Method for preparing a sterilization composition

Abstract

The present invention provides a sterilization composition comprising a conjugate of sodium hyaluronate or hyaluronic acid and a pharmaceutically active compound, and a preparation method comprising providing a conjugate of sodium hyaluronate or hyaluronic acid and a pharmaceutically active compound, and exposing the conjugate to ionizing radiation. The present invention further provides a sterilization method comprising exposing a conjugate of sodium hyaluronate or hyaluronic acid and a pharmaceutically active compound to ionizing radiation, the method comprising 10 -6 Or characterized by providing a better level of sterilization assurance. The present invention is a sterilization composition comprising a conjugate of sodium hyaluronate, or hyaluronic acid, and a pharmaceutically active compound, which can be obtained or obtained by the method of the present invention, and 10 -6 The present invention further provides a sterile composition comprising a conjugate of sodium hyaluronate or hyaluronic acid and a pharmaceutically active compound, characterized by a better level of sterility assurance.

Description

[Technical Field]

[0001] The present invention relates to a method for preparing a sterile composition comprising sodium hyaluronate, or a conjugate of hyaluronic acid, and a pharmaceutically active compound. The present invention also relates to a sterile composition, for example, a composition that can be prepared by the method of the present invention. [Background technology]

[0002] Hyaluronan is an anionic, non-sulfated glycosaminoglycan distributed throughout the connective tissue, epithelial tissue, and nerve tissue of humans and other vertebrates. Hyaluronic acid (HA) is a polysaccharide constructed from disaccharide repeat residues of β-D-glucuronic acid, N-acetyl-β-D-glucosamine (where the linkage is (1→3) glucuronic acid to glucosamine, (1→4) glucosamine to glucuronic acid). Hyaluronan refers to all physiological forms of hyaluronic acid, the most common of which is the sodium salt (sodium hyaluronate; NaHA). However, the term hyaluronic acid is commonly used in the literature to refer to any of its forms.

[0003] [ka]

[0004] It is a large molecule, with a molecular weight of several million daltons or more. Hyaluronan is present in the extracellular matrix of most mammalian tissues. In mammals, hyaluronan is found in greater quantities in the umbilical cord, and it is a component of the vitreous humor and articular cartilage. Hyaluronan is an important component of synovial fluid. It has high viscosity and provides lubrication to joints.

[0005] Hyaluronan and its modified derivatives are currently used in in vivo applications such as eye surgery, cosmetic injections, and intra-articular injections to treat osteoarthritis.

[0006] Furthermore, it is known that pharmaceutically active compounds are conjugated to hyaluronic acid, and the resulting conjugates are used for therapeutic, cosmetic, or other purposes. For example, such conjugates are known from WO2007 / 126154, Zhikui Dong et al., "Improved stability and tumor targeting of 5-fluorouracil by conjugation with hyaluronan," Journal of Applied Polymer Science, 130(2), 927-932, and WO2015 / 128787.

[0007] Medical materials and compounds, including hyaluronic acid derivatives and conjugates, must be sterilized before use. Common sterilization methods for hyaluronic acid-based medical materials include filtration, dry or wet heat treatment, ethylene oxide gas (EOG) sterilization, electron beam sterilization, and radiation sterilization. For further details, see, for example, "An Effective Translation: The Development of Hyaluronan-Based Medical Products From the Physicochemical, and Preclinical Aspects," Huerta-Angeles et al., Front Bioeng Biotechnol., 2018, 6, 62. However, the implementation of such sterilization methods typically results in the degradation of hyaluronic acid materials (a decrease in the weight-average molecular weight of hyaluronic acid), which can limit the usefulness of hyaluronic acid-based materials for some medical procedures.

[0008] Chemical sterilization (e.g., EOG sterilization) can result in chemical contaminants remaining in the hyaluronic acid. Heating methods can avoid chemical contamination, but they may decompose the hyaluronic acid and alter its structure (see, for example, US5621093 A).

[0009] While low molecular weight hyaluronic acid has been shown to have beneficial effects in certain medical applications, such as wound healing, many medical applications of hyaluronic acid utilize high molecular weight hyaluronic acid. Therefore, there is a need for sterilization methods for hyaluronic acid materials that cause no or very limited degradation of hyaluronic acid or hyaluronic acid materials. [Overview of the Initiative]

[0010] The present invention provides a method for preparing a sterile composition comprising a conjugate of sodium hyaluronate or hyaluronic acid and a pharmaceutically active compound, the method comprising providing a conjugate of sodium hyaluronate or hyaluronic acid and a pharmaceutically active compound, and exposing the conjugate to ionizing radiation.

[0011] The present invention also provides a sterile composition comprising a conjugate of sodium hyaluronate or hyaluronic acid and a pharmaceutically active compound, which can be obtained or obtained by the method of the present invention.

[0012] The present invention also provides a sterilization composition comprising a conjugate of sodium hyaluronate, or hyaluronic acid, and a pharmaceutically active compound, wherein the sterilization composition is 10 -6 Characterized by a level of sterility assurance that is better than or equal to that level. [Modes for carrying out the invention]

[0013] The present invention provides a method for preparing a sterile composition comprising a conjugate of sodium hyaluronate or hyaluronic acid and a pharmaceutically active compound, the method comprising exposing the conjugate of sodium hyaluronate or hyaluronic acid and a pharmaceutically active compound to ionizing radiation. This method is beneficial because it provides a conjugate of sodium hyaluronate or hyaluronic acid and a pharmaceutically active compound that is sterile as required for use as a medical material. Surprisingly, the degradation of sodium hyaluronate or hyaluronic acid that can occur under ionizing radiation occurs only to a minimal extent when the method of the present invention is carried out.

[0014] When sodium hyaluronate or hyaluronic acid is treated with ionizing radiation, it is typically degraded, and its average molecular weight decreases significantly. Irradiation methods such as gamma radiation are known to induce significant degradation of hyaluronic acid to the extent that gamma radiation is widely used for the intentional production of low molecular weight hyaluronic acid by degrading high molecular weight hyaluronic acid.

[0015] Huang et al. (Polymers, 2019, 11, 1214) prepared low molecular weight hyaluronic acid (LMWHA) powder for use in wound dressings from higher molecular weight (MW) hyaluronic acid. Treatment of hyaluronic acid (MW 3000 kDa) with 20 kGy of gamma radiation resulted in a decrease of more than 90% in the average molecular weight of the hyaluronic acid, and treatment with higher doses of gamma radiation resulted in even lower molecular weight.

[0016] Choi et al. (Carbohydrate Polymers, 2010, 79, 1080) describe the decomposition of high molecular weight hyaluronic acid (average molecular weight 1042 kDa) in powder form to low molecular weight fragments (200-230 kDa) by several methods, including electron beam irradiation, gamma ray irradiation, microwave irradiation, and heat treatment. Treatment with gamma radiation at a dose of 50 kGy produced LMWHA with an average molecular weight of 211 kDa.

[0017] US6,383,344 B1 describes a method for reducing the molecular weight of high molecular weight polymers such as hyaluronic acid, in which a solid-phase polymer is exposed to a certain dose of gamma radiation. Higher doses of gamma radiation exposure resulted in a lower molecular weight hyaluronic acid.

[0018] US9,011,894 B2 describes a method for sterilizing hyaluronic acid-derived materials using gamma radiation. This method produces high molecular weight hyaluronic acid-based materials sterilized by gamma radiation. However, the problem of hyaluronic acid degradation was recognized, and the inventors found that the addition of stabilizing excipients was necessary to avoid a significant decrease in molecular weight. The stabilizing excipients used were chelating agents, radical scavengers, antioxidants, solubilizers, and thiols, particularly ascorbic acid, dithiothreitol (DTT), ethylenediaminetetraacetic acid (EDTA), and sucrose (or mixtures thereof).

[0019] Such additives may need to be removed from the composition before they can be used for their intended purpose, adding another step to the preparation process that is costly, time-consuming, or may affect the sterility of the final product.

[0020] The inventors have surprisingly found that when sodium hyaluronate, or a conjugate of hyaluronic acid and a pharmaceutically active compound, is treated with ionizing radiation, the average molecular weight remains considerably higher than when hyaluronic acid alone is treated with ionizing radiation. Surprisingly, sterilization can be achieved using the method of the present invention without substantial degradation of the sodium hyaluronate or hyaluronic acid conjugate.

[0021] Furthermore, this is achieved without stabilizing excipients, such as chelating agents, radical scavengers, antioxidants, solubilizers, or thiols (e.g., without ascorbic acid, dithiothreitol (DTT), ethylenediaminetetraacetic acid (EDTA), and sucrose (or mixtures thereof)). In preferred embodiments of the present invention, the conjugate of hyaluronic acid and a pharmaceutically active compound is exposed to ionizing radiation in the absence of such stabilizers. In specific embodiments, stabilizers may be used to further improve the products of this method.

[0022] The present invention relates to a sterile composition comprising a conjugate of sodium hyaluronate, or hyaluronic acid, and a pharmaceutically active compound, and 10 -6 The present invention provides a sterile composition characterized by being treated by a sterilization method that provides a sterilization assurance level (SAL) or better.

[0023] The present invention also provides a sterile composition comprising sodium hyaluronate, or a conjugate of hyaluronic acid and a pharmaceutically active compound, wherein the conjugate of sodium hyaluronate or hyaluronic acid has a molecular weight of 16,000 to 2,400,000 Da, 40,000 to 1,200,000 Da, or 40,000 to 900,000 Da (e.g., about 120,000 to 750,000 Da, about 150,000 to 600,000 Da, about 300,000 to 750,000 Da, about 500,000 to 1,000,000 Da, or about 300,000 to 500,000, or 300,000 to 420,000 Da). In one embodiment, the present invention provides a sterile composition comprising sodium hyaluronate, or a conjugate of hyaluronic acid and a pharmaceutically active compound, wherein the conjugate of sodium hyaluronate or hyaluronic acid has a molecular weight of about 200,000 to 500,000 Da (e.g., about 200,000 to 400,000 Da, about 250,000 to 400,000 Da, about 250,000 to 420,000 Da, about 250,000 to 420,000 Da, about 250,000 to 400,000 Da, or about 300,000 to 400,000 Da). In one embodiment, the average molecular weight of the conjugate after exposure to ionizing radiation is about 250,000 to 400,000 Da or about 300,000 to 400,000 Da. The sterile composition may further be characterized by having a sterility assurance level (SAL) of 10 -6 , or better. As described below, when defined using M n , or M w , there is little difference in the average molecular weight of sodium hyaluronate, or hyaluronic acid, or the conjugate of sodium hyaluronate or hyaluronic acid. Preferably, the average molecular weight defined in this paragraph is M w . Alternatively, the average molecular weight may be M n . More preferably, the average molecular weight is M w , and AF4 is M wIt is used as a measurement method (for example, AF4 combined with a UV-FL-MALS-RI detector uses light scattering and concentration data to measure M w (Used for direct measurement)

[0024] A conjugate of hyaluronic acid and a pharmaceutically active compound. Hyaluronic acid is well-known and widely used in medical applications. Conjugates can be prepared using various pharmaceutically active compounds via conventional chemical synthesis routes. Numerous conjugates of hyaluronic acid and pharmaceutically active compounds are known in the art. For example, such conjugates are known from WO2007 / 126154, Zhikui Dong et al, "Improved stability and tumor targeting of 5-fluorouracil by conjugation with hyaluronan," Journal of Applied Polymer Science, 2013, 130(2), 927-932, and WO2015 / 128787.

[0025] Generally, in a conjugate of hyaluronic acid and a pharmaceutically active compound, the pharmaceutically active compound is linked to the hyaluronic acid by a linker group. Various linker groups have been proposed, and certain linkers have advantages in specific situations and uses. For example, for certain applications, it may be beneficial if the linker releases the pharmaceutically active compound from the hyaluronic acid when the conjugate is in a physiological environment. For other applications, it may be beneficial if the linker does not release the pharmaceutically active compound from the hyaluronic acid, or releases it very slowly, when the conjugate is in a physiological environment. In this way, the pharmaceutically active compound can exert its desired effect at the desired site over a long period of time.

[0026] Typically, a linker contains at least two atoms in its chain and optionally has side groups. For example, a linker may contain a chain of 2 to 15 atoms in length, linking hyaluronic acid with a pharmaceutically active compound.

[0027] Since hyaluronic acid contains acidic groups, linkers can most conveniently bind to hyaluronic acid polymers by bonding to the acidic groups, for example, by forming ester or amide groups.

[0028] Many pharmaceutically active compounds contain groups that can be used as linking sites for linkers. Examples of suitable linking groups include acid groups, alcohol groups, and amine groups.

[0029] Nonsteroidal anti-inflammatory drugs (NSAIDs) are among the pharmaceutically active compounds of interest in binding to hyaluronic acid. A prominent example of such a drug is diclofenac, which contains an acidic group. This acidic group can be conveniently used as a binding site to a linker. This can be achieved, for example, by the formation of an ester or amide group. Examples of conjugates of hyaluronic acid and diclofenac having this type of structure are known, for example, from WO2007 / 126154 and WO2015 / 128787.

[0030] For example, a conjugate of hyaluronic acid and a pharmaceutically active compound may include hyaluronic acid having a free hemiester group and a pharmaceutically active compound bonded to hyaluronan via the reacted hemiester group (which becomes an ester group or amide), thereby forming a linker with a chain length L of 2 to 9 atoms. Thus, in the hyaluronic acid conjugate, some of the hemiester groups are free, while others are bonded to the pharmaceutically active compound. In certain embodiments, the hyaluronic acid conjugate can be produced by providing hyaluronic acid in solution or gel form, reacting the hyaluronic acid in solution or gel form with an anhydride reagent (e.g., succinic anhydride) to provide a hyaluronic acid hemiester (referred to herein as activated hyaluronic acid) having a chain of length L between the hyaluronic acid and the ester group, and then bonding the hyaluronic acid hemiester to a pharmaceutically active compound.

