KR102599100B1 - Hyaluronidase inhibitor-incorporated crosslinked hyaluronic acid hydrogel - Google Patents

Abstract

The present invention provides a method for producing a cross-linked hyaluronic acid hydrogel introduced with a hyaluronic acid degrading enzyme inhibitor, a hyaluronic acid hydrogel produced thereby, and an injectable agent for subcutaneous injection containing the same.
The cross-linked hyaluronic acid hydrogel formulation containing the hyaluronic acid degrading enzyme inhibitor of the present invention and the subcutaneous injection containing the same can prolong retention in the subcutaneous tissue and successfully achieve sustained release of the drug. It can be useful as a formulation that can improve patient compliance by reducing the frequency of administration.

Description

Crosslinked hyaluronic acid hydrogel incorporating a hyaluronic acid degrading enzyme inhibitor {Hyaluronidase inhibitor-incorporated crosslinked hyaluronic acid hydrogel}

The present invention relates to a cross-linked hyaluronic acid hydrogel, and more particularly to a cross-linked hyaluronic acid hydrogel into which a hyaluronic acid degrading enzyme inhibitor has been introduced.

Hyaluronic acid (HA) has been extensively studied for biomedical applications, including drug delivery, tissue engineering, and cosmetics (e.g., dermal fillers) (Cho, 2020; Fakhari and Berkland, 2013; Highley et al. , 2016; Lee and Cho, 2019; Lee et al., 2018b, Lee et al., 2019a; Prestwich, 2011; Son et al., 2013). HA consists of D-glucuronic acid and N -acetyl-D-glucosamine linked by β-1,3-glycosidic bonds and β -1,4-glycosidic bonds. ) and is found in epithelial and connective tissues (Trombino et al., 2019). HA is synthesized by hyaluronan synthase in the plasma membrane and moves to the extracellular matrix. HA is decomposed by hyaluronidase (HAase), and has a half-life of several minutes to several weeks depending on the site of HA (Trombino et al., 2019). Endo-β- N -acetyl hexosaminidase is a major hyaluronic acid-degrading enzyme in mammals that can cleave β-glycosidic bonds in HA (Girish and Kemparaju, 2007; Girish et al., 2009). HA is essential for maintaining the mechanical and viscoelastic role of the body due to its high molecular weight and high water absorption capacity (Trombino et al., 2019). Due to the biocompatibility and biodegradability of HA, it is useful in a variety of clinical applications, including wound dressings, dermal fillers, and drug delivery.

Additionally, the insufficient mechanical and rheological properties of the HA dispersion itself can be controlled by physical interactions and chemical reactions (Trombino et al., 2019). Their negative charge can cause electrostatic interactions with cations, and their carboxylic acid and hydroxyl groups can react with other functional groups to form chemical crosslinks (Park et al., 2014). Various chemical reaction strategies (e.g. radical polymerization, click chemistry, disulfide cross-linking, and enzymatic cross-linking) have been used to design crosslinked HA (cHA) hydrogels (Trombino et al. ., 2019). carbodiimide, divinyl sulfone, hydrazide, methacrylamide, poly(ethylene glycol) diglycidyl ether, and 1 Among chemical crosslinking agents, including 1,4-butanediol diglycidyl ether (BDDE) (Fidalgo et al., 2018; Hwang et al., 2012), BDDE combines the hydroxyl group of HA with BDDE. It was most commonly used to form covalent ether linkages between epoxide groups (Kenne et al., 2013;; La Gatta et al., 2016; La Gatta et al., 2019). Since the deprotonated hydroxyl (under alkaline conditions) is a stronger nucleophile than other groups of HA (e.g. carboxylic acid groups), it can mainly participate in reactions with cross-linkers to form ether bonds. (Kenne et al., 2013). In addition, BDDE is applied to dermal injection preparations because it is more biodegradable and less toxic than other cross-linking agents with ether bonds (De Boulle et al., 2013). Therefore, various HA hydrogels cross-linked by BDDE are commercially available as cosmetics, and their applications in tissue engineering and drug delivery are being actively studied (La Gatta et al., 2016; La Gatta et al. , 2019; Schante et al., 2011). However, the amount of BDDE in the hydrogel must be optimized for clinical applications to minimize toxicity and improve in vivo retention.

Drug release mechanisms from HA hydrogels are diverse and complex, and include diffusion/dissolution and erosion, as commonly seen in other hydrogel delivery systems (Li and Mooney, 2016). Hyaluronic acid inhibitors can maintain the balance between anabolism and catabolism of HA, and various types of hyaluronic acid inhibitors (i.e. proteins, glycosaminoglycans, poly/oligosaccharides, fatty acids and and other chemicals) have been reported (Girish and Kemparaju, 2007; Girish et al., 2009). Therefore, introducing a hyaluronic acid lyase inhibitor into the HA hydrogel can protect the gel from degradation by hyaluronic acid lyase and improve the gel's retention time in the injected tissue (Lee et al., 2018a). The slow degradation of hydrogels may also contribute to the sustained release of encapsulated drugs (Li and Mooney, 2016). Various flavonoids were screened for hyaluronidase inhibitory activity, and quercetin (QCT) showed significant inhibitory activity against bovine testis HAase (Kuppusamy et al., 1990).

Republic of Korea Patent Registration No. 10-1554758 (2015.09.15.)

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The technical problem to be achieved by the present invention is to provide a hydrogel formulation suitable for various purposes, especially in designing a cross-linked hyaluronic acid hydrogel formulation for subcutaneous injection, using a cross-linking agent to a minimum, while maintaining a high degree of swelling and To provide a method for manufacturing a hydrogel optimized to have drug content.

The technical problem to be achieved by the present invention is not limited to the technical problem mentioned above, and other technical problems not mentioned can be clearly understood by those skilled in the art from the description below. There will be.

In order to achieve the above technical problem, according to one aspect of the present invention, mixing hyaluronic acid with a cross-linking agent in a basic aqueous solution to obtain a cross-linked hyaluronic acid hydrogel; and swelling the obtained cross-linked hyaluronic acid hydrogel in a medium containing a hyaluronic acid degrading enzyme inhibitor. A method for producing a cross-linked hyaluronic acid hydrogel into which a hyaluronic acid degrading enzyme inhibitor has been introduced is provided. .

