Hydrogel carrier, pharmaceutical composition, use thereof and process for their preparation

Abstract

The subject of the present invention is a hydrogel carrier, a pharmaceutical composition, use thereof and process for their preparation. The biopolymeric photocrosslinked delivery system for temozolomide obtained according to the invention should be particularly suitable for local therapy of brain glioblastoma.

Description

[0001] Hydrogel carrier, pharmaceutical composition, use thereof and process for their preparation

[0002] The subject of the present invention is a hydrogel carrier, a pharmaceutical composition, use thereof and process for their preparation. In particular, the present invention relates to a biopolymeric photocrosslinked delivery system for temozolomide, in particular a methacrylated derivative thereof, designed for the local treatment of brain glioblastoma. The developed material is based on methacrylated gelatin (Gel-MA), chitosan (Chit-MA) and hyaluronic acid (HA-MA) crosslinked in the presence of a lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP 405) photoinitiator.

[0003] Background art

[0004] Glioblastoma (GBL) is one of the most common (it includes about 60-70% of primary brain tumors) and the most malignant glial tumors [1]. GBL is atumor with an aggressive course and usually an unfavorable prognosis depending on many factors, such as the patient's age, general health, tumor location, stage of the disease and response to therapy [2], Due to its invasiveness, histological diversity and tendency to recurrence, the outcome of treatment is often unsatisfactory [3], The treatment pathway of diagnosed patients includes resections and subsequent adjuvant therapies, including chemo- and radiotherapy. Unfortunately, even then the median survival of patients is limited from 16 to 19 months [4], One procedure that gives better results is temozolomide chemotherapy (TMZ) (CAS no: 85622-93- 1), which is a first-line drug used in the treatment of GBL. Its effectiveness in a commonly used form is sometimes limited, which is, among others, a consequence of the existence of protective mechanisms of the central nervous system (CNS), mainly the blood-brain-barrier (BBB), which protects the CNS against the ingress of potentially harmful compounds, but also pharmaceuticals [5], Another difficulty is the instability of TMZ in the physiological pH at which it undergoes hydrolysis. The short half-life of TMZ (pH 7.4 ti / 2 = 1.83 h) means that it is absorbed and used by the body in only 20-30%, which requires repeated administration of high doses, exposing the patient to serious systemic side effects. Another challenge for GBL therapy is the inability to achieve therapeutic concentration of TMZ at the tumor site (insufficient accumulation of drug) [6] .

[0005] A promising aspect in the treatment of GBL seems to be the implantation therapy, in which a chemotherapeutic agent enclosed in a biodegradable carrier would be delivered directly to the operating room [6] . This approach allows to bypass the blood-brain barrier, provides a high local concentration of the drug at the target site, thanks to which systemic side effects are significantly minimized. Gliadel® is the only preparation available on the market used for local treatment of glioblastoma [7] . This material is based on biodegradable polymer plates and an anti -cancer drug, carmustine. This preparation has promising properties, yet studies have shown that after its implantation, there is a so-called "sink effect" - the drug is released from the system in an uncontrolled manner and leached into the circulatory system [8], At this time, there is no clinically acceptable formulation for topical administration of TMZ. Technical Problem Therefore, it is still highly desirable to develop a new delivery system for TMZ or a pharmaceutically active derivative thereof which would bypass the blood-brain barrier and extend the time of local drug release.

[0006] Surprisingly, the above described technical problems have been solved by the present invention.

[0007] Summary of Invention

[0008] The subject of the present invention is a hydrogel carrier, a pharmaceutical composition, use thereof and process fortheir preparation, as defined in the attached claims.

[0009] The present invention is a response to the above described clinical problems associated with the treatment of glioblastomas. The biopolymer hydrogel-based system proposed in the invention with enclosed / incorporated TMZ / TMZ-MA has been designed to be able to line the site after tumor resection and release the drug directly into the surrounding brain parenchyma, which is the most common site of tumor recurrence. Such a drug delivery strategy makes it possible to bypass the blood-brain barrier and significantly reduce systemic side effects while increasing local drug accumulation. Thus, the developed system has a real chance to increase patient's comfort of life and improve the effectiveness of chemotherapy.