[0031] According to certain embodiments of the present invention, the linker comprises a carbon skeleton and optionally contains one or two oxygen atoms in the skeleton. The carbon skeleton of the hemiester chain may optionally comprise one or more branches of alkyl, aryl, oxy-alkyl, or oxy-aryl.

[0032] In a more specific embodiment, the chain that binds to hyaluronan has the following formula: -C(O)-(CHR) n -(CH2) (m-n) -COO - In the formula, n is 0 or 1, m = 2-8, for example 2, 3, 4, 5, 6, 7, or 8, and R = alkyl, aryl, O-alkyl, or O-aryl. -C(O)-(CHR) n -(CH2) (p-1) -O-(CH2) q -COO - In the formula, n is 0 or 1, p and q are independently 1 to 4, for example 1, 2, 3, or 4, and R = alkyl, aryl, O-alkyl, or O-aryl.

[0033] In further embodiments, the linker that binds the pharmaceutically active compound to hyaluronan is of the following formula. -C(O)-(CH2) m -COO - In the formula, m = 2 - 8, for example, 2, 3, 4, 5, 6, 7, or 8. -C(O)-(CH2) p -O-(CH2) q -COO - In the formula, p and q are independently 1 to 4, for example, 1, 2, 3, or 4, or -C(O)-(CH2) r -O-(CH2) s -O-(CH2) t -COO - In the equation, r and t are independently 1 to 2, and s is 2.

[0034] Those skilled in the art will know that in the reaction of hyaluronan with an anhydride reagent, the activating intermediate may be in the form of a salt of an ester group, for example, a sodium salt, containing a free hemiester group, and in each of the above formulas, -COO - It is understood that this is -COONa.

[0035] In solution, it will be understood that many carboxylate groups exist in their ionized form, and their levels typically depend on the pH of the solution. It will also be understood that the conjugates can be in the form of sodium salts. In this specification, references to conjugates of hyaluronic acid and pharmaceutically active compounds should be understood to include all physiological forms of conjugates of hyaluronic acid and pharmaceutically active compounds (i.e., conjugates of hyaluronan and pharmaceutically active compounds), including conjugates of sodium hyaluronate (NaHA) and pharmaceutically active compounds (unless otherwise indicated in the context).

[0036] In one embodiment, hyaluronan is crosslinked to form a gel (as described, for example, in Laurent et al. (Acta. Chem. Scand., 1964, 18(1), 274-275) and Malson et al. (US4, 716, 154)), then activated by the formation of a hemiester and / or subsequently bound to a drug via an ester or amide bond.

[0037] In one embodiment, the formation of hyaluronan succinyl hemiester (HSE) and the subsequent binding of pharmaceutically active substances are by esterification. Anhydrides other than succinic anhydride, and esters formed therefrom, may also be used. In a particular embodiment, glutaryl hemiester is used.

[0038] The degree of ester substitution can be influenced by changing the ratio of the anhydrous reagent to the hyaluronane polymer, the reaction time, and the temperature.

[0039] Typically, without raising the temperature above room temperature, an average degree of substitution (DS) of hemisuccinate of up to 3 moles per mole of hyaluronane repeat disaccharide units can be obtained. In certain embodiments, the average degree of substitution is 0.5 to 3 per mole of hyaluronane repeat disaccharide units, and in more specific embodiments, 1 to 3, or 2 to 3 moles, of hemiesters, such as hemisuccinates.

[0040] Formula (I) shows a schematic diagram of an HSE-drug conjugate that may be used in the present invention.

[0041] [ka] In the formula, X is H, -CO-CH2CH2-COONa,-CO-CH2CH2-CO-NH-CH2CH2-O-CH2CH2-O-DRUG, or -CO-CH2CH2-CO-NH-CH2CH2-O-CH2CH2-O-CO-CH2CH2-CO-DRUG, where DRUG represents a pharmaceutically active compound.

[0042] For example, DRUG could be diclofenac, for instance, bonded via its acidic group.

[0043] Theoretically, a drug molecule (i.e., a pharmaceutically active compound) can occupy all carboxyl groups exposed by HSE; however, in practice, higher substitutions can adversely alter the polymer properties, especially when a solution suitable for injection is desired. For substitution with diclofenac, an average degree of substitution (DS) of 0.3 moles of drug or less per mole of hyaluronan disaccharide repeat unit is preferred for formulations of injection solutions. Depending on the intended use, an average degree of substitution of 0.01 to 0.3 moles of drug, particularly 0.05 to 0.2 moles per mole of hyaluronan disaccharide repeat unit, may be used. For drugs other than diclofenac, other degrees of substitution may be preferred. For the manufacture of solid formulations, e.g., films or particles, the intended use determines the preferred DS, and for applications requiring high doses, an average DS of up to 3 moles of drug per mole of hyaluronan is preferred.

[0044] In another specific embodiment of the present invention, the drug in the conjugate is dexamethasone. The preparation of a suitable HA dexamethasone conjugate is described in WO2015 / 128787.

[0045] In preferred embodiments of the present invention, the pharmaceutically active compound (e.g., DRUG of formula (I) above) is diclofenac, for example, diclofenac linked via its acid group.

[0046] In one embodiment of the present invention, the pharmaceutically active compound (i.e., the drug in the conjugate, e.g., the DRUG of formula (I) above) is a nonsteroidal anti-inflammatory drug (e.g., selected from the group consisting of diclofenac, ibuprofen, ketoprofen, bromfenac, aceclofenac, flunixin, and carprofen), a steroid (e.g., selected from the group consisting of dexamethasone and prednisolone), an antibiotic (e.g., selected from the group consisting of metronidazole, azithromycin, and levofloxacin), a plant alkaloid (e.g., podophyllotoxin), an antiviral drug (e.g., These include acyclovir, chemotherapy drugs (e.g., selected from the group consisting of paclitaxel, docetaxel, doxorubicin, and daunorubicin), retinoids (e.g., adapalene), immunosuppressants (e.g., selected from the group consisting of cyclosporine and tacrolimus), prostaglandin analogs (e.g., latanoprost), mast cell stabilizers (e.g., selected from the group consisting of cromoglycic acid, nedocromil, and olopatadine), antihistamines (e.g., selected from the group consisting of levocabastine and bepotastine), or analgesics (e.g., opioids such as morphine). In preferred embodiments of the present invention, the pharmaceutically active compound (i.e., the drug in the conjugate, e.g., the DRUG of formula (I) above) is a nonsteroidal anti-inflammatory drug (e.g., selected from the group consisting of diclofenac, ibuprofen, ketoprofen, bromfenac, and aceclofenac) or a steroid (e.g., selected from the group consisting of dexamethasone and prednisolone).

[0047] In another embodiment of the present invention, the drug in the conjugate is cisplatin. Further pharmaceutically active compounds that may be used include ibuprofen, ketoprofen, naproxen, bromfenac, aceclofenac, prednisolone, metronidazole, podophyllotoxin, paclitaxel, flunixin, carprofen, docetaxel, doxorubicin, daunorubicin, adapalene, azithromycin, levofloxacin, acyclovir, cyclosporine, tacrolimus, latanoprost, cromoglycic acid, levocabastine, nedocromil, olopatadine, bepotastine, and morphine.

[0048] In one embodiment of the present invention, the compound does not contain a sulfate group. In another embodiment of the present invention, the compound does not contain a sulfur-containing group. In another embodiment of the present invention, the compound of the present invention does not contain a sulfated sodium hyaluronate group or a sulfated hyaluronic acid group (for example, the compound does not contain an -OH group converted to a sulfate group by esterification with sulfuric acid, for example).

[0049] The compound of the present invention can be produced by providing hyaluronan in solution, reacting the hyaluronan in solution with an anhydrous reagent to provide a hyaluronan hemiester having a hemiester group, and then binding the hyaluronan hemiester to a pharmaceutically active compound.

[0050] In one embodiment, hyaluronan in solution is reacted with an anhydride reagent, such as succinic anhydride. The hyaluronan solution may be provided using a solvent suitable for solid sodium hyaluronate, such as formamide, with the addition of a tertiary amine, pyridine, or substituted pyridine. In certain embodiments, the solvent is pyridine, and optionally, 4-dimethylaminopyridine (DMAP) or 2,6-dimethyl-4-dimethylaminopyridine is added. This procedure allows for the dissolution of solid sodium hyaluronate without extra steps such as acidic forms or ion exchange to hyaluronic acid, which are typically used in the prior art.

[0051] In previously described methods, such as the one described in WO96 / 35720, dimethylformamide (DMF) is used as the solvent. However, sodium hyaluronate is not soluble in this solvent, and ion exchange of hyaluronan to its acidic form or conversion to an amine salt in water is required before it can be dissolved in DMF. After that, the water is removed by evaporation, and the hyaluronan is redissolved in DMF, after which the reagent is added.

[0052] In one embodiment, the conjugate is produced by dissolving it in formamide solvent and then directly adding the reagent, thus providing a simpler and shorter procedure for the synthesis of the hemiester of formula (II) than those commonly used in the prior art.

[0053] [ka] In the formula, R is H or an ester chain, for example, in the case of succinic anhydride, -CO-CH2-CH2-COO-Na.

[0054] In one embodiment, the conjugate is produced by dissolving it in formamide solvent and then directly adding the reagent, thus providing a simpler and shorter procedure for the synthesis of the hemiester of formula (I) than those commonly used in the prior art.

[0055] [ka] In the formula, X is H or an ester chain, for example, in the case of succinic anhydride, -CO-CH2-CH2-COO-Na.

[0056] A hemiester, such as succinylated hyaluronan (HSE), can then be reacted with an amino group-containing compound to obtain an amide on the carboxyl group exposed on the hyaluronan hemiester. The desired pharmaceutically active agent may possess an amino functional group. In certain embodiments, the amino functional group is combined with a longer portion to detach the pharmaceutically active agent from the hyaluronan and provide better access for in vivo degrading enzymes. Furthermore, in certain embodiments, the coupling of the amine-functionalized pharmaceutically active agent to the hyaluronan hemiester group can be carried out in a water-containing medium, i.e., water or an aqueous solvent, such as a DMF-water mixture or a suitable water-based buffer. This feature makes it possible to couple molecules that are poorly soluble in aprotic solvents.

[0057] In one embodiment of the present invention, the combined body is produced by the following: We provide hyaluronane in solution, and react the hyaluronane in solution with an anhydrous reagent to provide a hyaluronane hemiester having a hemiester group of the following formula. -C(O)-(CHR) n -(CH2) (m-n) -COO - In the formula, n is 0 or 1, m = 2 to 8, and R = C 1~4 Alkyl, C 6~10 Ariel, OC 1~4 Alkyl, or OC 6~10 It is Ariel, or -C(O)-(CHR) n -(CH2) (p-1) -O-(CH2) q -COO - In the equation, n is 0 or 1, p and q are independently 1 to 4, and R = C 1~4 Alkyl, C 6~10 Ariel, OC 1~4 Alkyl, or OC 6-10 It is Ariel, Next, the hyaluronane hemiester is bound to a pharmaceutically active compound.

[0058] In an alternative embodiment, the composite is generated by the following: We provide hyaluronane in solution, and react the hyaluronane in solution with an anhydrous reagent to provide a hyaluronane hemiester having a hemiester group of the following formula. -C(O)-(CH2) m -COO - In the formula, m is between 2 and 8. -C(O)-(CH2) p -O-(CH2) q -COO - In the formula, p and q are both 1 to 4, or -C(O)-(CH2) r -O-(CH2) s -O-(CH2) t -COO - In the formula, r and t are 1 to 2, and s is 2. Next, the hyaluronane hemiester is bound to a pharmaceutically active compound.

[0059] In another alternative embodiment, the composite is produced by: We provide hyaluronane in solution, and react the hyaluronane in solution with an anhydrous reagent to provide a hyaluronane hemiester having a hemiester group of the following formula. -CO-CH2CH2-COO - , -CO-CH2CH2-CO-NH-CH2CH2-O-CH2CH2-O - , or -CO-CH2CH2-CO-NH-CH2CH2-O-CH2CH2-O-CO-CH2CH2-CO - , Next, the hyaluronane hemiester is bound to a pharmaceutically active compound.

[0060] Sterilization method In the method of the present invention, a sterilization composition is prepared by exposing a composition comprising a conjugate of sodium hyaluronate, or hyaluronic acid, and a pharmaceutically active compound to ionizing radiation. Ionizing radiation is radiation, such as particles, X-rays, or gamma rays, that has sufficient energy to cause ionization in the medium through which it passes. In certain embodiments, the composition exposed to ionizing radiation consists of, or essentially consists of, sodium hyaluronate, or hyaluronic acid, and a pharmaceutically active compound.

[0061] In embodiments of the present invention, the ionizing radiation is beta, gamma, or X-ray radiation. In preferred embodiments of the present invention, the ionizing radiation is beta or gamma radiation. In particularly preferred embodiments of the present invention, the ionizing radiation is gamma radiation.

[0062] In embodiments of the present invention, the method may be carried out in air, or in an inert atmosphere such as nitrogen or argon, or under vacuum. It has been found that exposure of hyaluronic acid-based materials to ionizing radiation in an inert atmosphere reduces the degradation of hyaluronic acid compared to when the method is carried out in air. Therefore, in preferred embodiments of the present invention, the compound is exposed to ionizing radiation in an inert atmosphere. In more preferred embodiments of the present invention, the inert atmosphere is an argon atmosphere or a nitrogen atmosphere. The inert atmosphere is, for example, an argon atmosphere. Alternatively, the inert atmosphere is a nitrogen atmosphere.

[0063] The method of the present invention may be carried out on a conjugate of hyaluronic acid and a pharmaceutically active compound in solid form or in solution. In a preferred embodiment of the present invention, the method is carried out on a conjugate in solid form. In a particularly preferred embodiment of the present invention, the method is carried out on a conjugate in powder form.

