描述般。然而，此類程序需要額外步驟以提供非天然中間產物，諸如提供胺基。此外，如此獲得的共軛物不合意地包含醯胺鍵。期望經由酯鍵直接共軛天然多醣。In this context, it is believed that amide bonds can be formed by crosslinking polysaccharides having carboxylic acid residues and amine groups with each other. To achieve this purpose, polysaccharides, such as glycosaminoglycans, are modified to contain free amine groups conjugated to free carboxyl groups, as described in WO 2019/002369. However, such procedures require additional steps to provide non-natural intermediates, such as providing amine groups. In addition, the conjugates thus obtained undesirably contain amide bonds. It is desirable to conjugate natural polysaccharides directly via ester bonds.

在該技術領域中存在明顯偏見，即在多醣之間形成酯鍵需要嚴峻的活化劑，諸如碳二亞胺活化劑(包含結構組元-N=C=N-)或2-氯-甲基吡啶碘化物。例如，EP-A 0341745教導可藉由2-氯-甲基吡啶碘化物完成酯化。JP-A 2019019201教導使用碳二亞胺活化劑，諸如N,N'-二環己基碳二亞胺(DCC)、二異丙基碳二亞胺(DIC)和1-乙基-3-(3-二甲基胺基丙基)碳二亞胺(EDC)。此等反應劑為不合意地刺激物，或甚至有毒並可具有其他缺點。There is a clear bias in the art that the formation of ester bonds between polysaccharides requires harsh activators, such as carbodiimide activators (comprising the structural element -N=C=N-) or 2-chloro-methylpyridinium iodide. For example, EP-A 0341745 teaches that esterification can be accomplished by 2-chloro-methylpyridinium iodide. JP-A 2019019201 teaches the use of carbodiimide activators, such as N,N'-dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). Such reactants are undesirable irritants, or even toxic and may have other disadvantages.

另一方面，當省略此類活化劑時，酯化不會發生或極緩慢並無效，甚至在嚴峻的pH範圍下，如JP-A 2003/252905中所示般。On the other hand, when such activators are omitted, esterification does not occur or occurs very slowly and ineffectively, even in a severe pH range, as shown in JP-A 2003/252905.

鑑於上述，仍對用於製備交聯材料之有效方法存在未滿足的需求，該有效方法僅需要低程序工作量，避免異生素連接子部分導入水凝膠中並最小化有毒試劑的殘餘量。另外期望獲得多醣部分之間的酯鍵。特別理想地係提供用於顏面和身體重塑之可注射水凝膠(例如，可用作超級豐盈劑(super-volumizer))的方法。In view of the above, there remains an unmet need for an efficient method for preparing cross-linked materials that requires only low process effort, avoids the introduction of xenobiotic linker moieties into the hydrogel and minimizes residual amounts of toxic reagents. It is also desirable to obtain ester bonds between polysaccharide moieties. It would be particularly desirable to provide an injectable hydrogel for facial and body remodeling (e.g., useful as a super-volumizer).

令人驚訝地，已發現具有有益性質之交聯材料可由包括多醣部分之至少一些羧酸殘基與多醣部分之至少一些羥基殘基反應以形成酯鍵而使多醣部分彼此共價交聯的方法獲得，其中該反應係藉由一或多種基於三𠯤之活化劑觸發。Surprisingly, it has been found that cross-linked materials having beneficial properties can be obtained by a process comprising covalently cross-linking polysaccharide moieties to each other by reacting at least some of the carboxylic acid residues of the polysaccharide moieties with at least some of the hydroxyl residues of the polysaccharide moieties to form ester bonds, wherein the reaction is triggered by one or more trihydrium-based activators.

第一方面關於用於製備交聯材料之方法，該方法包括： (i) 使下列成分彼此接觸： (A) 多醣部分，其包含羧酸殘基或其鹽和羥基殘基， (B) 一或多種基於三𠯤之活化劑，其羧酸殘基與羥基殘基反應，從而形成酯鍵，和 (C) 一或多種溶劑；並且 (ii) 允許多醣部分之至少一些羧酸殘基與多醣部分之至少一些羥基殘基反應以形成酯鍵而使多醣部分彼此共價交聯；並且 (iii) 視情況純化獲自步驟(ii)之交聯材料， 其中多醣部分彼此共價交聯的酯鍵是多於多醣部分彼此共價交聯的醯胺鍵， 其中步驟(ii)係在無任何不同於基於三𠯤之活化劑的活化劑存在下進行，特別地，其中步驟(ii)係在無碳二亞胺活化劑、基於琥珀醯亞胺基之活化劑、基於環氧丙基之活化劑、對硝基酚酯和2-氯甲基吡啶碘化物的存在下進行。The first aspect relates to a method for preparing a crosslinking material, the method comprising:(i) contacting the following components with each other:(A) a polysaccharide portion comprising a carboxylic acid residue or a salt thereof and a hydroxyl residue,(B) one or more trihydric activators whose carboxylic acid residues react with hydroxyl residues to form ester bonds, and(C) one or more solvents; and(ii) allowing at least some of the carboxylic acid residues of the polysaccharide portion to react with at least some of the hydroxyl residues of the polysaccharide portion to form ester bonds to covalently crosslink the polysaccharide portions with each other; and(iii) optionally purifying the crosslinking material obtained from step (ii),wherein the number of ester bonds covalently crosslinking the polysaccharide moieties to each other is greater than the number of amide bonds covalently crosslinking the polysaccharide moieties to each other,wherein step (ii) is carried out in the absence of any activator different from the trioxane-based activator, in particular, wherein step (ii) is carried out in the absence of a carbodiimide activator, a succinimidyl-based activator, a glycidyl-based activator, p-nitrophenol ester and 2-chloromethylpyridinium iodide.

已發現此類交聯材料具有意想不到的有益性質。可廣泛地保持黏度。同樣地，亦可合意地減少酶降解性。令人驚訝地發現可避免現有技術中常用的異生素連接子結構。本發明交聯材料實質上可由亦可在自然界中發現之多醣部分組成。亦可將本發明交聯材料稱為”交聯多醣”、”自交聯材料”或”自交聯多醣”。可由本發明方法獲得之交聯材料可具有持久的穩定性。Such crosslinking materials have been found to have unexpected beneficial properties. Viscosity can be widely maintained. Likewise, enzymatic degradability can be desirably reduced. It has surprisingly been found that the xenobiotic linker structures commonly used in the prior art can be avoided. The crosslinking materials of the present invention can be substantially composed of polysaccharide parts that can also be found in nature. The crosslinking materials of the present invention can also be referred to as "crosslinking polysaccharides", "self-crosslinking materials" or "self-crosslinking polysaccharides". The crosslinking materials obtainable by the method of the present invention can have long-lasting stability.

在水性環境中，所獲得的交聯材料可形成水凝膠。In an aqueous environment, the obtained cross-linked materials can form hydrogels.

交聯材料亦可用於模擬細胞外基質，因此可誘導細胞增生及/或細胞遷移及/或可用作細胞的骨架。其可成功地用作可以細胞填充之填充劑(例如，真皮填充劑)。本發明交聯材料可具有良好的剪切減黏性質。其較佳可具有觸變減黏性質。因此，在經受應力時，其黏性可能較小。使其可很好注射，同時在其目標區域中仍相當黏稠(例如，當在皮下區域投予時)。需要相對較低的擠壓力。可獲得高黏度和低擠壓力的凝膠。其可視情況用作超級增容劑。The cross-linked material can also be used to simulate the extracellular matrix, thereby inducing cell proliferation and/or cell migration and/or can be used as a skeleton for cells. It can be successfully used as a filler that can be filled with cells (for example, a dermal filler). The cross-linked material of the present invention can have good shear viscosity-reducing properties. It can preferably have thixotropic viscosity-reducing properties. Therefore, when subjected to stress, its viscosity may be less. It can be injected well while still being quite viscous in its target area (for example, when administered in the subcutaneous area). Relatively low extrusion pressure is required. Gels with high viscosity and low extrusion pressure can be obtained. It can be used as a super-volume expander as appropriate.

本發明方法可在無過多負擔的情況下以相對較低的投入進行。The method of the invention can be carried out with relatively low investment without excessive burden.

相較於現有技術中所描述的程序，所主張的方法是特別有益的，因為除了溶劑之外，其僅需要兩種浸提物(原料)，即多醣部分和一或多種基於三𠯤之活化劑。不需要其他成分，諸如連接子。Compared to the procedures described in the prior art, the claimed method is particularly advantageous because, in addition to the solvent, it requires only two extracts (raw materials), namely the polysaccharide fraction and one or more trion-based activators. No other ingredients, such as linkers, are required.

省略異生素連接子結構，例如BDDE可使較大量之材料能夠投予受試者，例如經皮下方式注射。此外，其在投藥後可在受試者體內具有長壽命。Omitting the xenobiotic linker structure, such as BDDE, allows larger amounts of the material to be administered to a subject, such as by subcutaneous injection. In addition, it may have a long life in the subject after administration.

由於避免反應性基團，本發明可獲得的交聯材料可具有長保存期限和儲存性。其亦相對較熱穩定。Due to the avoidance of reactive groups, the cross-linked materials obtainable by the present invention can have long shelf life and storability. They are also relatively thermally stable.

如本文所使用，如該技術領域中一般所理解，可以最廣泛的意義來理解酯鍵。典型地，酯鍵具有結構-O-CO-或-CO-O-，包括其互變異構結構。As used herein, an ester bond may be understood in the broadest sense as generally understood in the art. Typically, an ester bond has the structure -O-CO- or -CO-O-, including its tautomeric structures.

如上所指示，多醣部分較佳主要係經由酯鍵彼此連接。換言之，基於多醣部分之間的所有鍵(多醣部分之間的互連基團)，超過50 莫耳%，超過75 莫耳%，或甚至超過80 莫耳%較佳可為酯鍵。As indicated above, the polysaccharide moieties are preferably linked to each other primarily via ester bonds. In other words, based on all bonds between the polysaccharide moieties (interconnecting groups between the polysaccharide moieties), more than 50 mol%, more than 75 mol%, or even more than 80 mol% may preferably be ester bonds.

在較佳具體實施態樣中，在交聯材料中形成比醯胺鍵更多之酯鍵。在較佳具體實施態樣中，交聯材料包含至少25：1，較佳為至少50：1，特別是至少100：1之酯基：醯胺基的莫耳比。在較佳具體實施態樣中，交聯材料(實質上)不具胺基。In a preferred embodiment, more ester bonds are formed in the crosslinking material than amide bonds. In a preferred embodiment, the crosslinking material comprises a molar ratio of ester groups:amide groups of at least 25:1, preferably at least 50:1, in particular at least 100:1. In a preferred embodiment, the crosslinking material has (substantially) no amine groups.

如本文所使用，術語”殘基”和”基團”可交換理解。As used herein, the terms "residue" and "group" are understood interchangeably.

如本文所用，術語”羧酸殘基”和”羧基殘基”可如該技術領域中一般所理解般，即如殘基-COOH或其鹽-COO-可交換地理解。As used herein, the terms "carboxylic acid residue" and "carboxyl residue" may be understood as generally understood in the art, ie, as the residue -COOH or its salt -COO- interchangeably.

在用作本發明方法之浸提物的多醣部分中，較佳係羥基殘基(-OH)多於一級胺基團(-NH₂，包括其鹽，即-NH₃⁺(和相對離子))。In the polysaccharide fraction used as the extract in the process of the present invention, preferably there are more hydroxyl residues (-OH) than primary amine groups (_-NH2 , including its salts, i.e._-NH3⁺ (and relative ions)).

在較佳具體實施態樣中，多醣部分包含至少25：1，較佳為至少50：1，特別是至少100：1之羥基殘基：一級胺基的莫耳比。在較佳具體實施態樣中，多醣部分(實質上)不具胺基。In a preferred embodiment, the polysaccharide portion comprises a molar ratio of hydroxyl residues:primary amine groups of at least 25:1, preferably at least 50:1, especially at least 100:1. In a preferred embodiment, the polysaccharide portion has (substantially) no amine groups.

如本發明背景下所使用，可以最廣泛的意義將術語”多醣部分” 理解為該技術領域中已知包含羧酸殘基或其鹽和羥基殘基之任何多醣部分。如本發明背景下所使用，可以最廣泛的意義將術語”多醣”理解為該技術領域中之任何多醣。根據本發明，至少一個多醣部分包含至少一個羧酸殘基或其鹽。多醣部分另外包含羥基殘基。多醣可為可經修飾之天然多醣或可為合成多醣。在此背景下，多醣可經分枝或未分枝。應理解術語“多醣部分”亦可包括其鹽和修飾形式。在較佳具體實施態樣中，多醣未經氧化。As used in the context of the present invention, the term "polysaccharide portion" can be understood in the broadest sense as any polysaccharide portion known in the art that contains a carboxylic acid residue or its salt and a hydroxyl residue. As used in the context of the present invention, the term "polysaccharide" can be understood in the broadest sense as any polysaccharide in the art. According to the present invention, at least one polysaccharide portion contains at least one carboxylic acid residue or its salt. The polysaccharide portion further contains a hydroxyl residue. The polysaccharide may be a natural polysaccharide that may be modified or may be a synthetic polysaccharide. In this context, the polysaccharide may be branched or unbranched. It should be understood that the term "polysaccharide portion" may also include its salts and modified forms. In a preferred embodiment, the polysaccharide is not oxidized.

多醣部分較佳係重量平均分子量(M_w)為至少1 kDa(1000 Da)，更佳為至少5 kDa，甚至更佳為至少10 kDa之聚合物部分。在較佳具體實施態樣中，多醣部分的重量平均分子量為至少50 kDa，較佳為至少200 kDa，特別是在500至4000 kDa之範圍內。在較佳具體實施態樣中，多醣部分的重量平均分子量(M_w)係在10至10000 kDa之範圍內，更佳係在25至7500 kDa之範圍內，特別是在50至4000 kDa之範圍內。The polysaccharide moiety is preferably a polymer moiety with a weight average molecular weight (_Mw ) of at least 1 kDa (1000 Da), more preferably at least 5 kDa, even more preferably at least 10 kDa. In preferred embodiments, the weight average molecular weight of the polysaccharide moiety is at least 50 kDa, preferably at least 200 kDa, in particular in the range of 500 to 4000 kDa. In preferred embodiments, the weight average molecular weight (_Mw ) of the polysaccharide moiety is in the range of 10 to 10000 kDa, more preferably in the range of 25 to 7500 kDa, in particular in the range of 50 to 4000 kDa.

多醣部分可具有任何固有黏度(例如藉由流變計測得，例如下方實驗部分中所描述般)。在較佳具體實施態樣中，多醣部分可具有1至4 m³/kg(20℃，1013 hPa，水)之固有黏度。在較佳具體實施態樣中，多醣部分可具有1.0至3.3 m³/kg(20℃，1013 hPa，水)之固有黏度。在較佳具體實施態樣中，多醣部分可具有1.1至3.2 m³/kg、2.0至3.1 m³/kg或2.5至3.0 m³/kg(分別在20℃、1013 hPa、水)之固有黏度。The polysaccharide portion may have any intrinsic viscosity (e.g., as measured by a rheometer, such as described in the experimental section below). In preferred embodiments, the polysaccharide portion may have an intrinsic viscosity of 1 to 4 m³ /kg (20° C., 1013 hPa, water). In preferred embodiments, the polysaccharide portion may have an intrinsic viscosity of 1.0 to 3.3 m³ /kg (20° C., 1013 hPa, water). In preferred embodiments, the polysaccharide portion may have an intrinsic viscosity of 1.1 to 3.2 m³ /kg, 2.0 to 3.1 m³ /kg, or 2.5 to 3.0 m³ /kg (at 20° C., 1013 hPa, water, respectively).

在較佳具體實施態樣中，多醣部分的重量平均分子量為1500至3500 kDa(1.5和3.5 MDa)。其重量平均分子量更佳可在100至5000 kDa之範圍內，在200至2000 kDa之範圍內，在250至1500 kDa之範圍內，在300至1000 kDa之範圍內，在400至900 kDa之範圍內或在500至900 kDa之範圍內。In preferred embodiments, the weight average molecular weight of the polysaccharide portion is 1500 to 3500 kDa (1.5 and 3.5 MDa). More preferably, the weight average molecular weight is in the range of 100 to 5000 kDa, in the range of 200 to 2000 kDa, in the range of 250 to 1500 kDa, in the range of 300 to 1000 kDa, in the range of 400 to 900 kDa or in the range of 500 to 900 kDa.

在較佳具體實施態樣中，多醣部分包含一或多種類型之糖酸部分或其鹽。In preferred embodiments, the polysaccharide portion comprises one or more types of sugar acid moieties or salts thereof.

在較佳具體實施態樣中，多醣部分包含一或多種類型之糖酸部分或其鹽，其中一或多種類型之糖酸部分係選自由下列各者組成之群組： (B1) 一或多個醣醛酸部分，特別是選自由葡萄糖醛酸部分、半乳糖醛酸部分、艾杜糖醛酸部分及其兩種或多種之組合組成之群組； (B2) 一或多種醛糖酸部分，特別是選自由甘油酸部分、木糖酸部分、葡萄糖酸部分、抗壞血酸部分及其兩種或多種之組合組成之群組； (B3) 一或多種酮醣酸部分，特別是選自由神經胺糖酸部分、酮去氧辛酮糖酸部分及其組合組成之群組；及/或 (B4) 一或多種醛醣二酸部分，特別是選自由酒石酸部分、內消旋半乳糖二酸部分、葡萄糖二酸部分及其兩種或多種之組合組成之群組。In a preferred embodiment, the polysaccharide portion comprises one or more types of sugar acid moieties or salts thereof, wherein the one or more types of sugar acid moieties are selected from the group consisting of:(B1) one or more uronic acid moieties, in particular selected from the group consisting of glucuronic acid moieties, galacturonic acid moieties, iduronic acid moieties and combinations of two or more thereof;(B2) one or more aldonic acid moieties, in particular selected from the group consisting of glyceric acid moieties, xylonic acid moieties, gluconic acid moieties, ascorbic acid moieties and combinations of two or more thereof;(B3) one or more ketone acid moieties, in particular selected from the group consisting of neuraminic acid moieties, keto-deoxyoctulose acid moieties and combinations thereof; and/or(B4) One or more aldose diacid moieties, in particular selected from the group consisting of tartaric acid moieties, meso-galactosaric acid moieties, glucaric acid moieties and combinations of two or more thereof.