[0010] Detailed description of the invention

[0011] The clinically used prodrug temozolomide (TMZ) was introduced into the polymer matrix in two ways (i) in the unmodified initial form and (ii) in the form of a derivative functionalized with methacrylic groups (TMZ-MA) enabling its covalent attachment to the polymer network. The conducted comparative studies for both types of formulations confirm that the approach involving the use of the TMZ-MA derivative significantly improves the therapeutic effectiveness of the system manifested by (i) higher encapsulation efficiency (ii) prolongation of the drug release kinetics (iii) increased cytotoxicity to cancer cells of astrocytoma glioblastoma multiforme. Due to the innovative approach of connecting the drug to the biopolymer carrier network and the proven properties of such a system, the essence of the present invention is a photocrosslinked system with incorporated TMZ-MA.

[0012] In an exemplary embodiment, photocrosslinked hydrogels are formed as a result of the photopolymerization reaction occurring in the presence of a photoinitiator and under the influence of radiation, most often in the UV-Vis range. In order for polymers to participate in the photocrosslinking process, the presence of appropriate functional groups e.g. methacrylic groups (MA) in the macromolecules, is necessary; CH2=C(CH3)CO-. These groups are commonly used in the synthesis of polymers and hydrogels with various applications, including in tissue engineering or for controlled drug delivery. MA groups undergo polymerization reactions under the influence of UV radiation, which allows for the formation of a network of covalent bonds [9] . The methacrylation process is often used to modify not only the biopolymers themselves, but also the active substances introduced into the system

[0010] , This approach has also been used to develop the present invention. Fig. 1 presents a diagram of interaction of TMZ with a hydrogel carrier in the form of free TMZ and methacrylated TMZ-MA. Methacrylic groups present in the TMZ-MA derivative are involved in the radical reaction enabling covalent attachment of the drug to the polymer network.

[0013] To better explain the invention, it is presented in the below described embodiments and illustrated by the attached figures.

[0014] Figure 1 shows a diagram of interaction of TMZ with a hydrogel carrier (A) an unmodified form of TMZ, (B) a methacrylated derivative of TMZ-MA.

[0015] Figure 2 shows1H-NMR spectra for gelatin (Gel) and methacrylation product of gelatin (Gel- MA) (A) for hyaluronic acid (HA) and methacrylated hyaluronic acid (HA-MA) (B).

[0016] Figure 3 shows a diagram of photocrosslinking reaction of methacrylated biopolymers with Gel- MA as an example (A) Photos of series of Gel-MA / Chit-MA / HA-MA hydrogel matrices after photocrosslinking and lyophilization process in dry form (white discs) and after wetting (B).

[0017] Figure 4 shows gel fraction (%) of obtained hydrogel matrices.

[0018] Figure 5 shows degree of swelling (%) of hydrogel matrices after 24 hours of incubation in PBS.

[0019] Figure 6 shows degradation of hydrogel matrices over time expressed as remaining mass of material (%).

[0020] Figure 7 shows1H NMR (A) and13C NMR (B) spectra for TMZ (red), TMZ-COOH (grey) and methacrylated derivative, TMZ-MA (blue).

[0021] Figure 8 shows cumulative release profile of TMZ / TMZ-MA over time.

[0022] Figure 9 shows results of U-251 cell viability test for tested materials.

[0023] Figure 10 shows optical microscope images showing morphologies of U-251 cells after contact with tested materials.