[0064] Medical devices and materials that enter the body must be sterilized. In this field, materials must pass verified sterility tests before being made available for medical use (Ph..Eur.,11 thedition,2.6.1.monograph and USP,43 rd edition, <71> (monograph). Ph.Eur., 11 th As stated in edition 5.1.1. monograph, "The sterility of a product cannot be guaranteed by testing, but must be guaranteed by the implementation of a properly validated manufacturing process. To ensure the effectiveness and integrity of the product, it is essential to investigate the effect of the selected sterilization procedure on the product (including its final container or packaging) and to validate the procedure before actually implementing it." Therefore, sterilization assurance levels are used to express sterilization. The sterilization assurance level (SAL) is the probability that a single unit subjected to sterilization remains non-sterile, i.e., the probability that any microorganism will survive after sterilization. 10 -6 SAL stands for non-sterile unit, meaning a probability of 1 / 1,000,000. 10 -6 SAL is generally required for medical materials used in the body, but 10 -3 SAL may be acceptable for materials intended only for contact with intact skin (Ph.Eur.,11 th (Edition, 5.1.1. monograph and ISO 11137-1:2006,-2:2013 and-3:2017 guidelines). The SAL for a specific sterilization process for a specific material is established by corresponding validation studies of the process.

[0065] Ph.Eur.,11 th Edition 5.1.1. monograph states that when ionizing radiation is used as a sterilization method, the standard absorbed dose is 25 kGy. USP, 43 rdedition, <1211> The monograph states, "For a complete description of process development, validation, and routine control of ionizing radiation processes, readers should refer to ISO 11137-1, -2, and -3." ISO 11137-2:2013 (Sterilization of Healthcare Products) specifies 25 kGy (Verification Dose Max (VDmax25) method) as the acceptable standard dose and recommends it for products with a maximum bioburden of 1000 CFU. Validation methods for processes using ionizing radiation as a sterilization method are provided in ISO 11137-2:2013.

[0066] The degree of degradation of hyaluronic acid-based materials upon exposure to ionizing radiation is related to the dose of radiation they receive. Treatment with higher doses of gamma radiation typically results in a lower average molecular weight of the sterilized hyaluronic acid material. In Huang et al., Polymers, 2019, 11, 1214, the authors found that treatment of hyaluronic acid with a MW of 3000 kDa with 20 kGy of gamma radiation resulted in a reduction of over 90% of the average molecular weight of HA, treatment with 40 kGy resulted in a reduction of over 95%, and treatment with 60 kGy resulted in a reduction of over 98%. The inventors found that when a conjugate of hyaluronic acid and a pharmaceutically active compound was exposed to ionizing radiation, a higher average molecular weight was retained in the sterilized conjugate than when unconjugated hyaluronic acid was exposed to the same dose of radiation. The inventors found that sterilization was achieved while degradation remained at an acceptable low level.

[0067] In one embodiment of the present invention, the dose of ionizing radiation to which a conjugate of hyaluronic acid and a pharmaceutically active compound is exposed is approximately 5 to 40 kGy. In another embodiment of the present invention, the dose of ionizing radiation is approximately 6 to 40 kGy. In yet another embodiment of the present invention, the dose of ionizing radiation is approximately 8 to 40 kGy. In a preferred embodiment of the present invention, the dose of ionizing radiation is approximately 15 to 40 kGy. In a more preferred embodiment of the present invention, the dose of ionizing radiation is approximately 20 to 40 kGy. In a more preferred embodiment of the present invention, the dose of ionizing radiation is approximately 20 to 35 kGy. In a more preferred embodiment of the present invention, the dose of ionizing radiation is approximately 20 to 30 kGy. In a particularly preferred embodiment of the present invention, the dose of ionizing radiation is approximately 25 kGy. In a particularly preferred embodiment of the present invention, the dose of ionizing radiation is approximately 25 kGy and is verified by the VDmax25 method of ISO 11137-2:2013.

[0068] In another embodiment of the present invention, the dose of ionizing radiation to which a conjugate of hyaluronic acid and a pharmaceutically active compound is exposed is about 25-40 kGy, for example, 25-32 kGy. In a preferred embodiment of the present invention, the dose of ionizing radiation is about 30-40 kGy. In a more preferred embodiment of the present invention, the dose of ionizing radiation is about 30-35 kGy. In another embodiment of the present invention, the dose of ionizing radiation is 35-40 kGy.

[0069] In another embodiment of the present invention, the dose of ionizing radiation to which a conjugate of hyaluronic acid and a pharmaceutically active compound is exposed is about 10 to 30 kGy. In a preferred embodiment of the present invention, the dose of ionizing radiation is about 10 to 25 kGy. In a more preferred embodiment of the present invention, the dose of ionizing radiation is about 10 to 20 kGy. In a more preferred embodiment of the present invention, the dose of ionizing radiation is about 10 to 15 kGy. In another embodiment of the present invention, the dose of ionizing radiation is about 15 to 20 kGy.

[0070] The method of the present invention can be carried out at temperatures up to ambient temperature. It has been found that applying ionizing radiation to a hyaluronic acid-based material at low temperatures results in a higher average molecular weight of the resulting sterilized material.

[0071] In a preferred embodiment of the present invention, the composite is exposed to ionizing radiation at a temperature of -120°C to 30°C. In a preferred embodiment of the present invention, the composite is exposed to ionizing radiation at a temperature of -80°C to 30°C, for example, -80°C to 0°C. In a preferred embodiment of the present invention, the composite is exposed to ionizing radiation at a temperature of -80°C to -20°C. In a more preferred embodiment of the present invention, the composite is exposed to ionizing radiation at a temperature of -80°C to -40°C. In a particularly preferred embodiment of the present invention, the composite is exposed to ionizing radiation at a temperature of -78°C.

[0072] The molecular weight of polymers such as hyaluronic acid and hyaluronic acid conjugates (including sodium hyaluronate conjugates) is expressed as the average molecular weight, or as a molecular mass distribution, or molecular weight distribution, and the polymer consists of a distribution of many molecular weights, or chain lengths. The average, or distribution, can be defined in different ways depending on the statistical method used. For example, it can be the number-average molar mass (M) which is simply the average of the molecular weights of individual polymer lengths. n (Generally expressed using the unit Da), and weight-average molar mass (M w M can generally be defined as (expressed using the unit g / mol, or Da), where larger molecules contribute more to the average than smaller molecules. w Mw can be measured, for example, under the assumption that the fraction is homogeneous, and M n It can be converted to M. n , or M w When defined using M, there is little difference between the values ​​of “average molecular weight” for hyaluronic acid or hyaluronic acid conjugates (including sodium hyaluronate conjugates), and both can be used to define the “average molecular weight” for these polymers. Where molecular weight or average molar mass is referred to herein, it is Mn , or M w It is possible. Preferably, it is M w (For example, M w AF4 is used as the measurement method. w More preferably, AF4 combined with a UV-FL-MALS-RI detector uses light scattering and concentration data to perform M w (Used for direct measurement)

[0073] The average molecular weight of the conjugate can be evaluated by various methods known in the art. For example, the average molecular weight of the conjugate can be measured by asymmetric flow-field-flow fractionation (AF4) (Kwon et al., Depolymerization study of sodium hyaluronate by flow field-flow fractionation / multiangle light scattering, Anal. Bioanal. Chem., 2009, 395, 519-525). However, other methods, such as viscosity measurement, conventional size exclusion chromatography (conventional SEC), size exclusion chromatography using multiangle laser light scattering detectors (SEC-MALLS), or gel electrophoresis can also be used (Cowman and Mendichi, Methods for Determination of Hyaluronan Molecular Weight, Chemistry and Biology of Hyaluronan, chapter 3, Elsevier Science Ltd., 2004, pp. 41-69). Naturally, the molar masses of the two compositions being compared must be established using the same method.

[0074] For the compounds of the present invention, AF4 has been found to be the most reliable method for evaluating the average molar mass (particularly the weight-average molar mass), and in particular, using light scattering and concentration data, M w This is AF4, combined with a UV-FL-MALS-RI detector, used to directly obtain [the desired result].

[0075] The method of the present invention enables the use of ionizing radiation for sterilization of hyaluronic acid conjugates incorporating pharmaceutically active compounds, while maintaining the average molecular weight of the resulting sterilized conjugate without requiring the addition of stabilizing additives that may need to be removed from the composition in further processing steps, and without impairing or limiting the usefulness of the conjugate as a medical material.

[0076] In embodiments of the present invention, the average molecular weight of the conjugate after exposure to ionizing radiation is more than 40% of the starting average molecular weight of the conjugate before exposure to ionizing radiation. In preferred embodiments of the present invention, the average molecular weight of the conjugate after exposure to ionizing radiation is more than 45%, preferably more than 50%, for example more than 55%, or more than 60%, of the starting average molecular weight of the conjugate before exposure to ionizing radiation. In particularly preferred embodiments of the present invention, the average molecular weight of the conjugate after exposure to ionizing radiation is more than 60% of the starting average molecular weight of the conjugate before exposure to ionizing radiation. That is, the average molecular weight of the conjugate after exposure to ionizing radiation is at least 60% of the average molecular weight before the irradiation process.

[0077] In embodiments of the present invention, the decrease in the average molecular weight of the conjugate after exposure to ionizing radiation is less than 60% of the starting average molecular weight of the conjugate before exposure to ionizing radiation. In preferred embodiments of the present invention, the decrease in the average molecular weight of the conjugate after exposure to ionizing radiation is less than 55%, preferably less than 50%, for example, less than 45% or 40%, of the starting average molecular weight of the conjugate before exposure to ionizing radiation. In particularly preferred embodiments of the present invention, the decrease in the average molecular weight of the conjugate after exposure to ionizing radiation is less than 40% of the starting average molecular weight of the conjugate before exposure to ionizing radiation.

[0078] As mentioned above, M n , or M w When defined using, there is little difference between the average molecular weights of sodium hyaluronate, hyaluronic acid, or conjugates of sodium hyaluronate or hyaluronic acid. Preferably, the average molecular weight change defined above is Mw This is a change in M. Alternatively, the change in average molecular weight is M n This can be a change. More preferably, the change in average molecular weight is M w It is a change, and AF4 is M w It is used as a measurement method (for example, AF4 combined with a UV-FL-MALS-RI detector uses light scattering and concentration data to measure M w (Used for direct measurement)

[0079] In one embodiment, the starting average molecular weight of the conjugate before exposure to ionizing radiation is about 40,000 to 4,000,000 Da. In a preferred embodiment, the starting average molecular weight of the conjugate before exposure to ionizing radiation is about 100,000 to 2,000,000 Da. In a more preferred embodiment, the starting average molecular weight of the conjugate before exposure to ionizing radiation is about 100,000 to 1,500,000 Da, for example, about 200,000 to 1,250,000 Da, or about 250,000 to 1,000,000 Da. In a particularly preferred embodiment, the starting average molecular weight of the conjugate before exposure to ionizing radiation is about 500,000 to 1,250,000 Da. In another particularly preferred embodiment, the starting average molecular weight of the conjugate before exposure to ionizing radiation is about 500,000 to 1,000,000 Da. In another particularly preferred embodiment, the starting average molecular weight of the conjugate before exposure to ionizing radiation is about 500,000 to 700,000 Da. As described above, M n , or M w When defined using, there is little difference between the average molecular weights of sodium hyaluronate, hyaluronic acid, or conjugates of sodium hyaluronate or hyaluronic acid. Preferably, the starting average molecular weight as defined in this paragraph is the starting M w Therefore, the starting average molecular weight is the starting M. n It is possible. More preferably, the starting average molecular weight is the starting M w And AF4 is M w It is used as a measurement method (for example, AF4 combined with a UV-FL-MALS-RI detector, using light scattering and concentration data, Mw (used to directly measure).

[0080] In a preferred embodiment of the present invention, the average molecular weight of the compound after exposure to ionizing radiation is more than 40% of the starting average molecular weight of the compound before exposure to ionizing radiation, and the starting average molecular weight of the compound before exposure to ionizing radiation is about 40,000 to 4,000,000 Da, about 100,000 to 2,000,000 Da, or about 100,000 to 1,500,000 Da (preferably about 200,000 to 1,250,000 Da, or about 250,000 to 1,000,000 Da, more preferably about 500,000 to 1,250,000 Da, or about 500,000 to 1,000,000 Da (for example, about 500,000 to 700,000 Da)). In a preferred embodiment of the present invention, the average molecular weight of the conjugate after exposure to ionizing radiation is more than 45%, preferably more than 50%, for example more than 55%, or more than 60%, of the starting average molecular weight of the conjugate before exposure to ionizing radiation, and the starting average molecular weight of the conjugate before exposure to ionizing radiation is about 40,000 to 4,000,000 Da, about 100,000 to 2,0 The value is 00,000 Da, or approximately 100,000 to 1,500,000 Da (preferably approximately 200,000 to 1,250,000 Da, or approximately 250,000 to 1,000,000 Da, more preferably approximately 500,000 to 1,250,000 Da, or approximately 500,000 to 1,000,000 Da (for example, approximately 500,000 to 700,000 Da)). In a particularly preferred embodiment of the present invention, the average molecular weight of the conjugate after exposure to ionizing radiation is more than 60% of the starting average molecular weight of the conjugate before exposure to ionizing radiation, and the starting average molecular weight of the conjugate before exposure to ionizing radiation is about 40,000 to 4,000,000 Da, about 100,000 to 2,000,000 Da, or about 100,000 to 1,500,000 Da (preferably about 200,000 to 1,250,000 Da, or about 250,000 to 1,000,000 Da, more preferably about 500,000 to 1,250,000 Da, or about 500,000 to 1,000,000 Da (for example, about 500,000 to 700,000 Da)). In other words, the average molecular weight of the compound after exposure to ionizing radiation is at least 60% of the average molecular weight before the irradiation process. As described above, M n , or Mw When defined using, there is little difference in the average molecular weight of sodium hyaluronate, or hyaluronic acid, or a conjugate of sodium hyaluronate, or hyaluronic acid. Preferably, the starting average molecular weight defined in this paragraph is the starting M w is. Alternatively, the starting average molecular weight is the starting M n can be. More preferably, the starting average molecular weight is the starting M w is, and AF4 is M w is used as a measurement method for (for example, AF4 combined with a UV-FL-MALS-RI detector uses light scattering and concentration data to directly measure M w ).