According to another aspect of the present invention, a cross-linked hyaluronic acid hydrogel produced by the above production method and introduced with a hyaluronic acid degrading enzyme inhibitor is provided.

According to another aspect of the present invention, a subcutaneous injection comprising a cross-linked hyaluronic acid hydrogel into which the hyaluronic acid degrading enzyme inhibitor is introduced is provided.

The cross-linked hyaluronic acid hydrogel into which the hyaluronic acid degrading enzyme inhibitor was introduced, prepared by the present invention, was optimized to have high swelling degree and drug content, and the hyaluronic acid degrading enzyme inhibitor and the introduced drug were successfully absorbed by anti-solvent precipitation method. was introduced as The optimized cross-linked hyaluronic acid hydrogel exhibits rheological properties suitable for subcutaneous injection, can extend retention in the subcutaneous tissue, does not show serious toxicity, and increases drug release while reducing the initial maximum release when introduced in vivo. It was confirmed that it could be continued successfully. This formulation can be a new approach to improve patient compliance by reducing the frequency of drug administration and can therefore be useful in clinical applications.

The effects of the present invention are not limited to the effects described above, and should be understood to include all effects that can be inferred from the configuration of the invention described in the description or claims of the present invention.

Figure 1 is a strategy of cross-linked hyaluronic acid (cHA) hydrogel formulation for subcutaneous injection. HA is cross-linked with a minimal amount of BDDE to have rheological properties suitable for subcutaneous injection and high swelling properties to maximize drug content, and hyaluronic acid (cHA) hydrogel formulation is shown in Figure 1. The introduction of a lone acid lyase (HAase) inhibitor contributes to sustaining drug release by delaying the decomposition of cHA.
Figure 2 shows the manufacturing process of cHA hydrogel, in which quetiapine (QTP) and quercetin (QCT) were introduced into cHA hydrogel as a model drug and hyaluronic acid lyase inhibitor, respectively.
Figure 3 shows the optimization process of the cross-linked hydrogel formulation. (A) Solubility of QTP hemifumarate and QTP precipitate in DW, PBS, and EtOH [each value represents mean ± SD (n = 4). ]. (B) Swelling ratio of cHA hydrogel according to BDDE amount and EtOH percentage [each value is expressed as mean ± SD (n = 3)]. (C) cHA hydrogel (BDDE/HA=1.1, w/w) before and after swelling for 24 h with various ratios (0, 50, 60, 70, 80, and 100%) of EtOH mixed with DW or PBS. It's a photo. (D) Swelling ratio calculated after swelling cHA hydrogel (BDDE/HA=1.1, w/w) with various ratios (0, 50, 60, 70, and 80%) of EtOH mixed with PBS for 24 hours [ Each value is expressed as mean ± SD (n = 3)]. (E) QTP content of cHA/QTP hydrogels swollen for 24 h in EtOH solutions at various ratios (50, 60, and 70%) in DW and then incubated in PBS for 24 h [each value is the mean ± SD ( indicated as n=4)]. (F) QCT content of cHA/QCT1, cHA/QCT3, and cHA/QCT5 hydrogels [each value is expressed as mean ± SD (n = 4)].
Figure 4 shows the rheological characteristics of cross-linked hydrogel formulations: (A) gelation characteristics of HA, cHA, cHA/QTP, and cHA/QTP/QCT5 hydrogels tested by inversion method, (B) cHA/QTP/QCT5 Syringe injectability of hydrogels, (C) Viscoelastic properties of cHA/QTP and cHA/QTP/QCT5 hydrogels according to frequency and deformation.
Figure 5 shows the in vitro degradation patterns of HA, cHA/QCT1, cHA/QCT3, and cHA/QCT5 hydrogels, expressed as a percentage (%) of the remaining weight of each hydrogel compared to the initial weight [each value is the mean ± SD ( indicated as n=3)].
Figure 6 is a graph of the release of QTP from cHA/QTP and cHA/QTP/QCT5 hydrogels. The hydrogel or QTP solution was placed in a dialysis membrane bag and placed in PBS containing 0.5% SLS and then measured [each value is the mean ± SD. (represented as (n=3)].
Figure 7 shows the in vivo degradation pattern after subcutaneous injection of cHA/QTP and cHA/QTP/QCT5 hydrogels into mice and a photograph of the remaining hydrogel after 14 days, expressed as a percentage (%) of the remaining weight of the hydrogel compared to the initial weight. [Each value is expressed as mean ± SD (n = 3)].
Figure 8 shows the blood chemistry test results of the HA, cHA, cHA/QCT5, cHA/QTP and cHA/QTP/QCT5 injection groups [each value is expressed as mean ± SD (n = 4)].
Figure 9 is a photo of hematoxylin and eosin (H&E) staining of HA, cHA, cHA/QCT5, cHA/QTP and cHA/QTP/QCT5 injection groups [scale bar length is 100 μm].
Figure 10 shows the plasma concentration pattern of QTP after subcutaneous injection of cHA/QTP or cHA/QTP/QCT5 hydrogel (20 mg/kg as QTP) in rats [each value is expressed as mean ± SD (n = 4)] .

The present invention includes the steps of mixing hyaluronic acid with a cross-linking agent in a basic aqueous solution to obtain a cross-linked hyaluronic acid hydrogel; and swelling the obtained cross-linked hyaluronic acid hydrogel in a medium containing a hyaluronic acid degrading enzyme inhibitor. .

The mechanism for forming the hydrogel network in the production method of the present invention is shown in Figure 1. In the above manufacturing method, the focus was on a strategy to reduce the decomposition rate of the cross-linked hyaluronic acid hydrogel by introducing a hyaluronic acid degrading enzyme inhibitor into the hydrogel network, and included a strategy of delaying hydrogel decomposition and sustained drug release after subcutaneous injection. It is done. For the formulation for subcutaneous injection, quetiapine (QTP), one of the atypical antipsychotic drugs, was encapsulated in the cHA hydrogel, and to extend the maintenance period of the hydrogel in the subcutaneous tissue, it was used as a hyaluronic acid degrading enzyme inhibitor. QCT was used. During production, the cHA hydrogel composition and swelling conditions were optimized, and then both QTP and QCT were introduced by applying an anti-solvent precipitation method. The cHA hydrogel incorporating a hyaluronic acid degrading enzyme inhibitor of the present invention is the first attempt to delay hydrogel decomposition and prolong drug release.