在較佳具體實施態樣中，多醣部分包含醣醛酸部分。在較佳具體實施態樣中，多醣部分包含葡萄糖醛酸部分。在較佳具體實施態樣中，多醣部分包含D-葡萄糖醛酸部分。In a preferred embodiment, the polysaccharide portion comprises a uronic acid portion. In a preferred embodiment, the polysaccharide portion comprises a glucuronic acid portion. In a preferred embodiment, the polysaccharide portion comprises a D-glucuronic acid portion.

在較佳具體實施態樣中，多醣部分包含或由D-糖部分組成。在替代具體實施態樣中，多醣部分包含或由L-糖部分組成。在替代具體實施態樣中，多醣部分包含或由D-糖部分和L-糖部分之組合組成。例如，在此種組合中，可包含糖部分之外消旋混合物，或特定糖部分為D-糖部分，而其他為L-糖部分。在較佳具體實施態樣中，多醣部分包含或由一或多個醣胺聚醣部分組成。In preferred embodiments, the polysaccharide moiety comprises or consists of a D-sugar moiety. In alternative embodiments, the polysaccharide moiety comprises or consists of an L-sugar moiety. In alternative embodiments, the polysaccharide moiety comprises or consists of a combination of a D-sugar moiety and an L-sugar moiety. For example, in such a combination, a racemic mixture of sugar moieties may be included, or certain sugar moieties may be D-sugar moieties and others may be L-sugar moieties. In preferred embodiments, the polysaccharide moiety comprises or consists of one or more glycosaminoglycan moieties.

在較佳具體實施態樣中，多醣部分係選自由玻尿酸(HA)部分、肝素聚醣部分、肝素、硫酸軟骨素及其兩種或多種之混合物組成之群組。在較佳具體實施態樣中，多醣部分包含或由玻尿酸、經甘油接枝之玻尿酸、肝素聚醣、硫酸軟骨素和羧甲基纖維素組成。此類包含羧酸基之多醣亦可商業購得。In a preferred embodiment, the polysaccharide portion is selected from the group consisting of a hyaluronic acid (HA) portion, a heparin polysaccharide portion, heparin, chondroitin sulfate, and a mixture of two or more thereof. In a preferred embodiment, the polysaccharide portion comprises or consists of hyaluronic acid, glycerol-grafted hyaluronic acid, heparin polysaccharide, chondroitin sulfate, and carboxymethyl cellulose. Such polysaccharides containing carboxylic acid groups are also commercially available.

在較佳具體實施態樣中，多醣部分包含或由玻尿酸、經甘油接枝之玻尿酸、肝素聚醣、硫酸軟骨素和羧甲基纖維素組成。應理解此等物亦可包含其鹽。In a preferred embodiment, the polysaccharide portion comprises or consists of hyaluronic acid, hyaluronic acid grafted with glycerol, heparin polysaccharide, chondroitin sulfate and carboxymethylcellulose. It should be understood that these substances may also include their salts.

在較佳具體實施態樣中，多醣部分包含或由一或多個玻尿酸部分組成。應理解此亦可包含其鹽。In a preferred embodiment, the polysaccharide portion comprises or consists of one or more hyaluronic acid portions. It should be understood that this may also include salts thereof.

在較佳具體實施態樣中，本發明交聯材料為凝膠。在較佳具體實施態樣中，本發明交聯材料為基於多醣之凝膠。在較佳具體實施態樣中，本發明交聯材料為基於玻尿酸之凝膠(HA凝膠)。視情況亦可將此材料稱為自交聯玻尿酸(HA)。In a preferred embodiment, the crosslinking material of the present invention is a gel. In a preferred embodiment, the crosslinking material of the present invention is a polysaccharide-based gel. In a preferred embodiment, the crosslinking material of the present invention is a hyaluronic acid-based gel (HA gel). This material may also be referred to as self-crosslinking hyaluronic acid (HA) as the case may be.

可以最廣泛的意義將玻尿酸(亦：HA、玻尿酸鹽或醣醛酸)理解為該技術領域中之任何玻尿酸。其可為包含玻尿酸部分(亦為玻尿酸單元)之多醣部分，較佳係相對於多醣中醣部分之全部含量，包含至少50 莫耳%之玻尿酸部分，更佳為至少75 莫耳%，甚至更佳為至少80 莫耳%，甚至更佳為至少90 莫耳%之玻尿酸部分。玻尿酸可視情況包含一或多種異於玻尿酸之醣部分。Hyaluronic acid (also: HA, hyaluronate or uronic acid) can be understood in the broadest sense as any hyaluronic acid in the art. It can be a polysaccharide portion comprising a hyaluronic acid portion (also a hyaluronic acid unit), preferably comprising at least 50 mol% of hyaluronic acid portion relative to the total content of sugar portions in the polysaccharide, more preferably at least 75 mol%, even more preferably at least 80 mol%, even more preferably at least 90 mol% of hyaluronic acid portion. Hyaluronic acid may optionally contain one or more sugar portions different from hyaluronic acid.

在較佳具體實施態樣中，玻尿酸係由N-乙醯基-D-葡萄糖胺和D-葡萄糖醛酸([α-1,4-D-葡萄糖醛酸-β-1,3 -N-乙醯基-D-葡萄糖胺]_n)之重複單元連接所組成的天然醣胺聚醣。因此，玻尿酸之重複/單體單元可為下列範例或其鹽：In a preferred embodiment, hyaluronic acid is a natural glycosaminoglycan composed of repeating units of N-acetyl-D-glucosamine and D-glucuronic acid ([α-1,4-D-glucuronic acid-β-1,3-N-acetyl-D-glucosamine]_n ). Therefore, the repeating/monomer units of hyaluronic acid can be the following examples or their salts:

因此，玻尿酸之重複結構組元為下列範例或其鹽：Therefore, the repeating structural components of hyaluronic acid are the following examples or their salts:

玻尿酸亦可涵蓋經甘油接枝之玻尿酸。Hyaluronic acid can also include glycerol-grafted hyaluronic acid.

可如WO 2022/268871中所描述般使用玻尿酸。在本發明背景下，玻尿酸的重量平均分子量(M_w)較佳為至少1 kDa(1000 Da)，更佳為至少5 kDa，甚至更佳為至少10 kDa，甚至更佳為至少50 kDa，甚至更佳為至少100 kDa，甚至更佳為至少200 kDa，甚至更佳為至少300 kDa或更高。Hyaluronic acid can be used as described in WO 2022/268871. In the context of the present invention, the weight average molecular weight (_Mw ) of the hyaluronic acid is preferably at least 1 kDa (1000 Da), more preferably at least 5 kDa, even more preferably at least 10 kDa, even more preferably at least 50 kDa, even more preferably at least 100 kDa, even more preferably at least 200 kDa, even more preferably at least 300 kDa or more.

在較佳具體實施態樣中，多醣部分具有如上展示之重量平均分子量。In preferred embodiments, the polysaccharide portion has a weight average molecular weight as shown above.

可以最廣泛的意義將肝素聚醣理解為任何肝素聚醣。在較佳具體實施態樣中，其可為諸如WO 2015/149941中所描述般。肝素聚醣(HEP)係屬於多醣之醣胺聚醣(GAG)族的生物聚合物。Heparin polysaccharide can be understood in the broadest sense as any heparin polysaccharide. In a preferred embodiment, it can be as described in WO 2015/149941. Heparin polysaccharide (HEP) is a biopolymer belonging to the glycosaminoglycan (GAG) family of polysaccharides.

在人體內，其為肝素和硫酸肝素之生物合成中的中間產物。肝素聚醣的結構係與玻尿酸(HA)高度相似，因為其具有與玻尿酸相同的單醣成分糖，與HA不同之處僅在HA中葡萄糖醛酸(GlcUA)與N-乙醯基葡萄糖胺(GlcNAc)之間的β-(1,3)醣苷鍵係被HEP中之β-(1,4)醣苷鍵取代，以及HA中N-乙醯基葡萄糖胺(GlcNAc)與葡萄糖醛酸(GlcUA)之間的β-(1,4)醣苷鍵係被HEP中之α-(1,4)醣苷鍵取代： GlcUA-β-(1-4)-[GlcNAc-α-(1-4)-GlcUA-β-(1-4)]_n-GlcNAc   HEPIn the human body, it is an intermediate in the biosynthesis of heparin and heparan sulfate. The structure of heparin polysaccharide is highly similar to hyaluronic acid (HA) because it has the same monosaccharide component sugar as HA, and differs from HA only in that the β-(1,3) glycosidic bond between glucuronic acid (GlcUA) and N-acetylglucosamine (GlcNAc) in HA is replaced by the β-(1,4) glycosidic bond in HEP, and the β-(1,4) glycosidic bond between N-acetylglucosamine (GlcNAc) and glucuronic acid (GlcUA) in HA is replaced by the α-(1,4) glycosidic bond in HEP: GlcUA-β-(1-4)-[GlcNAc-α-(1-4)-GlcUA-β-(1-4)]_n -GlcNAc HEP

典型地，肝素聚醣具有優異的生物相容性。肝素聚醣攜帶大量負電荷和羥基殘基，因此具有高度親水性，從而提高組織相容性。此外，由於肝素聚醣聚合物即使在修飾之後仍包含天然硫酸乙醯肝素和肝素聚合物中出現的伸展，肝素聚醣典型地係非免疫性的(例如，不會誘導抗體)。此外，由於肝素聚醣與玻尿酸之間結構相似，可在官能基上進行相同的化學修飾，包括氧化成醛，如玻尿酸已知之化學修飾。本發明背景下所使用之肝素聚醣聚合物的分子量(M_w)可具有任何分子量。Typically, heparin polysaccharides have excellent biocompatibility. Heparin polysaccharides carry a large number of negative charges and hydroxyl residues and are therefore highly hydrophilic, thereby improving tissue compatibility. In addition, since heparin polysaccharide polymers still contain the stretches present in native acetyl heparin sulfate and heparin polymers even after modification, heparin polysaccharides are typically non-immunogenic (e.g., do not induce antibodies). In addition, due to the structural similarity between heparin polysaccharides and hyaluronic acid, the same chemical modifications can be performed on the functional groups, including oxidation to aldehydes, as are known chemical modifications of hyaluronic acid. The molecular weight (_Mw ) of the heparin polysaccharide polymers used in the context of the present invention can have any molecular weight.

硫酸軟骨素可如該技術領域中一般所理解之最廣泛的意義來理解。在水性環境中，其可為包含下列重複/單體單元(相對離子未描繪並可為任何陽離子，例如鈉)之結構：Chondroitin sulfate can be understood in the broadest sense as generally understood in the art. In an aqueous environment, it can be a structure comprising the following repeating/monomer units (the relative ions are not depicted and can be any cation, such as sodium):

多醣部分可為一或多種類型之多醣部分。此等物的分子大小可能不同，及/或其單體單元的組成可能不同，及/或其化學結構可能不同。視情況而定，不同的多醣部分可為不同類型之多醣部分。The polysaccharide moiety may be one or more types of polysaccharide moieties. These may have different molecular sizes, and/or different monomer unit compositions, and/or different chemical structures. Depending on the circumstances, different polysaccharide moieties may be different types of polysaccharide moieties.

在一個具體實施態樣中，多醣部分，特別是玻尿酸部分具有至少兩種不同的分子量，其各別包含一級胺基殘基或其鹽。換言之，多醣部分亦可為不同分子量之多醣部分的混合物。在較佳具體實施態樣中，多醣部分具有至少兩種不同的分子量，並且至少一個多醣部分較佳具有至少兩個多醣部分，特別是所有多醣部分，其分子量分別係在10至10000 kDa之範圍內，在100至10000 kDa之範圍內，或在100至5000 kDa之範圍內，在100至3500 kDa之範圍內，在200至2000 kDa之範圍內，在250至1500 kDa之範圍內，在300至1000 kDa之範圍內，在400至900 kDa之範圍內，或在500至900 kDa之範圍內。In one embodiment, the polysaccharide portion, in particular the hyaluronic acid portion, has at least two different molecular weights, each of which contains a primary amino residue or a salt thereof. In other words, the polysaccharide portion may also be a mixture of polysaccharide portions of different molecular weights. In preferred embodiments, the polysaccharide moieties have at least two different molecular weights, and at least one polysaccharide moiety, preferably at least two polysaccharide moieties, in particular all polysaccharide moieties, have molecular weights in the range of 10 to 10,000 kDa, in the range of 100 to 10,000 kDa, or in the range of 100 to 5,000 kDa, in the range of 100 to 3,500 kDa, in the range of 200 to 2,000 kDa, in the range of 250 to 1,500 kDa, in the range of 300 to 1,000 kDa, in the range of 400 to 900 kDa, or in the range of 500 to 900 kDa.

在較佳具體實施態樣中，多醣部分具有至少兩種不同的分子量，並且至少一個多醣部分較佳具有至少兩個多醣部分，特別是所有多醣部分，其分子量分別係在1500至3500 kDa之範圍內。In a preferred embodiment, the polysaccharide moieties have at least two different molecular weights, and at least one polysaccharide moiety, preferably at least two polysaccharide moieties, in particular all polysaccharide moieties, have molecular weights in the range of 1500 to 3500 kDa, respectively.

在較佳具體實施態樣中，基於多醣部分之總量，多醣部分之總量不包含超過20 重量%，不包含超過10 重量%，不包含超過5 重量%，不包含超過1 重量%，不包含超過0.5 重量%，或不包含超過0.1 重量%之分子量小於200 kDa的多醣部分。In a preferred embodiment, based on the total amount of the polysaccharide portion, the total amount of the polysaccharide portion does not contain more than 20 wt%, does not contain more than 10 wt%, does not contain more than 5 wt%, does not contain more than 1 wt%, does not contain more than 0.5 wt%, or does not contain more than 0.1 wt% of a polysaccharide portion having a molecular weight of less than 200 kDa.

在較佳具體實施態樣中，多醣部分包含或由至少兩個具有至少兩種不同分子量之玻尿酸部分組成，並且至少一個玻尿酸部分具有較佳至少兩個玻尿酸部分，其二者的分子量，特別是所有玻尿酸部分的分子量分別係在10至10000 kDa之範圍內，在100至10000 kDa之範圍內，或在100至5000 kDa之範圍內，在100至3500 kDa之範圍內，在200至2000 kDa之範圍內，在250至1500 kDa之範圍內，在300至1000 kDa之範圍內，在400至900 kDa之範圍內，或在500至900 kDa之範圍內。在較佳具體實施態樣中，多醣部分包含或由至少兩個具有至少兩種不同分子量之玻尿酸部分組成，並且至少一個玻尿酸部分的的分子量，較佳至少兩個玻尿酸部分二者的分子量，特別是所有玻尿酸部分的分子量分別係在1500至3500 kDa之範圍內。In a preferred embodiment, the polysaccharide portion comprises or consists of at least two hyaluronic acid portions having at least two different molecular weights, and at least one hyaluronic acid portion has preferably at least two hyaluronic acid portions, the molecular weights of both, in particular all hyaluronic acid portions, are in the range of 10 to 10,000 kDa, in the range of 100 to 10,000 kDa, or in the range of 100 to 5,000 kDa, in the range of 100 to 3,500 kDa, in the range of 200 to 2,000 kDa, in the range of 250 to 1,500 kDa, in the range of 300 to 1,000 kDa, in the range of 400 to 900 kDa, or in the range of 500 to 900 kDa. In a preferred embodiment, the polysaccharide portion comprises or consists of at least two hyaluronic acid portions having at least two different molecular weights, and the molecular weight of at least one hyaluronic acid portion, preferably the molecular weights of at least two hyaluronic acid portions, in particular the molecular weights of all hyaluronic acid portions are in the range of 1500 to 3500 kDa.

基於三𠯤之活化劑的使用含量可為任何適用於使多醣部分之羧酸殘基與多醣部分之至少一些羥基殘基交聯以形成酯鍵，從而使多醣部分彼此共價交聯。The trihydroxide-based activator may be used in any amount suitable for cross-linking the carboxylic acid residues of the polysaccharide moieties with at least some of the hydroxyl residues of the polysaccharide moieties to form ester bonds, thereby covalently cross-linking the polysaccharide moieties to each other.

典型地，基於三𠯤之活化劑活化多醣部分之羧酸殘基。因此，可將基於三𠯤之活化劑的含量合理定義為相對於多醣部分之羧酸殘基的莫耳當量(eq.)。在此背景下，應理解羧酸殘基亦涵蓋其鹽。換言之，基於三𠯤之活化劑的莫耳當量(eq.)係定義為[基於三𠯤之活化劑]：[多醣部分之羧酸殘基]的莫耳比。為達此目的，在玻尿酸(HA)鈉鹽的背景下，近似地可假設以鈉鹽(分子量(M_W)近402 g/mol)使用時，各別HA單體/重複單元具有一個羧酸殘基。可以不同莫耳當量(eq.)之基於三𠯤之活化劑調整交聯度。Typically, trihedral based activators activate carboxylic acid residues of the polysaccharide portion. Therefore, the amount of trihedral based activator can be reasonably defined as the molar equivalent (eq.) relative to the carboxylic acid residues of the polysaccharide portion. In this context, it should be understood that carboxylic acid residues also encompass their salts. In other words, the molar equivalent (eq.) of trihedral based activator is defined as the molar ratio of [trihedral based activator]:[carboxylic acid residues of the polysaccharide portion]. For this purpose, in the context of hyaluronic acid (HA) sodium salt, it can be approximately assumed that each HA monomer/repeat unit has one carboxylic acid residue when the sodium salt (molecular weight (_MW ) approximately 402 g/mol) is used. The degree of crosslinking can be adjusted by using different molar equivalents (eq.) of the trihedron-based activator.

在較佳具體實施態樣中，[基於三𠯤之活化劑]：[多醣部分之羧酸殘基]的莫耳比係在1：100至100：1之範圍內，較佳係在1：10至10：1之範圍內，更佳係在1：5至5：1之範圍內，或在1：2至2：1之範圍內。In a preferred embodiment, the molar ratio of [trioxane-based activator]:[carboxylic acid residue of the polysaccharide moiety] is in the range of 1:100 to 100:1, preferably in the range of 1:10 to 10:1, more preferably in the range of 1:5 to 5:1, or in the range of 1:2 to 2:1.

如本發明背景下所使用，基於三𠯤之活化劑可為任何基於三𠯤核心結構之化合物，該核心結構招致羧酸殘基與羥基殘基反應，從而形成酯鍵。應理解其主要意指活化劑係招致多醣部分之羧酸殘基與多醣部分之羥基殘基反應，從而形成酯鍵的化合物。As used in the context of the present invention, a trioxane-based activator may be any compound based on a trioxane core structure that causes a carboxylic acid residue to react with a hydroxyl residue to form an ester bond. It is understood that this primarily means that the activator is a compound that causes a carboxylic acid residue of a polysaccharide moiety to react with a hydroxyl residue of a polysaccharide moiety to form an ester bond.