[0081] In one embodiment of the present invention, the average molecular weight of the conjugate after exposure to ionizing radiation is about 16,000 - 2,400,000 Da, about 40,000 - 1,200,000 Da, or about 40,000 - 900,000 Da (for example, about 120,000 - 750,000 Da, about 150,000 - 600,000 Da, about 300,000 - 750,000 Da, about 500,000 - 1,oo0,000 Da, or about 300,000 - 500,000, or 300,000 - 420,000 Da). In one embodiment, the average molecular weight of the conjugate after exposure to ionizing radiation is about 200,000 - 500,000 Da (for example, about 200,000 - 400,000 Da, about 250,000 - 400,000 Da, about 250,000 - 420,000 Da, about 250,000 - 420,000 Da, about 250,000 - 400,000 Da, or about 300,000 - 400,os000 Da). In one embodiment, the average molecular weight of the conjugate after exposure to ionizing radiation is about 250,000 - 400,000 Da, or about 300,000 - 400,000 Da. As described above, M n , or M w When defined using, there is little difference in the average molecular weight of sodium hyaluronate, or hyaluronic acid, or a conjugate of sodium hyaluronate, or hyaluronic acid. Preferably, the average molecular weight defined in this paragraph is M wTherefore, the average molecular weight is M n It may be. More preferably, the average molecular weight is M w And AF4 is M w It is used as a measurement method (for example, AF4 combined with a UV-FL-MALS-RI detector uses light scattering and concentration data to measure M w (Used for direct measurement)

[0082] The method of the present invention may be performed on the conjugate either before or after packaging the conjugate in a container for use in individual medical procedures.

[0083] Therefore, the method of the present invention may further include the step of dividing the sterilized composition into containers. In one embodiment of the present invention, the sterilized composition is divided into vials.

[0084] Alternatively, the conjugate of hyaluronic acid and a pharmaceutically active compound is divided into containers for use in medical procedures before the container containing the conjugate is exposed to ionizing radiation.

[0085] In the method of the present invention, the composite can be packaged in one or more bags. If there are two or more bags, the bags can be made of the same material or different materials. Suitable materials include polyethylene (PE) and aluminum. In one embodiment, at least one PE bag (e.g., 1, 2, 3, 4, or 5) and at least one (e.g., 1, 2, or 3) aluminum bag are used, for example, three PE bags and one aluminum bag are used.

[0086] In one embodiment of the present invention, a conjugate of hyaluronic acid and a pharmaceutically active compound (optionally in a container) is packaged in a PE bag under an argon atmosphere, and then the PE bag is packaged in a heat-sealed aluminum bag under an argon atmosphere. In a particular embodiment of the present invention, the conjugate of hyaluronic acid and a pharmaceutically active compound is removed from storage below 15°C and placed at ambient temperature to reach equilibrium over 2 to 4 hours. The outside of the first package is wiped clean of dust before opening. A desired amount of the conjugate is transferred to the primary PE bag. Air is squeezed out of the PE bag and replaced with argon. Argon is squeezed out and the primary PE bag is closed. The primary PE bag is placed in the secondary PE bag. Air is squeezed out of the secondary PE bag and replaced with argon. Argon is squeezed out and the secondary PE bag is closed. The secondary PE bag is placed in the tertiary PE bag. Air is squeezed out of the tertiary PE bag and replaced with argon. Argon is squeezed out and the tertiary PE bag is closed. Place the tertiary PE bag into an aluminum bag. Squeeze the air out of the aluminum bag and replace it with argon. Squeeze out the argon, heat seal the aluminum bag, and attach a label.

[0087] In another embodiment of the present invention, a conjugate of hyaluronic acid and a pharmaceutically active compound (optionally in a container) is packaged in a PE bag under an air atmosphere, and then the PE bag is packaged in a heat-sealed aluminum bag under an air atmosphere. In a particular embodiment of the present invention, the conjugate of hyaluronic acid and a pharmaceutically active compound is removed from storage below 15°C and placed at ambient temperature to reach equilibrium over 2-4 hours. The outside of the first package is wiped clean of dust before opening. A desired amount of the conjugate is transferred to the primary PE bag. Air is squeezed out of the PE bag and the primary PE bag is closed. The primary PE bag is placed in the secondary PE bag. Air is squeezed out of the secondary PE bag and the secondary PE bag is closed. The secondary PE bag is placed in the tertiary PE bag. Air is squeezed out of the tertiary PE bag and the tertiary PE bag is closed. The tertiary PE bag is placed in the aluminum bag. Air is squeezed out of the aluminum bag, the aluminum bag is heat-sealed, and labeled.

[0088] The method of the present invention may further include a step of filtering a composition, the composition being in the form of an aqueous liquid composition. Preferably, after sterilizing the composition, it is mixed with an aqueous solution (preferably a sterile aqueous solution, e.g., glucose for injection, saline solution for injection, or water for injection) to form an aqueous liquid composition, and then the aqueous liquid composition is filtered. Filtration of the composition may be carried out by membrane filter filtration. In such embodiments, a membrane filter (e.g., a commercially available membrane filter) may be optionally used in conjunction with a sterile container, sterile syringe, syringe barrel, etc. For example, a membrane filter having a pore size of 0.05 μm to 20 μm, for example, 0.4 to 8.0 μm (e.g., 0.4 μm, 0.5 μm, 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, or 8 μm), 0.5 to 6 μm, or 1 to 5 μm, in particular a pore size of 5 μm, may be used.

[0089] In certain embodiments, the method of the present invention includes the step of mixing a sterile conjugate composition with an aqueous solution of a sugar or sugar alcohol (e.g., an aqueous solution of sugar) to provide an aqueous liquid composition. In preferred embodiments, the sugar is glucose, sucrose, fructose, or trehalose, and the sugar alcohol is mannitol, ethylene glycol, glycerol, sorbitol, or xylitol. For example, the sugar is glucose, sucrose, or trehalose, and the sugar alcohol is mannitol. Alternatively, the sugar is glucose or trehalose. Alternatively, the sugar is glucose or fructose. In the most preferred embodiment, the sugar is glucose. In another preferred embodiment, the sugar is not sucrose.

[0090] In the context of the present invention, “aqueous liquid composition” includes any mixture resulting from a mixture or combination of water with a component defined to be present in the composition, whether or not it is completely dissolved. In preferred embodiments, the component is completely dissolved.

[0091] In one embodiment, the aqueous solution of sugar or sugar alcohol for use in the method may be in the form of a sterile composition, for example, a sterile glucose solution (for example, a glucose solution for injection), for example, the composition may be 10 -3 Or better, for example, 10 -3 Or better, for example, 10 -5 Or better, or for example, 10 -6 Or it has a better sterility assurance level (SAL). In one embodiment, an aqueous solution of sugar or sugar alcohol is sterile, and 10 -6 , or having a better SAL, for example, the sugar is glucose, in the form of a glucose solution for injection.

[0092] In embodiments further comprising the step of mixing the sterilization composition with an aqueous solution of sugar or sugar alcohol, the concentration of the sterilization conjugate in the aqueous liquid composition is preferably 2 to 50 mg / mL, for example, 10 to 40 mg / mL, 12 to 30 mg / mL, or 12 to 21 mg / mL. In more preferred embodiments, the concentration of the conjugate in the composition is 15 to 21 mg / mL, for example, 21 mg / mL.

[0093] In embodiments further comprising the step of mixing the sterilization composition with an aqueous solution of sugar or sugar alcohol, the concentration of sugar or sugar alcohol (e.g., glucose) in the aqueous liquid composition is preferably 10 to 100 mg / mL, for example 20 to 100 mg / mL, 35 to 70 mg / mL, or 40 to 60 mg / mL. In more preferred embodiments, the concentration of sugar or sugar alcohol (e.g., glucose) in the composition is 45 to 55 mg / mL, for example 50 mg / mL.

[0094] In embodiments further comprising the step of mixing the sterilization composition with an aqueous solution of sugar or sugar alcohol, the aqueous liquid composition may contain additional components in addition to the conjugate and sugar. For example, the aqueous liquid composition may contain NaCl or another salt (e.g., NaCl, KCl, CaCl2, NaBr, MgCl2, choline chloride, NaHCO2, NaHPO4, KH2PO4, or a combination thereof), and / or citrate buffer, phosphate-buffered saline, or Ringer's solution. In preferred embodiments, the aqueous liquid composition contains NaCl. In one embodiment, the concentration of NaCl or another salt in the composition is 0.1 to 50 mg / mL, 0.5 to 3 mg / mL, or 1 to 2 mg / mL. In particularly preferred embodiments, the concentration of NaCl or another salt in the composition is 1.5 mg / mL. In one embodiment, the aqueous liquid composition comprises a sugar or sugar alcohol (e.g., glucose) at a concentration of 30 mg / mL and NaCl or another salt (e.g., NaCl) at a concentration of 1.5 mg / mL, wherein the concentrations of the sugar and NaCl or other salt are such that the aqueous liquid composition is isotonic. In one embodiment, the mass ratio of the sugar or sugar alcohol to the NaCl or other salt is 2:1 to 900:1. In a preferred embodiment, the mass ratio of the sugar or sugar alcohol to the NaCl or other salt is 5:1 to 120:1. In another preferred embodiment, the mass ratio of the sugar or sugar alcohol to the NaCl or other salt is 10:1 to 50:1. In a more preferred embodiment, the mass ratio of the sugar or sugar alcohol to the NaCl or other salt is 16:1 to 40:1. In a still more preferred embodiment, the mass ratio of the compound to the sugar or sugar alcohol is 20:1. In such embodiments, preferably, the aqueous solution of NaCl or another salt is a solution of NaCl. In such embodiments, preferably, the sugar or sugar alcohol is glucose.

[0095] Additionally, or alternatively, in one embodiment, NaCl or another salt for use in the method may be in the form of a sterile composition, e.g., a sterile solution, e.g., sodium chloride solution for injection (also called saline solution for injection). For example, NaCl or another salt for use in the method may be 10 -3Or better, for example, 10 -3 Or better, for example, 10 -5 Or better, or for example, 10 -6 Or it may have a better sterility assurance level (SAL). In one embodiment, NaCl, or another salt, is sterile and 10 -6 Alternatively, it may have a better SAL, such as NaCl, or another salt which is NaCl, in the form of a saline solution for injection.

[0096] In embodiments that include the step of mixing a sterilization composition with an aqueous solution, such as an aqueous solution of sugar or sugar alcohol, or an aqueous solution of NaCl, the method may include mixing the sterilization composition and / or mixing the aqueous solution of sugar or sugar alcohol, or an aqueous solution of NaCl, with sterile water, such as water for injection.

[0097] Sterilized composition of the present invention The present invention provides a sterilization composition comprising a conjugate of hyaluronic acid and a pharmaceutically active compound. For example, the sterilization composition can be obtained by the sterilization method of the present invention.

[0098] In a preferred embodiment of the present invention, the sterilization composition is 10 -6 Or it has a better level of sterility assurance (SAL).

[0099] In one embodiment of the present invention, the sterilization composition is in solid form. In a preferred embodiment, the sterilization composition is in powder form.

[0100] In one embodiment of the present invention, the sterilization composition comprises a conjugate of sodium hyaluronate, or hyaluronic acid, and a pharmaceutically active compound obtained or obtainable by the method of the present invention as described herein, and further comprises a sugar, or a sugar alcohol, for example, the composition is a sterilization aqueous liquid composition. In such embodiments, the sugar is preferably glucose, sucrose, fructose, or trehalose, and the sugar alcohol is mannitol, ethylene glycol, glycerol, sorbitol, or xylitol. For example, the sugar is glucose, sucrose, or trehalose, and the sugar alcohol is mannitol. Alternatively, the sugar is glucose or trehalose. Alternatively, the sugar is glucose or fructose. In the most preferred embodiment, the sugar is glucose. In another preferred embodiment, the sugar is not sucrose.

[0101] The composition may contain additional components in addition to the hyaluronic acid conjugate and sugar. For example, an aqueous liquid composition may contain NaCl or another salt (e.g., NaCl, KCl, CaCl2, NaBr, MgCl2, choline chloride, NaHCO2, NaHPO4, KH2PO4, or a combination thereof), and / or citrate buffer, phosphate-buffered saline, or Ringer's solution. In preferred embodiments, the composition may contain NaCl. In embodiments where the composition is a sterile aqueous liquid composition, the concentrations of the sterile conjugate, sugar, or sugar alcohol, and optionally, NaCl or another salt, may be present in the concentrations defined above with respect to the method of the present invention.

[0102] The present invention also provides a method for producing the sterile compositions and / or aqueous liquid compositions described herein, comprising mixing a sterile conjugate composition with an aqueous solution of a sugar or sugar alcohol. The present invention also provides a method for producing the aqueous liquid compositions described herein, comprising mixing a sterile conjugate composition with an aqueous solution of a sugar or sugar alcohol and an aqueous solution of NaCl or another salt (e.g., NaCl, KCl, CaCl2, NaBr, MgCl2, choline chloride, NaHCO2, NaHPO4, KH2PO4, or a combination thereof, preferably an aqueous solution of NaCl, KCl, CaCl2, NaHCO2, NaHPO4, KH2PO4, or a combination thereof, more preferably an aqueous solution of NaCl). In one embodiment, the method comprises mixing a sterile conjugate composition with an aqueous solution of a sugar or sugar alcohol having a concentration of 10 to 100 mg / mL. In a preferred embodiment, the aqueous solution of sugar or sugar alcohol has a concentration of 35 to 70 mg / mL. In a more preferred embodiment, the aqueous solution of sugar or sugar alcohol has a concentration of 40 to 60 mg / mL. In a more preferred embodiment, the aqueous solution of sugar or sugar alcohol has a concentration of 45 to 55 mg / mL. In a particularly preferred embodiment, the aqueous solution of sugar or sugar alcohol has a concentration of 50 mg / mL.