The production method of the present invention includes mixing hyaluronic acid with 1,4-butanediol diglycidyl ether in a basic aqueous solution to obtain a cross-linked hyaluronic acid hydrogel. In the above step, hyaluronic acid is commonly used in the technical field to which the present invention pertains, and has an average molecular weight sufficient to form a hydrogel. Preferably, the average molecular weight ranges from 10 to 8000 kDa, and further. Preferably it may be 100 to 800 kDa.

In one embodiment, the crosslinking agent is a material containing a diglycidyl ether group and can be used without limitation as long as it can form an ether bond by reacting with the hydroxyl group of hyaluronic acid through an epoxy group. For example, 1,4-butanediol diglycidyl ether; 1,2-bis(2,3-epoxypropoxy)ethylene; 1-(2,3-epoxypropyl)-2,3-epoxycyclohexane; and a combination thereof, but is not limited thereto.

When using a cross-linking agent, the cross-linking ratio with hyaluronic acid can be adjusted to suit the purpose of use of the formed hydrogel, and the degree of cross-linking can be adjusted to maximize the inclusion of substances introduced (encapsulated) into the cross-linked structure. In an alkaline state, the epoxy group of a material containing a diglycidyl ether group preferentially reacts with the hydroxyl group (primary alcohol) of hyaluronic acid to form an ether bond, and the ether bond is known to be more stable than the ester bond or amide bond. . However, a high degree of cross-linking increases rigidity and reduces injectability through a syringe needle. Therefore, it is important to design a hyaluronic acid hydrogel with minimal crosslinking to have rheological properties suitable for subcutaneous injection and a high degree of swelling to maximize drug content. To achieve the above object, in one embodiment of the present invention, the hyaluronic acid and the cross-linking agent may be mixed at a weight ratio of 1:0.5 to 1:3, preferably at a weight ratio of 1:0.8 to 1:2.5. Preferably, the weight ratio may be 1:1 to 1:1.5.

The manufacturing method of the present invention includes the step of swelling the obtained cross-linked hyaluronic acid hydrogel in a medium containing a hyaluronic acid degrading enzyme inhibitor. This step is a step of introducing a hyaluronic acid degrading enzyme inhibitor into the hydrogel network in order to reduce the decomposition rate of the cross-linked hyaluronic acid hydrogel. This is linked to the delayed hydrogel degradation in vivo after subsequent subcutaneous injection and the sustained drug release strategy.

In this step, flavonoids can be used as the hyaluronic acid degrading enzyme inhibitor, and the flavonoids include luteolin, apigenin, tangeritin, quercetin, and kaempferol ( kaempferol, myricetin, fisetin, isorhamnetin, pachypodol, rhamnazin, hesperetin, naringenin, eriodic Thiol (eriodictyol), hemoeriodictyol (homieriodictyol), taxifolin (taxifolin), dihydroquercetin, dihydrokaempferol, tannic acid, tannis, concentrated tannis, hydrolyzable tannis and these It may be one type selected from the group consisting of a combination of.

In one embodiment, the hyaluronic acid degrading enzyme inhibitor may be included in a weight ratio of 0.1 to 2, preferably 0.2 to 1, relative to the hyaluronic acid.

In one embodiment, the medium hydrates and swells the cross-linked hyaluronic acid hydrogel, while at the same time dissolving the hyaluronic acid degrading enzyme inhibitor, and optionally dissolving additionally included substances (e.g., drugs). It must be a solvent that can be used. Therefore, if these conditions are met, it can be used without limitation, and an appropriate solvent must be selected depending on the combination of substances used (hyaluronic acid degrading enzyme inhibitor, drug, etc.). In one embodiment of the present invention, a 50 to 80% (EtOH/water, v/v) EtOH aqueous solution can be used, and preferably a 60 to 70% (v/v) EtOH aqueous solution can be used.

In one embodiment, the medium may include a drug. The drug is contained in a form dissolved in the medium and can be introduced into the hydrogel network in a dissolved state in the medium during the process of swelling (hydration) of the hydrogel. Therefore, the drug can be used without limitation as long as it is a material that can be dissolved in the medium and penetrate into the hydrogel network in a solution state, and preferably, it can be a drug that meets the purpose of sustained release by the hydrogel of the present invention. For example, it may be, but is not limited to, an antipsychotic that targets schizophrenia, depressive disorder, and bipolar disorder.

In one embodiment, the swelling may be performed until the cross-linked hyaluronic acid hydrogel no longer expands in volume, and may preferably be performed for 1 to 48 hours.

In one embodiment, the step of incubation in a phosphate buffered salt solution may be further included after the swelling step. This is to minimize the influence of the presence of pharmaceutical salts and different pH values on the swelling behavior of the cross-linked hyaluronic acid hydrogel. In other words, by inducing changes that occur in vivo when the obtained hydrogel is administered in vivo, the purpose is to minimize potential side effects that may occur due to changes in the formulation (or substances introduced into the hydrogel) after in vivo administration. am. Incubation in the phosphate buffered salt solution can be performed until the hydrogel swollen in the medium is no longer changed, and is preferably performed for 1 to 48 hours.

The present invention also provides a cross-linked hyaluronic acid hydrogel prepared by the above production method and into which a hyaluronic acid degrading enzyme inhibitor is introduced. After subcutaneously injecting the cross-linked hyaluronic acid hydrogel of the present invention into mice, the in vivo biodegradation rate of the hydrogel was measured. As a result, the decomposition rate when a hyaluronic acid degrading enzyme inhibitor was introduced was hyaluronic acid degrading enzyme inhibitor. When it was not included, it was significantly lower (p <0.05), which means that the function of the hyaluronic acid degrading enzyme inhibitor was expressed as intended. Accordingly, by introducing a hyaluronic acid degrading enzyme inhibitor, decomposition of the hydrogel at the injection site can be reduced and maintenance for a long time can be extended, thereby contributing to the sustained release of the drug introduced therein.