[0024] Example 1. Preparation of biopolymeric photocrosslinked delivery system for temozolomide

[0025] Methacrylation of biopolymers

[0026] Synthesis of materials was preceded by functionalization (methacrylation) of biopolymers - gelatin and hyaluronic acid. The biopolymers were modified with methacrylic anhydride, then dialysed against water and lyophilized to dryness. Structure of the obtained products was confirmed based on the recorded1H NMR spectra (Fig. 2). For Gel-MA, the degree of substitution with methacrylic groups was 83% and for HA-MA, 57%

[0027] Synthesis of methacrylated polymers

[0028] Methacrylated gelatin (Gel-MA) was obtained based on the procedure described in [GRAULUS, G.-J., et al. Cross-linkable alginate -graft-gelatin copolymers for tissue engineering applications. European Polymer Journal, 2015, 72: 494-506], Briefly, 10 g of type B gelatin (GelB, Sigma-Aldrich) was dissolved in 100 m of phosphate buffer at pH 7.8 at 40°C. Further, 1.43 m of methacrylate anhydride (MAA, >94%, Sigma-Aldrich) was added dropwise to the solution. After adding the entire volume of MAA, the system was stirred for 1 h at 40°C. Further, the reaction mixture was diluted twice with distilled water and transferred to a dialysis tube (MWCO 12000). Dialysis was performed at 40°C for 48 hours. The resulting product was frozen and lyophilized. At each stage of synthesis, the reaction mixture / product was protected from light.

[0029] Methacrylated hyaluronic acid (HA-MA). 1 g of sodium hyaluronate (HA, 1.5 - 1.8 IO6Da, Sigma- Aldrich) was dissolved in 100 mL of DI. 22.64 mL of MAA was added dropwise to the dissolved HA. The pH of the reaction mixture was controlled for 2h with 5M NaOH (34mL). The solution was then allowed to stir at room temperature for 24 h. The product was precipitated in ethanol. The resulting sludge was repeatedly washed with ethanol to get rid of MAA residues. The isolated product was suspended in DI and dialysed against water until the conductivity of the dialysate was close to that of water (1-2 pS). The product was then frozen and lyophilized.

[0030] Methacrylated chitosan (Chit-MA) was purchased from POLBIONICA (product catalogue number TNT-04) and was used in the purchased form. The degree of methacrylation of 81% and the degree of deacetylation of >75% specified by the manufacturer were determined based on measurements of1HNMR spectra,

[0031] Preparation of methacrylated derivative of temozolomide acid (TMZ-MA)

[0032] In order to effectively incorporate the drug into the biopolymer matrix, it was decided to obtain a methacrylated derivative of TMZ, TMZ-MA, so that the appropriately modified cytostatics molecules could be incorporated into the system at the stage of material formation in the photocrosslinking process. In order to obtain TMZ-MA, it was necessary to convert TMZ to its acid derivative, TMZ-COOH, which served as a substrate for the synthesis of TMZ-MA.1H-NMR and13C NMR spectra generated for TMZ and synthesized derivatives are presented in Fig. 7. The obtained results unequivocally confirm the obtaining of the desired compounds of high purity.

[0033] (i) Synthesis of temozolomide acid. TMZ-COOH

[0034] 2g of TMZ (Sigma- Aldrich) were dissolved in 16mL of concentrated sulfuric acid (VI). 10.4 mL of aqueous NaNO2 solution at a concentration of 255mg / mL was slowly added to the resulting mixture placed in an ice bath. The system was left for 16 hours on a magnetic stirrer in an ice bath. TMZ-COOH was then precipitated in frozen water. The product was isolated by filtration and dried under vacuum at room temperature.

[0035] (ii) Synthesis of a methacrylated derivative of temozolomide acid TMZ-MA

[0036] Synthesis of TMZ-MA derivative was carried out based on the protocol presented in the publication [SKINNER, Matthew; WARD, Sarah M.; EMRICK, Todd. Versatile synthesis of polymertemozolomide conjugates. ACS Macro Letters, 2017, 6.3: 215-218], This synthesis consisted of two steps of (i) obtaining temozolomide acid (TMZ-COOH) (ii) attaching 2-hydroxyethyl methacrylate (HEMA) to form an ester bond in the presence of (l-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC).