[0103] In alternative embodiments, the method involves mixing a sterile conjugate composition with an aqueous solution of sugar or sugar alcohol in a mass ratio of 1:50 to 5:1 of conjugate to sugar or sugar alcohol. In preferred embodiments, the mass ratio of conjugate to sugar or sugar alcohol is 1:12 to 5:2. In another preferred embodiment, the mass ratio of conjugate to sugar or sugar alcohol is 6:25 to 3:2. In a more preferred embodiment, the mass ratio of conjugate to sugar or sugar alcohol is 1:4 to 1:1. In an even more preferred embodiment, the mass ratio of conjugate to sugar or sugar alcohol is 1:3 to 1:1.5. In a particularly preferred embodiment, the mass ratio of conjugate to sugar or sugar alcohol is 1:2.

[0104] In addition or alternatively, in one embodiment, the method includes the step of mixing a sterile conjugate composition with an aqueous solution of sugar or sugar alcohol and an aqueous solution of NaCl or another salt having a concentration of 0.1 to 50 mg / mL. In a preferred embodiment, the aqueous solution of NaCl or another salt has a concentration of 0.5 to 3 mg / mL. In a more preferred embodiment, the aqueous solution of NaCl or another salt has a concentration of 1 to 2 mg / mL. In a more preferred embodiment, the aqueous solution of NaCl or another salt has a concentration of 1.5 mg / mL. In such embodiments, preferably, the aqueous solution of NaCl or another salt is a solution of NaCl. In such embodiments, preferably, the sugar or sugar alcohol is glucose.

[0105] In addition, or alternatively, in one embodiment, the method comprises mixing a sterile conjugate composition with an aqueous solution of sugar or sugar alcohol and an aqueous solution of NaCl or another salt in a mass ratio of sugar or sugar alcohol to NaCl or another salt of 2:1 to 900:1. In a preferred embodiment, the mass ratio of sugar or sugar alcohol to NaCl or another salt is 5:1 to 120:1. In another preferred embodiment, the mass ratio of sugar or sugar alcohol to NaCl or another salt is 10:1 to 50:1. In a more preferred embodiment, the mass ratio of sugar or sugar alcohol to NaCl or another salt is 16:1 to 40:1. In an even more preferred embodiment, the mass ratio of the conjugate to sugar or sugar alcohol is 20:1. In such embodiments, preferably, the aqueous solution of NaCl or another salt is a solution of NaCl. In such embodiments, preferably, the sugar or sugar alcohol is glucose.

[0106] In certain embodiments, the sugar or sugar alcohol for use in the manufacturing method may be in the form of a sterile composition, for example, a sterile glucose solution. In one embodiment, the sugar or sugar alcohol for use in the manufacturing method may be in the form of a sterile composition, for example, a sterile glucose solution (for example, a glucose solution for injection), for example, the composition may be 10 -3 Or better, for example, 10 -3Or better, for example, 10 -5 Or better, or for example, 10 -6 Or it has a better sterility assurance level (SAL). In one embodiment, the aqueous liquid composition is sterile and 10 -6 Or it has a better SAL.

[0107] Additionally, or alternatively, in one embodiment, NaCl or another salt for use in the manufacturing method may be in the form of a sterile composition, for example, a sterile solution (e.g., sterile NaCl solution, for example, sodium chloride solution for injection (also called physiological saline solution for injection)), for example, the composition is 10 -3 Or better, for example, 10 -3 Or better, for example, 10 -5 Or better, or for example, 10 -6 Or it has a better sterility assurance level (SAL). In one embodiment, the aqueous liquid composition is sterile and 10 -6 Or it has a better SAL.

[0108] Additionally, or alternatively, the manufacturing method may further include a step of filtration of an aqueous liquid composition comprising a sterile conjugate composition and a sugar or sugar alcohol. Filtration of the composition may be carried out by membrane filtration. In such embodiments, a membrane filter (e.g., a commercially available membrane filter) may be optionally used in conjunction with a sterile container, sterile syringe, syringe barrel, etc. For example, membrane filters with pore sizes of 0.05 μm to 20 μm, e.g., 0.4 to 8.0 μm (e.g., 0.4 μm, 0.5 μm, 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, or 8 μm), 0.5 to 6 μm, or 1 to 5 μm, particularly 5 μm, may be used.

[0109] The compositions of the present invention can be used in various medical settings and can be provided to patients in various ways, for example, by injection.

[0110] In certain embodiments of the present invention, the pharmaceutically active compound is diclofenac, and the sterile conjugate finds specific uses in the treatment of joint diseases, such as osteoarthritis and / or other joint conditions (e.g., osteoarthritis of the knee). For example, the composition of the present invention can be prepared into an injectable formulation and administered by injection to a joint (e.g., the knee). The patient may be a human patient. The composition of the present invention also finds uses in veterinary medicine, for example, in the treatment of horses.

[0111] In certain embodiments, the present invention provides a composition for use as a pharmaceutical (i.e., sodium hyaluronate, or hyaluronic acid, and, for example, obtained or obtainable by the method of the present invention, and / or 10 -6 The present invention also provides a sterilization composition (e.g., a sterilization aqueous liquid composition) comprising a conjugate of a pharmaceutically active compound according to the present invention, which has a better level of sterilization assurance.

[0112] Further aspects of the present invention relate to compositions of the present invention (i.e., sodium hyaluronate or hyaluronic acid) in humans or veterinary medicine, which are obtained or can be obtained, for example, by the method of the present invention, and / or 10 -6 The present invention includes the use of a sterile composition (e.g., a sterile aqueous liquid composition) comprising a conjugate of the pharmaceutically active compound according to the present invention, or having a better level of sterilization assurance.

[0113] The present invention also relates to a method for treating or preventing a disease or disorder in a subject, comprising a therapeutically effective amount of the composition of the present invention (i.e., sodium hyaluronate or hyaluronic acid, and, for example, obtained or obtainable by the method of the present invention, and / or 10 -6 The present invention provides a method comprising administering a sterile composition (e.g., a sterile aqueous liquid composition) comprising a conjugate of a pharmaceutically active compound according to the present invention, or having a better level of sterility assurance. Preferably, the composition is administered by injection. In a particularly preferred embodiment, the composition is administered by intra-articular injection.

[0114] According to a particular embodiment, the disease or disorder is a joint disease, such as osteoarthritis (e.g., osteoarthritis of the knee).

[0115] According to a particular embodiment, the disease or disorder is cataract.

[0116] According to a particular embodiment, the disease or disorder is cancer.

[0117] The present invention also provides the use of aqueous liquid compositions for the manufacture of pharmaceuticals for use in humans or veterinary medicine.

[0118] In certain embodiments, the pharmaceutical is intended for use in the treatment of joint diseases, such as osteoarthritis (e.g., osteoarthritis of the knee).

[0119] In another embodiment of this design, the pharmaceutical product is intended for use in cataract surgery.

[0120] In a further embodiment of this pattern, the pharmaceutical product is intended for use in cancer therapy.

[0121] In certain embodiments of the present invention, the pharmaceutically active compound is diclofenac, and the composition finds specific uses in the treatment of osteoarthritis and other joint conditions. For example, the composition of the present invention can be prepared into an injectable formulation and administered by injection to a joint (e.g., the knee). The patient may be a human patient. The composition of the present invention also finds uses in veterinary medicine, for example in the treatment of horses, for example in the treatment of equine osteoarthritis (e.g., knee osteoarthritis). [Examples]

[0122] Example 1 - Synthesis of hyaluronic acid diclofenac conjugate (conjugate 1) Step 1: Synthesis of [2-(2,6-dichlorophenylamino)-phenyl]-acetic acid 2-(2-tert-butoxycarbonylamino-ethoxy)-ethyl ester (compound 1) Diclofenac (50.0 g, 0.169 mol, 1.0 equivalent) and 2-[2-(BOC-amino)ethoxy]ethanol (69.5 g, 0.339 mol, 2.0 equivalents) were mixed in DCM (331 g), and the suspension was cooled to 1°C. 4-dimethylaminopyridine (DMAP) (3.0 g, 0.025 mol, 0.15 equivalents) was added, and the mixture was stirred at 1°C for 10-20 minutes. Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC HCl) (40.5 g, 0.211 mol, 1.25 equivalents) was added over 5 hours at 1°C. The mixture was stirred for a further 4 hours at 1°C, then warmed to 20°C and stirred for 12 hours. The mixture was quenched with water, and the two phases were separated. The organic phase was washed twice with water and concentrated to dryness under vacuum. The residue was purified by column chromatography on silica gel (1.5 kg). The yield was 72 g (88%), as a bright yellow oily substance, which solidified at ambient temperature.

[0123] Step 2: Synthesis of 2-(2,6-dichlorophenylamino)-phenyl]-acetic acid 2-(2-amino-ethoxy)-ethyl ester HCl salt (Compound 2) [2-(2,6-dichlorophenylamino)-phenyl]-acetic acid 2-(2-tert-butoxycarbonylamino-ethoxy)-ethyl ester (Compound 1, 65 g, 0.134 mol, 1.0 equivalent) from Step 1 was dissolved in DCM (665 g). HCl (2 M, 232 g, 0.650 mol, 4.9 equivalents) in diethyl ether was added, and the mixture was stirred at 20-25°C for 1 hour. The crude product was cooled to 3-7°C and stirred for 1 hour. The precipitated final product was isolated by filtration. The filtration cake was washed with a cold mixture of DCM / Et2O. The wet filtration cake was vacuum-dried at 20-30°C. The yield was 49 g (80%) as a white solid.

[0124] Step 3: Synthesis of hyaluronan succinyl ester (HSE) The sodium hyaluronate (NaHA) used in the synthesis is produced by bacterial fermentation (streptococcus) and ferments at 25°C to 1.54 ml. 3It had an intrinsic viscosity (IV) of / kg. The weight-average molar mass (M) was measured by AF4. w The value was 667 kDa (see Example 5 below).

[0125] Sodium hyaluronate (200 g, 0.50 mol, 1.0 equivalent) was stirred in formamide (22.6 kg). Pyridine (393 g, 5.0 mol, 10 equivalents), DMAP (6.1 g, 0.05 mol, 0.1 equivalent), and succinic anhydride (500 g, 5.0 mol, 10 equivalents) were added, and the reaction mixture was stirred at room temperature for 16 hours. The reaction was quenched by adding 25% NaCl aqueous solution (0.6 kg). The crude product was precipitated by adding ethanol, and the solid was separated from the liquid. The solid was stirred in 1% NaCl aqueous solution (20 kg). The crude product was precipitated by adding ethanol, and the product was separated from the liquid. The solid was stirred in 1% NaCl aqueous solution (20 kg). The viscous solution was filtered through a filter cloth for clarification, and the filter was washed with 1% NaCl aqueous solution (20 kg). The product was precipitated by the addition of ethanol, isolated by filtration, and washed with ethanol and acetone. The wet cake was vacuum dried at 22-28°C. The yield was 220 g (68%) as a white solid. The weight-average molar mass (M) was measured by HSE AF4. w The value was 579 kDa (see Example 5 below).

[0126] Step 4: Synthesis of HSE-diclofenac (conjugate 1) Hyaluronan succinyl ester (HSE) (220 g, 0.34 mol, 1.0 equivalent) from step 3 was stirred in purified water (5.5 kg). Dimethylformamide (DMF) (15.6 kg) was added, and the solution was stirred. N-methylmorpholine (17.7 g) was added, followed by the addition of a solution of 2-(2,6-dichlorophenylamino)-phenyl]-acetic acid 2-(2-amino-ethoxy)-ethyl ester HCl salt (compound 2, 38.8 g, 0.085 mol, 0.25 equivalents) in DMF (520 g). Hydroxybenzotriazole hydrate (HOBT) (1.16 g, 0.009 mol, 0.025 equivalents) was dissolved in DMF (160 g) and added to the reaction mixture. EDC HCl (16.2 g, 0.085 mol, 0.25 equivalents) was dissolved in DMF (260 g) and purified water (275 g) and added to the reactants. The mixture was stirred for 16 hours. The reaction was stopped by adding 25% NaCl aqueous solution (0.68 kg). The crude product was precipitated by adding ethanol. The solid was stirred in purified water (22 kg) for 16 hours. The pH was adjusted to 5.5-6.0 by adding 0.1 M NaOH. 25% NaCl aqueous solution (0.6 kg) was added, and the crude product was precipitated by adding ethanol. The product was separated from the liquid, and the solid was stirred in purified water (22 kg). The viscous solution was diluted with purified water (17.0 kg), filtered through a filter cloth for clarification, and the filter was washed with purified water (5.0 kg). A 25% NaCl aqueous solution (1.2 kg) was added to the filtrate, and the product was precipitated by the addition of ethanol. The solid product was isolated by filtration and washed with ethanol and acetone. The wet cake was dried under vacuum at 33-37°C. The yield was 225 g (90%) as a white solid. The diclofenac content was 6.3% w / w based on analytical UV spectrophotometric analysis using a calibration curve from diclofenac stock solution. The weight-average molar mass (M) of conjugate 1 was measured by AF4. w The value was 611 kDa (see Example 5 below).

[0127] Example 2a - Irradiation of compound 1 under argon (25 kGy) A sample of compound 1, prepared as described in Example 1, was irradiated with gamma rays. Step 1: Compound 1 was packaged in a primary polyethylene (PE) bag (as powder, sample size 1.0 g ± 0.1 g). Air was squeezed out of the primary PE bag and replaced with argon. The argon was squeezed out and the primary PE bag was closed with a zip tie. The primary PE bag was placed in an aluminum bag. Air was squeezed out of the aluminum bag and replaced with argon, the argon was squeezed out and the aluminum bag was closed with a heat seal (also called heat sealing) and labeled. The bags were then packaged in a carton box of approximately 32 × 21 × 21 cm.