The present invention also provides a subcutaneous injection comprising a cross-linked hyaluronic acid hydrogel into which the hyaluronic acid degrading enzyme inhibitor has been introduced. In order to be used as a hypodermic injection, it must take the form of a hydrogel and at the same time have physicochemical and rheological properties that allow it to be injected from the needle of a hypodermic syringe. The hydrogel formulation of the present invention exhibited viscoelasticity and injectability suitable for subcutaneous injection, did not cause serious acute toxicity in blood chemistry and histological staining studies when administered in vivo, and did not contain a hyaluronic acid degrading enzyme inhibitor. It was confirmed that it can be used as a biocompatible subcutaneous injection formulation for sustained drug release by exhibiting lower maximum drug concentration (C _max ), longer half-life (t _1/2 ), and mean residence time (MRT) values compared to the previous case. .

Hereinafter, the present invention will be described in more detail through examples. However, the following examples are for illustrating the present invention, and the scope of the present invention is not limited thereto.

<Example>

1. Materials and experimental methods

1.1. matter

HA (molecular weight range 0.15–0.6 MDa) was supplied by SK Bioland Co., Ltd. (Cheonan, Korea). BDDE, hyaluronic acid degrading enzyme (from bovine testis), polyethylene glycol (PEG) 400, and QCT were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). QTP hemifumarate and sodium lauryl sulfate (SLS) were purchased from Tokyo Chemical Industry (Tokyo, Japan). All other reagents were analytical grade supplied from commercial sources.

1.2. Solubility evaluation of QTP in various media

To select a solvent to hydrate (i.e., swell) the cHA gel, the solubility of QTP hemifumarate and QTP base in distilled water (DW), phosphate buffered saline (PBS, pH 7.4), and ethanol (EtOH). was quantitatively analyzed using a high-performance liquid chromatography (HPLC) system. Briefly, an excess of QTP hemifumarate or QTP base was added to 1 mL of each solvent, thoroughly vortexed, and incubated in a shaking water bath (50 rpm, Lab Companion, BS-21, Jeiotech Co.) at 37°C for 24 hours. Ltd., Korea). When steady-state was reached, each sample was centrifuged and filtered to remove undissolved fractions. The prepared sample was injected onto a reversed-phase C ₁₈ column (Kinetex C ₁₈ ; 250 × 4.6 mm, 5 μm; Phenomenex, Torrance, CA, USA). QTP concentration in each solvent was measured using an HPLC system equipped with a pump (Waters 1515; Waters Company, Milford, MA, USA), UV/Vis detector (Waters 2487), and automatic injector (Waters 717 plus). The mobile phase consisted of 30 mM ammonium acetate in DW (pH 6.0) and methanol (30:70, v/v). The flow rate and injection volume were 1.0 mL/min and 20 μL, respectively. The absorption wavelength for detection was set at 238 nm, and the retention time of QTP was 14 min (Li et al., 2016).

QTP base precipitate was prepared by slightly modifying the reported desalting method (Sugihara and Taylor, 2018). Briefly, QTP hemifumarate (10 mg) was dissolved in 1 N HCl solution (0.7 mL), and then 1 N NaOH solution (0.8 mL) was added to precipitate the QTP base. The mixture was centrifuged at 16,000 g for 5 minutes and the supernatant was removed. After adding 1 mL of DW to the precipitate, it was centrifuged at 16,000 g for 5 minutes to remove the supernatant. Finally, the QTP base precipitate was freeze-dried at -70 °C for 48 hours for further use (FDCF-12003, Operon, Gimpo, Korea).

1.3. Optimization of crosslinking and swelling conditions of HA hydrogels

The epoxy group of BDDE reacts with the hydroxyl group of HA under alkaline conditions, and BDDE and HA are cross-linked by ether bonds (Xue et al., 2020). The amount of BDDE and the percentage of EtOH for cross-linking and swelling were optimized to maximize the content of QTP in the HA hydrogel, respectively. Briefly, HA (50 mg) was dissolved in 0.25 N NaOH solution (1 mL), mixed with various amounts of BDDE (55, 110, 165 mg), and then each aliquot (0.1 mL) was spread in a 96 well plate. added to each well. After the cross-linking reaction was performed at room temperature for 24 hours, fragments of each gel were swollen in 10 mL of 0%, 50%, 60%, 70%, and 80% EtOH in DW or PBS for 24 hours. They were then further incubated in PBS (20 mL) for 24 hours. The swelling degree according to the amount of BDDE and the percentage of EtOH was calculated using the following equation 1.

<Calculation 1>

where W _a and W _b are the weight of the hydrogel after and before swelling with PBS for 24 hours, respectively.

The QTP content in cHA hydrogel (BDDE/HA=1.1, w/w) was determined by precipitating QTP base (100 mg) in DW solution (10 mL) mixed with various ratios (50%, 60% and 70%) of EtOH. was added for 24 hours and then incubated in PBS (20 mL) for an additional 24 hours before measurement. The QCT content of cHA hydrogels (BDDE/HA=1.1, w/w) [cHA/QCT1, cHA/QCT3, and cHA/QCT5, respectively] containing 10 mg, 30 mg, or 50 mg of QCT was measured in EtOH/DW (70 :30, v/v) solution (10 mL) for 24 hours, followed by incubation in PBS (20 mL) for 24 hours.

The QTP and QCT contents of cHA hydrogel were quantitatively analyzed using HPLC method. Briefly, each hydrogel was placed in a mixture of DMSO and DW (7:3, v/v) and homogenized by vortex mixing and sonication for 1 h. Next, the mixture was passed through a syringe filter (Wattman syringe filter; nylon; 0.20 μm pore size), and QTP and QCT were appropriately diluted with a mobile phase and quantitatively analyzed by HPLC. The HPLC system for QCT and QTP consisted of injecting the QCT sample onto a reversed-phase C ₁₈ column (Fortis C18; 150 , v/v), except that it was the same as above. The absorption wavelength and retention time for QCT were 354 nm and 5.6 min, respectively (Kumari et al., 2010).