[0037] A weighted amount of TMZ-COOH (592.5mg) was dispersed in 20mL of dichloromethane (DCM). the milky suspension. The reaction mixture was left on a magnetic stirrer under nitrogen for 14 hours. The resulting solution was then filtered and the filtrate diluted with the addition of 30mL of DCM. The system was rinsed five times with 0.1 M HC1 (5x 50 mL) and the organic layer was dried with Na2SC>4. The organic solvent was evaporated on a vacuum evaporator and the resulting product was dried under vacuum.

[0038] Preparation of photocrosslinked hydrogel matrices

[0039] The following biopolymer solutions were prepared: 100 mg / mL Gel-MA in deionized water (DI), 10 mg / mL Chit-MA in 0.5% CH3COOH, 10 mg / mL HA-MA in lOxPBS (composed of: NaCl (c=1.37 M), KC1 (c=27 mM), Na2HPO4 (c=43 mM), KH2PO4 (c=14 mM), pH set to 7.4 with concentrated (c=35%) chloric acid HC1 solution). The biopolymers were dissolved at 37°C for 24 h. Appropriate volumes of solutions corresponding to the weight ratios of Gel-MA / Chit-MA / HA-MA to a total volume of 900 pL in each tube were introduced into Eppendorf tubes (see Table 1). After adding subsequent volumes, the contents of the tube were mixed each time (Vortex, 30 sec.). Then, 50 pL of dimethyl sulfoxide (DMSO) and 50 pL of an aqueous solution of a lithium phenyl-2,4,6-trimethylbenzoylphosphinate photoinitiator (LAP 405, POLBIONICA) at a concentration of 27 mg / mL) were added to the biopolymer mixture. The prepared sol samples were transferred to silicone molds with an inner diameter of 1.6 cm. Photocrosslinking was carried out using the POLBIONICA UV VIS Lamp, at a power of 13.5 mW / cm2and a wavelength of 365 nm. After 6 min of photopolymerization, the resulting hydrogels were transferred to a 24-well plate and flushed three times with DI (2 mL, 5 min, 100 rpm), then frozen at - 20°C and lyophilized. The obtained materials in the form of dry flakes were used for subsequent characterization.

[0040] Table 1. Polymer composition of developed photocrosslinked systems

[0041] Preparation of photocrosslinked hydrogel matrices with encapsulated / attached TMZ / TMZ-MA Hydrogel materials with TMZ / TMZ-MA were prepared according to the above procedure with minor changes. A 50 pL solution of TMZ in DMSO (25.27 mg / mL) or TMZ-MA in DMSO (40.00 mg / mL), respectively, was introduced into the biopolymer mixture instead of DMSO alone, as in the case of the matrices. Then, 50 pL of LAP 405 photoinitiator solution (27 mg / mL in DI) was added and the procedure was continued as for hydrogels. The obtained materials are in the form of dry flakes.

[0042] Physicochemical characteristics of photocrosslinked hydrogel matrices

[0043] In the photocrosslinking reaction using the LAP photoinitiator 405 (Fig. 3A), series of hydrogel matrices with different weight ratios of Gel-MA / Chit-MA / HA-MA were obtained. In total, 10 systems were created. Photos of cross-linked, lyophilized biopolymer flakes of varying composition, dry and moistened, respectively, are shown in Fig. 3B.

[0044] The obtained matrices were characterized in terms of physicochemical properties, including gel fractions and degree of swelling, as well as the degradation process over time.

[0045] Gel fraction

[0046] In order to assess the degree of cross-linking of individual materials, their gel fractions were determined. Lyophilisates with different mass ratios of biopolymers were obtained in 4 replicates for each system and then weighed (Wd). The materials were transferred to a 24-well plate, 1 mL of DI was introduced into each well, and incubated for 24 h at 37°C, shaking at 100 rpm. After the specified time, the entire volume of liquid was drawn off, the materials were frozen, lyophilized and reweighed (Wd). The values of the gel fraction (GF%) were calculated based on formula (1): The results, expressed as mean values with corresponding standard deviations, are shown in Fig. 4.