[0128] Before the irradiation process, a carton containing one conjugate sample was placed inside a larger box filled with dry ice. The larger box measured approximately 46 x 46 x 57 cm and weighed 12-13 kg.

[0129] Step 2: The irradiation position is Cobalt 60 ( 60 Gamma-ray irradiation, emitted by Co), was used. A dose of 25 kGy was employed. Cobalt-60 was contained in stainless steel cylinders ("pencils") placed on racks and stored in an irradiation bunker in a pool 6 meters deep. The irradiation plant (Gammatom Srl, Italy) used a batch mode with totes. The distribution of pencils to the source racks, as well as the exposure time based on the requested dose and product density, was managed by validated software.

[0130] Example 2b - Irradiation of compound 1 under argon (32 kGy) The process of Example 2a was repeated, but a dose of 32 kGy was used.

[0131] Example 2c - Irradiation of compound 1 under air conditions (25 kGy) The process of Example 2a was repeated, but the following differences were observed in step 1. One sample of compound 1, 1.0 g ± 0.1 g, was packaged in a primary polyethylene (PE) bag. Air was squeezed out of the primary PE bag, and the primary PE bag was closed with a zip tie. The primary PE bag was placed in an aluminum bag. Air was squeezed out of the aluminum bag, the aluminum bag was closed with a heat seal, and a label was attached. This bag was then packaged in a carton box.

[0132] Example 2d - Irradiation of compound 1 under air conditions (32 kGy) The process of Example 2c was repeated, but in step 2, a dose of 32 kGy was used.

[0133] Example 2e - Irradiation of diclofenac, compound 1, and compound 2 under air (25 kGy) One sample of diclofenac (100 mg ± 10 mg), one sample of [2-(2,6-dichloro-phenylamino)-phenyl]-acetic acid 2-(2-tert-butoxycarbonylamino-ethoxy)-ethyl ester (compound 1) (100 mg ± 10 mg), and one sample of 2-(2,6-dichloro-phenylamino)-phenyl]-acetic acid 2-(2-amino-ethoxy)-ethyl ester HCl salt (compound 2) (100 mg ± 10 mg) were each packaged in a polyethylene (PE) bag. Air was squeezed out of the PE bags and the PE bags were closed with zip ties. Each PE bag was then placed in an aluminum bag. Air was squeezed out of the aluminum bags, the aluminum bags were closed with heat seals, and labels were applied. The three bags were then packaged in a carton box.

[0134] Before undergoing the irradiation process, carton boxes containing samples of diclofenac, compound 1, and compound 2 were placed inside a larger box containing dry ice. The larger box measured 46 x 46 x 57 cm and weighed 12-13 kg.

[0135] The irradiation position is Cobalt-60 ( 60Gamma-ray irradiation, emitted by Co), was used. A dose of 25 kGy was employed. Cobalt-60 was contained in stainless steel cylinders ("pencils") placed on racks and stored in an irradiation bunker in a pool 6 meters deep. The irradiation plant (Gammatom Srl, Italy) used a batch mode with totes. The distribution of pencils to the source racks, as well as the exposure time based on the requested dose and product density, was managed by validated software.

[0136] Example 2f - Irradiation with sodium hyaluronate under air conditions (32 kGy) The sodium hyaluronate (NaHA) sample used in the synthesis of conjugate 1 described in Example 1 (Step 1) was irradiated with gamma rays. Step 1: NaHA was packaged in a polyethylene (PE) bag (as a powder, sample size 1.0 g ± 0.1 g). Air was squeezed out of the primary PE bag. The PE bag was closed with a zip tie. The PE bag was placed in an aluminum bag. Air was squeezed out of the aluminum bag, the aluminum bag was sealed with a heat seal, and a label was attached. The bags were then packaged in a carton box measuring approximately 32 x 21 x 21 cm.

[0137] Before undergoing the irradiation process, one box containing the NaHA sample was placed inside a larger box containing dry ice. The larger box measured approximately 46 x 46 x 57 cm and weighed 12-13 kg.

[0138] Step 2: The irradiation position is Cobalt 60 ( 60 Gamma-ray irradiation, emitted by Co), was used. A dose of 32 kGy was employed. Cobalt-60 was contained in stainless steel cylinders ("pencils") placed on racks and stored in an irradiation bunker in a pool 6 meters deep. The irradiation plant (Gammatom Srl, Italy) used a batch mode with totes. The distribution of pencils to the source racks, as well as the exposure time based on the requested dose and product density, was managed by validated software.

[0139] Example 2g - Irradiation with HSE under air (32kGy) The HSE sample used in the synthesis of compound 1 described in Example 1 (Step 3) was irradiated with gamma rays. Step 1: HSE was packaged in polyethylene (PE) bags (as powder, sample size 1.0g ± 0.1g). Air was squeezed out of the primary PE bag. The primary PE bag was closed with a zip tie. The PE bag was placed in an aluminum bag. Air was squeezed out of the aluminum bag, the aluminum bag was sealed with a heat seal, and a label was attached. The bags were then packaged in a carton box measuring approximately 32 x 21 x 21 cm.

[0140] Before undergoing the irradiation process, one box containing the HSE sample was placed inside a larger box containing dry ice. The larger box measured approximately 46 x 46 x 57 cm and weighed 12-13 kg.

[0141] Step 2: The irradiation position is Cobalt 60 ( 60 Gamma-ray irradiation, emitted by Co), was used. A dose of 32 kGy was employed. Cobalt-60 was contained in stainless steel cylinders ("pencils") placed on racks and stored in an irradiation bunker in a pool 6 meters deep. The irradiation plant (Gammatom Srl, Italy) used a batch mode with totes. The distribution of pencils to the source racks, as well as the exposure time based on the requested dose and product density, was managed by validated software.

[0142] Example 3a - Sterilization of compound 1 by irradiation under argon (25 kGy) The sample of compound 1, prepared as described in Example 1, was sterilized by gamma irradiation as follows. Step 1: Compound 1 was packaged (as powder) in three PE bags and one aluminum bag in sample sizes of 20.0 g ± 0.1 g, 1.0 g ± 0.1 g, and 2 × 130 mg ± 5 mg, respectively. More specifically, each sample of compound 1 was packaged in a primary polyethylene (PE) bag. Air was squeezed out of the primary PE bag and replaced with argon. Argon was squeezed out and the primary PE bag was closed with a zip tie. The primary PE bag was placed in a secondary PE bag. Air was squeezed out of the secondary PE bag and replaced with argon. Argon was squeezed out and the secondary PE bag was closed with a heat seal. The secondary PE bag was placed in a tertiary PE bag. Air was squeezed out of the tertiary PE bag and replaced with argon. Argon was squeezed out and the tertiary PE bag was closed with a heat seal. The tertiary PE bag was placed in an aluminum bag. Air was squeezed out of the aluminum bags, replaced with argon, the argon was squeezed out, the aluminum bags were heat-sealed and labeled. Four bags (20.0g ± 0.1g, 1.0g ± 0.1g, and 2 × 130mg ± 5mg) were then packaged in a carton box measuring 32.0 × 21.0 × 21.0 cm. The total weight of the box was 0.5 kg, and the apparent density was 0.035 g / cm³. 3 This process was repeated until five carton boxes were produced, each containing four bags of packaged samples of conjugate 1 (20.0 g ± 0.1 g, 1.0 g ± 0.1 g, and 2 × 130 mg ± 5 mg).

[0143] It should be noted that the orientation of the four bags of packaged samples within the box was not important to the method and therefore was not described. However, it was preferable to maintain the same orientation of the samples within the box, and the same orientation of the four bags of packaged samples within each box was maintained as similarly as possible. (See reference ISO 11137-3 9.2.1.3. Low-density products tend to be fairly homogeneous so that the orientation of individual products within the irradiation chamber is unlikely to have a significant effect on the dose distribution when irradiated with gamma rays.)

[0144] Before undergoing the irradiation process, one box containing one conjugate sample was placed inside a larger box containing dry ice. The larger box measured 46.0 × 46.0 × 57.0 cm, had a total weight of 12.6 kg, and an apparent density of 0.104 g / cm³. 3 That was the case.

[0145] Step 2: The irradiation position is Cobalt 60 ( 60 Gamma-ray irradiation, emitted by Co), was used. A dose of 25 kGy was employed. Cobalt-60 was contained in stainless steel cylinders ("pencils") placed on racks and stored in an irradiation bunker in a pool 6 meters deep. The irradiation plant (Gammatom Srl, Italy) used a batch mode with totes. The distribution of pencils to the source racks, as well as the exposure time based on the requested dose and product density, was managed by validated software.

[0146] Example 3b: Verification of the sterilization process of conjugate 1 The sterilization method was validated according to ISO 11137-2:2013, VDmax 25 (single batch validation). The unified standard ISO 11137 requires that products labeled as sterilized undergo at least 10 -6 The achievement of the Sterility Assurance Level (SAL) is required. 10 -6 SAL stands for non-sterile unit, meaning a probability of 1 / 1,000,000.

[0147] Verification procedure protocol and results The sterilization method was validated using a 130 ± 5 mg sample of conjugate 1, packaged as described in Example 2a, Step 1.

[0148] 46 samples of compound 1 were individually packaged (as powder) in three PE bags and an aluminum bag, each with a sample size of 130 mg ± 5 mg. More specifically, each sample of compound 1 was packaged in a primary polyethylene (PE) bag. Air was squeezed out of the primary PE bag and replaced with argon. Argon was squeezed out and the primary PE bag was closed with a zip tie. The primary PE bag was placed in a secondary PE bag. Air was squeezed out of the secondary PE bag and replaced with argon. Argon was squeezed out and the secondary PE bag was closed with a heat seal. The secondary PE bag was placed in a tertiary PE bag. Air was squeezed out of the tertiary PE bag and replaced with argon. Argon was squeezed out and the tertiary PE bag was closed with a heat seal. The tertiary PE bag was placed in an aluminum bag. Air was squeezed out of the aluminum bag, replaced with argon, argon was squeezed out, the aluminum bag was closed with a heat seal, and a label was applied.

[0149] The usage of each of the 46 samples is outlined below. • Five samples for validation of the bioburden test • 10 samples for bioburden determination • 11 samples for verification of sterility testing • Irradiation at the verification dose and 10 samples for sterility testing using TSB medium. • Irradiation at the verification dose and 10 samples for sterility testing using FTM medium.

[0150] Validation of the Bioburden trial Bioburden validation was performed using an inoculation method with a Staphylococcus aureus suspension, and five samples of 130±5 mg of conjugate 1 were tested.

[0151] Biobaden decided Ten samples of 130±5 mg of conjugate 1 were tested using triptycase soy agar (TSA) and (SDA) agar. Conjugate 1 (solid) in the primary packaging (PE bag) was tested as one unit.

[0152] Total Aerobic Microbial Count (TAMC): A sample containing 10 microorganisms was mixed in a sterile bag with 20 mL of MRD (Maximum Recovery Dilution) solution. The bag containing the sample and MRD solution was shaken at 240 rpm for 30 minutes. 10 mL of this solution was placed in an empty 14 cm petri dish. Liquid TSA was added, and the solution was homogeneously mixed through agar. After the mixture solidified, the plate was incubated. The incubation conditions for TSA were as follows: 32.5°C ± 2.5°C for 3 to 5 days.

[0153] Total number of yeasts and molds (TYMC): The same procedure was followed for testing yeasts and molds using SDA agar, with 10 samples of 130±5 mg of conjugate 1. The incubation conditions for SDA were as follows: 22.5℃±2.5℃ for 5-7 days.

[0154] Table 1 below shows the total bioburden, which is the total aerobic microbial count (TAMC) and the total number of yeasts and fungi (TYMC) for each of the 10 samples.

[0155] [Table 1]

[0156] Calculation of average bioburden and verification dose The average total bioburden for the verification dose was calculated to be 4.9 CFU / unit. Then, using the VDmax25 method and Table 9 of ISO 11137-2:2013, the appropriate verification dose was determined, and the calculated verification dose was as follows: Minimum: 5.67 kGy, Average: 6.3 kGy, Maximum: 6.93 kGy

[0157] SAL 10 -6 To test the dose (VDmax25) for this, it is necessary to irradiate 1 million samples and perform a sterility test. Using Table 9 (Verification Dose) of ISO 11137-2:2013, 10 -1The SAL is determined first for the validation dose. The experiment requires only 10 samples. If zero or one positive result is obtained in the sterility test of these samples after using the validation dose, a sterility dose of 25 kGy can be confirmed.

[0158] Irradiation of 20 samples at verification doses. Twenty samples of conjugate 1 containing 130±5 mg were irradiated. The irradiation location was Cobalt-60. 60 Gamma-ray irradiation emitted by Co) was used. The minimum required dose of 5.67 kGy was used. Actual doses were allowed to vary by up to ±10% from the selected dose. The irradiation plant (TBI 8450, Ionisos Baltics) was used in batch mode. The required dose and exposure time based on product density were managed by validated software. Irradiation was carried out in accordance with the requirements of ISO 11137:2015 and ISO 13485:2016. Absorbed dose was controlled by a dosimetry system.

[0159] The results from the dose measurement system are shown below. Applied verification doses: minimum: 5.83 kGy, maximum: 6.24 kGy

[0160] Verification of sterility tests To demonstrate the effectiveness of the sterility test, a so-called conformity test (bacteriostatic / fungal (B&F) test) was performed in accordance with ISO 11737-2:2013 to ensure that the ability to maintain microbial growth was not affected. To verify the sterility test, a growth-promoting check was performed to eliminate false negative results.