1.4. Physicochemical and rheological properties of cHA hydrogel

The gelation behavior of cHA hydrogel was evaluated through various physicochemical and rheological characterization analyses. In the inversion test, HA, cHA, cHA/QTP, and cHA/QTP/QCT5 specimens were prepared in plastic tubes and inverted to check for cross-linking and gelation (Lee et al., 2020). To evaluate the injectability of the cHA hydrogel (i.e., cHA/QTP/QCT5), it was pushed through a syringe needle (23 gauge). The rheological properties of the hydrogel were evaluated by plate-plate geometry (diameter 25 mm) using a rheometer (Advanced Rheometric Expansion System; Rheometric Scientific, Inc., Piscataway, NJ, USA) (Lee et al., 2020). The frequency sweep test (frequency sweep: 1251 rad/s frequency range and 20 % strain) and strain sweep test (1200 % strain range and 10 rad/s frequency) measurements were performed at 25 °C.

The in vitro degradation properties of the cHA hydrogel structure were investigated by monitoring the change in weight of the hydrogel relative to the initial weight using a slightly modified method reported (Khaleghi et al., 2020). Briefly, to evaluate the weight change of cHA and QCT-loaded cHA hydrogels (i.e., cHA/QCT1, cHA/QCT3, and cHA/QCT5), each gel specimen (approximately 1,100 mg) was incubated with 5 U/H of hyaluronic acid lyase. It was added to 10 mL of PBS solution containing mL. The hydrogel was immersed in the medium and the weight of the hydrogel was measured at set time intervals (2, 4, 6, and 8 hours). The percentage (%) of the remaining weight of each hydrogel compared to the initial weight was recorded.

In vitro QTP release from cHA hydrogel was measured using a modified dialysis bag method (Lee et al., 2020). Briefly, QTP solutions (mixture of ethanol, PEG 400, and PBS (1:1:2, v/v/v)) and QTP-encapsulated hydrogels (i.e., cHA/QTP and cHA/QTP/QCT5) (12 mg of QTP) An aliquot of dialysis membrane (corresponding to Each dialysis membrane bag was submerged in PBS (20 mL) containing 0.5% SLS and then stirred at 50 rpm in a stirred water bath (37 °C). Aliquot aliquots from the release medium for 15 days at defined time intervals (0.5, 1, 2, 4, 6, 8, 24, 48, 72, 96, 144, 168, 192, 288, 336, and 360 hours) and distribute equal volumes. was replenished with new release medium. The QTP concentration in the release medium was quantitatively analyzed by the HPLC method described above.

1.5. In vivo ( in vivo ) Decomposition study

The in vivo biodegradation rate of cHA hydrogels (i.e., cHA/QTP and cHA/QTP/QCT5) was measured in ICR mice (5 weeks old, male, Orient Bio, Seongnam, Korea) according to the reported method with slight modifications. was performed (Hahn et al., 2007). Each hydrogel (0.2 mL) was injected subcutaneously into mice, and the remaining gel weight was measured after resection at 2, 4, 7, and 14 days. The relative remaining weight (%) was calculated compared to the initial weight of the hydrogel.

1.6. In vivo ( in vivo ) Toxicity study

The in vivo toxicity of the cross-linked hydrogel formulation was evaluated by blood chemistry and histological staining analysis (Lee et al., 2020). Aliquots (0.2 mL) of HA, cHA, cHA/QCT5, cHA/QTP, and cHA/QTP/QCT5 were injected subcutaneously into the back of mice, and blood samples were prepared by cardiac blood sampling on day 7. The amounts of albumin, alanine transaminase (ALT), aspartate transaminase (AST), and blood urea nitrogen (BUN) in serum samples were measured using a Cobas 8000 C702 automatic chemical analyzer (Roche Diagnostics, Manheim, Germany) and quantitative measurement was performed. Major organs (i.e., heart, kidney, liver, lung, and spleen) were excised from mice for histological analysis. To fix them, they were incubated in a solution of formaldehyde (4%, v/v), and tissue sections (approximately 6 μm thick) were paraffinized and hydrated in a series of ethanolic solutions. Each specimen was then stained with hematoxylin & eosin (H&E) reagent according to standard protocols.

1.7. In vivo pharmacokinetic studies

Pharmacokinetic studies of QTP were performed in rats after subcutaneous injection of cHA/QTP or cHA/QTP/QCT5 hydrogel (QTP at a dose of 20 mg/kg). Male rats (Sprague-Dawley, SD) were purchased from Orient Bio (Sungnam, Korea). Aliquots (~ 250 μL) of blood samples were taken from the tail vein at 0.5, 1, 2, 4, 6, 8, 24, 48, 72, 120, and 168 hours after subcutaneous injection of each hydrogel. After centrifugation at 16,000 g for 2.5 minutes at 4°C, an aliquot (50 μL) of the supernatant was obtained and stored at -20°C.

Plasma samples (50 μL) were vortexed for 5 min with methanol (200 μL) containing carbamazepine (200 ng/mL; internal standard (IS)) and centrifuged at 16,000 g for 5 min. An aliquot (5 μL) of the supernatant was injected into a liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) system. An HPLC system (Agilent Technologies) equipped with an LC-MS system (Agilent Technologies 6430 Triple Quad) and coupled to a C ₁₈ column (Synergi Hydro-RP; 4 μm, 80 Å, 75 mm × 2.0 mm; Phenomenex, Torrance, CA, USA). 1260 Infinity, Agilent Technologies, Santa Clara, CA, USA). The mobile phase consisted of 80% acetonitrile and 20% water containing 5 mM ammonium acetate, and the flow rate was maintained at 0.4 mL/min. The m/z values of precursor versus product ions, fragment voltage, collision energy, and cell accelerator voltage for QTP were 384.2 versus 253.1, 144 V, 17 eV, and 1 V, respectively. Carbamazepine (IS) was 237 vs. 194, 126 V, 18 eV, and 1 V, respectively. The retention times of QTP and IS were 1.7 and 0.7 min, respectively.