[0047] The obtained differences in the gel fraction values of individual systems are closely correlated with the varying degree of methacrylation of biopolymers used for the synthesis of matrices. The highest gel fraction (> 74%), and thus the highest degree of cross-linking, was shown by materials with the highest content of Gel-MA (methacrylation degree 84%) and at the same time the lowest content of HA -MA (methacrylation degree 57%). These are systems with 50%, 60%, 70% and 80% Gel-MA content and 10%HA-MA content, successively 5 / 4 / 1, 6 / 3 / 1, 7 / 2 / 1 and 8 / 1 / 1 systems. Accordingly, the lowest values of the gel fraction (about 50%) were shown by materials with a high HA-MA content (30 and 40%), i.e. 5 / 2 / 3 and 5 / 1 / 4 systems.

[0048] Degree of swelling

[0049] Lyophilized materials in 4 replicates for each system were weighed ). The samples were transferred to a 24-well plate, 1 mL of PBS (pH 7.4) was introduced into each well, and incubated for 24 h at 37°C, shaking at 100 rpm. After the specified time, the materials were reweighed (W ). The degree of swelling (SR%) for each material was calculated by Formula (2):

[0050] Fig. 5 depicts mean values of degree of swelling with corresponding standard deviations.

[0051] The results of the experiment confirm that all developed systems have the ability to absorb water (> 490%). Analyzing the obtained data, one can notice a correlation with the results of gel fraction tests - the more cross-linked the system (higher GF), the lower the swelling capacity of the material. Thus, the lowest values of the degree of swelling (492-534%) characterize materials with the highest gel fraction (5 / 4 / 1, 6 / 3 / 1, 5 / 1 / 9 systems). The exception is the 8 / 1 / 1 system, for which the highest degree of swelling was observed (748%). This is probably due to the dominant share of gelatin in the composition of this system (80%), which, due to the presence of numerous hydrophilic groups, has a natural ability to swell. The obtained results are consistent with literature data confirming that the swelling of the hydrogel decreases with increasing cross-linking of the material.

[0052] Degradation

[0053] Lyophilized materials in 4 replicates for each system were placed in a 24-well plate. 1 mL of DI was introduced into each well, and incubated for 2 h at 37°C, shaking at 100 rpm. After 2 hours, the DI was drawn off, the materials were gently squeezed from the excess water, weighed, and the resulting mass was assumed to be 100% (Wo). 1 mL of PBS (pH 7.4) was then introduced into each well, and incubated under the same conditions. Materials were weighed at time points of 4, 24, 48, 72, and 144 h (Wt), respectively. The remaining weight of the material at appropriate time points was determined from the formula (3): remaining weight of material(° / o) = — w0x 100% (3)

[0054] The results obtained, expressed as the average remaining weight of the material along with the values of standard deviations, are presented in Fig. 6. All of the developed systems gradually degrade over time, and this process is clearly differentiated depending on the composition of the materials. Materials with the highest gelatin content degrade the fastest, 8 / 1 / 1, 7 / 1 / 2, 7 / 2 / 1 systems, respectively, reaching the lowest value of the remaining mass of the material (<20%) after 144 h. This is the result of gelatin's natural ability to denature above 30°C. Of the remaining materials, the slowest degradation course was observed for the 5 / 4 / 1 and 6 / 3 / 1 systems - after 72 hours of incubation in PBS, these hydrogels retained more than 80% of the initial mass. These systems also exhibited high gel fraction values and limited swelling capacity.

[0055] Taking into account the experiments carried out, it was found that biopolymer photocrosslinked flakes having the proposed compositions can mimic the features of hemostatic neurosurgical materials available on the market (swelling ability, degradation course). What is also important, changes in the composition of biopolymers make it possible to adjust their degradation profile to the desired level.

[0056] The systems with the highest gel fraction (the highest degree of cross-linking) were selected for further research due to the expected maximum efficiency of TMZ encapsulation. Thus, on the basis of the obtained results, the 5 / 4 / 1, 6 / 3 / 1, 7 / 2 / 1 and 8 / 1 / 1 systems were selected as the matrices for the introduction of TMZ / TMZ-MA.