[0161] For each product / culture medium combination, 0.1 mL of each microbial suspension (1-100 cfu) was inoculated according to Table 2.

[0162] [Table 2]

[0163] The incubation conditions were as follows: For PA, SA, BA, and CS, the samples were incubated at 30°C to 35°C for 7 days, or until growth was observed. For CA and AB, the samples were incubated at 20°C to 25°C for 7 days, or until growth was observed.

[0164] All sterilization tests showed growth. This means the product did not show any inhibitory effect on sterility testing. Therefore, it can be concluded that the sterilization tests were effective.

[0165] Sterility test of irradiated samples Twenty irradiated samples of conjugate 1 were individually subjected to sterility testing according to ISO 11737-2:2013. According to ISO, this can be carried out using one medium, tripty soy broth (TSB). In this case, the test was carried out using two different media (TSB and liquid thioglycolate medium (FTM)). Sterility testing was performed by direct inoculation according to ISO 11737-2:2013.

[0166] The following results were observed after 14 days of incubation for 10 irradiated samples subjected to sterility testing using TSB medium. Number of positive results: 0 Number of negative results: 10

[0167] Ten irradiated samples were subjected to sterility testing using FTM medium, and the following results were observed after 14 days of incubation. Number of positive results: 0 Number of negative results: 10

[0168] Summary of the determination of the minimum sterilization dose (kGy) The average bioburden was found to be 4.9 CFU / unit, which resulted in a validation dose of approximately 6.3 kGy (i.e., 6.3 kGy ± 10%). This validation dose was successfully applied, and the product units were tested for sterility. All 20 tests were observed to be negative. Therefore, the validation experiment was successful.

[0169] Acceptance of the minimum dose as a sterilization dose A typical sterilization dose of approximately 25 kGy is 10 kGy per conjugate. -6 It was shown that the Sterility Assurance Level (SAL) could be achieved.

[0170] Example 4: Diclofenac, Compound 1, and Compound 2 before and after irradiation (25 kGy) 1 H-NMR, and 13 Analysis by C-NMR and HPLC-UV method As described in Example 2e above, samples of diclofenac, [2-(2,6-dichlorophenylamino)-phenyl]-acetic acid 2-(2-tert-butoxycarbonylamino-ethoxy)-ethyl ester (compound 1), and 2-(2,6-dichlorophenylamino)-phenyl]-acetic acid 2-(2-amino-ethoxy)-ethyl ester HCl (compound 2) before and after irradiation were prepared by dissolving each compound (33-36 mg) in deuterated dimethyl sulfoxide (DMSO-d6). 1 H, and 13 The 1C-NMR spectra were recorded using a Bruker Advance spectrometer at 400 MHz and 100 MHz, respectively. 1 H-NMR, and 13 Analysis was performed by 13C-NMR and HPLC-UV.

[0171] result 1 H, and 13No differences were observed in the 1C-NMR spectra, nor in the spectra of each sample before and after irradiation. The HPLC purity of diclofenac, compound 1, and compound 2 did not differ by more than 0.04% before and after irradiation (see Table 3 below). The results of this example demonstrate that diclofenac, compound 1, and compound 2 are stable under gamma-ray irradiation conditions.

[0172] [Table 3] * Average of 2 injections

[0173] Example 5: Analysis of compound 1 before and after irradiation method i) FT-IR: As described in Examples 2b and 2d above, under air or argon, gamma rays (32 kGy, 60 The FT-IR spectra of the solid of conjugate 1 before and after irradiation with Co) were obtained from European Pharmacoepia 11. th Recordings were made at room temperature using a Spectrum 100 FT-IR (Perkin Elmer) spectrometer, following the procedure described in edition 2.2.24 monograph.

[0174] ii) Diclofenac content and degree of substitution: As described in Examples 2b and 2d above, before irradiation, gamma rays (32 kGy, 60 The composite 1 after irradiation with Co), and as described in Example 2a above, under argon, gamma rays (25 kGy, 60 The total content of diclofenac and the degree of substitution by diclofenac in conjugate 1 after irradiation with Co) were analyzed by UV spectrophotometric analysis using a calibration curve from a diclofenac stock solution. The quantitative measurement of diclofenac (w / w%) was calculated using diclofenac as a reference, and the absorbance of the sample was measured dually at 275 nm.

[0175] The amounts of free diclofenac and all diclofenac-related impurities were determined by HPLC analysis using the conditions shown in Tables 4 and 5 below. The amount of bound diclofenac in conjugate 1 was calculated by subtracting the amount of free diclofenac (HPLC) from the total amount of diclofenac (UV).

[0176] [Table 4]

[0177] [Table 5]

[0178] iii) Molecular weight (average molar mass) Analysis of molecular weight (average molar mass, more specifically, weight-average molar mass (M) w ) Measured as and expressed in kDa): • Before irradiation, composite 1, As described in Examples 2b and 2d above, under air or argon, gamma rays (32 kGy, 60 After irradiation with Co, the combined body 1, As described in Example 2a above, under argon, gamma rays (25 kGy, 60 After irradiation with Co, the combined body 1, • Before irradiation, HSE (succinyl-substituted sodium hyaluronate from step 3 of Example 1a), • In air, gamma rays (32 kGy, 60 After irradiation with Co (Example 2g), HSE, • Before irradiation, the unsubstituted sodium hyaluronate (NaHA) salt, which was used as the starting material in step 3 of Example 1, and • In air, gamma rays (32 kGy, 60 NaHA after irradiation with Co (Example 2f), Asymmetric flow-field flow fractionation (AF4) analysis was performed as follows:

[0179] Sample preparation: Stock solutions of each sample were prepared using a carrier liquid (0.2 M aqueous NaCl containing 3 mM NaN3) to a typical concentration of 1 mg / mL. The samples were placed on a magnetic stirrer at room temperature for 6 hours, and then stored at 2–8°C (Day 0). On Day 2, dilutions of each sample were prepared in glass vials using the carrier liquid (0.2 mg / mL), and these were then placed on a rocking table for 30 minutes before analysis.

[0180] Asymmetric flow-field flow fractionation (AF4) analysis was performed using an Eclipse III (Wyatt technology) connected to an 1100 series LC system consisting of an ERC-3415 vacuum degasser (ERC), a G1311A pump, a G1329A autosampler, and a G1315B diode array (UV) detector (all from Agilent Technologies). A Dawn Heleos II multi-angle light scattering (MALS) detector and an Optilab t-Rex differential refractive index (dRI) detector (both from Wyatt technology) were connected online after the channel. The UV detector was monitored at 280 nm. MALS used a laser with a wavelength of 658 nm, and scattered light was measured using 17 detectors in an aqueous mobile phase. The dRI detector used a lamp monitored at a wavelength of 658 nm. Data acquisition was performed using an Astra 6.2 (Wyatt technology). The autosampler was set to maintain the sample vial at 8°C. Fractionation was performed at ambient temperature (approximately 22°C). Separation was performed using a detector flow rate of 0.50 mL / min and a system pressure of approximately 9 bar. Performance tests for AF4 separation and UV-FL-MALS-RI detection were conducted by analyzing a solution of bovine serum albumin. The carrier liquid was 0.2 M NaCl containing 3 mM NaN3 to avoid bacterial growth in the system. Centrifugation or filtration was not performed on the samples. Unless otherwise specified, measurements were performed in triple replication.

[0181] Data were evaluated using Astra 6.1 (Wyatt technology). The dRI detector showed a nonlinear background signal, compensated by subtracting the signal from the blank analysis (i.e., carrier liquid). Molar mass calculations were performed according to the Berry method, with a first-order fit to scattering detectors 8-15 and 0.167 mL·g. -1 The analysis was performed using the refractive index increment dn / dc. Using AF4 in combination with a UV-FL-MALS-RI detector, light scattering and concentration data were used to determine the weight-average molar mass (M w The ) was obtained directly. The radius of gyration of the cross section (Rg) was obtained from MALS and its angle dependence. The quadratic virial coefficient term was assumed to be negligible.

[0182] result i) FT-IR Irradiation under air or argon (32 kGy, 60 FT-IR of compound 1 was recorded before and after irradiation (Co). The spectral bands are as follows: before irradiation, after irradiation in air (32 kGy), and after irradiation in argon (32 kGy). 60 Regarding conjugate 1 after Co), it remains identical, indicating that there was no structural change in conjugate 1 after irradiation.

[0183] ii) Diclofenac content and degree of substitution: Before irradiation, the combined body 1 was subjected to gamma rays (32 kGy, under air or argon). 60 The combined body 1 after irradiation with Co, and under argon, gamma rays (25 kGy, 60 For conjugate 1 after irradiation with Co), the degree of substitution of diclofenac, free diclofenac, and diclofenac in the conjugate, as well as the total weight percentage of total impurities, were measured. The results are shown in Table 6 below.

[0184] [Table 6] a Contains free diclofenac.

[0185] The results in Table 6 show that irradiation of conjugate 1 with gamma rays under air or argon conditions does not significantly change the amount of bound diclofenac in conjugate 1, indicating that gamma irradiation does not significantly affect the structure of conjugate 1, particularly the binding of diclofenac to the conjugate.

[0186] iii) Molecular weight (average molar mass) The average molar mass of compound 1 (more specifically, the weight-average molar mass, M) w ) before irradiation and under argon, gamma rays (32kGy, 60 Co), or gamma rays (25 kGy, under argon, 60 The measurement was taken by AF4 after irradiation with Co). For comparison, the average molar mass (specifically, M) of succinyl-substituted sodium hyaluronate from step 3 of Example 1a and the unsubstituted sodium hyaluronate salt used as a starting material in step 3 of Example 1 (NaHA) was measured. w ) also emits gamma rays (32 kGy, 60 Measurements were taken before and after irradiation with Co) (Examples 2g and 2f). The results were as follows.

[0187] NaHA before irradiation: The sample eluted as a very broad peak between 5 and 47 minutes in dRI and MALS. The sample did not show a UV signal. The weight-average molar mass (M) of the sample was... w The peak was 667 (±41) kDa. From the MALS and angle dependence, the z-mean cross-sectional radius of gyration (Rg) of the peak was obtained, which was 83 (±9) nm.

[0188] NaHA after irradiation (32 kGy in air) The sample eluted in dRI and MALS within 2–15 minutes. The sample did not have a UV signal. The weight-average molar mass (M) of the sample was... w The element's amplitude was 79 (±4) kDa. Since the element's size is not angularly dependent when it approaches the lower limit of the MALS detector size (10 nm), there is insufficient radius data to determine the cross-sectional radius of rotation (Rg).

[0189] Comparison of NaHA before and after irradiation When comparing NaHA before and after irradiation, it can be observed that the molar mass decreased. The main peak shifted to a lower retention time, indicating a size change most likely due to depolymerization by irradiation.

[0190] HSE before irradiation The sample eluted between 5 and 40 minutes in dRI and MALS. The sample had no UV signal. The weight-average molar mass (M w ) was 579 (±48) kDa. From MALS and the angle dependence, the z-average cross-sectional radius of gyration (Rg) of the peak could be obtained, which was 77 (±3) nm.

[0191] HSE after irradiation (32 kGy in air)<0**00898>The sample eluted between 2 and **15 minutes in RI and MALS. The sample had no UV signal. The weight-average molar mass (M<** w ) was 94 (±3) kDa. When the size of the component approached the size lower limit of the MALS detector, which is 10 nm, there was no angle dependence, so there was not enough radius data for the determination of the cross-sectional radius of gyration (Rg). <** <**

[0192] <** Comparison of HSE before and after irradiation<** When comparing HSE before and after irradiation, it was observed that the molar mass decreased. The main peak shifted to a lower retention time, indicating a size change most likely due to depolymerization by irradiation. <** <**

[0193] <** Conjugate 1 before irradiation<** The sample eluted as one very broad peak in dRI, MALS, and UV from 5 minutes to the end of the analysis. The sample had a UV signal characteristic of substitution by diclofenac. The weight-average molar mass (M<** w ) was 611 (±58) kDa. The z-average cross-sectional radius of gyration (Rg) could not be determined due to the quality of the radius data.

[0194] Conjugate 1 after irradiation (32 kGy under argon) The sample eluted between 2 and 30 minutes in dRI, MALS, and UV. Some large components eluted between 30 and 50 minutes, but their concentrations were very low (no dRI or UV signal in this region, only MALS). Since the concentrations of these large components were low, it was impossible to accurately determine their molar masses. The weight-average molar mass (M w ) of the sample was 354 (±37) kDa. From MALS and the angular dependence, the z-average cross-sectional radius of gyration (Rg) of the peak could be obtained, which in this case was 34 (±5) nm.

[0195] [[ID=A]] Comparison of Conjugate 1 before and after irradiation (32 kGy under argon) When Conjugate was compared before and after irradiation (32 kGy under argon), it was observed that the molar mass decreased, but to a lesser extent compared to NaHA and HSE after irradiation. The main peak also shifted to a lower retention time, indicating a size change most likely due to depolymerization by irradiation.

[0196] Conjugate 1 after irradiation (25 kGy under argon) The sample eluted between 2 and 40 minutes as one very broad peak in dRI, MALS, and UV. The MALS signal tailed to the end of the chromatogram. The noise or "spikes" in the MALS signal after 30 minutes suggested that large components or insufficiently dissolved components were eluting. The amount of material in the tail of the peak was very small. Reliable molar mass data could only be obtained up to 25 minutes. The weight-average molar mass (M w ) of the sample was 379 (±56) kDa. Since there was no angular dependence when the size of the components approached 10 nm, the lower size limit of the MALS detector, there was not enough radius data for the determination of the cross-sectional radius of gyration (Rg).

[0197] Comparison of Conjugate 1 before and after irradiation (25 kGy under argon) Comparing conjugate 1 before and after irradiation (25 kGy under argon), the molar mass decreased, but to a lesser extent compared to NaHA and HSE after irradiation. The main peak also shifted to a lower retention time, indicating a size change most likely attributable to depolymerization due to irradiation. The molar mass decreased to a similar level as that of conjugate 1 sampled after 32 kGy irradiation under argon.