在較佳具體實施態樣中，活化劑典型地非共價包含在交聯材料中。因此，典型地可視情況藉由任何方式，例如洗滌、過濾等將其自本發明交聯材料中去除。In preferred embodiments, the activator is typically non-covalently contained in the cross-linking material. Therefore, it can typically be removed from the cross-linking material of the present invention by any means, such as washing, filtering, etc.

基於三𠯤之活化劑可具有任何包含三𠯤核心的結構，該三𠯤核心係適用於使多醣部分之羧酸殘基與多醣部分之至少一些羥基殘基交聯以形成酯鍵，從而多醣部分彼此共價交聯。例如，基於三𠯤之活化劑可具有下列結構：其中R¹、R²和R³係彼此獨立地為任何殘基，例如各別獨立地為選自由下列各者組成之群組的殘基：氫、氘、C₆-C₁₀-芳基、C₂-C₁₀-雜芳基、線性或分枝C₁-C₁₀-(環)烷基、線性或分枝C₁-C₁₀-(環)烷氧基、線性或分枝C₁-C₁₀-雜(環)烷基、線性或分枝C₂-C₁₀-(環)烯基、C₂-C₁₀-雜(環)烯基、線性或分枝C₂-C₁₀-(環)炔基和C₂-C₁₀-雜(環)烯基， 其中上述殘基各別視情況經一或多個選自由下列各者組成之群組的殘基取代：鹵素、C₆-C₁₀-芳基、C₂-C₁₀-雜芳基、線性或分枝C₁-C₁₀-(環)烷基、線性或分枝C₁-C₁₀-雜(環)烷基、線性或分枝C₁-C₁₀-(環)烷氧基、線性或分枝C₂-C₁₀-(環)烯基、C₂-C₁₀-雜(環)烯基、線性或分枝C₂-C₁₀-(環)炔基和C₂-C₁₀-雜(環)烯基， 或其鹽。The tris-based activator may have any structure comprising a tris-based core suitable for cross-linking the carboxylic acid residues of the polysaccharide moieties with at least some of the hydroxyl residues of the polysaccharide moieties to form ester bonds, thereby covalently cross-linking the polysaccharide moieties to each other. For example, the tris-based activator may have the following structure: wherein R¹ , R² and R³ are independently any residue, for example, are independently a residue selected from the group consisting of hydrogen, deuterium, C₆ -C₁₀ -aryl, C₂ -C₁₀ -heteroaryl, linear or branched C₁ -C₁₀ -(cyclo)alkyl, linear or branched C₁ -C₁₀ -(cyclo)alkoxy, linear or branched C₁ -C₁₀ -hetero(cyclo)alkyl, linear or branched C₂ -C₁₀ -(cyclo)alkenyl, C₂ -C₁₀ -hetero(cyclo)alkenyl, linear or branched C₂ -C₁₀ -(cyclo)alkynyl and C₂ -C₁₀ -hetero(cyclo)alkenyl, wherein the above-mentioned residues are optionally substituted by one or more residues selected from the group consisting of halogen, C₆ -C₁₀ -aryl, C₂ -C₁₀ -heteroaryl, linear or branched C₁ -C₁₀ -(cyclo)alkyl, linear or branched C₁ -C₁₀ -hetero(cyclo)alkyl, linear or branched C₁ -C₁₀ -(cyclo)alkoxy, linear or branched C₂ -C₁₀ -(cyclo)alkenyl, C₂ -C₁₀ -hetero(cyclo)alkenyl, linear or branched C₂ -C₁₀ -(cyclo)alkynyl and C₂ -C₁₀ -hetero(cyclo)alkenyl, or a salt thereof.

在較佳具體實施態樣中，殘基R¹、R²和R³中之至少一者是視情況經取代之嗎啉鎓殘基。在較佳具體實施態樣中，殘基R¹、R²和R³中之至少一者為C₁-C₄-烷基-嗎啉鎓殘基。在較佳具體實施態樣中，殘基R¹、R²和R³中之至少一者為甲基嗎啉鎓殘基。在較佳具體實施態樣中，殘基R¹、R²和R³中之至少一者為視情況經取代之線性或分枝C₁-C₁₀-(環)烷氧基殘基。在較佳具體實施態樣中，殘基R¹、R²和R³中之至少一者為視情況經取代之線性或分枝C₁-C₄-烷氧基殘基。在較佳具體實施態樣中，殘基R¹、R²和R³中之至少一者為視情況經取代之線性或分枝甲氧基殘基。In a preferred embodiment, at least one of the residues R¹ , R² and R³ is an optionally substituted morpholinium residue. In a preferred embodiment, at least one of the residues R¹ , R² and R³ is a C₁ -C₄ -alkyl-morpholinium residue. In a preferred embodiment, at least one of the residues R¹ , R² and R³ is a methylmorpholinium residue. In a preferred embodiment, at least one of the residues R¹ , R² and R³ is an optionally substituted linear or branched C₁ -C₁₀ -(cyclo)alkoxy residue. In a preferred embodiment, at least one of the residues R¹ , R² and R³ is an optionally substituted linear or branched C₁ -C₄ -alkoxy residue. In a preferred embodiment, at least one of the residues R¹ , R² and R³ is an optionally substituted linear or branched methoxy residue.

在較佳具體實施態樣中，基於三𠯤之活化劑的分子量不超過1500 g/mol，不超過1000 g/mol，不超過500 g/mol，不超過400 g/mol。In preferred embodiments, the molecular weight of the trihydroxide-based activator is no more than 1500 g/mol, no more than 1000 g/mol, no more than 500 g/mol, and no more than 400 g/mol.

在較佳具體實施態樣中，一或多種活化劑係選自由下列各者組成之群組：4-(4,6-二甲氧基-1,3,5-三𠯤-2-基)-4-甲基嗎啉鎓(DMTMM)或其鹽及/或2-氯-4,6-二甲氧基-1,3,5-三𠯤或其鹽以及其組合。In a preferred embodiment, the one or more activators are selected from the group consisting of 4-(4,6-dimethoxy-1,3,5-trioxan-2-yl)-4-methylmorpholinium (DMTMM) or a salt thereof and/or 2-chloro-4,6-dimethoxy-1,3,5-trioxan-2-yl or a salt thereof and combinations thereof.

在較佳具體實施態樣中，活化劑為4-(4,6-二甲氧基-1,3,5-三𠯤-2-基)-4-甲基嗎啉鎓(DMTMM)或其鹽。In a preferred embodiment, the activating agent is 4-(4,6-dimethoxy-1,3,5-trioxan-2-yl)-4-methylmorpholinium (DMTMM) or a salt thereof.

DMTMM之鹽較佳為相對離子是美容及/或製藥上可接受之陰離子，例如氯離子、乙酸根、碳酸氫根(碳酸氫根)或兩種或多種陰離子之混合物的鹽。The salt of DMTMM is preferably a salt having a cosmetically and/or pharmaceutically acceptable anion as the relative ion, such as chloride, acetate, bicarbonate (hydrogen carbonate) or a mixture of two or more anions.

在較佳具體實施態樣中，活化劑為4-(4,6-二甲氧基-1,3,5-三𠯤-2-基)-4-甲基嗎啉氯化物(CAS No 3945-69 -5)。In a preferred embodiment, the activating agent is 4-(4,6-dimethoxy-1,3,5-trioxan-2-yl)-4-methylmorpholinium chloride (CAS No 3945-69-5).

認為DMTMM在一般使用量下具有相對較低和實質上可忽略的毒性，不會致癌，不會誘發突變並且不會形成畸胎/生殖毒性。因此，其特別適用於製備軟組織填充劑，諸如真皮或結締組織填充劑。DMTMM is considered to have relatively low and essentially negligible toxicity at typical usage levels, is non-carcinogenic, non-mutagenic and non-teratogenic/reproductive. It is therefore particularly suitable for the preparation of soft tissue fillers, such as dermal or connective tissue fillers.

當使用DMTMM或其鹽作為活化劑時，可以形成4-甲基嗎啉(NMM)及/或4,6-二甲氧基-1,3-5-三𠯤-2-醇(DMT)作為降解產物。例如，DMTMM之氯鹽可用作活化劑：When DMTMM or its salts are used as activators, 4-methylmorpholine (NMM) and/or 4,6-dimethoxy-1,3-5-trioxan-2-ol (DMT) can be formed as degradation products. For example, the chloride salt of DMTMM can be used as an activator:

在較佳具體實施態樣中，該方法的特徵另外在於其不包括下列至少一者： (a) 使用插入多醣部分之羧酸殘基或其鹽與羥基殘基之間的互連連接子部分，特別是無胜肽或異生素連接子部分； (b) 使用基於碳二亞胺之活化劑，特別是選自由下列各者組成之群組：N,N'-二環己基碳二亞胺(DCC)、二異丙基碳二亞胺(DIC)、1-乙基-3-(3-二甲基胺基丙基)碳二亞胺(EDC)及其兩種或多種之組合； (c) 使用選自由環氧丙基醚、順丁烯二醯亞胺(maleiimide)、酸酐、烷氧化物及其兩種或多種之組合組成之群組的反應性基團； (d) 具有一級胺基、硫醇基、醯亞胺基、亞胺基或環氧基之多醣部分。In a preferred embodiment, the method is further characterized in that it does not include at least one of the following:(a) the use of an interconnecting linker moiety inserted between the carboxylic acid residue or its salt and the hydroxyl residue of the polysaccharide moiety, in particular a peptide-free or isobiotic-free linker moiety;(b) the use of a carbodiimide-based activator, in particular selected from the group consisting of: N,N'-dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and combinations of two or more thereof;(c) the use of a reactive group selected from the group consisting of glycidyl ethers, maleimides, anhydrides, alkoxides and combinations of two or more thereof;(d) A polysaccharide moiety having a primary amine group, thiol group, amide group, imine group or epoxy group.

在較佳具體實施態樣中，該方法的特徵另外在於其不包括下列至少兩項，更佳為至少三項，特別是所有項： (a) 使用插入多醣部分之羧酸殘基或其鹽與羥基殘基之間的互連連接子部分，特別是無胜肽或異生素連接子部分； (b) 使用基於碳二亞胺之活化劑，特別是選自由下列各者組成之群組：N,N'-二環己基碳二亞胺(DCC)、二異丙基碳二亞胺(DIC)、1-乙基-3-(3-二甲基胺基丙基)碳二亞胺(EDC)及其兩種或多種之組合； (c) 使用選自由環氧丙基醚、順丁烯二醯亞胺、酸酐、烷氧化物以及其兩種或多種之組合組成之群組的反應性基團； (d) 具有一級胺基、硫醇基、醯亞胺基、亞胺基或環氧基之多醣部分。In a preferred embodiment, the method is further characterized in that it does not include at least two, preferably at least three, and in particular all of the following:(a) use of an interconnecting linker moiety inserted between a carboxylic acid residue or a salt thereof and a hydroxyl residue of the polysaccharide moiety, in particular a peptide-free or xenobiotic-free linker moiety;(b) use of a carbodiimide-based activator, in particular selected from the group consisting of: N,N'-dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and combinations of two or more thereof;(c) Using a reactive group selected from the group consisting of glycidyl ether, cis-butylene diimide, acid anhydride, alkoxide and a combination of two or more thereof;(d) a polysaccharide moiety having a primary amine group, a thiol group, an imido group, an imine group or an epoxide group.

溶劑可為任何適用於使多醣部分之羧酸殘基或其鹽與羥基殘基反應的溶劑。在較佳具體實施態樣中，溶劑係適用於溶解多醣部分或使其懸浮以及溶解基於三𠯤之活化劑。在較佳具體實施態樣中，溶劑係適用於溶解多醣和基於三𠯤之活化劑。在較佳具體實施態樣中，溶劑為極性溶劑。在較佳具體實施態樣中，溶劑為質子溶劑。在較佳具體實施態樣中，溶劑為質子極性溶劑。The solvent can be any solvent suitable for reacting the carboxylic acid residue or its salt with the hydroxyl residue of the polysaccharide portion. In a preferred embodiment, the solvent is suitable for dissolving or suspending the polysaccharide portion and dissolving the trihydron-based activator. In a preferred embodiment, the solvent is suitable for dissolving polysaccharides and trihydron-based activators. In a preferred embodiment, the solvent is a polar solvent. In a preferred embodiment, the solvent is a protic solvent. In a preferred embodiment, the solvent is a protic polar solvent.

在較佳具體實施態樣中，相對於溶劑之總質量，溶劑包含超過50 重量%，至少60 重量%，至少70 重量%，至少80 重量%，至少90 重量%，95 重量%或甚至100 重量%之一或多種選自由下列各者組成之群組的成分： 水； 一或多種醇，較佳為一或多種C₁-C₅-醇，更佳為一或多種選自由下列各者組成之群組的C₁-C₅-醇：甲醇、乙醇、正丙醇、異丙醇、正丁醇(1-丁醇)、二級丁醇(2-丁醇)、異丁醇、(2-甲基丙-1-醇)、三級丁醇(2-甲基丙醇)、戊-1-醇、2-甲基丁-1-醇、3-甲基丁-1-醇、2,2-二甲基丙-1-醇、戊-2-醇、3-甲基丁-2-醇、戊-3-醇及/或2-甲基丁-2-醇，以及其兩種或多種之組合，特別是甲醇及/或乙醇； 一或多種一級胺，特別是一或多種C₁-C₅-胺；一或多種碳酸，較佳為一或多種選自由下列各者組成之群組的C₁-C₅-碳酸：甲酸、乙酸、丙酸、丁酸、戊酸、異戊酸，特別是甲酸及/或乙酸； 一或多種一級或二級醯胺，較佳為一或多種C₁-C₅-醯胺，特別是甲醯胺； 一或多種亞碸，較佳為一或多種C₁-C₅-醯胺，特別是二甲基亞碸(DMSO)； 以及其兩種或多種之組合。In a preferred embodiment, the solvent comprises more than 50 wt. %, at least 60 wt. %, at least 70 wt. %, at least 80 wt. %, at least 90 wt. %, 95 wt. % or even 100 wt. %, relative to the total mass of the solvent, of one or more components selected from the group consisting of: water; one or more alcohols, preferably one or more C₁ -C₅ -alcohols, more preferably one or more C₁ -C₅ -ols selected from the group consisting of: -alcohols: methanol, ethanol, n-propanol, isopropanol, n-butanol (1-butanol), di-butanol (2-butanol), isobutanol, (2-methylpropan-1-ol), tertiary butanol (2-methylpropanol), pentan-1-ol, 2-methylbutan-1-ol, 3-methylbutan-1-ol, 2,2-dimethylpropan-1-ol, pentan-2-ol, 3-methylbutan-2-ol, pentan-3-ol and/or 2-methylbutan-2-ol, and combinations of two or more thereof, in particular methanol and/or ethanol; one or more primary amines, in particular one or more C₁ -C₅ -amines; one or more carbonic acids, preferably one or more C₁ -C₅ -carbonic acids selected from the group consisting of formic acid, acetic acid, propionic acid, butyric acid, valeric acid, isovaleric acid, in particular formic acid and/or acetic acid; one or more primary or secondary amides, preferably one or more C₁ -C₅ -amides, especially formamide; one or more sulfoxides, preferably one or more C₁ -C₅ -amides, especially dimethyl sulfoxide (DMSO); and combinations of two or more thereof.

在較佳具體實施態樣中，溶劑為水性溶液。可以最廣泛的意義地將水性溶劑理解為包含水之溶劑，其中相對於溶劑之總質量，水含量以重量計係超過50 重量%，至少60 重量%，至少70 重量%，至少80 重量%，至少90 重量%，至少95 重量%，或甚至100 重量%。在本發明一個具體實施態樣中，除水之外，水性緩衝液還包含一或多種選自由下列各者組成之群組的成分：一或多種醇(特別是一或多種C₁-C₅-醇，例如甲醇、乙醇、正丙醇、異丙醇、正丁醇(1-丁醇)、二級丁醇(2-丁醇)、異丁醇、(2-甲基丙-1-醇)、三級丁醇(2-甲基丙醇)、戊-1-醇、2-甲基丁-1-醇、3-甲基丁-1-醇、2,2-二甲基丙-1-醇、戊-2-醇、3-甲基丁-2-醇、戊-3-醇及/或2-甲基丁-2-醇)、一或多種一級胺(特別是一或多種C₁-C₅-胺)、一或多種碳酸(特別是一或多種C₁-C₅-碳酸，例如甲酸、乙酸、丙酸、丁酸、戊酸、異戊酸)、一或多種一級或二級醯胺(特別是一或多種C₁-C₅-醯胺，例如甲醯胺)、一或多種亞碸(特別是一或多種C₁-C₅-醯胺，例如二甲基亞碸(DMSO))、一或多種無機或有機陽離子(特別是一或多種分子量小於1000 Da之無機或有機陽離子，特別是鹼金屬陽離子或鹼土金屬陽離子、其他金屬陽離子、質子、銨陽離子等)、一或多種無機或有機陰離子(特別是一或多種分子量小於1000 Da之無機或有機陰離子，特別是氯、硫酸鹽等)、一或多種矽酸鹽及其兩種或多種之組合。In a preferred embodiment, the solvent is an aqueous solution. An aqueous solvent can be understood in the broadest sense as a solvent comprising water, wherein the water content by weight relative to the total mass of the solvent is more than 50 wt %, at least 60 wt %, at least 70 wt %, at least 80 wt %, at least 90 wt %, at least 95 wt %, or even 100 wt %. In one embodiment of the present invention, the aqueous buffer comprises, in addition to water, one or more components selected from the group consisting of: one or more alcohols (especially one or more C₁ -C₅ -alcohols, such as methanol, ethanol, n-propanol, isopropanol, n-butanol (1-butanol), di-butanol (2-butanol), isobutanol, (2-methylpropan-1-ol), tertiary butanol (2-methylpropanol), pentan-1-ol, 2-methylbutan-1-ol, 3-methylbutan-1-ol, 2,2-dimethylpropan-1-ol, pentan-2-ol, 3-methylbutan-2-ol, pentan-3-ol and/or 2-methylbutan-2-ol), one or more primary amines (especially one or more C₁ -C₅ -amines), one or more carbonic acids (especially one or more C₁ -C₅ -carbonic acid, such as formic acid, acetic acid, propionic acid, butyric acid, valeric acid, isovaleric acid), one or more primary or secondary amides (especially one or more C₁ -C₅ -amides, such as formamide), one or more sulfoxides (especially one or more C₁ -C₅ -amides, such as dimethyl sulfoxide (DMSO)), one or more inorganic or organic cations (especially one or more inorganic or organic cations with a molecular weight of less than 1000 Da, especially alkali metal cations or alkali earth metal cations, other metal cations, protons, ammonium cations, etc.), one or more inorganic or organic anions (especially one or more anions with a molecular weight of less than 1000 Da ... Da inorganic or organic anions, especially chloride, sulfate, etc.), one or more silicates and combinations of two or more thereof.