[0057] Example 2. Characterization of materials with enclosed / attached TMZ / TMZ-MA

[0058] Temozolomide in free form (TMZ) and methacrylated form (TMZ -MA) was introduced into selected hydrogel matrices. The resulting systems were characterized in terms of (i) encapsulation efficiency (ii) drug release profile (iii) cytostatic properties in vitro.

[0059] Encapsulation efficiency (EE)

[0060] The obtained materials with TMZ were rinsed three times with DI (2 mb, 5 min, 100 rpm). Absorption spectra of solutions collected from above the samples were recorded. The free TMZ concentrations in each sample were determined based on the TMZ calibration curve. The encapsulation efficiency was determined based on the formula (4): total weight of TMZ-weight of free TMZ

[0061] EE(%) = x 100% total! weight of TMZ (4)

[0062] The EE results (%), together with the corresponding standard deviation values, are summarized in Table

[0063] 2.

[0064] Table 2. Encapsulation efficiency values with standard deviations for individual materials.

[0065] In each of the selected materials, the active substance was enclosed with a high encapsulation efficiency in the range of 54-75% and 96-97% for TMZ and TMZ-MA, respectively. Significantly higher EE values were obtained for systems with the methacrylated form of TMZ-MA temozolomide. As shown schematically in Fig. IB, due to the methacrylic groups present in the derivative being tested, TMZ-MA can participate in the photocrosslinking process as a result of which it is incorporated / cross-linked into the biopolymer matrix. Consequently, at the stage of material purification, its elution is significantly reduced (in comparison with unmodified TMZ). In addition, the presence of methacrylic groups in TMZ- MA contributes to improving its photostability under the applied irradiation conditions.

[0066] Examination of release profiles

[0067] The TMZ / TMZ-MA release kinetics study was performed for the 5 / 4 / 1 system. Lyophilized materials of 5 / 4 / 1 series in 3 replicates for each type (control-empty matrix, TMZ in matrix, TMZ-MA in matrix) were placed in glass bottles, 4 mb of PBS (pH 7.4) was introduced, and incubated at 37°C, shaking at 100 rpm. At specific time points, 2 mb of liquid was collected and replaced with a fresh portion of PBS (2 mb). The TMZ concentrations at individual time points were determined based on the calibration curve for the hydrolyzed form of the 5-aminoimidazole-4-carboxamide (AIC) drug. Results of the cumulative percentage of drug release over time are shown in Fig. 8.

[0068] When analyzing the results of release experiments for both formulations tested, with TMZ and TMZ- MA, respectively, significant differences can be observed. In the case of a system with introduced TMZ, after 30 minutes from the start of the experiment, about 80% of the closed drug is ejected, while at the same time point, less than 24% of TMZ-MA is released. Comparing the two systems, the further course of release for TMZ-MA is much slower than for TMZ, thanks to which the release is more controlled. After about 24 h, the release for the system with TMZ reaches a stable level (>97%), with increments within the error limits at subsequent measurement points. Meanwhile, for the system with incorporated TMZ-MA, approximately 65% of the drug was released after 24 hours. The visible differences in the release kinetics of TMZ / TMZ-MA are the result of the chemical structure and the method of incorporating both forms into the hydrogel matrix. For TMZ systems, a burst release of small drug molecules that have not been chemically incorporated into the polymer network was observed, so that the maximum amount of drug released is reached already within an hour. In the case of systems with TMZ-MA, which, thanks to additional MA groups, is cross-linked into a polymer matrix, the release occurs much slower. At first, particles from the surface of the material or those that are not cross-linked are released. After 24 h, the degradation of the material begins, which results in an increase in the release observed until the end of the conducted experiment. Summing up, the release profile for TMZ-MA systems is of a controlled and prolonged release character.