[0198] Summary and comparison of results for NaHA, HSE, and conjugates. The summarized results are shown in Table 7 below.

[0199] [Table 7] a The percentage of the weight-average molar mass retained after irradiation, compared to the weight-average molar mass of the substance before irradiation.

[0200] The results in Table 4 show that for compound 1, the average molecular weight (more specifically, the weight-average molar mass) of the compound decreased by 44% after irradiation in air compared to the average molecular weight (more specifically, the weight-average molar mass) before irradiation. When irradiation was performed under an argon atmosphere, the average molecular weight (more specifically, the weight-average molar mass) decreased by 42% or 38% compared to the average molecular weight (more specifically, the weight-average molar mass) before irradiation.

[0201] In contrast, the decrease in the average molecular weight (more specifically, the weight-average molar mass) of hyaluronic acid caused by gamma irradiation was far more significant in hyaluronic acid materials that did not contain diclofenac. Succinyl-substituted HA showed an 84% decrease in weight-average molar mass after irradiation compared to the pre-irradiation value, while unsubstituted HA showed an even higher decrease of 88% in weight-average molar mass after irradiation.

[0202] The results of this experiment show that hyaluronic acid substitution significantly reduces the susceptibility of conjugate 1 to depolymerization. Irradiated NaHA and irradiated HSE have nearly similar retention times and weight-average molar masses of 79 and 94 kDa, respectively. On the other hand, irradiated conjugate 1 has weight-average molar masses of 342 kDa (32 kGy in air), 354 kDa (32 kGy in argon), or 379 kDa (25 kGy in argon).

[0203] Example 6: Study of the release of diclofenac and other compounds from conjugate 1 before and after irradiation. The release of diclofenac (compound A) and compounds B, C, D, E, and F from conjugate 1 before and after irradiation was compared by incubating conjugate 1 in two different media: a) 150 mM PBS buffer and b) human plasma (1 μg / mL) at 37°C for 48 hours. The release of diclofenac and the other two compounds was monitored and quantified by ULC-MS at LLOQ = 10 nM (see Tables 11 and 12, and Tables 13 and 14 below).

[0204] method As described in Example 3a above, the bond 1 before irradiation, or the bond 1 after irradiation under argon (25 kGy, 604 μL of a 100 μM stock solution of (Co) in 50% DMSO was added to 396 μL of PBS and human gender plasma to obtain a 1 μM incubation concentration of conjugate 1. The spiked study matrix was incubated at 37 °C with continuous shaking at 600 rpm for 48 hours, and samples were taken at 0, 2, 6, 24, and 48 hour time points. The samples were quenched after collection by precipitation with two volumes of acetonitrile containing 100 mg / mL of warfarin as an internal standard (50 μL of sample + 100 μL of precipitation solution containing internal standard), and then stored at -20 °C until analysis. For analysis, the precipitated samples were centrifuged at 2272 × g for 10 minutes at room temperature. The supernatant was transferred to a 96-well plate for analysis and subjected to LC / MS analysis. LC-MS methods for the analysis and quantification of compounds A (diclofenac), B, C, D, E, and F are shown in Tables 9 and 10 below. Standard samples were prepared in PBS and human plasma at concentrations of 10 - 50000 nM of compounds A (diclofenac), B, C, D, E, and F, which are possible degradation products of conjugate 1, shown in Table 8 below, by spiking the matrix with 1 volume of spiking solution (50% DMSO) and 9 volumes of matrix, and otherwise preparing them for analysis as samples containing conjugate 1.

[0205]

Table 8

[0206]

Table 9

[0207]

Table 10

[0208] Results During the 48-hour study period, the concentration profiles of compounds A, B, C, D, E, and F released from conjugate 1 before and after irradiation were nearly identical in both PBS and human plasma (see Tables 11 and 12 (PBS buffer results) and Tables 13 and 14 (human plasma results) below). This suggests that conjugate 1, both before and after irradiation, exhibits a similar release pattern over time, regardless of the irradiation process. The results also demonstrate, as shown in Example 5, that gamma irradiation and changes in molecular weight (weight-average molar mass) do not alter the amount of bound diclofenac in the molecule or the release of its individual components.

[0209] [Table 11]

[0210] [Table 12]

[0211] [Table 13]

[0212] [Table 14]

[0213] Example 7: Formulation of conjugate 1 after irradiation and sterile filling The formulation solvent was prepared in a 2 L polyethylene terephthalate glycol (PETG) medium bottle by mixing 250 mL of 0.9% sterile sodium chloride solution with 350 mL of water for injection (WFI) to obtain a 0.375% physiological saline solution. Then, the resulting 0.375% physiological saline solution (600 mL) was mixed with 900 mL of 5% sterile glucose solution to obtain a final 3% glucose, 0.15% sodium chloride mixture. A solution of conjugate 1 was then mixed with 19.6 g of sterile conjugate 1 (irradiated under argon at 25 kGy, as described in Example 3a above). 60Co) was prepared in a new 2 L PETG medium bottle by adding it to 880 mL of formulation solvent in four separate additions. The mixture was placed on a shaker and gently shaken at room temperature for 2–3 hours. The bottle was then transferred to a refrigerator and stored overnight at 2–8°C. The next day, the mixture was shaken again at room temperature for 1–3 hours. The resulting homogeneous mixture was then filled into glass vials (6 mL per vial) using an electronic pipette / dispenser and a sterile 10 mL combitip, followed by the attachment of a stopper and cap, and then crimping. The vials were packaged and stored at 2–8°C.

[0214] Example 8: Analysis of irradiated conjugate 1 after formulation i) Sterilization after formulation method The sterility of the solution of conjugate 1, formulated according to Example 7, was determined to be in accordance with the European Pharmacopoeia, the United States Pharmacopoeia, and the Japanese Pharmacopoeia (Ph.Eur.11 th edition 2.6.1:Sterility monograph;USP 43 rd edition, <71> :Sterility Tests monograph;and Japanese Pharmacopoeia 18 th The tests were performed according to the procedure for microbial testing via direct inoculation, as per edition 4.06: Sterility Test monograph.

[0215] result After formulation (as in Example 7 above), the solution was subjected to a sterilization test. After 21 weeks, the solution remained sterile (maintained at 2-8°C).

[0216] ii) Diclofenac content, degree of substitution, and impurities after formulation method Total diclofenac content (mg / mL) determined by UV light The total amount of diclofenac in the sample was evaluated at the following time points: at the time of formulation, and at 1 month, 3 months, and 6 months after formulation. Diclofenac concentrations were obtained by measuring the absorbance of the solution using spectrophotometric (UV) spectroscopy. For quantitative measurement of diclofenac (w / v), diclofenac was calculated using a 6-point calibration with diclofenac as a reference, and the absorbance of the sample was measured twice at 275 nm.

[0217] Content of free diclofenac and related impurities (w / w%) of diclofenac as determined by HPLC-UV The content of free diclofenac and related impurities (w / w%) of diclofenac in samples of conjugate 1, formulated according to Example 7 and maintained at 2–8°C, was measured at the following time points: at formulation, and at 1 month, 3 months, and 6 months post-formulation. Unknown related substances were quantified against the standard peak response of diclofenac, as these impurities were assumed to contain a diclofenac moiety. This method used an XBridge BEH C18 column (3.5 μm, 150 mm × 4.6 mm, 130 Å), mobile phase A: 0.1 v / v% TFA in water, mobile phase B: 0.1 v / v% TFA in acetonitrile. The sample solvent for the reference standard was acetonitrile / water (78:22 v / v). The sample solvent for the test substance was acetonitrile. Table 15 below shows the gradient profile used in this analytical method.

[0218] [Table 15]

[0219] Degree of substitution by diclofenac (calculated value) The compound was formulated according to Example 7, and at the following time points: at formulation, and at 1 month, 3 months, and 6 months after formulation, the degree of substitution of diclofenac in conjugate 1 in the sample of conjugate 1, maintained at 2-8°C, was calculated by subtracting the amount of free diclofenac (HPLC) from the total amount of diclofenac (UV).

[0220] result The results are shown in Table 16 below. As can be seen from Table 16, total diclofenac, free diclofenac, degree of substitution, and total impurities did not change significantly over the test period. This experiment indicates that irradiated conjugate 1, when formulated, was stable at 2–8°C for at least 6 months.

[0221] [Table 16] a Contains free diclofenac.

Claims

1. A method for preparing a sterile composition comprising a conjugate of sodium hyaluronate or hyaluronic acid with a pharmaceutically active compound, To provide a conjugate of sodium hyaluronate or hyaluronic acid with a pharmaceutically active compound, A preparation method comprising exposing the aforementioned compound to ionizing radiation.

2. The method according to claim 1, wherein the ionizing radiation is beta, gamma, or X-ray radiation.

3. The method according to claim 2, wherein the ionizing radiation is gamma radiation.

4. The method according to any one of claims 1 to 3, wherein the composite is exposed to the ionizing radiation in an inert atmosphere.

5. The method according to claim 4, wherein the inert atmosphere is an argon atmosphere or a nitrogen atmosphere.

6. The method according to any one of claims 1 to 5, wherein the compound is provided in the form of a solid, for example, a powder.

7. The method according to any one of claims 1 to 6, wherein the dose of the ionizing radiation is 5 to 40 kGy, preferably 8 to 40 kGy, or 20 to 35 kGy, for example, 25 kGy or 32 kGy.

8. The method according to any one of claims 1 to 7, wherein the composite is exposed to the ionizing radiation at a temperature of -80°C to 30°C, for example, -80°C to -40°C, for example, -78°C.

9. The method according to any one of claims 1 to 8, wherein the decrease in the average molecular weight of the conjugate after exposure to the ionizing radiation is less than 60%, for example, less than 50% or less than 45%.

10. The method according to any one of claims 1 to 9, wherein the pharmaceutically active compound is bonded to sodium hyaluronate or hyaluronic acid via a linker bonded to the alcohol group of hyaluronic acid.

11. The linker is -CO-(CH ２ ) ａ The method according to any one of claims 1 to 10, comprising a -CO- group (wherein a is 1 to 5).

12. The aforementioned linker, -CO-CH ２ CH ２ -CO-NH-CH ２ CH ２ -O-CH ２ CH ２ The method according to claim 11, comprising -O-.

13. The conjugate of hyaluronic acid and the pharmaceutically active compound is 【Chemistry 1】 (wherein X is H, -CO-CH ２ CH ２ -COONa, -CO-CH ２ CH ２ -CO-NH-CH ２ CH ２ -O-CH ２ CH ２ -O-DRUG, or -CO-CH ２ CH ２ -CO-NH-CH ２ CH ２ -O-CH ２ CH ２ -O-CO-CH ２ CH ２ -CO-DRUG, wherein DRUG represents a pharmaceutically active compound), the method according to any one of claims 1 to 12.

14. The method according to claim 13, wherein the drug is linked to sodium hyaluronate or hyaluronic acid via an ester bond.

15. The method according to any one of claims 1 to 14, wherein the pharmaceutically active compound is a nonsteroidal anti-inflammatory drug, a steroid, an antibiotic, a plant alkaloid, an antiviral drug, a chemotherapeutic drug, a retinoid, an immunosuppressant, a prostaglandin analog, a mast cell stabilizer, an antihistamine, or an analgesic, preferably a nonsteroidal anti-inflammatory drug or a steroid, more preferably diclofenac or dexamethasone, for example, the pharmaceutically active compound is diclofenac.

16. The method according to any one of claims 1 to 14, wherein the ionizing radiation is gamma radiation, the pharmaceutically active compound is diclofenac, and the drug is connected to sodium hyaluronate or hyaluronic acid via an ester bond.

17. The method according to any one of claims 1 to 16, further comprising the step of dividing the sterilization composition into vials / containers.

18. The method according to any one of claims 1 to 17, further comprising the step of mixing the sterilization composition with a sugar or sugar alcohol (e.g., glucose) and optionally an aqueous solution of NaCl or another salt to provide an aqueous liquid composition.

19. The method according to claim 18, wherein the concentration of the sugar in the aqueous liquid composition is 10 to 100 mg / mL, the concentration of the compound in the aqueous liquid composition is 2 to 50 mg / mL, and optionally the concentration of the NaCl or another salt in the aqueous liquid composition is 0.1 to 50 mg / mL.

20. A sterilization method comprising the step of exposing a conjugate of sodium hyaluronate, or hyaluronic acid, and a pharmaceutically active compound to ionizing radiation, wherein the method comprises 10 －６ A sterilization method characterized by providing a better level of sterilization assurance than or equal to that provided thereunder.

21. A sterilization composition comprising a conjugate of sodium hyaluronate or hyaluronic acid and a pharmaceutically active compound, which can be obtained or obtained by the method described in any one of claims 1 to 20.

22. A sterilization composition comprising a conjugate of sodium hyaluronate or hyaluronic acid and a pharmaceutically active compound, a sugar or sugar alcohol, and optionally NaCl or another salt, which can be obtained or obtained by the method of any one of claims 1 to 20, wherein the composition is, for example, a sterilization aqueous liquid composition.

23. A sterile composition comprising sodium hyaluronate, or a conjugate of hyaluronic acid and a pharmaceutically active compound, 10 －６ A sterile composition characterized by a better level of sterilization assurance.

24. A sterilization composition comprising a conjugate of sodium hyaluronate or hyaluronic acid and a pharmaceutically active compound, wherein the conjugate of sodium hyaluronate or hyaluronic acid is characterized by having a molecular weight of 200,000 to 500,000 Da (e.g., 250,000 to 400,000 Da, 250,000 to 400,000 Da, or 300,000 to 400,000 Da), and optionally, 10 －６ A sterile composition further characterized by a better level of sterilization assurance.

25. The sterilization composition according to claim 23 or 24, wherein the compound is in the form of a powder.

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