在較佳具體實施態樣中，溶劑為水性緩衝液(例如，經磷酸鹽緩衝之鹽水(PBS)、Tris緩衝液、硼酸鹽緩衝液、經乙酸緩衝之緩衝液等)。在較佳具體實施態樣中，使用水醇溶劑，例如水與乙醇、甲醇、丙醇、丁醇及/或戊醇之混合物。在較佳具體實施態樣中，使用水作為溶劑。在較佳具體實施態樣中，溶劑為pH在5至9之範圍內，特別是6至8之水性緩衝液。例如，溶劑可為pH在6.1至7.9之範圍內，6.5至7.7，6.7至7.5或7.0至7.5之水性緩衝液。In a preferred embodiment, the solvent is an aqueous buffer (e.g., phosphate-buffered saline (PBS), Tris buffer, borate buffer, acetic acid buffer, etc.). In a preferred embodiment, a hydroalcoholic solvent is used, such as a mixture of water and ethanol, methanol, propanol, butanol and/or pentanol. In a preferred embodiment, water is used as the solvent. In a preferred embodiment, the solvent is an aqueous buffer with a pH in the range of 5 to 9, particularly 6 to 8. For example, the solvent may be an aqueous buffer having a pH in the range of 6.1 to 7.9, 6.5 to 7.7, 6.7 to 7.5 or 7.0 to 7.5.

令人驚訝地發現包括預溶脹之反應步驟不需要鹼化，並且反應在實質上中性的pH下進行。此反駁在本技術領域中指示需要強酸性或鹼性pH範圍之偏見。Surprisingly, it was found that the reaction step including pre-dissolution does not require alkalization and the reaction proceeds at a substantially neutral pH. This contradicts the bias in the art indicating that a strongly acidic or alkaline pH range is required.

可以最廣泛的意義理解本文所使用的水。水較佳為去離子水、蒸餾水或自來水，特別是去離子水或蒸餾水。Water as used herein can be understood in the broadest sense. Water is preferably deionized water, distilled water or tap water, especially deionized water or distilled water.

在較佳具體實施態樣中，該方法不包括純化步驟(iii)。在另一個具體實施態樣中，該方法包括至少一個純化步驟(iii)。在一個具體實施態樣中，該方法包括至少一個藉由過濾、洗滌及/或透析，特別是錯流過濾、滲濾及/或終端過濾純化交聯材料的步驟(iii)；In a preferred embodiment, the method does not include a purification step (iii). In another embodiment, the method includes at least one purification step (iii). In one embodiment, the method includes at least one step (iii) of purifying the crosslinking material by filtration, washing and/or dialysis, in particular cross-flow filtration, osmosis and/or terminal filtration;

透析可以任何方式進行。在較佳具體實施態樣中，透析係使用截留分子量(MWCO)為1至25 kDa，5至20 kDa或10至15 kDa之透析膜。純化時間可依分子結構進行調整並可在至少1小時、至少6小時、12至78小時、1至7天或1至3天之範圍內。可視情況藉由膜之重量控制溶脹比，一旦產物顯示期望的交聯材料濃度就可停止透析。Dialysis can be performed in any manner. In preferred embodiments, dialysis is performed using a dialysis membrane with a molecular weight cutoff (MWCO) of 1 to 25 kDa, 5 to 20 kDa, or 10 to 15 kDa. The purification time can be adjusted according to the molecular structure and can be in the range of at least 1 hour, at least 6 hours, 12 to 78 hours, 1 to 7 days, or 1 to 3 days. The swelling ratio can be controlled by the weight of the membrane as appropriate, and the dialysis can be stopped once the product shows the desired concentration of cross-linked material.

應理解過濾可為錯流過濾、終端過濾或二者之組合。過濾可藉由任何方式進行。在過濾背景下，過濾器可具有任何適用於純化交聯材料，即較佳保留交聯材料並允許反應物和視情況存在之未反應多醣通過的孔徑。視情況而定，孔徑可在5 nm至2 μm之範圍內，更特別地孔徑為30 nm至600 nm，更特別地孔徑為80 nm至300 nm，特別地孔徑為5 nm至60 nm。過濾器可為任何材料，例如陶瓷、金屬、聚合物材料或其組合。It should be understood that the filtration may be cross-flow filtration, terminal filtration or a combination of the two. Filtration may be performed by any means. In the context of filtration, the filter may have any pore size suitable for purifying the cross-linked material, i.e. preferably retaining the cross-linked material and allowing the reactants and, if applicable, unreacted polysaccharides to pass through. As the case may be, the pore size may be in the range of 5 nm to 2 μm, more particularly the pore size is 30 nm to 600 nm, more particularly the pore size is 80 nm to 300 nm, and particularly the pore size is 5 nm to 60 nm. The filter may be any material, such as ceramic, metal, polymer material or a combination thereof.

視情況而定，過濾可為動態過濾，例如WO 2020/030629中所描述般。因此，步驟(iii)可視情況包括交聯材料之動態過濾，視情況其包括下列步驟： a) 將交聯材料轉移至配備有半滲透濾盤之動態過濾裝置中並滲濾凝膠，其包括下列步驟： i) 藉由施加在20 1/min至500 1/min之範圍內的旋轉速度和在0.5至6 bar之範圍內的過壓，將交聯材料濃縮至預定濃度；或將交聯材料直接泵入動態過濾裝置之處理室中； ii) 藉由施加在20 1/min至500 1/min之範圍內的旋轉速度和在0.5至6 bar之範圍內的過壓進行滲濾以減少不需要的分子； b) 視情況將包含非交聯材料和水之混合物加入交聯材料中。Optionally, the filtering may be dynamic filtering, such as described in WO 2020/030629. Therefore, step (iii) may include dynamic filtration of the cross-linked material as appropriate, which may include the following steps:a) Transferring the cross-linked material to a dynamic filtration device equipped with a semi-permeable filter disc and filtering the gel, which may include the following steps:i) Concentrating the cross-linked material to a predetermined concentration by applying a rotation speed in the range of 20 1/min to 500 1/min and an overpressure in the range of 0.5 to 6 bar; or pumping the cross-linked material directly into the processing chamber of the dynamic filtration device;ii) Concentrating the cross-linked material to a predetermined concentration by applying a rotation speed in the range of 20 1/min to 500 1/min and an overpressure in the range of 0.5 to 6 bar; bar to reduce unwanted molecules;b) adding a mixture comprising non-crosslinked material and water to the crosslinked material as appropriate.

在一個具體實施態樣中，動態過濾裝置配備有1至10個半滲透濾盤。在DCF中，可使用任何旋轉速度和壓力，例如在20 1/min至500 1/min之範圍內的旋轉速度和在0.5至3 bar之範圍內的壓力。在DCF中，可使用任何濃度，例如10至70 mg/g。In a specific embodiment, the dynamic filtration device is equipped with 1 to 10 semi-permeable filter discs. In DCF, any rotation speed and pressure can be used, for example, a rotation speed in the range of 20 1/min to 500 1/min and a pressure in the range of 0.5 to 3 bar. In DCF, any concentration can be used, for example, 10 to 70 mg/g.

在本發明具體實施態樣中，錯流過濾(crossflow filtration)(亦：錯流過濾(cross-flow filtration))為動態錯流過濾(DCF)。因此，在具體實施態樣中，該方法的特徵另外在於其包括藉由DCF純化交聯材料的步驟(iii)。此係舉例說明於下。視情況而定，DCF可為諸如WO 2020/030629中所描述般。In a specific embodiment of the present invention, the crossflow filtration (also: cross-flow filtration) is dynamic cross-flow filtration (DCF). Therefore, in a specific embodiment, the method is further characterized in that it includes a step (iii) of purifying the crosslinked material by DCF. This is illustrated below by way of example. As the case may be, the DCF may be as described in WO 2020/030629.

視情況而定，可在進行步驟(iii)藉由過濾、洗滌及/或透析純化交聯材料之前、期間或之後，添加一或多種另外成分(例如，一或多種局部麻醉劑(例如，如下展示例如利多卡因)、一或多種細胞增生因子、一或多種染料及其兩種或多種之組合)。Optionally, one or more additional components (e.g., one or more local anesthetics (e.g., lidocaine, as shown below), one or more cell proliferation factors, one or more dyes, and combinations of two or more thereof) may be added before, during, or after step (iii) of purifying the cross-linked material by filtration, washing, and/or dialysis.

在一個具體實施態樣中，步驟(i)和(ii)係在單一批次中進行。在另一個具體實施態樣中，步驟(i)和(ii)係分批進行。In one embodiment, steps (i) and (ii) are performed in a single batch. In another embodiment, steps (i) and (ii) are performed in batches.

步驟(i)和(ii)及視情況選用的步驟(iii)各別可在任何溫度範圍下進行。在較佳具體實施態樣中，步驟(i)係在5至90℃之範圍內，較佳係在18至60℃，特別更佳係在18至25℃或在20℃至50℃之溫度下進行。在較佳具體實施態樣中，步驟(ii)係在5至90℃之範圍內，較佳係在18至60℃，特別係在20℃至50℃之溫度下進行。在較佳具體實施態樣中，步驟(iii)係在5℃至90℃之範圍內，或在18℃至60℃，或在20℃至50℃之溫度下進行。在較佳具體實施態樣中，步驟(i)和(ii)及視情況選用的步驟(iii)係在5至90°C之範圍內，較佳係在18至60°C，特別是在20°C至50°C之溫度下進行。在較佳具體實施態樣中，步驟(i)和(ii)係在差異不超過10℃，差異不超過5℃，或差異不超過2℃之溫度下進行。例如，步驟(i)及/或(ii)及/或步驟(iii)可在(約)18°C、(約)19°C、(約)20°C、(約)21°C、(約)22°C、(約)23°C、(約)24°C、(約)25°C、(約)26°C、(約)27°C、(約)28°C、(約)29°C或(約)30°C之溫度下進行。步驟(i)和(ii)及視情況選用的步驟(iii)可在任何壓力下進行。例如，壓力可為環境壓力(例如，經常近970至1100 hPa外部壓力)。Steps (i) and (ii) and optionally step (iii) can be carried out at any temperature range. In a preferred embodiment, step (i) is carried out at a temperature in the range of 5 to 90°C, preferably 18 to 60°C, more preferably 18 to 25°C or 20 to 50°C. In a preferred embodiment, step (ii) is carried out at a temperature in the range of 5 to 90°C, preferably 18 to 60°C, more preferably 20 to 50°C. In a preferred embodiment, step (iii) is carried out at a temperature in the range of 5 to 90°C, or 18 to 60°C, or 20 to 50°C. In a preferred embodiment, steps (i) and (ii) and optionally step (iii) are carried out at a temperature in the range of 5 to 90° C., preferably 18 to 60° C., in particular 20 to 50° C. In a preferred embodiment, steps (i) and (ii) are carried out at a temperature that differs by no more than 10° C., by no more than 5° C., or by no more than 2° C. For example, step (i) and/or (ii) and/or step (iii) may be carried out at a temperature of (about) 18°C, (about) 19°C, (about) 20°C, (about) 21°C, (about) 22°C, (about) 23°C, (about) 24°C, (about) 25°C, (about) 26°C, (about) 27°C, (about) 28°C, (about) 29°C or (about) 30°C. Steps (i) and (ii) and optionally step (iii) may be carried out at any pressure. For example, the pressure may be ambient pressure (e.g., typically approximately 970 to 1100 hPa external pressure).

使成分彼此接觸的步驟(i)可藉由任何方式進行。在較佳具體實施態樣中，該方法的特徵另外在於步驟(i)包括混合各成分，即多醣和一或多種活化劑及一或多種溶劑以及視情況選用的一或多種其他成分。此種混合可藉由任何方式進行，例如藉由攪拌及/或搖動方式進行。The step (i) of bringing the ingredients into contact with each other can be carried out by any means. In a preferred embodiment, the method is further characterized in that step (i) comprises mixing the ingredients, i.e. the polysaccharide and one or more activators and one or more solvents and optionally one or more other ingredients. Such mixing can be carried out by any means, for example by stirring and/or shaking.

在較佳具體實施態樣中，多醣部分係在使其與一或多種活化劑接觸之前先與溶劑或部分溶劑混合。此步驟可包括多醣部分與溶劑或部分溶劑之混合物的培養(即：預溶脹)。例如，此類視情況選用的培養步驟可進行(例如，在4至40°C的溫度下)至少10 min，30 min至7天，1小時至2天，6至36小時或12至24小時。一或多種活化劑可視情況與部分溶劑預混，隨後添加至多醣部分與溶劑或部分溶劑之混合物中。In a preferred embodiment, the polysaccharide portion is mixed with a solvent or a portion of a solvent before contacting it with one or more activators. This step may include incubation (i.e., pre-dissolution) of the mixture of the polysaccharide portion and the solvent or a portion of a solvent. For example, such an optional incubation step may be performed (e.g., at a temperature of 4 to 40° C.) for at least 10 min, 30 min to 7 days, 1 hour to 2 days, 6 to 36 hours or 12 to 24 hours. One or more activators may be premixed with a portion of a solvent as appropriate and then added to the mixture of the polysaccharide portion and the solvent or a portion of a solvent.

只要適用於完成羧酸殘基或其鹽與羥基殘基之期望反應，可進行反應步驟(ii)。可進行步驟(ii)達任何適合此目的之時間。視情況而定，步驟(ii)可進行1 min至1週或更長時間，2 min至5天，3 min至4天，5 min至72小時，5 min至24小時，10 min至12小時，30 min至6小時，1小時至5小時或2至4小時。步驟(ii)可在任何適合此目的之溫度下進行，例如在0°C至100°C下，在4°C至95°C下，在10°C至70°C下，在15°C至30°C下，在18至25°C下，在20°C至70°C下，在20°C至40°C下或在60°C至70°C下進行。在較佳具體實施態樣中，步驟(ii)進行不超過72 h、不超過48 h或不超過24 h。在較佳具體實施態樣中，步驟(ii)進行至少10 min，較佳為15 min至48 h，特別是30 min至18 h。在較佳具體實施態樣中，該方法的特徵另外在於步驟(ii)係在5至90℃之範圍內，較佳為18至60℃，特別是20℃至50℃之溫度下進行至少10 min，較佳為15 min至48 h，特別是30 min至18 h。在較佳具體實施態樣中，該方法的特徵另外在於步驟(ii)係在5至90℃之範圍內的溫度下進行至少10 min。在較佳具體實施態樣中，該方法的特徵另外在於步驟(ii)係在18至60℃之範圍內的溫度下進行30 min至18 h。步驟(ii)可在任何pH下進行。例如，其可在5至9之範圍內，特別是6至8、6.1至7.9、6.5至7.7、6.7至7.5或7.0至7.5之pH下進行。The reaction step (ii) may be carried out for as long as is suitable for achieving the desired reaction of the carboxylic acid residue or its salt with the hydroxyl residue. Step (ii) may be carried out for any time suitable for this purpose. Depending on the circumstances, step (ii) may be carried out for 1 min to 1 week or longer, 2 min to 5 days, 3 min to 4 days, 5 min to 72 hours, 5 min to 24 hours, 10 min to 12 hours, 30 min to 6 hours, 1 hour to 5 hours or 2 to 4 hours. Step (ii) can be carried out at any temperature suitable for this purpose, for example at 0°C to 100°C, at 4°C to 95°C, at 10°C to 70°C, at 15°C to 30°C, at 18 to 25°C, at 20°C to 70°C, at 20°C to 40°C or at 60°C to 70°C. In a preferred embodiment, step (ii) is carried out for no more than 72 h, no more than 48 h or no more than 24 h. In a preferred embodiment, step (ii) is carried out for at least 10 min, preferably 15 min to 48 h, especially 30 min to 18 h. In a preferred embodiment, the method is further characterized in that step (ii) is carried out at a temperature in the range of 5 to 90°C, preferably 18 to 60°C, in particular 20°C to 50°C for at least 10 min, preferably 15 min to 48 h, in particular 30 min to 18 h. In a preferred embodiment, the method is further characterized in that step (ii) is carried out at a temperature in the range of 5 to 90°C for at least 10 min. In a preferred embodiment, the method is further characterized in that step (ii) is carried out at a temperature in the range of 18 to 60°C for 30 min to 18 h. Step (ii) can be carried out at any pH. For example, it may be carried out at a pH in the range of 5 to 9, in particular 6 to 8, 6.1 to 7.9, 6.5 to 7.7, 6.7 to 7.5 or 7.0 to 7.5.

在反應步驟(ii)期間，可使用任何濃度範圍之多醣部分。在較佳具體實施態樣中，使用0.01至800 mg/mL或0.1至500 mg/mL或0.5至200 mg/mL之多醣部分。在較佳具體實施態樣中，使用1至100 mg/mL或2至75 mg/mL或5至50 mg/mL或20-25 mg/mL或25至35 mg/mL之多醣部分。During reaction step (ii), any concentration range of the polysaccharide fraction can be used. In preferred embodiments, 0.01 to 800 mg/mL, or 0.1 to 500 mg/mL, or 0.5 to 200 mg/mL of the polysaccharide fraction is used. In preferred embodiments, 1 to 100 mg/mL, or 2 to 75 mg/mL, or 5 to 50 mg/mL, or 20-25 mg/mL, or 25 to 35 mg/mL of the polysaccharide fraction is used.

視情況選用的純化步驟(iii)，若進行，則可進行任何適合此目的之時間。視情況而定，步驟(iii)可進行1 min至1週或更長時間，2 min至5天，3 min至4天，5 min至72小時，5 min至24小時，10 min至12小時，30 min至6小時，1小時至5小時或2至4小時。步驟(iii)可在任何適合此目的之溫度下進行，例如在0°C至100°C下，在4°C至95°C下，在10°C至70°C下，在15°C至30°C下，在18°C至25°C下，在20°C至70°C下，在20°C至40°C下或在60°C至70°C下進行。The optional purification step (iii), if performed, may be performed for any time suitable for that purpose. Step (iii) may be performed for 1 min to 1 week or longer, 2 min to 5 days, 3 min to 4 days, 5 min to 72 hours, 5 min to 24 hours, 10 min to 12 hours, 30 min to 6 hours, 1 hour to 5 hours or 2 to 4 hours, as appropriate. Step (iii) may be carried out at any temperature suitable for this purpose, for example at 0°C to 100°C, at 4°C to 95°C, at 10°C to 70°C, at 15°C to 30°C, at 18°C to 25°C, at 20°C to 70°C, at 20°C to 40°C or at 60°C to 70°C.