Area under the plasma concentration-time curve (AUC) from 0 hours to infinity, maximum plasma concentration (C _max ), time to reach C _max (T _max ), half-life (t _1/2 ) and mean residence time (MRT) The same pharmacokinetic parameters were obtained based on noncompartmental analysis using WinNonlin (version 3.1, NCA 201; Pharsight, Mountain View, CA, USA).

1.8. statistical analysis

All experiments were performed at least three times, and data were expressed as mean ± standard deviation (SD). Statistical analysis was performed using analysis of variance (ANOVA) and two-tailed Student's t -test. A p-value of less than 0.05 was considered statistically significant.

2. Results and discussion

2.1. Preparation and optimization of cross-linked hydrogel formulations

Figure 1 shows a schematic diagram of the cHA hydrogel formulation and includes strategies for delaying hydrogel decomposition and sustained drug release after subcutaneous injection. Figure 2 summarizes the cHA hydrogel preparation process and the introduction of QCT and QTP by anti-solvent precipitation method. In alkaline conditions, the epoxy group of BDDE preferentially reacts with the hydroxyl group (primary alcohol) of HA to form an ether bond (Xue et al., 2020). Ether bonds are known to be more stable than ester or amide bonds, so BDDE cross-linked HA-based fillers can exhibit high persistence in clinical applications (De Boulle et al., 2013). However, a high degree of cross-linking increases rigidity and reduces injectability through a syringe needle. Therefore, a cHA hydrogel with minimal BDDE cross-linking was designed to have rheological properties suitable for subcutaneous injection and high swelling to maximize drug content. Additionally, the in vivo retention time of the cHA hydrogel was extended by encapsulating a hyaluronic acid lyase inhibitor (i.e., QCT in this study) into the cHA hydrogel structure (Figure 1). Hyaluronic acid lyase inhibitors interfere with the decomposition process of HA and can therefore prolong the retention of HA hydrogel in the subcutaneous tissue after injection.

In this example, QTP, an antipsychotic targeting schizophrenia, depressive disorder, and bipolar disorder, was encapsulated in a hydrogel network (Ravindran et al., 2010). As shown in Figure 3A, because the solubility of QTP hemifumarate in DW, PBS, and EtOH was not sufficient for hydrogel preparation, the hemifumarate salt was removed to prepare a base precipitate form. Since the solubility of QTP base in EtOH was more than 50 mg/mL (Figure 3A), it was selected as the solvent for QTP in the preparation of hydrogel formulations. In addition, QCT is virtually insoluble in water (<10 μg/mL), but well soluble in EtOH (17.64±1.05 mg/mL), suggesting that EtOH solution is suitable for the preparation of cHA hydrogel formulations containing both QTP and QCT. confirmed. Therefore, by changing the ratio of the amount of BDDE and EtOH to optimize the crosslinking and swelling conditions of the cHA hydrogel, a high swelling degree was obtained that maximizes the QTP and QCT contents in the cHA hydrogel.

Figure 3B shows the effect of BDDE amount and EtOH percentage on the swelling degree of the hydrogel. Except for the 80% EtOH group in DW, the swelling ratio of cHA hydrogel prepared with a weight ratio of BDDE and HA of 1.1 (BDDE/HA=1.1, w/w) was higher than that of the weight ratios of 2.2 and 3.3. Therefore, a BDDE to HA ratio of 1.1 weight ratio was chosen to crosslink the HA hydrogel. The effect of EtOH ratio on the swelling degree of cHA hydrogel is shown in Figures 3C and 3D, where various ratios of EtOH mixed with DW or PBS were used as swelling media. The swelling degree of cHA gel was highest in DW (0% EtOH), but it was not suitable as a swelling solution due to the insufficient water solubility of QTP and QCT. Above 80% EtOH (in both DW and PBS), the swelling of the cHA hydrogel was almost negligible and was therefore excluded from the data. The EtOH group in DW showed higher swelling properties than the EtOH group in PBS (Figures 3C and 3D). The presence of pharmaceutical salts and different pH values were shown to influence the swelling behavior. Therefore, the cHA hydrogel was swollen in various ratios of EtOH in the DW solution, then further incubated with PBS to precipitate the drug (precipitation within the hydrogel), and then the QTP content in the cHA hydrogel was measured to swell the cHA hydrogel. The optimal solution was selected (Figure 3E). There was no significant difference in the QTP content of cHA hydrogels between the 50%, 60%, and 70% EtOH groups in DW. However, when QCT was dissolved in 50%, 60%, and 70% EtOH solutions in DW at a concentration of 5 mg/mL, it was dissolved transparently only in the 70% EtOH solution. Therefore, 70% EtOH in DW was selected as the hydration solution, and then the actual content of QCT in various cHA hydrogels (cHA/QCT1, cHA/QCT3, and cHA/QCT5) was quantitatively analyzed after swelling and precipitation (Figure 3F). The QCT content in the cHA hydrogel was linearly dependent on the loading amount of QCT, with the cHA/QCT5 hydrogel showing the highest QCT content. Therefore, cHA/QTP/QCT5 hydrogel was finally selected for further evaluation, prepared with 50 mg HA, 55 mg BDDE, 100 mg QTP base, and 50 mg QCT. The cHA hydrogel was swollen in 10 mL of EtOH/DW (70:30, v/v) solution containing QTP and QCT for 24 h and then precipitated by incubation in 20 mL of PBS for 24 h. The final cHA/QTP/QCT5 hydrogel was transparent with the yellow color of QCT, but changed to a cloudy color after the formation of precipitates of QTP and QCT (Figure 2). The actual contents of QTP and QCT in cHA/QTP/QCT5 were 16.4 ± 1.1 μg/mg gel and 7.3 ± 0.9 μg/mg gel, respectively.