[0069] In vitro bioassays

[0070] In vitro bioassays were performed on human astrocytoma glioblastoma multiforme cells, of U-251 cell line . In order to demonstrate the biological activity of the drug, the Alamar Blue viability test was carried out. Cell survival was tested for the empty hydrogel matrix (KON, control), TMZ materials and TMZ- MA materials for the four selected systems (5 / 4 / 1, 6 / 3 / 1, 7 / 2 / 1, 8 / 1 / 1). The cells were incubated with the appropriate materials for 72 h, then the assay was performed. The survival results, together with the corresponding standard deviation values, are shown in Fig. 9. Additionally, Fig. 10 compiles optical microscope images showing the morphologies of U-251 cells after contact with the test materials.

[0071] The obtained results indicate that all the tested systems comprising TMZ or TMZ-MA cause a reduction in the viability of U-251 cells in comparison with the empty control matrix (KON). However, the highest cytotoxicity to U-251 cells was obtained for 5 / 4 / 1 and 6 / 3 / 1 materials comprising TMZ or TMZ-MA. In addition, the lowest viability (at the level of about 60% of the control - empty matrix of 5 / 4 / 1 or 6 / 3 / 1) was observed for TMZ-MA. This confirms the improved anticancer properties of the modified form of TMZ over the unmodified drug. A key observation is also the change in the morphology of U- 251 cells caused by the modified drug, observed using an optical microscope. As can be seen in Fig. 10, cells incubated with 5 / 4 / 1, 6 / 3 / 1 or 7 / 2 / 1 systems comprising TMZ-MA have a folding cell membrane in the form of apoptotic bodies. This phenomenon is associated with the late phase of cell death in a process called apoptosis. In the case of U-251 cells incubated with 5 / 4 / 1, 6 / 3 / 1 or 7 / 2 / 1 matrices comprising unmodified TMZ, only a reduction in cell density was observed. This phenomenon is associated with the inhibition of the cell cycle, which did not lead to direct cell death during 72-hour incubation with TMZ -comprising systems. Again, this result confirms the improved properties of TMZ- MA in comparison with unmodified TMZ. The analysis of the viability of U-251 cells shows strong anti -cancer properties of the tested systems with TMZ-MA.

[0072] References

[0073] [1] UOUIS, David N., et al. The 2021 WHO classification of tumors of the central nervous system: a summary. Neuro-oncology, 2021, 23.8: 1231-1251 [2] WELLER, Michael, et al. EANO guideline for the diagnosis and treatment of anaplastic gliomas and glioblastoma. The lancet oncology, 2014, 15.9: e395-e403.

[0074] [3] HANIF, Farina, et al. Glioblastoma multiforme: a review of its epidemiology and pathogenesis through clinical presentation and treatment. Asian Pacific journal of cancer prevention: APJCP, 2017, 18.1: 3

[0075] [4] ITURRIOZ-RODRIGUEZ, Nerea; SAMPRON, Nicolas; MATHEU, Ander. Current advances in temozolomide encapsulation for the enhancement of glioblastoma treatment. Theranostics, 2023, 13.9: 2734.

[0076] [5] ALIMOHAMMADI, Ehsan, et al. The impact of extended adjuvant temozolomide in newly diagnosed glioblastoma multiforme: a meta-analysis and systematic review. Oncology Reviews, 2020, 14.1.

[0077] [6] KRAJCER, Aleksandra; GRZYWNA, Ewelina; LEWANDOWSKA-LANCUCKA, Joanna. Strategies increasing the effectiveness of temozolomide at various levels of anti-GBL therapy. Biomedicine & Pharmacotherapy, 2023, 165: 115174.

[0078] [7] CHOWDHARY, Sajeel A.; RYKEN, Timothy; NEWTON, Herbert B. Survival outcomes and safety of carmustine wafers in the treatment of high-grade gliomas: a meta-analysis. Journal of neurooncology, 2015, 122: 367-382.

[0079] [8] BOTA, Daniela A., et al. Interstitial chemotherapy with biodegradable BCNU (Gliadel®) wafers in the treatment of malignant gliomas. Therapeutics and clinical risk management, 2007, 3.5: 707-715.