在較佳具體實施態樣中，該方法的特徵另外在於： (a) 其包括藉由過濾、洗滌及/或透析，特別是錯流過濾、滲濾及/或終端過濾純化交聯材料之步驟(iii)； (b) 步驟(i)和(ii)係在單一批次中進行； (c) 步驟(i)和(ii)及視情況選用的步驟(iii)係在5至90℃之範圍內，較佳為18至60℃，特別是20℃至50℃之溫度下進行；及/或 (d) 步驟(ii)係進行至少10 min，較佳為15 min至48 h，特別是30 min至18 h。In a preferred embodiment, the method is further characterized in that:(a) it comprises a step (iii) of purifying the cross-linked material by filtration, washing and/or dialysis, in particular cross-flow filtration, osmosis and/or terminal filtration;(b) steps (i) and (ii) are carried out in a single batch;(c) steps (i) and (ii) and optionally step (iii) are carried out at a temperature in the range of 5 to 90°C, preferably 18 to 60°C, in particular 20 to 50°C; and/or(d) step (ii) is carried out for at least 10 min, preferably 15 min to 48 h, in particular 30 min to 18 h.

在較佳具體實施態樣中，該方法包括： (i) 使下列成分彼此接觸： (A) 重量平均分子量為至少50 kDa之多醣部分，其包含羧酸殘基或其鹽和羥基殘基， (B) 一或多種基於三𠯤之活化劑，其引起羧酸殘基與羥基殘基反應，從而形成酯鍵，特別地，其中活化劑為4-(4,6-二甲氧基-1,3,5-三𠯤-2-基)-4-甲基嗎啉鎓或其鹽；和 (C) 水性溶劑；較佳為pH在5至9之範圍內，特別是6至8之水性溶劑，並 (ii) 允許至少一些羧酸殘基與至少一些羥基殘基反應以形成酯鍵，而使多醣部分彼此共價交聯；以及 (iii) 視情況純化獲自步驟(ii)之交聯材料。In a preferred embodiment, the method comprises:(i) contacting the following components with each other:(A) a polysaccharide portion having a weight average molecular weight of at least 50 kDa, which comprises a carboxylic acid residue or a salt thereof and a hydroxyl residue,(B) one or more trioxane-based activators, which cause the carboxylic acid residue to react with the hydroxyl residue to form an ester bond, in particular, wherein the activator is 4-(4,6-dimethoxy-1,3,5-trioxane-2-yl)-4-methylmorpholinium or a salt thereof; and(C) an aqueous solvent; preferably an aqueous solvent having a pH in the range of 5 to 9, in particular 6 to 8, and(ii) allowing at least some of the carboxylic acid residues to react with at least some of the hydroxyl residues to form ester bonds, thereby covalently cross-linking the polysaccharide moieties to each other; and(iii) optionally purifying the cross-linked material obtained from step (ii).

在視情況選用之其他步驟中，可視情況進一步處理所獲得的交聯材料。例如，可視情況均勻化之及/或使其通過篩(篩選)。在此背景下，可交換地理解術語篩、過濾器和篩網。此可導致特別均勻的材料(例如水凝膠)。在視情況選用之其他步驟中，可使交聯材料進行滅菌。In an optional further step, the crosslinked material obtained can optionally be further processed. For example, it can optionally be homogenized and/or passed through a sieve (screening). In this context, the terms sieve, filter and screen are to be understood interchangeably. This can result in particularly homogenous materials (e.g. hydrogels). In an optional further step, the crosslinked material can be sterilized.

在較佳具體實施態樣中，該方法包括下列步驟： (i) 使下列成分彼此接觸： (A) 多醣部分，其包含羧酸殘基或其鹽和羥基殘基， (B) 一或多種基於三𠯤之活化劑，其引起羧酸殘基與羥基殘基反應，從而形成酯鍵，和 (C) 水性溶劑；較佳為水性溶劑， 其中多醣部分係與部分水性溶劑預混合並培養(例如30 min至7天)； (ii) 允許至少一些羧酸殘基與至少一些羥基殘基反應以形成酯鍵，而使多醣部分彼此在20至60℃之溫度下共價交聯10 min至2天；並 (iii) 視情況藉由透析純化獲自步驟(ii)之交聯材料；並 (iv) 視情況均勻化及/或通過篩及/或混合麻醉劑(例如利多卡因)及/或視情況滅菌。In a preferred embodiment, the method comprises the following steps:(i) contacting the following components with each other:(A) a polysaccharide portion comprising a carboxylic acid residue or a salt thereof and a hydroxyl residue,(B) one or more trihydric activators that cause the carboxylic acid residue to react with the hydroxyl residue to form an ester bond, and(C) an aqueous solvent; preferably an aqueous solvent,wherein the polysaccharide portion is premixed with a portion of the aqueous solvent and incubated (e.g., for 30 min to 7 days);(ii) allowing at least some of the carboxylic acid residues to react with at least some of the hydroxyl residues to form an ester bond, thereby covalently crosslinking the polysaccharide portions with each other at a temperature of 20 to 60°C for 10 min to 2 days; and(iii) The cross-linked material obtained from step (ii) is purified by dialysis as appropriate; and(iv) is homogenized as appropriate and/or by sieving and/or mixing with an anesthetic (e.g. lidocaine) and/or sterilized as appropriate.

在較佳具體實施態樣中，該方法包括： i) 使下列成分彼此接觸： (A) 重量平均分子量為至少50 kDa之多醣部分，其包含羧酸殘基或其鹽和羥基殘基， 其中多醣部分係與部分水性溶劑預混合並培養(例如30 min至7天)， (B) 一或多種基於三𠯤之活化劑，其引起羧酸殘基與羥基殘基反應，從而形成酯鍵，特別地，其中活化劑為4-(4,6 -二甲氧基-1,3,5-三𠯤-2-基)-4-甲基嗎啉鎓或其鹽；和 (C) 水性溶劑；較佳為pH在5至9之範圍內，特別是6至8之水性溶劑；並 (ii) 允許至少一些羧酸殘基與至少一些羥基殘基反應以形成酯鍵，而使多醣部分彼此在20至60℃之溫度下共價交聯10 min至2天；和 (iii) 視情況藉由透析純化獲自步驟(ii)之交聯材料；並 (iv) 視情況均勻化及/或通過篩及/或混合麻醉劑(例如利多卡因)及/或視情況滅菌。In a preferred embodiment, the method comprises: i) contacting the following components with each other: (A) a polysaccharide portion having a weight average molecular weight of at least 50 kDa, which comprises a carboxylic acid residue or a salt thereof and a hydroxyl residue, wherein the polysaccharide portion is premixed with a portion of an aqueous solvent and incubated (e.g., for 30 min to 7 days), (B) one or more trioxane-based activators, which cause the carboxylic acid residue to react with the hydroxyl residue to form an ester bond, in particular, wherein the activator is 4-(4,6-dimethoxy-1,3,5-trioxane-2-yl)-4-methylmorpholinium or a salt thereof; and (C) an aqueous solvent; preferably an aqueous solvent having a pH in the range of 5 to 9, in particular 6 to 8; and(ii) allowing at least some of the carboxylic acid residues to react with at least some of the hydroxyl residues to form ester bonds, thereby covalently crosslinking the polysaccharide moieties to each other at a temperature of 20 to 60°C for 10 min to 2 days; and(iii) purifying the crosslinked material obtained from step (ii) by dialysis, as appropriate; and(iv) homogenizing and/or passing through a sieve and/or mixing with an anesthetic (e.g. lidocaine), as appropriate, and/or sterilizing, as appropriate.

應理解可由本發明方法獲得之交聯材料具有特殊技術特徵，諸如結構特徵且無嚴峻的(雙)官能連接子和活化劑存在。It is understood that the crosslinked materials obtainable by the process of the invention have specific technical features, such as structural features and the absence of critical (bi)functional linkers and activators.

因此，本發明另一方面關於可由本發明方法獲得之交聯材料。Therefore, another aspect of the present invention relates to the crosslinked material obtainable by the process of the present invention.

應理解在本發明方法之背景下所展示的定義和較佳具體實施態樣在經必要修正後施用於本發明交聯材料。It is to be understood that the definitions and preferred embodiments presented in the context of the method of the present invention apply mutatis mutandis to the cross-linking material of the present invention.

在本發明具體實施態樣中，交聯材料已藉由使用DMTMM作為活化劑而製得。在本發明具體實施態樣中，交聯材料(特別是在最終純化之前)包含： (a) 4-(4,6-二甲氧基-1,3,5-三𠯤-2-基)-4-甲基嗎啉鎓或其鹽， (b) N-甲基嗎啉鎓或其鹽；及/或 (c) 4,6-二甲氧基-1,3,5-三𠯤-2-醇或其互變異構物或鹽。 在本發明具體實施態樣中，交聯材料(特別是在最終純化之前)包含NMM及/或DMT。在本發明具體實施態樣中，相對於交聯物質(凝膠)之總重量，交聯材料(特別是在最終純化之前)包含至多0.1 重量%，較佳為0.01至1000 ppm，0.1至100 ppm或1至50 ppm之NMM及/或DMT。In a specific embodiment of the present invention, the crosslinking material has been prepared by using DMTMM as an activator. In a specific embodiment of the present invention, the crosslinking material (particularly before final purification) comprises:(a) 4-(4,6-dimethoxy-1,3,5-trioxan-2-yl)-4-methylmorpholinium or a salt thereof,(b) N-methylmorpholinium or a salt thereof; and/or(c) 4,6-dimethoxy-1,3,5-trioxan-2-ol or a tautomer or salt thereof.In a specific embodiment of the present invention, the crosslinking material (particularly before final purification) comprises NMM and/or DMT. In a specific embodiment of the present invention, the crosslinking material (especially before final purification) contains up to 0.1% by weight, preferably 0.01 to 1000 ppm, 0.1 to 100 ppm or 1 to 50 ppm of NMM and/or DMT relative to the total weight of the crosslinking substance (gel).

本發明另一方面關於交聯材料，其包含或由一或多個重量平均分子量為至少50 kDa之玻尿酸部分組成，其中玻尿酸部分係經由酯鍵而無互連連接子結構地彼此共價交聯，其特徵另外在於交聯材料不包含二醯亞胺基、環氧基或異生素連接子部分。Another aspect of the invention relates to a crosslinking material comprising or consisting of one or more hyaluronic acid moieties having a weight average molecular weight of at least 50 kDa, wherein the hyaluronic acid moieties are covalently crosslinked to each other via ester bonds without interconnecting linker structures, and further characterized in that the crosslinking material does not contain diimide, epoxy or xenobiotic linker moieties.

如本文所使用，可以最廣泛的意義將術語”無互連連接子結構”理解為無非源自(亦：不存在於)多醣部分(例如，玻尿酸部分)之其他化學部分導入以酯鍵共軛多醣部分的化學結構中。換言之，酯鍵較佳係由源自多醣部分(例如玻尿酸部分)之氧原子的夾雜物和源自多醣部分(例如玻尿酸部分)之碳原子的夾雜物形成。As used herein, the term "no interconnected linker structure" can be understood in the broadest sense as no other chemical moieties not derived from (i.e., not present in) the polysaccharide moiety (e.g., hyaluronic acid moiety) are introduced into the chemical structure of the polysaccharide moiety conjugated with an ester bond. In other words, the ester bond is preferably formed by a hybrid of an oxygen atom derived from the polysaccharide moiety (e.g., hyaluronic acid moiety) and a hybrid of a carbon atom derived from the polysaccharide moiety (e.g., hyaluronic acid moiety).

在較佳具體實施態樣中，交聯材料的特徵另外在於其不包含醯亞胺基或二醯亞胺基。在較佳具體實施態樣中，交聯材料的特徵另外在於其不包含亞胺基。在較佳具體實施態樣中，交聯材料的特徵另外在於其不包含環氧基。在較佳具體實施態樣中，交聯材料的特徵另外在於其不包含異生素連接子部分基團。In preferred embodiments, the crosslinking material is further characterized in that it does not include an imide group or a diimide group. In preferred embodiments, the crosslinking material is further characterized in that it does not include an imine group. In preferred embodiments, the crosslinking material is further characterized in that it does not include an epoxy group. In preferred embodiments, the crosslinking material is further characterized in that it does not include an xenobiotic linker moiety group.

在較佳具體實施態樣中，交聯材料的特徵另外在於其不包含： (a) 二醯亞胺基 (b) 醯亞胺基； (c) 亞胺基； (d) 環氧基；及/或 (e) 異生素連接子部分， 互連多醣部分。In preferred embodiments, the crosslinking material is further characterized in that it does not include:(a) diimide groups(b) imide groups;(c) imine groups;(d) epoxy groups; and/or(e) xenobiotic linker moieties,interconnected polysaccharide moieties.

在較佳具體實施態樣中，交聯材料的特徵另外在於其不包含： (a) 二醯亞胺基 (b) 醯亞胺基； (c) 亞胺基； (d) 環氧基；和 (e) 異生素連接子部分， 互連多醣部分。In preferred embodiments, the crosslinking material is further characterized in that it does not include:(a) diimide groups(b) imide groups;(c) imine groups;(d) epoxy groups; and(e) xenobiotic linker moieties,interconnected polysaccharide moieties.

在較佳具體實施態樣中，交聯材料係由本發明方法獲得。In a preferred embodiment, the crosslinked material is obtained by the method of the present invention.

視交聯材料的預定用途而定，可立即使用或可儲存之。例如，可將其儲存在任何適合此目的之條件下，例如在環境溫度下(例如，18至30°C，較佳為18至25°C)，在冰箱中(例如，在0至15°C下，較佳為3至10°C)，在冷凍庫中(例如，-30至0°C，較佳為-25至-10°C)，在超低溫冷凍庫中(例如，-100至-200°C，較佳為- 90至-55°C)，在液態氮上，在乾冰上，或甚至在一或多種液態稀有氣體中。其可在乾燥狀態下以水凝膠、包含其他非水性溶劑之凝膠及/或懸浮液、乳液、膠體或溶液形式儲存。Depending on the intended use of the crosslinking material, it can be used immediately or can be stored. For example, it can be stored under any conditions suitable for this purpose, such as at ambient temperature (e.g., 18 to 30°C, preferably 18 to 25°C), in a refrigerator (e.g., at 0 to 15°C, preferably 3 to 10°C), in a freezer (e.g., -30 to 0°C, preferably -25 to -10°C), in an ultra-low temperature freezer (e.g., -100 to -200°C, preferably -90 to -55°C), on liquid nitrogen, on dry ice, or even in one or more liquid noble gases. They may be stored in a dry state as hydrogels, gels containing other non-aqueous solvents and/or suspensions, emulsions, colloids or solutions.

視情況可將步驟(i)和(ii)及/或視情況選用之步驟(iii)中所使用的溶劑部分或完全去除。因為所使用的成分，包括活化劑可能具有相對較低毒性，步驟(i)和(ii)及/或視情況選用之步驟(iii)中所使用的溶劑亦或可視情況保持在交聯材料中。若去除，步驟(i)和(ii)及/或視情況選用之步驟(iii)中所使用的溶劑可藉由任何方式去除，例如藉由另一種溶劑(例如，另一種水性緩衝液)取代之及/或藉由蒸發去除。The solvent used in steps (i) and (ii) and/or optionally step (iii) may be partially or completely removed as appropriate. Because the ingredients used, including the activator, may have relatively low toxicity, the solvent used in steps (i) and (ii) and/or optionally step (iii) may also be retained in the cross-linking material as appropriate. If removed, the solvent used in steps (i) and (ii) and/or optionally step (iii) may be removed by any means, such as by replacing it with another solvent (e.g., another aqueous buffer) and/or by evaporation.

交聯材料可用於任何目的中並可呈任何形式。例如，當其空隙中包含液體或黏性介質時，其可為凝膠。一般而言，此種液體或黏性介質可為任何不會崩解交聯材料之液體或黏性介質。液體或黏性介質較佳為水性液體。如上所指示般，本發明交聯材料可用於製備水凝膠。The crosslinking material can be used for any purpose and in any form. For example, it can be a gel when it contains a liquid or viscous medium in its interstices. Generally speaking, such a liquid or viscous medium can be any liquid or viscous medium that does not disintegrate the crosslinking material. The liquid or viscous medium is preferably an aqueous liquid. As indicated above, the crosslinking material of the present invention can be used to prepare a hydrogel.

因此，本發明另一方面關於水凝膠，其包含： (A) 本發明交聯材料；和 (B) 水性溶液；及 (C) 視情況選用之一或多種其他在製藥上或美容上可接受的試劑，特別是一或多種局部麻醉劑。Therefore, another aspect of the present invention relates to a hydrogel comprising:(A) a crosslinking material of the present invention; and(B) an aqueous solution; and(C) optionally one or more other pharmaceutically or cosmetically acceptable agents, in particular one or more local anesthetics.

應理解在本發明方法和交聯材料之背景下所展示的定義和較佳具體實施態樣在經必要修正後施用於本發明水凝膠中。It is to be understood that the definitions and preferred embodiments presented in the context of the methods and crosslinking materials of the present invention apply mutatis mutandis to the hydrogels of the present invention.

因此，本發明亦關於製備水凝膠之方法。視情況而定，此種方法可包括將水性緩衝液添加至經純化之交聯材料中的步驟。Therefore, the present invention also relates to a method for preparing a hydrogel. Optionally, such a method may include the step of adding an aqueous buffer to the purified cross-linked material.

本發明交聯材料及/或水凝膠可用於任何目的。例如，可使用此等物作為可注射組成物。The cross-linked materials and/or hydrogels of the present invention can be used for any purpose. For example, they can be used as injectable compositions.

因此，本發明另一方面關於包含本發明交聯材料或水凝膠之可注射組成物，其中交聯材料較佳為超級增容劑。Therefore, another aspect of the present invention relates to an injectable composition comprising the crosslinking material or hydrogel of the present invention, wherein the crosslinking material is preferably a super-volume expander.

應理解在本發明方法、交聯材料和水凝膠之背景下所展示的定義和較佳具體實施態樣在經必要修正後施用於本發明可注射組成物中。It will be appreciated that the definitions and preferred embodiments presented in the context of the methods, cross-linking materials and hydrogels of the present invention apply mutatis mutandis to the injectable compositions of the present invention.

在較佳具體實施態樣中，本發明交聯材料、水凝膠及/或可注射組成物可用作軟組織填充劑，特別是真皮填充劑或結締組織填充劑。In a preferred embodiment, the cross-linked material, hydrogel and/or injectable composition of the present invention can be used as a soft tissue filler, in particular a dermal filler or a connective tissue filler.