2.2. Physicochemical and rheological properties of hydrogels

The gelation behavior of the optimized hydrogel formulation was tested by the inversion method (Figure 4A) (Lee et al., 2020). Compared to the unbound HA group, the cHA group showed that the cross-linked hydrogel mass remained in the reverse position. This indicates an increase in BDDE-initiated crosslinking and viscoelastic properties of the HA dispersion. Both cHA/QTP and cHA/QTP/QCT5 samples also did not flow and stayed in the reverse position, indicating that the introduction of QTP and QCT into the hydrogel system did not interfere with the BDDE-mediated cross-linking of the HA structure. The injectability of the cHA/QTP/QCT5 hydrogel was tested with a plastic syringe connected to a needle (Figure 4B). The cross-linked hydrogel could easily pass a 23-gauge syringe needle, indicating that it is suitable for minimally invasive subcutaneous injection. The viscoelastic properties of cHA hydrogels (i.e., cHA/QTP and cHA/QTP/QCT5) were investigated more accurately using a rheometer (Figure 4C) (Lee et al., 2020). Both cHA/QTP and cHA/QTP/QCT5 hydrogels showed higher storage modulus (G') compared to loss modulus (G") in frequency and strain change test results. Elasticity higher than viscosity is a typical behavior of viscoelastic solids. As observed in the inversion test (Figure 4A), the stay of the crosslinked hydrogel in the inverted position can be further explained by the viscoelastic properties as above.

The hyaluronic acid lyase inhibitory effect of QCT was investigated by measuring the in vitro degradation rate of QCT-loaded cHA hydrogels (i.e., HB/QCT1, HB/QCT3, and HB/QCT5) (Figure 5). QCT was selected as a hyaluronic acid lyase inhibitor in this study to slow down the degradation rate of HA hydrogel. As shown in Figure 5, after 8 hours of incubation, the relative proportions of the remaining gel weight of the cHA, cHA/QCT1, cHA/QCT3, and cHA/QCT5 groups were 9.3%, 49.2%, 63.0%, and 91.2%, respectively. In the case without QCT, more than 90% of the cHA hydrogel was lost over 8 hours, whereas QCT inhibited hyaluronic acid degrading enzyme in proportion to the dose, so in the cHA/QCT5 hydrogel, more than 90% remained for the same time. These in vitro degradation results imply that encapsulation of QCT in cHA can effectively delay the degradation rate of HA hydrogel.

Figure 6 shows the QTP release rate of cHA hydrogel. 0.5% SLS was added to the release medium to maintain sink conditions during the release experiment. In the single-substance QTP group, more than 90% of QTP was released from the dialysis membrane within 8 hours, confirming that the dialysis membrane did not act as a barrier to release. However, the cHA/QTP and cHA/QTP/QCT5 groups showed a sustained release pattern, reaching complete (almost 100%) release of QTP at 15 days. There was no significant difference in QTP release rate between cHA/QTP and cHA/QTP/QCT5 groups, meaning that QCT of cHA did not affect the release of QTP and sustained release of QTP was achieved for up to 15 days. .

2.3. biodegradable

The in vivo biodegradation rate of cHA/QTP and cHA/QTP/QCT5 hydrogels was evaluated after subcutaneous injection into mice (Figure 7). The remaining weight of cHA/QTP hydrogel decreased more rapidly than that of cHA/QTP/QCT5. At 14 days, the relative residual weight of cHA/QTP hydrogel (22%) was significantly lower (p <0.05) than that of cHA/QTP/QCT5 (63%), which was consistent with in vitro degradation studies (Figure 5). . The slower degradation rate of cHA/QTP/QCT5 compared to cHA/QTP is explained by the inhibitory effect of QCT on hyaluronic acid degrading enzyme. Therefore, by introducing a hyaluronic acid degrading enzyme inhibitor, maintenance of the cHA/QTP hydrogel at the injection site can be further prolonged, contributing to the sustained release of the introduced drug.

2.4. In vivo toxicity of cross-linked hydrogels

The in vivo toxicity of the cHA hydrogel formulation was evaluated 7 days after subcutaneous injection in mice. Blood chemistry test results (i.e., albumin, ALT, AST, and BUN concentrations) and H&E staining photos of HA, cHA, cHA/QCT5, cHA/QTP, and cHA/QTP/QCT5 groups (i.e., heart, kidney, liver, lung, and Spleen) is shown in Figures 8 and 9. It can be seen that there is no significant difference in albumin, ALT, AST, and BUN concentrations between the control group and the cHA/QTP/QCT5 hydrogel group (FIG. 8). In addition, there was no significant difference in the cHA/QTP/QCT5 group compared to the control group in the H&E staining photos (FIG. 9). From these results, it can be seen that subcutaneous injection of the cHA hydrogel formulation into mice does not cause serious toxicity to each organ and tissue.

HA is known to be a biocompatible and biodegradable polymer and is widely used in biomedical applications (Prestwich, 2011). Various BDDE-crosslinked HA hydrogels are also being applied as dermal fillers and can be easily used in subcutaneous injection clinics by controlling the BDDE concentration (La Gatta et al., 2016; Xue et al., 2020). The ether bond of cHA can be cleaved by cytochrome P450, while BDDE is known to be hydrolyzed to glycerol and butanediol and then eliminated through urine (De Boulle et al., 2013). A low degree of BDDE cross-linking may reduce the possible toxicity of HA hydrogels, but rapid degradation after injection may result in a shorter retention time in the subcutaneous tissue. Therefore, incorporating QCT as a hyaluronic acid lyase inhibitor into cHA hydrogels could be an alternative strategy to extend retention time while minimizing the toxicity associated with BDDE cross-linking.

2.5. In vivo pharmacokinetics

The plasma concentration profile of QTP after subcutaneous injection of cHA hydrogel formulation (20 mg/kg as QTP) in rats is shown in Figure 10, and the pharmacokinetic parameters are summarized in Table 1 below.

parameter cHA/QTP cHA/QTP/QCT5 AUC (ng·h/mL) 15772.0±5439.3 15875.1±3531.2 C _max (ng/mL) 1827.6±481.3 782.6±174.4 ^* T _max (h) 2.4±1.9 3.0±3.4 t _1/2 (h) 13.4±4.9 23.5±2.7 ^* MRT (h) 14.3±4.8 30.9±3.9 ^{* *} p <0.05, compared to cHA/QTP group.
Data are expressed as mean ± SD (n = 4).