[0080] [9] SOULLARD, Lenaic, et al. Optimization of the methacrylation of carboxymethylcellulose and use forthe design ofhydrogels and cryogels with controlled structure and properties. Cellulose, 2023, 30.10: 6203-6217.

[0081]

[0010] SKINNER, Matthew; WARD, Sarah M.; EMRICK, Todd. Versatile synthesis of polymertemozolomide conjugates. ACS Macro Letters, 2017, 6.3: 215-218.

Claims

Claims1. A hydrogel carrier comprising temozolomide or a derivative thereof, characterized in that the hydrogel is a photocrosslinked mixture of methacrylated derivatives of gelatin (Gel-MA), chitosan (Chit-MA) and hyaluronic acid (HA-MA), wherein preferably the derivative of temozolomide is a methacrylic derivative of temozolomide (TMZ-MA).

2. The hydrogel carrier according to claim 1, characterized in that the molecules of temozolomide or a derivative thereof are suspended in the hydrogel.

3. The hydrogel carrier according to claim 1 , characterized in that the molecules of the methacrylic derivative of temozolomide are covalently bound to the hydrogel.

4. The hydrogel carrier according to claim 1, characterized in that the mixture of methacrylated derivatives of gelatin (Gel-MA), chitosan (Chit-MA) and hyaluronic acid (HA-MA) was selected from the mixtures with a weight ratio of Gel-MA / Chit-MA / HA-MA equal to, respectively: 5 / 4 / 1, 6 / 3 / 1, 7 / 2 / 1 and 8 / 1 / 1.

5. A pharmaceutical composition comprising temozolomide or a derivative thereof, characterized in that temozolomide or the derivative thereof is comprised in the hydrogel carrier as defined in claims 1-4.

6. The hydrogel carrier comprising temozolomide or a derivative thereof as defined in claims 1-4, or the pharmaceutical composition as defined in claim 5, for use in the treatment or prevention of tumors, particularly brain glioblastoma.

7. A method for producing a hydrogel carrier comprising temozolomide or a derivative thereof, characterized in that: a) a solution of methacrylated gelatin, a solution of methacrylated chitosan and a solution of methacrylated hyaluronic acid is obtained, b) based on these solutions, a mixture of solutions of methacrylated derivatives of gelatin (Gel- MA), chitosan (Chit-MA) and hyaluronic acid (HA-MA) is obtained, preferably selected from the mixtures as defined in claim 4, c) a solution comprising temozolomide (TMZ) or methacrylated temozolomide (TMZ-MA) and a photoinitiator solution, preferably lithium phenyl-2,4,6-trimethylbenzophosphinate, is added to the mixture obtained in step b), d) the mixture obtained in step c) is photocrosslinked, preferably with 365 nm wavelength light, at a power of 13.5 mW / cm2for 6 minutes e) the hydrogel obtained in step d) is purified, then it is frozen and lyophilized.

8. The method of claim 7, characterized in that in step a) a solution of methacrylated gelatin at a concentration of lOOmg / mL, preferably in DI, a solution of methacrylated chitosan at a concentration of lOmg / mL, preferably in 0.5% v / v CH3COOH and a solution of methacrylated hyaluronic acid at a concentration of lOmg / mL, preferably in lOxPBS buffer, ofthe following composition: NaCl (c=1.37 M), KC1 (c=27 mM), Na2HPC>4 (c=43 mM), KH2PO4 (c=14 mM), is obtained, with pH set to 7.4 with concentrated (c=35%) chloric acid HC1 solution.

9. The method of claim 7, characterized in that in step c) a solution comprising temozolomide (TMZ) or methacrylated temozolomide (TMZ-MA) at a concentration of 25.27 mg / mL (130mM for TMZ) or 40mg / mL (130mM for TMZ-MA), respectively, preferably in DMSO, and a solution comprising lithium phenyl-2,4,6-trimethylbenzophosphinate at a concentration of 27mg / mL, preferably in DMSO, is used.

10. The method of claim 7, characterized in that in step e) the obtained hydrogel is purified by rinsing it three times in deionized water and then subjected to freezing at -20°C and lyophilization.

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