如可注射組成物中所包含的根據本發明液體或黏性載體可為任何可注射載體。典型地，液體或黏性載體為製藥上及/或美容上可接受的載體，因此，當投予本發明意義上之哺乳動物時，該載體對哺乳動物，特別是人類是無毒的。液體或黏性載體較佳可包含或由一或多種溶劑，例如水、水性緩衝液(例如鹽水或磷酸鹽緩衝鹽水)、二甲基亞碸(DMSO)、乙醇、植物油、石蠟油或其組合組成。液體或黏性載體更佳係包含或由無熱原等張緩衝液，更特別地為生理食鹽水溶液或緩衝生理食鹽水溶液組成。According to the present invention, the liquid or viscous carrier as included in the injectable composition can be any injectable carrier. Typically, the liquid or viscous carrier is a pharmaceutically and/or cosmetically acceptable carrier, so that when administered to a mammal in the sense of the present invention, the carrier is non-toxic to the mammal, especially humans. The liquid or viscous carrier preferably comprises or consists of one or more solvents, such as water, an aqueous buffer (such as saline or phosphate buffered saline), dimethyl sulfoxide (DMSO), ethanol, vegetable oil, paraffin oil or a combination thereof. The liquid or viscous carrier more preferably comprises or consists of a pyrogen-free isotonic buffer, more particularly a physiological saline solution or a buffered physiological saline solution.

視情況存在之其他成分可為任何成分。例如，此種其他成分可選自由以下各者組成之群組：一或多種局部麻醉劑、一或多種細胞增生因子、一或多種染料及其兩種或多種之組合。The optional additional ingredients may be any ingredients. For example, such additional ingredients may be selected from the group consisting of: one or more local anesthetics, one or more cell proliferation factors, one or more dyes, and combinations of two or more thereof.

可在任何時間點，諸如在純化交聯材料之前、期間或之後添加此類其他成分。例如，可在進行純化步驟(iii)期間添加一或多種其他成分。在本發明另一個具體實施態樣中，可將一或多種其他成分添加至所製備並視情況純化之交聯材料中。Such additional ingredients may be added at any point in time, such as before, during or after the purification of the crosslinked material. For example, one or more additional ingredients may be added during the purification step (iii). In another embodiment of the present invention, one or more additional ingredients may be added to the prepared and optionally purified crosslinked material.

局部麻醉劑可使注射入個體中更舒適。細胞增生因子可改善細胞侵入投予之本發明交聯材料中。染料可改善注射之定位(例如，製藥上可接受的螢光染料，如螢光黃或玫瑰紅)，或可改善原本發白的交聯材料之隱形性(例如，藉由使其呈肉色)。亦可添加任何其他製藥上活性化合物。然後，本發明交聯材料亦可用作投藥之緩釋形式。Local anesthetics can make injection into an individual more comfortable. Cell proliferation factors can improve cell invasion into the administered cross-linked material of the present invention. Dyes can improve the localization of injection (e.g., pharmaceutically acceptable fluorescent dyes such as fluorescent yellow or rose bengal), or can improve the invisibility of an originally whitish cross-linked material (e.g., by making it flesh-colored). Any other pharmaceutically active compound can also be added. The cross-linked material of the present invention can then also be used as a sustained release form for administration.

本文適合使用之局部麻醉劑包括(但不限於)氨布卡因(ambucaine)、阿美索卡因(amolanone)、阿米卡因(amylocaine)、奧布卡因(benoxinate)、苯唑卡因(benzocaine)、貝托卡因(betoxycaine)、苯海拉明(biphenamine)、布比卡因(bupivacaine)、布他卡因(butacaine)、氨苯丁酯(butamben)、布坦卡因(butanilicaine)、丁胺卡因(butethamine)、丁氧基卡因(butoxycaine)、卡鐵卡因(carticaine)、氣普魯卡因(chloroprocaine)、己基苯酸愛康因(cocaethylene)、古柯鹼(cocaine)、環美卡因(cyclomethycaine)、二丁卡因(dibucaine)、二甲異喹(dimethysoquin)、二甲卡因(dimethocaine)、地呱冬(diperodon)、雙環胺(dicyclomine)、去水芽子鹼(ecgonidine)、愛哥寧(ecgonine)、氯乙烷(ethyl chloride)、依替卡因(etidocaine)、β-優卡因(beta-eucaine)、尤普羅辛(euprocin)、非那可明(fenalcomine)、福莫卡因(formocaine)、海克卡因(hexylcaine)、羥丁卡因(hydroxytetracaine)、對胺基苯甲酸異丁酯(isobutyl p-aminobenzoate)、甲磺酸亮氨卡因(leucinocaine mesylate)、左沙屈爾(levoxadrol)、利多卡因(lidocaine)、甲脈卡因(mepivacaine)、美普卡因(meprylcaine)、美布卡因(metabutoxycaine)、氯甲烷(methyl chloride)、麥替卡因(myrtecaine)、納依卡因(naepaine)、奧他卡因(octacaine)、鄰卡因(orthocaine)、奧昔卡因(oxethazaine)、配侖薩思卡因(parethoxycaine)、非那卡因(phenacaine)、酚、匹派魯卡因(piperocaine)、匹多卡因(piridocaine)、聚桂醇(polidocanol)、普莫卡因(pramoxine)、丙胺卡因(prilocaine)、普魯卡因(procaine)、丙泮卡因(propanocaine)、丙美卡因(proparacaine)、丙哌卡因(propipocaine)、丙氧卡因(propoxycaine)、假卡因(psuedococaine)、吡咯卡因(pyrrocaine)、羅哌卡因(ropivacaine)、柳醇、四卡因(tetracaine)、托利卡因(tolycaine)、三甲卡因(trimecaine)、左拉敏(zolamine)及其鹽類。本文亦可使用兩種或多種所提麻醉劑之組合，例如利多卡因與其他”卡因”麻醉劑，如丙胺卡因之組合。Local anesthetics suitable for use herein include, but are not limited to, ambucaine, amolanone, amylocaine, benoxinate, benzocaine, betoxycaine, biphenamine, bupivacaine, butacaine, butamben, butanilicaine, butethamine, butoxycaine, toxycaine, carticaine, chloroprocaine, cocaethylene, cocaine, cyclomethycaine, dibucaine, dimethysoquin, dimethocaine, diperodon, dicyclomine, ecgonidine, ecgonine, ethyl chloride chloride), etidocaine, beta-eucaine, euprocin, fenalcomine, formocaine, hexylcaine, hydroxytetracaine, isobutyl p-aminobenzoate, leucinocaine mesylate, levoxadrol, lidocaine, mepivacaine, meprylcaine, metabutoxycaine, methyl chloride chloride), myrtecaine, naepaine, octacaine, orthocaine, oxethazaine, parethoxycaine, phenacaine, phenol, piperocaine, piridocaine, polidocanol, pramoxine, prilocaine e), procaine, propanocaine, proparacaine, propipocaine, propoxycaine, pseudocaine, pyrrocaine, ropivacaine, salbutamol, tetracaine, tolycaine, trimecaine, zolamine and their salts. Combinations of two or more of the mentioned anesthetics may also be used herein, for example, lidocaine in combination with other "caine" anesthetics, such as prilocaine.

視本發明交聯材料、水凝膠及/或可注射組成物之預定用途而定，其可以不同包裝提供。其可儲存在任何適合此目的之條件下，例如在環境溫度下(例如，18至30°C，較佳為18至25°C)，在冰箱中(例如，在0至15°C，較佳為3至10°C)，在冷凍庫中(例如，-30至0°C，較佳為-25至-10°C)，在超低溫冷凍庫中(例如，-100至-200°C，較佳為-90至-55°C)，在液態氮上，在乾冰上，或甚至在一或多種液態稀有氣體中。例如，其可以小玻璃瓶、注射器提供。可經由注射(例如，經由注射器或點滴)將其投予受試者。其可在乾燥狀態下以水凝膠、包含其他非水性溶劑之凝膠及/或懸浮液、乳液、膠體或溶液形式儲存。Depending on the intended use of the crosslinking material, hydrogel and/or injectable composition of the present invention, it can be provided in different packaging. It can be stored under any conditions suitable for this purpose, such as at ambient temperature (e.g., 18 to 30°C, preferably 18 to 25°C), in a refrigerator (e.g., at 0 to 15°C, preferably 3 to 10°C), in a freezer (e.g., -30 to 0°C, preferably -25 to -10°C), in an ultra-low temperature freezer (e.g., -100 to -200°C, preferably -90 to -55°C), on liquid nitrogen, on dry ice, or even in one or more liquid noble gases. For example, it can be provided in a small glass bottle, a syringe. It can be administered to a subject by injection (e.g., via a syringe or drip). It can be stored in a dry state as a hydrogel, a gel containing other non-aqueous solvents and/or a suspension, emulsion, colloid or solution.

本發明亦意指根據本發明可注射組成物用於美容應用之用途。本發明更佳亦意指根據本發明可注射組成物用於包括顏面和身體重塑及回春之美容應用的用途。The invention also relates to the use of the injectable composition according to the invention for cosmetic applications. The invention also relates to the use of the injectable composition according to the invention for cosmetic applications including facial and body remodeling and rejuvenation.

如本文所使用，可以最廣泛的意義將美容應用和美容用途理解為任何預定用於改善外表之非治療性應用。亦可將其分別稱為美學應用和美學用途。在本發明背景下，應理解美容應用和美容用途可包括任何投藥途徑，該途徑可視情況包括個別材料之注射及/或個別材料之局部投藥或另一種合適的投藥途徑。As used herein, cosmetic applications and cosmetic uses may be understood in the broadest sense as any non-therapeutic application intended for improving appearance. They may also be referred to as aesthetic applications and aesthetic uses, respectively. In the context of the present invention, it is understood that cosmetic applications and cosmetic uses may include any route of administration, which may include injection of the individual material and/or topical administration of the individual material or another suitable route of administration, as appropriate.

本發明另一方面關於本發明可注射組成物用於美容應用之用途，該美容應用包括顏面和身體重塑及回春，較佳包括填平皺紋、改善顏面細紋、乳房重建或隆胸、皮膚回春、豐臀、顴骨重塑、軟組織增補、填平顏面皺紋、改善眉間紋、改善法令紋、改善木偶紋、改善頰部連合、改善唇緣皺紋、改善魚尾紋、改善眉、顴頰脂肪墊之皮下支持、改善淚溝、改善鼻子外觀、豐唇、豐頰、口周區域增大、眶下區域增大、解決顏面不對稱、改善下顎輪廓、墊下巴或其兩種或多種之組合。Another aspect of the present invention relates to the use of the injectable composition of the present invention for cosmetic applications, which include facial and body remodeling and rejuvenation, preferably including filling wrinkles, improving facial fine lines, breast reconstruction or breast augmentation, skin rejuvenation, buttocks augmentation, cheekbone remodeling, soft tissue augmentation, filling facial wrinkles, improving glabellar lines, improving nasolabial lines, improving marionette lines, improving cheek commissures, improving lip wrinkles, improving crow's feet, improving eyebrows, subcutaneous support of cheek fat pads, improving tear troughs, improving nose appearance, lip augmentation, cheek augmentation, perioral area enlargement, infraorbital area enlargement, resolving facial asymmetry, improving jaw contour, chin augmentation or a combination of two or more thereof.

應理解在本發明方法、交聯材料、水凝膠和可注射組成物之背景下所展示的定義和較佳具體實施態樣在經必要修正後施用於本發明用途。It is to be understood that the definitions and preferred embodiments presented in the context of the methods, cross-linking materials, hydrogels and injectable compositions of the present invention apply mutatis mutandis to the present use.

此用途可為治療及/或美容用途。在較佳具體實施態樣中，本發明關於本發明可注射組成物用於減少顏面皺摺的用途。在具體實施態樣中，本發明用途可為美容用途，較佳為非治療用途。本發明用途可藉由美容、美容專家或健康護理專家進行。This use may be therapeutic and/or cosmetic. In a preferred embodiment, the present invention relates to the use of the injectable composition of the present invention for reducing facial wrinkles. In a specific embodiment, the use of the present invention may be cosmetic, preferably non-therapeutic. The use of the present invention may be performed by a beauty therapist, a beauty specialist or a health care specialist.

在較佳具體實施態樣中，本發明可注射組成物的用途係用於改善皮膚品質、治療細紋、治療深紋或體積恢復或作為乳房或豐臀之超級豐盈填充劑。本發明亦關於顏面和身體重塑及回春之方法(較佳包括上述特定用途)，該方法包括投予根據本發明可注射組成物。In a preferred embodiment, the injectable composition of the present invention is used for improving skin quality, treating fine lines, treating deep lines or volume restoration or as a super-volume filler for breasts or buttocks. The present invention also relates to a method for facial and body remodeling and rejuvenation (preferably including the above-mentioned specific uses), which method comprises administering the injectable composition according to the present invention.

當意指治療用途時，本發明關於根據本發明可注射組成物在用於下列各者之方法中的用途：顏面和身體重塑及回春，較佳包括填平皺紋、改善顏面細紋、乳房重建或隆胸、皮膚回春、豐臀、顴骨重塑、軟組織增補、填平顏面皺紋、改善眉間紋、改善法令紋、改善木偶紋、改善頰部連合、改善唇緣皺紋、改善魚尾紋、改善眉、顴頰脂肪墊之皮下支持、改善淚溝、改善鼻子外觀、豐唇、豐頰、口周區域增大、眶下區域增大、解決顏面不對稱、改善下顎輪廓、墊下巴或其兩種或多種之組合。When therapeutic use is intended, the present invention relates to the use of an injectable composition according to the present invention in a method for: facial and body remodeling and rejuvenation, preferably including wrinkle filling, improvement of facial fine lines, breast reconstruction or breast augmentation, skin rejuvenation, buttocks augmentation, cheekbone remodeling, soft tissue augmentation, filling of facial wrinkles, improvement of frown lines, Improve nasolabial folds, improve marionette lines, improve cheek commissures, improve lip wrinkles, improve crow's feet, improve eyebrows, subcutaneous support of cheek fat pads, improve tear troughs, improve nose appearance, lip augmentation, cheek augmentation, enlargement of periorbital area, enlargement of infraorbital area, solve facial asymmetry, improve jaw contour, chin augmentation or a combination of two or more.

換言之，本發明亦關於用於下列各者之方法的使用方法：顏面和身體重塑及回春，較佳包括填平皺紋、改善顏面細紋、乳房重建或隆胸、皮膚回春、豐臀、顴骨重塑、軟組織增補、填平顏面皺紋、改善眉間紋、改善法令紋、改善木偶紋、改善頰部連合、改善唇緣皺紋、改善魚尾紋、改善眉、顴頰脂肪墊之皮下支持、改善淚溝、改善鼻子外觀、豐唇、豐頰、口周區域增大、眶下區域增大、解決顏面不對稱、改善下顎輪廓、墊下巴或其兩種或多種之組合，其中將足夠量之根據本發明可注射組成物投予有其需要之受試者。In other words, the present invention also relates to methods of use for the following: facial and body remodeling and rejuvenation, preferably including wrinkle filling, facial fine line improvement, breast reconstruction or breast augmentation, skin rejuvenation, buttocks augmentation, cheekbone remodeling, soft tissue augmentation, facial wrinkle filling, glabellar lines improvement, nasolabial lines improvement, marionette lines improvement, cheek commissure improvement, lip improvement Wrinkles, crow's feet, eyebrows, subcutaneous support of cheek fat pads, tear troughs, nose appearance, lip and cheek augmentation, perioral area enlargement, infraorbital area enlargement, facial asymmetry, jaw contour improvement, chin augmentation or a combination of two or more thereof, wherein a sufficient amount of the injectable composition according to the present invention is administered to a subject in need thereof.

如本文所使用，受試者(亦：個體)可為任何動物，典型地為哺乳動物，較佳為馴養的哺乳動物或人類。個體特佳為人類。亦可將經治療之人類稱為患者，與他/她的健康狀況無關。As used herein, a subject (also: individual) can be any animal, typically a mammal, preferably a domesticated mammal or a human. The individual is particularly preferably a human. A human being treated may also be referred to as a patient, regardless of his/her health condition.

可藉由任何方式進行投藥。在較佳具體實施態樣中，投藥係經由針投藥。在較佳具體實施態樣中，投藥係經由注射器投藥，特別經由注射器皮內或皮下投藥。投藥可為手動投藥、使用機械泵投藥或甚至自動投藥。例如，Syringe One系統可用於投藥。The administration can be carried out by any means. In a preferred embodiment, the administration is via a needle. In a preferred embodiment, the administration is via a syringe, particularly intradermally or subcutaneously via a syringe. The administration can be manual, using a mechanical pump, or even automatic. For example, the Syringe One system can be used for administration.

本發明交聯材料以及本發明可注射組成物可用於任何目的。視情況而定，本發明交聯材料以及本發明可注射組成物可用於美容及/或治療用途。可注射組成物可為填充劑，特別是軟組織填充劑，例如軟組織填充劑，特別是真皮填充劑或結締組織填充劑。The cross-linked material of the invention and the injectable composition of the invention can be used for any purpose. As appropriate, the cross-linked material of the invention and the injectable composition of the invention can be used for cosmetic and/or therapeutic purposes. The injectable composition can be a filler, in particular a soft tissue filler, such as a soft tissue filler, in particular a dermal filler or a connective tissue filler.

本發明亦意指根據本發明可注射組成物作為填充劑，諸如軟組織填充劑，特別是真皮填充劑或結締組織填充劑的用途。其可用作超級增容劑。在此背景下，其可用作水凝膠。The present invention also relates to the use of the injectable composition according to the invention as a filler, such as a soft tissue filler, in particular a dermal filler or a connective tissue filler. It can be used as a supervolume agent. In this context, it can be used as a hydrogel.

如本文所使用，可以最廣泛的意義將術語”填充劑”理解為任何可用於填充空腔或用作軟組織填充劑，較佳為軟組織填充劑的試劑。可以最廣泛的意義將軟組織填充物理解為設計用於增加軟組織缺陷區域之體積的材料。可將填充劑投予至任何位置中並藉由任何類型之注射進行，並且適合用於美容/美學應用以及治療目的中。通常填充劑可為任何添加、取代或增大皮下體積，從而導致例如使皮膚皺紋平滑、豐唇、改善皮膚外觀或治療疤痕的組成物。其通常係用於真皮區域，諸如表皮下方或皮下層上方，因此可以皮下(subcutaneously)、皮下(hypodermically)或皮內或一些組合的方式注射。As used herein, the term "filler" may be understood in the broadest sense as any agent that can be used to fill a cavity or as a soft tissue filler, preferably a soft tissue filler. Soft tissue fillers may be understood in the broadest sense as materials designed to increase the volume of an area of soft tissue defect. Fillers may be administered in any location and by any type of injection and are suitable for use in cosmetic/aesthetic applications as well as therapeutic purposes. In general, a filler may be any composition that adds, replaces or increases subcutaneous volume, resulting in, for example, smoothing of skin wrinkles, lip plumping, improvement of skin appearance or treatment of scars. It is usually used in the dermal region, i.e., below the epidermis or above the subcutaneous layer, and can therefore be injected subcutaneously, hypodermically, or intradermally, or some combination.