Since the biological half-life of QTP (intragastric administration) in rats was reported to be 2.1 hours (Chen et al., 2019), it is suggestive that QTP plasma concentration in the cHA hydrogel group showed a slow disappearance pattern for up to 7 days. Additionally, it should be noted that the cHA/QTP/QCT5 group shows a slower rate of disappearance than the cHA/QTP group. There was no significant difference in AUC and T _max between cHA/QTP and cHA/QTP/QCT5 groups (p> 0.05) (Table 1). However, t _1/2 and MRT of the cHA/QTP/QCT5 group were higher than those of the cHA/QTP group (p <0.05), while C _max of cHA/QTP was significantly higher than that of the cHA/QTP/QCT5 group. These results indicate that introducing QTP into cHA hydrogel formulations can successfully prolong QTP release while reducing the initial burst release. Figure 6 shows that the in vitro release of QTP was not affected by the addition of QCT to the cHA hydrogel, while the degradation of cHA in vitro and in vivo was inhibited by QCT (Figures 5 and 7). . Therefore, it appears that the addition of QCT to the cHA hydrogel successfully inhibits hyaluronic acid degrading enzyme activity and delays the decomposition of the cHA hydrogel, thereby contributing to the sustained release of QTP.

3. Conclusion

The composition and preparation of cHA hydrogel with minimal BDDE cross-linking were optimized to have high swelling degree and drug content. Hyaluronic acid catabolism inhibitor (i.e., QCT) was introduced into the cHA hydrogel to delay the rapid degradation of cHA. QCT and QTP were successfully introduced by antisolvent precipitation method. The optimized cHA hydrogel exhibited rheological properties suitable for subcutaneous injection and did not exhibit significant toxicity. Pharmacokinetic results obtained after subcutaneous injection of cHA hydrogel in rats show that the introduction of QCT into cHA hydrogel formulation can successfully sustain the release of QTP while reducing the initial maximum release. The delayed degradation of cHA hydrogels, confirmed in in vitro and in vivo evaluations, may contribute to the sustained release of QTP. In summary, introducing a hyaluronic acid lyase inhibitor into cHA hydrogel can prolong its retention in the subcutaneous tissue and may be a suitable strategy to sustain the release of the introduced drug. Since these formulations could be a new approach to improve patient compliance by reducing the frequency of drug administration, it is necessary to investigate their feasibility for clinical application.

4. Summary

In the present invention, in order to implement a hydrogel formulation for subcutaneous injection with high swelling degree and drug content, hyaluronic acid (HA) cross-linked with 1,4-butanediol diglycidyl ether (BDDE) ) The composition ratio of the hydrogel was optimized, and a hyaluronic acid degrading enzyme (HAase) inhibitor was introduced. With the hydrogel formulation for subcutaneous injection of the present invention, long-term retention of the hydrogel and sustained release of drug in subcutaneous tissue were demonstrated, and crosslinked HA (crosslinked HA, cHA)-based hydrogel has viscoelasticity and viscoelasticity suitable for subcutaneous injection. Possibility of injection was indicated. When QCT was introduced into the cHA hydrogel formulation, the in vitro and in vivo degradation rates were reduced compared to the case where QCT was not introduced, and a single dose of the optimized hydrogel was administered to rats by subcutaneous route. Blood chemistry and histological staining studies performed after treatment showed no serious acute toxicity. In a pharmacokinetic study performed after subcutaneous injection in rats, QCT-incorporated cHA hydrogels showed lower maximum QTP concentration (C _max ), longer half-life (t _1/2 ), and mean residence time (MRT) values compared to cHA hydrogels. indicated. All these results demonstrated that the cHA hydrogel encapsulated with a hyaluronic acid lyase inhibitor can be a biocompatible subcutaneous injection formulation for sustained drug release.

The description of the present invention described above is for illustrative purposes, and those skilled in the art will understand that the present invention can be easily modified into other specific forms without changing the technical idea or essential features of the present invention. will be. Therefore, the embodiments described above should be understood in all respects as illustrative and not restrictive. For example, each component described as unitary may be implemented in a distributed manner, and similarly, components described as distributed may also be implemented in a combined form.

The scope of the present invention is indicated by the claims described below, and all changes or modified forms derived from the meaning and scope of the claims and their equivalent concepts should be construed as being included in the scope of the present invention.

Claims (14)

Obtaining a cross-linked hyaluronic acid hydrogel by mixing hyaluronic acid with 1,4-butanediol diglycidyl ether, a cross-linking agent, in a basic aqueous solution at a weight ratio of 1:1 to 1:1.5; and
Swelling the obtained cross-linked hyaluronic acid hydrogel in a 50 to 70% (EtOH/water, v/v) EtOH aqueous solution containing a hyaluronic acid degrading enzyme inhibitor in a weight ratio of 0.1 to 2 relative to the weight of the hyaluronic acid in the step. ;
A method for producing a cross-linked hyaluronic acid hydrogel containing a hyaluronic acid degrading enzyme inhibitor. The method of claim 1, wherein the hyaluronic acid has an average molecular weight range of 10 to 8000 kDa. delete delete The production method according to claim 1, wherein the hyaluronic acid degrading enzyme inhibitor is a flavonoid. The method of claim 5, wherein the flavonoid is luteolin, apigenin, tangeritin, quercetin, kaempferol, myricetin, blood fisetin, isorhamnetin, pachypodol, rhamnazin, hesperetin, naringenin, eriodictyol, hemoeriodictyol ( homoeriodictyol), taxifolin, dihydroquercetin, dihydrocaremferol, tannic acid, tannis, concentrated tannis, hydrolyzable tannis, and combinations thereof. Characterized manufacturing method. delete delete The method of claim 1, wherein the medium contains a drug. The manufacturing method according to claim 9, wherein the drug is an antipsychotic agent. The manufacturing method according to claim 1, wherein the swelling is performed for 1 to 48 hours. The manufacturing method according to claim 1, further comprising the step of incubating in a phosphate buffered salt solution after the swelling step. A cross-linked hyaluronic acid hydrogel into which a hyaluronic acid degrading enzyme inhibitor is introduced, manufactured by the production method of any one of claims 1, 2, 5, 6, and 9 to 12. A subcutaneous injection comprising a cross-linked hyaluronic acid hydrogel into which the hyaluronic acid degrading enzyme inhibitor of claim 13 has been introduced.