在本發明意義內之可注射組成物可在正常條件及正常壓力下藉由注射器(從注射器配藥)的方式投予。此外，本發明填充劑組成物較佳(實質上)為無菌。可注射組成物較佳係適合注入哺乳動物內，特別是人體內。Injectable compositions within the meaning of the present invention can be administered by means of a syringe (dispensing from a syringe) under normal conditions and normal pressure. In addition, the filler composition of the present invention is preferably (substantially) sterile. The injectable composition is preferably suitable for injection into a mammal, especially into the human body.

可基於美容目的進行重塑，或可在組織損失，例如因意外事故或手術介入導致組織損失之後進行。例如，一部分顏面可能因意外事故受傷。另一方面，可藉由皮下填充顴骨區域使顴骨突出。乳房或其一部分可以手術方式去除。另一方面，乳房重建或隆乳亦具美學理由。Reshaping may be done for cosmetic purposes or after tissue loss, such as from an accident or surgical intervention. For example, part of the face may be injured in an accident. On the other hand, the cheekbones may be made more prominent by filling the cheekbones with subcutaneous fillers. The breast or part of it may be surgically removed. On the other hand, breast reconstruction or breast augmentation may also have aesthetic reasons.

本發明可注射組成物較佳可藉由注射，諸如藉由皮下或皮內注射將有效量投予個體中。在較佳具體實施態樣中，在此用途的背景下，可注射組成物為填充劑。在更佳具體實施態樣中，在此用途的背景下，可注射組成物為填充劑，特別是超級增容劑，並且該用途包括包含對關注之組織投予本發明交聯材料之組成物，特別是以皮下或皮內方式。例如，可使用連續穿刺技術以皮內或皮下方式注射可注射組成物。有效量意指足以引起有益或期望之美容(美學)或治療結果的(可注射)軟組織填充劑組成物的量。The injectable composition of the present invention is preferably administered to an individual by injection, such as by subcutaneous or intradermal injection. In a preferred embodiment, in the context of this use, the injectable composition is a filler. In a more preferred embodiment, in the context of this use, the injectable composition is a filler, particularly a supervolumizer, and the use includes administering a composition of the crosslinking material of the present invention to a tissue of interest, particularly subcutaneously or intradermally. For example, the injectable composition can be injected intradermally or subcutaneously using a continuous puncture technique. An effective amount means an amount of a (injectable) soft tissue filler composition sufficient to cause a beneficial or desired cosmetic (aesthetic) or therapeutic result.

在特佳具體實施態樣中，在此用途的背景下，可注射組成物為填充劑，其可為超級增容劑，特別是軟組織填充劑，並且該用途包括包含本發明交聯材料之組成物以皮下或皮內方式投藥。對於此等用途，根據本發明交聯材料係特別有益的，因為交聯材料在水性環境，諸如體液中較穩定，並由於其結構和特徵而能侵入細胞中。In a particularly preferred embodiment, in the context of this use, the injectable composition is a filler, which may be a supervolume agent, in particular a soft tissue filler, and the use comprises administering the composition comprising the crosslinking material of the invention subcutaneously or intradermally. For such uses, the crosslinking material according to the invention is particularly beneficial because the crosslinking material is relatively stable in aqueous environments, such as body fluids, and can penetrate into cells due to its structure and characteristics.

本發明另一方面關於根據本發明交聯材料或可注射組成物在用於再生有其需要之個體組織的方法中的用途。Another aspect of the invention relates to the use of the cross-linked material or the injectable composition according to the invention in a method for regenerating tissue in a subject in need thereof.

換言之，本發明亦關於用於再生有其需要之個體組織的方法，該方法包括對有其需要之個體投予根據本發明交聯材料或可注射組成物。基於治療及/或美容目可進行有其需要之個體的組織再生。In other words, the present invention also relates to a method for regenerating tissue in an individual in need thereof, the method comprising administering to an individual in need thereof a crosslinked material or an injectable composition according to the present invention. Tissue regeneration in an individual in need thereof may be performed for therapeutic and/or cosmetic purposes.

應理解在上述交聯材料、水凝膠、方法和可注射組成物之背景下所展示的定義和較佳具體實施態樣在經必要修正後施用於個體組織再生的用途。It is to be understood that the definitions and preferred embodiments presented in the context of the above cross-linking materials, hydrogels, methods and injectable compositions apply mutatis mutandis to the use for tissue regeneration in an individual.

欲再生之組織可為任何組織。在一個較佳具體實施態樣中，組織為軟組織。在更佳具體實施態樣中，組織為選自由真皮組織(包括真皮和皮下之組織)和結締組織組成之群組的軟組織。然後，該方法可用於重塑和回春，包括如上所描述之用途。在本發明另一個較佳具體實施態樣中，該組織為關節組織。視情況而定，對於此種用途，交聯材料可包含一或多種刺激個別組織增生之細胞增生因子。The tissue to be regenerated can be any tissue. In a preferred embodiment, the tissue is a soft tissue. In a more preferred embodiment, the tissue is a soft tissue selected from the group consisting of dermal tissue (including dermal and subcutaneous tissue) and connective tissue. Then, the method can be used for remodeling and rejuvenation, including the uses described above. In another preferred embodiment of the present invention, the tissue is an articular tissue. As appropriate, for such uses, the crosslinking material may include one or more cell proliferation factors that stimulate the proliferation of individual tissues.

在替代較佳具體實施態樣中，組織為骨組織。然後，可將本發明交聯材料投予至預期生長之骨組織的位置，例如骨折間隙中或用於骨伸長。視情況而定，對於此用途，交聯材料可包含一或多種刺激骨細胞增生之細胞增生因子。In an alternative preferred embodiment, the tissue is bone tissue. The cross-linking material of the present invention can then be administered to the location of bone tissue growth expected, such as in a fracture gap or for bone elongation. Optionally, for this use, the cross-linking material may include one or more cell proliferation factors that stimulate bone cell proliferation.

視特定用途而定，在該技術領域中熟諳此技者將使用根據本發明微粒交聯材料或將使用根據本發明交聯材料塊。Depending on the particular application, one skilled in the art will use microparticles of crosslinked material according to the invention or will use blocks of crosslinked material according to the invention.

基於上述治療和美容用途，根據本發明交聯材料係特別有益的，因為交聯材料在水性環境，諸如體液中較穩定，並由於其交聯結構和表面特徵而能夠侵入細胞。如上所指示，本發明交聯材料係藉由多醣部分共軛時所獲得的本發明材料獲得。此共軛物本身亦具有意料之外的有益性質。Based on the above mentioned therapeutic and cosmetic uses, the crosslinked materials according to the present invention are particularly beneficial because the crosslinked materials are relatively stable in aqueous environments, such as body fluids, and can penetrate cells due to their crosslinked structure and surface characteristics. As indicated above, the crosslinked materials of the present invention are obtained by conjugating the polysaccharide moieties to the material of the present invention. This conjugate itself also has unexpected beneficial properties.

如本文所使用，可將術語”近”和”約”理解為包括個別數值之高達+/-10%偏差的範圍。應理解亦可明確揭露特定值。As used herein, the terms "near" and "about" may be understood to include a range of up to +/- 10% deviation of the individual numerical values. It should be understood that specific values may also be explicitly disclosed.

應進一步理解範圍涵蓋以涵蓋整個捨入限制之一般捨入值所提供的數值。例如，”1mg”的範圍涵蓋0.50至1.49 mg之範圍。It is further understood that ranges cover values provided with generally rounded values to cover the entire rounding limit. For example, a range of "1 mg" covers the range of 0.50 to 1.49 mg.

然而，本發明數值亦更詳細地揭露一或多個數量級之更詳細值。因此，例如“1 mg”亦可包括“1.0 mg”之特定揭露值。However, the numerical values of the present invention may also disclose more detailed values of one or more orders of magnitude. Therefore, for example, "1 mg" may also include a specific disclosed value of "1.0 mg".

實施例和圖示說明本發明具體實施態樣。The embodiments and drawings illustrate specific implementation aspects of the present invention.

實施例Embodiment

實施例1Embodiment 1

方法method

根據本發明交聯玻尿酸材料之製備Preparation of cross-linked hyaluronic acid material according to the present invention

令1.5 g 固有黏度為2.85 m³/kg 之玻尿酸鈉鹽(HA)(乾重)溶於45 mL pH 7.4之磷酸鹽緩衝鹽水(PBS)緩衝液中。使將獲所的混合物預溶脹近15 小時。注意到此為亦可省略之視情況選用的步驟。1.5 g of sodium hyaluronate (HA) (dry weight) with an intrinsic viscosity of 2.85 m³ /kg was dissolved in 45 mL of phosphate buffered saline (PBS) at pH 7.4. The resulting mixture was pre-swelled for approximately 15 hours. Note that this is an optional step that can be omitted if necessary.

令不同含量(例如相對於HA之羧基，近0、0.2、0.5、1.0、1.5或2.0 莫耳當量(eq.))之基於三𠯤之活化劑4-(4,6-二甲氧基-1,35-三𠯤-2-基)-4-甲基嗎啉氯化物(DMTMM))各別溶於5 mL pH 7.4 之PBS緩衝液中。使用1.03 g DMTMM以獲得近1 eq意指HA之羧基。Different amounts (e.g., approximately 0, 0.2, 0.5, 1.0, 1.5, or 2.0 molar equivalents (eq.) relative to the carboxyl groups of HA) of the trithion-based activator 4-(4,6-dimethoxy-1,35-trithion-2-yl)-4-methylmorpholinium chloride (DMTMM) were dissolved in 5 mL of PBS buffer at pH 7.4. 1.03 g of DMTMM was used to obtain approximately 1 eq of carboxyl groups of HA.

令45mL包含(HA)之PBS緩衝液和5 mL包含DMTMM之PBS緩衝液彼此混合。此可獲得30 mg/mL之HA濃度。進行混合直至獲得視覺上均勻的組成物。此典型地係在近10 min之後獲得。45 mL of PBS buffer containing (HA) and 5 mL of PBS buffer containing DMTMM were mixed with each other. This gave a HA concentration of 30 mg/mL. Mixing was performed until a visually homogeneous composition was obtained. This was typically obtained after approximately 10 min.

在本文所描繪的實驗中，樣品係在40°C下保持3小時。然而，注意到反應在其他溫度，諸如近20℃-22℃之環境溫度下亦可良好地進行。進一步觀察到較短的反應時間亦產生可行的交聯材料。In the experiments described herein, the samples were maintained at 40°C for 3 hours. However, it was noted that the reaction proceeded well at other temperatures, such as near ambient temperature of 20°C-22°C. It was further observed that shorter reaction times also produced viable crosslinked materials.

隨後，再次使交聯材料(以水凝膠形式存在)均勻化30 min並使其通過篩(篩選)。注意到此為亦可省略之視情況選用的步驟。Subsequently, the cross-linked material (in the form of hydrogel) is homogenized again for 30 min and passed through a sieve (screening). Note that this is an optional step that can also be omitted.

隨後，將交聯材料(以水凝膠形式存在)置於透析膜(截留分子量(MWCO)12-14 kDa)中並純化之近2天以去除DMTMM的副產物。溶脹比係由該膜的重量控制，並且一旦產物顯示期望交聯材料濃度就停止透析。注意到此為亦可省略之視情況選用的步驟。Subsequently, the cross-linked material (in the form of a hydrogel) was placed in a dialysis membrane (MWCO 12-14 kDa) and purified for nearly 2 days to remove the DMTMM byproduct. The swelling ratio was controlled by the weight of the membrane, and dialysis was stopped once the product showed the desired cross-linked material concentration. Note that this is an optional step that can also be omitted.

視情況而定，將局部麻醉劑，諸如利多卡因添加至交聯材料(以水凝膠形式存在)中。視情況而定，使交聯材料(以水凝膠形式存在)進行滅菌並視情況填充至注射器中。A local anesthetic, such as lidocaine, is added to the cross-linking material (in the form of a hydrogel) as appropriate. The cross-linking material (in the form of a hydrogel) is sterilized as appropriate and filled into a syringe as appropriate.

性質之測定Determination of properties

使用302模組化精簡型流變儀(Anton Paar GmbH, Graz, Austria)測定流變學(包括固有黏度)。簡而言之，使用在25°C下呈振盪模式(PP20)具有1.0 mm間隙尺寸之板/板；Tau(應力)設定為10 Pa，振盪頻率範圍為10至0.1 Hz。記載的G'值相當於1 Hz。Rheology (including intrinsic viscosity) was determined using a 302 modular compact rheometer (Anton Paar GmbH, Graz, Austria). Briefly, a plate/plate with a gap size of 1.0 mm in oscillatory mode (PP20) at 25 °C was used; Tau (stress) was set to 10 Pa and the oscillation frequency range was 10 to 0.1 Hz. The reported G' values correspond to 1 Hz.

使用TA.XT.(Stable Micro Systems) Plus組織分析儀測量擠壓力。簡而言之，以定速(12.6 mm/min)擠壓注射器，並隨時間測量擠壓力(以N為單位)。-½ 英吋27G TSK(TSK Laboratory, Japan)針，The extrusion pressure was measured using a TA.XT. (Stable Micro Systems) Plus tissue analyzer. Briefly, the syringe was extruded at a constant speed (12.6 mm/min) and the extrusion pressure (in N) was measured over time. -½ inch 27G TSK (TSK Laboratory, Japan) needle,

酶降解：在36°C下經由Anton Paar MCR 302流變儀、板CP50-1以振盪模式評估材料降解。每分鐘記錄一次測量點達1小時。令一支注射器(1 mL)的內容物沉積在測量板上，並使150 μL 150 U(活性單位)之玻尿酸酶溶液在凝膠中混合30 秒。將1 Hz時的G’值轉換成降解%以報告該值。Enzymatic degradation: Material degradation was evaluated at 36°C in oscillatory mode on an Anton Paar MCR 302 rheometer, plate CP50-1. Measurement points were recorded every minute for 1 hour. The contents of a syringe (1 mL) were deposited on the measuring plate and 150 μL of 150 U (activity units) of hyaluronidase solution were mixed in the gel for 30 seconds. The G' values at 1 Hz were converted to % degradation to report the values.

藉由經聚三葡萄糖標準品校正之凝膠滲透層析法(GPC)使用在PBS 緩衝液(pH 7.4)中濃度為5 mg/mL之樣品測定分子量值和分佈。Molecular weight values and distribution were determined by gel permeation chromatography (GPC) calibrated with polytriglyceride standards using a sample concentration of 5 mg/mL in PBS buffer (pH 7.4).

結果result

發現可以各種不同的含量比獲得具有有益性質的交聯材料。使用小規模實驗的結果摘要係顯示於下表 1中。流變參數係與其他基於多醣之水凝膠(例如 HA填充劑)一致，並且在滅菌後，G’值下降約50%。與1.5 eq 活性劑(DMTMM)交聯之凝膠的代表性擠壓曲線係顯示於圖 1中。不受此理論的約束，交聯材料由於低擠壓力似乎表現出剪切稀化效應。It was found that cross-linked materials with beneficial properties can be obtained at various content ratios. A summary of the results using small-scale experiments is shown in Table 1 below. The rheological parameters are consistent with other polysaccharide-based hydrogels (e.g. HA fillers) and the G' value decreases by about 50% after sterilization. A representative extrusion curve of the gel cross-linked with 1.5 eq of active agent (DMTMM) is shown in Figure 1. Without being bound by this theory, the cross-linked material appears to exhibit a shear thinning effect due to low extrusion pressures.

進一步研究不同產物之酶降解。結果係描繪於圖 2 中。在此，顯而易見可使用寬含量比之多醣部分和活化劑。The enzymatic degradation of the different products was further investigated. The results are depicted in Figure 2. Here, it is evident that a wide range of content ratios of polysaccharide fractions and activators can be used.

發現使用較少活化劑(DMTMM)當量之交聯材料顯示較低交聯程度。具有較低交聯程度之交聯材料實質上可在一小時之後以該酶完全降解，而具有較高交聯程度之交聯材料則較穩定。此顯示本發明方法非常適合有目的地調整交聯程度以達期望的目的。It was found that cross-linked materials using less activator (DMTMM) equivalents showed lower cross-linking degrees. Cross-linked materials with lower cross-linking degrees were substantially completely degraded by the enzyme after one hour, while cross-linked materials with higher cross-linking degrees were more stable. This shows that the method of the present invention is very suitable for purposefully adjusting the cross-linking degree to achieve the desired purpose.

實施例 2Embodiment 2

根據本發明交聯硫酸軟骨素材料之製備Preparation of cross-linked chondroitin sulfate material according to the present invention

以與實施例 1相同的方式，令2.0 g 重量平均分子量為100 kDa之硫酸軟骨素(CS)溶於PBS緩衝液(pH 7.4)中。將不同含量(相對於CS之羧基，近0.0、0.5、1.0或1.5 莫耳當量(eq.))之DMTMM溶於pH 7.4之PBS緩衝液中並與包含CS之PBS緩衝液混合，直至獲得視覺上均勻的組成物，並令混合物在23℃下反應17 h。隨後，藉由透析使用PBS緩衝液純化交聯之水凝膠。使用1.15 g DMTMM以獲得近1 eq意指CS之羧基。如上文實施例 1所描述般測定流變性質 並使其顯示於表 2中。 表 1. 使用兩個不同批次之玻尿酸(HA)(每批30 mg/mL)和不同分子當量(eq.)之活化劑DMTMM的結果摘要。結果顯示滅菌和未滅菌產物的流變值實驗編號使用含量範圍未滅菌滅菌測定 HA實驗編號 (HA 批次)使用 HADMTMM (交聯)Iη*IG'tan (δ)G' 下降Iη*IG'tan (δ)擠壓力HA 濃度游離 HA[g][eq.][1.0 Hz][%][1.0 Hz][N][mg/g][%]純 HA – A (4368C)1.444(-)77.84410.47955.142.71980.9176.2純 HA – B (20011C)1.35(-)95.1547.50.43741.561.13200.6675.5HAHA221101-A (20011C)1.6270.5 (+)131.5822.40.10065.348.82850.39115.524.447.7HAHA221101-B (20011C)1.6231 (+)135.6849.80.07244.675.94700.16617.624.412.5HAHA221101-C (20011C)1.6211.5 (+)149.4937.10.06455.167.5421.30.11120.921.713.1HAHA221101-D (4368C)1.7320.5 (+)120.1748.70.12661.9367.82850.27614.5625.022.9HAHA221101-E (4368C)1.7321 (+)130.31816.30.07749.7846.29410.000.203----25.719.4HAHA221101-F (4368C)1.7281.5 (+)140.37880.30.06147.4974.23462.300.133----19.59.4表 2. 在使用不同分子當量(eq.)之活化劑DMTMM交聯之前和交聯之後，硫酸軟骨素之流變性質摘要(未滅菌產物)。實驗編號使用含量範圍未滅菌實驗編號使用 CSDMTMM (交聯)Iη*IG'tan (δ)[g][eq.][1.0 Hz]純硫酸軟骨素 (交聯之前)2.0(-)0.050.047.06A2.00.55.5330.35B2.01.035.12200.09C2.01.549.63110.05In the same manner as in Example 1, 2.0 g of chondroitin sulfate (CS) with a weight average molecular weight of 100 kDa was dissolved in PBS buffer (pH 7.4). Different amounts of DMTMM (approximately 0.0, 0.5, 1.0 or 1.5 molar equivalents (eq.) relative to the carboxyl groups of CS) were dissolved in PBS buffer at pH 7.4 and mixed with PBS buffer containing CS until a visually uniform composition was obtained, and the mixture was reacted at 23° C. for 17 h. Subsequently, the cross-linked hydrogel was purified by dialysis using PBS buffer. 1.15 g of DMTMM was used to obtain approximately 1 eq of carboxyl groups of CS. The rheological properties were determined as described in Example 1 above and are shown in Table 2. Table 1. Summary of results using two different batches of hyaluronic acid (HA) (30 mg/mL each) and different molecular equivalents (eq.) of the activator DMTMM. Results show rheological values for sterilized and non-sterilized products. Experiment number Usage content range Not sterilized Sterilization Determination of HA Experimental No. (HA Batch) Using HA DMTMM (cross-linked) Iη*I G' tan (δ) G' down Iη*I G' tan (δ) Squeeze pressure HA concentration Free HA [g] [eq.] [1.0 Hz] [%] [1.0 Hz] [N] [mg/g] [%] Pure HA – A (4368C) 1.444 (-) 77.8 441 0.479 55.1 42.7 198 0.917 6.2 Pure HA – B (20011C) 1.35 (-) 95.1 547.5 0.437 41.5 61.1 320 0.667 5.5 HAHA221101-A (20011C) 1.627 0.5 (+) 131.5 822.4 0.100 65.3 48.8 285 0.391 15.5 24.4 47.7 HAHA221101-B (20011C) 1.623 1 (+) 135.6 849.8 0.072 44.6 75.9 470 0.166 17.6 24.4 12.5 HAHA221101-C (20011C) 1.621 1.5 (+) 149.4 937.1 0.064 55.1 67.5 421.3 0.111 20.9 21.7 13.1 HAHA221101-D (4368C) 1.732 0.5 (+) 120.1 748.7 0.126 61.93 67.8 285 0.276 14.56 25.0 22.9 HAHA221101-E (4368C) 1.732 1 (+) 130.31 816.3 0.077 49.78 46.29 410.00 0.203 ---- 25.7 19.4 HAHA221101-F (4368C) 1.728 1.5 (+) 140.37 880.3 0.061 47.49 74.23 462.30 0.133 ---- 19.5 9.4 Table 2. Summary of rheological properties of chondroitin sulfate before and after cross-linking using different molecular equivalents (eq.) of the activator DMTMM (unsterilized product). Experiment number Usage range Not sterilized Experiment number Using CS DMTMM (cross-linked) Iη*I G' tan (δ) [g] [eq.] [1.0 Hz] Pure Chondroitin Sulfate (Before Cross-linking) 2.0 (-) 0.05 0.04 7.06 A 2.0 0.5 5.5 33 0.35 B 2.0 1.0 35.1 220 0.09 C 2.0 1.5 49.6 311 0.05

無without

圖 1 顯示與1.5 eq DMTMM交聯之玻尿酸(HA)產物的擠壓力測量實施例。測量係使用TSK 27G ½”針以12.6 mm/min進行。圖示顯示多次測量之疊圖。可見出測量結果具有可比較性，並且沒發現顯著不均勻性。Figure 1 shows an example of extrusion pressure measurement of a hyaluronic acid (HA) product cross-linked with 1.5 eq DMTMM. The measurement was performed using a TSK 27G ½” needle at 12.6 mm/min. The figure shows an overlay of multiple measurements. It can be seen that the measurement results are comparable and no significant inhomogeneity was found.

圖 2 顯示各別基於玻尿酸(HA)之交聯材料的降解動力學，該玻尿酸(HA)係以不同含量之DMTMM進行交聯。降解係以個別交聯材料與150 單位之玻尿酸酶接觸並在37℃下培養的方式進行。上圖顯示由第一供應商獲得之HA。下圖顯示由另一家供應商獲得之HA。向下三角形指示非交聯HA。方塊指示與0.5 當量(eq.) DMTMM交聯之HA。圓圈指示與1.0 當量(eq.) DMTMM交聯之HA。向上三角形指示與1.5 當量(eq.) DMTMM交聯之HA。Figure 2 shows the degradation kinetics of various crosslinking materials based on hyaluronic acid (HA) crosslinked with different amounts of DMTMM. Degradation was performed by exposing each crosslinking material to 150 units of hyaluronidase and incubating at 37°C. The upper panel shows HA obtained from the first supplier. The lower panel shows HA obtained from another supplier. Downward-pointing triangles indicate non-crosslinked HA. Squares indicate HA crosslinked with 0.5 eq. DMTMM. Circles indicate HA crosslinked with 1.0 eq. DMTMM. Upward-pointing triangles indicate HA crosslinked with 1.5 eq. DMTMM.

無without

Claims (15)

Translated fromChinese

一種製備交聯材料之方法，該方法包括： (i) 使下列成分彼此接觸： (A) 多醣部分，其包含羧酸殘基或其鹽和羥基殘基， (B) 一或多種基於三𠯤之活化劑，其引起羧酸殘基與羥基殘基反應，從而形成酯鍵，和 (C) 一或多種溶劑；並 (ii) 允許多醣部分之至少一些羧酸殘基與多醣部分之至少一些羥基殘基反應以形成酯鍵，而使多醣部分彼此共價交聯；並且 (iii) 視情況純化獲自步驟(ii)之交聯材料， 其中多醣部分彼此共價交聯的酯鍵是多於多醣部分彼此共價交聯的醯胺鍵， 其中步驟(ii)係在無任何不同於基於三𠯤之活化劑的活化劑存在下進行，特別地，其中步驟(ii)係在無碳二亞胺活化劑、基於琥珀醯亞胺基之活化劑、基於環氧丙基之活化劑、對硝基酚酯和2-氯甲基吡啶碘化物的存在下進行。A method for preparing a crosslinking material, the method comprising:(i) contacting the following components with each other:(A) a polysaccharide portion comprising a carboxylic acid residue or a salt thereof and a hydroxyl residue,(B) one or more trihydric activators which cause the carboxylic acid residue to react with the hydroxyl residue to form an ester bond, and(C) one or more solvents; and(ii) allowing at least some of the carboxylic acid residues of the polysaccharide portion to react with at least some of the hydroxyl residues of the polysaccharide portion to form an ester bond, thereby covalently crosslinking the polysaccharide portions with each other; and(iii) optionally purifying the crosslinking material obtained from step (ii),wherein the number of ester bonds covalently crosslinking the polysaccharide moieties to each other is greater than the number of amide bonds covalently crosslinking the polysaccharide moieties to each other,wherein step (ii) is carried out in the absence of any activator different from the trioxane-based activator, in particular, wherein step (ii) is carried out in the absence of a carbodiimide activator, a succinimidyl-based activator, a glycidyl-based activator, p-nitrophenol ester and 2-chloromethylpyridinium iodide.如請求項1之方法，其中多醣部分包含至少25：1，較佳為至少50：1，特別是至少100：1之羥基殘基：一級胺基的莫耳比，較佳地，其中多醣部分包含或由玻尿酸、經甘油接枝之玻尿酸、肝素聚醣、硫酸軟骨素和羧甲基纖維素組成。The method of claim 1, wherein the polysaccharide portion comprises a molar ratio of hydroxyl residue:primary amine group of at least 25:1, preferably at least 50:1, and especially at least 100:1, and preferably, the polysaccharide portion comprises or consists of hyaluronic acid, glycerol-grafted hyaluronic acid, heparin polysaccharide, chondroitin sulfate and carboxymethyl cellulose.如請求項1或2中任一項之方法，其中多醣部分包含或由一或多個玻尿酸部分組成。The method of any one of claims 1 or 2, wherein the polysaccharide portion comprises or consists of one or more hyaluronic acid portions.如請求項1至3中任一項之方法，其中多醣部分的重量平均分子量為至少50 kDa，較佳為至少200 kDa，特別是在500至4000 kDa之範圍內。A method as claimed in any one of claims 1 to 3, wherein the weight average molecular weight of the polysaccharide portion is at least 50 kDa, preferably at least 200 kDa, in particular in the range of 500 to 4000 kDa.如請求項1至4中任一項之方法，其中一或多種活化劑係選自由下列各者組成之群組：4-(4,6-二甲氧基-1,3,5-三𠯤-2-基)-4-甲基嗎啉鎓或其鹽，及/或2-氯-4,6,-二甲氧基-1,3,5-三𠯤或其鹽及其組合。The method of any one of claims 1 to 4, wherein the one or more activating agents are selected from the group consisting of: 4-(4,6-dimethoxy-1,3,5-trioxan-2-yl)-4-methylmorpholinium or a salt thereof, and/or 2-chloro-4,6-dimethoxy-1,3,5-trioxan-2-yl or a salt thereof, and combinations thereof.如請求項1至5中任一項之方法，其中活化劑為4-(4,6-二甲氧基-1,3,5-三𠯤-2-基)-4-甲基嗎啉鎓或其鹽，特別是4-(4,6-二甲氧基-1,3,5-三𠯤-2-基)-4-甲基嗎啉氯化物。The method of any one of claims 1 to 5, wherein the activating agent is 4-(4,6-dimethoxy-1,3,5-trioxan-2-yl)-4-methylmorpholinium or a salt thereof, in particular 4-(4,6-dimethoxy-1,3,5-trioxan-2-yl)-4-methylmorpholinium chloride.如請求項1至6中任一項之方法，其中溶劑為水性溶液，特別是水性緩衝液，較佳係pH在5至9之範圍內，特別是6至8之水性緩衝液。The method of any one of claims 1 to 6, wherein the solvent is an aqueous solution, especially an aqueous buffer, preferably an aqueous buffer having a pH in the range of 5 to 9, especially 6 to 8.如請求項1至7中任一項之方法，其中方法的特徵另外在於其不包括下列至少一者： (a) 使用插入多醣部分之羧酸殘基或其鹽與羥基殘基之間的互連連接子部分，特別是無胜肽或異源連接子(xenobiotic linker)部分； (b) 使用基於碳二亞胺之活化劑； (c) 使用選自由環氧丙基醚、順丁烯二醯亞胺、酸酐、烷氧化物及其兩種或多種之組合組成之群組的反應性基團； (d) 具有一級胺基、硫醇基、醯亞胺基、亞胺基或環氧基之多醣部分。A method as claimed in any one of claims 1 to 7, wherein the method is further characterized in that it does not include at least one of the following:(a) the use of an interconnecting linker moiety, particularly a non-peptide or xenobiotic linker moiety, inserted between a carboxylic acid residue or a salt thereof and a hydroxyl residue of the polysaccharide moiety;(b) the use of a carbodiimide-based activator;(c) the use of a reactive group selected from the group consisting of glycidyl ethers, cis-butylene diimides, acid anhydrides, alkoxides and combinations of two or more thereof;(d) a polysaccharide moiety having a primary amine, thiol, imide, imine or epoxy group.如請求項1至8中任一項之方法，其中方法的特徵另外在於： (a) 其包括藉由過濾、洗滌及/或透析，特別是錯流過濾、滲濾及/或終端過濾純化交聯材料之步驟(iii)； (b) 步驟(i)和(ii)係在單一批次中進行； (c) 步驟(i)和(ii)及視情況選用的步驟(iii)係在5至90℃之範圍內，較佳為18至60℃，特別是20℃至50℃之溫度下進行；及/或 (d) 步驟(ii)係進行至少10 min，較佳為15 min至48 h，特別是30 min至18 h。A method as claimed in any one of claims 1 to 8, wherein the method is further characterized in that:(a) it comprises a step (iii) of purifying the cross-linked material by filtration, washing and/or dialysis, in particular cross-flow filtration, osmosis and/or terminal filtration;(b) steps (i) and (ii) are carried out in a single batch;(c) steps (i) and (ii) and optionally step (iii) are carried out at a temperature in the range of 5 to 90°C, preferably 18 to 60°C, in particular 20 to 50°C; and/or(d) step (ii) is carried out for at least 10 min, preferably 15 min to 48 h, in particular 30 min to 18 h.如請求項1至9中任一項之方法，其中方法包括： (i) 使下列成分彼此接觸： (A) 重量平均分子量為至少50 kDa之多醣部分，其包含羧酸殘基或其鹽和羥基殘基， (B) 一或多種基於三𠯤之活化劑，其引起羧酸殘基與羥基殘基反應，從而形成酯鍵，特別地，其中活化劑為4-(4,6-二甲氧基-1,3,5-三𠯤-2-基)-4-甲基嗎啉鎓或其鹽；和 (C) 水性溶劑；較佳為pH在5至9之範圍內，特別是6至8之水性溶劑，並 (ii) 允許至少一些羧酸殘基與至少一些羥基殘基反應以形成酯鍵，而使多醣部分彼此共價交聯；以及 (iii) 視情況純化獲自步驟(ii)之交聯材料。A method as claimed in any one of claims 1 to 9, wherein the method comprises:(i) contacting the following components with each other:(A) a polysaccharide moiety having a weight average molecular weight of at least 50 kDa, which comprises a carboxylic acid residue or a salt thereof and a hydroxyl residue,(B) one or more trioxane-based activators which cause the carboxylic acid residue to react with the hydroxyl residue to form an ester bond, in particular, wherein the activator is 4-(4,6-dimethoxy-1,3,5-trioxane-2-yl)-4-methylmorpholinium or a salt thereof; and(C) an aqueous solvent; preferably an aqueous solvent having a pH in the range of 5 to 9, in particular 6 to 8, and(ii) allowing at least some of the carboxylic acid residues to react with at least some of the hydroxyl residues to form ester bonds, thereby covalently cross-linking the polysaccharide moieties to each other; and(iii) optionally purifying the cross-linked material obtained from step (ii).一種交聯材料，其可由請求項1至10中任一項之方法獲得。A cross-linked material, which can be obtained by the method of any one of claims 1 to 10.一種交聯材料，其包含或由一或多個重量平均分子量為至少50 kDa之玻尿酸部分組成，其中玻尿酸部分係經由酯鍵而無互連連接子結構地彼此共價交聯， 其特徵另外在於交聯材料不包含二醯亞胺基、環氧基或異源連接子部分， 較佳地，其中交聯材料包含： (a) 4-(4,6-二甲氧基-1,3,5-三𠯤-2-基)-4-甲基嗎啉鎓或其鹽， (b) N-甲基嗎啉鎓或其鹽；及/或 (c) 4,6-二甲氧基-1,3,5-三𠯤-2-醇或其互變異構物或鹽，並且 較佳地，其中交聯材料係由請求項1至10中任一項之方法獲得。A crosslinking material comprising or consisting of one or more hyaluronic acid moieties having a weight average molecular weight of at least 50 kDa, wherein the hyaluronic acid moieties are covalently crosslinked to each other via ester bonds without interconnecting linker structures, further characterized in that the crosslinking material does not contain diimide, epoxy or heterologous linker moieties, preferably, wherein the crosslinking material comprises: (a) 4-(4,6-dimethoxy-1,3,5-trioxan-2-yl)-4-methylmorpholinium or a salt thereof, (b) N-methylmorpholinium or a salt thereof; and/or (c) 4,6-dimethoxy-1,3,5-trioxan-2-ol or a tautomer or salt thereof, and Preferably, the cross-linked material is obtained by the method of any one of claims 1 to 10.一種水凝膠，其包含： (A) 請求項11或12中任一項之交聯材料；和 (B) 水性溶液；及 (C) 視情況選用之一或多種其他在製藥上或美容上可接受的試劑，特別是一或多種局部麻醉劑。A hydrogel comprising:(A) a crosslinking material as claimed in any one of claims 11 or 12; and(B) an aqueous solution; and(C) optionally one or more other pharmaceutically or cosmetically acceptable agents, in particular one or more local anesthetics.一種包含請求項11至12中任一項之交聯材料或請求項13之水凝膠之水凝膠的可注射組成物，其中交聯材料為超級增容劑。An injectable composition of a hydrogel comprising the crosslinking material of any one of claims 11 to 12 or the hydrogel of claim 13, wherein the crosslinking material is a super expander.一種請求項14之可注射組成物用於美容應用之用途，其中該美容應用包括顏面和身體重塑及回春，較佳包括填平皺紋、改善顏面細紋、乳房重建或隆胸、皮膚回春、豐臀、顴骨重塑、軟組織增補、填平顏面皺紋、改善眉間紋、改善法令紋、改善木偶紋、改善頰部連合、改善唇緣皺紋、改善魚尾紋、改善眉、顴頰脂肪墊之皮下支持、改善淚溝、改善鼻子外觀、豐唇、豐頰、口周區域增大、眶下區域增大、解決顏面不對稱、改善下顎輪廓、墊下巴或其兩種或多種之組合。A use of the injectable composition of claim 14 for cosmetic applications, wherein the cosmetic applications include facial and body remodeling and rejuvenation, preferably including wrinkle filling, improvement of facial fine lines, breast reconstruction or breast augmentation, skin rejuvenation, buttocks augmentation, cheekbone remodeling, soft tissue augmentation, filling of facial wrinkles, improvement of glabellar lines, improvement of nasolabial lines, improvement of marionette lines, improvement of cheek commissures, improvement of lip wrinkles, improvement of crow's feet lines, improvement of eyebrows, subcutaneous support of cheek fat pads, improvement of tear troughs, improvement of nose appearance, lip augmentation, cheek augmentation, enlargement of the periorbital area, enlargement of the infraorbital area, resolution of facial asymmetry, improvement of jaw contour, chin augmentation or a combination of two or more thereof.