PL249145B1 - Method for obtaining micellar ionic conjugates based on linear polymers for co-delivery of antituberculosis drugs and their use - Google Patents

Abstract

The subject of the application is a method for obtaining micellar ionic conjugates based on linear copolymers, characterized in that the conjugate with p-aminosalicylate in an amount of 0.01 g to 1 g is dissolved in a 100- to 120-fold excess of polar solvent relative to the volume of the conjugate, after which isoniazid is added in a 1:1 weight ratio, then deionized water is added dropwise, a two-fold excess relative to the volume of the solvent used, the mixture is stirred for 12-48 h, preferably for 24 h at room temperature, the solvent is evaporated, and lyophilized. The application also covers the use of micellar ionic conjugates based on linear copolymers obtained by the described method in combination therapy for the treatment of diseases caused by Mycobacterium tuberculosis requiring multidrug therapy.

Description

Description of the invention

The subject of the invention is a method for obtaining micellar ionic conjugates based on linear polymers for co-delivery of antituberculosis drugs, for combination therapy, treatment of diseases caused by Mycobacterium tuberculosis, requiring multidrug therapy.

Conjugates of polymers covalently bound to the drug through various types of linkers are known from non-patent literature and are capable of micellization and encapsulation of the drug (Bae, Y., Diezi, T. A., Zhao, A., Kwon, G. S. Mixedpolymeric micelles for combination cancer chemotherapy through the concurrent delivery of multiple chemotherapeutic agents. Journal of Controlled Release, 2007, 122(3), 324330. Lomkova, E. A., Chytil, P., Janouskova, O., Mueller, T., Lucas, H., Filippov, S. K., Trhlikova, O., Aleshunin, P., Yury A. Skorik, Y., Ulbrich, K., Etrych, T. Biodegradable Micellar HPMA-Based Polymer Drug Conjugates with Betulinic Acid for Passive Tumor Targeting. Biomacromolecules. 2016, 17(11), 3493-3507; Bansal, D., Bhadra, D., Gupta, U., Jain, N. PEGylated methotrexate based micellar conjugates for anticancer chemotherapy. Asian Journal of Pharmaceutics. 2015, 9(1), 60; Yokoyama, M., Kwon, G. S., Okano, T., Sakurai, Y., Seto, T., Kataoka, K. Preparation of micelle-forming polymer-drug conjugates. Bioconjugate Chemistry. 1992, 3(4), 295-301). Conjugates of ionically bonded polymers with the drug capable of self-assembly are also known, whose linear or grafted structure was based on choline poly(ionic liquids). These carriers were used to transport ionic drugs such as salicylate, p-aminosalicylate, clavulanate, piperacillin, fusidate (Niesyto, K.; Neugebauer, D. Linear Copolymers Based on Choline Ionic Liquid Carrying Anti-Tuberculosis Drugs: Influence of Anion Type on Physicochemical Properties and Drug Release. International Journal of Molecular Sciences. 2021, 22, 284; Niesyto, K.; Neugebauer, D. Synthesis and Characterization of Ionic Graft Copolymers: Introduction and In Vitro Release of Antibacterial Drug by Anion Exchange. Polymers. 2020, 12, 2159), which were tested for cytotoxicity showing promising properties, i.e. no cytotoxic effect against human bronchial and lung epithelial cells, but cytotoxicity against cancer cells (Niesyto K., Skonieczna, M., Adamiec-Organisciok, M., Neugebauer, D. Toxicity evaluation of choline ionic liquid-based nanocarriers of pharmaceutical agents for lung treatment. Journal of Biomedical Materials Research. 2023, 111(7), 1374-1385; Niesyto, K., Łyżniak, W., Skonieczna, M., Neugebauer, D. Biological in vitro evaluation of PIL graft conjugates: cytotoxicity characteristics. Int. J. Mol. Sci., 2021, 22, 7741).

Poly(ionic liquids) contain ions in their structure and exhibit specific properties of ionic liquids, such as high thermal stability, high solvation capacity and low vapor pressure (Lei, Z., Chen, B., Koo, M-K., MacFarlane, D.R. Introduction: Ionic Liquids. Chem. Rev. 2017, 117, 10, 6633-6635; Forsyth, S. A., Pringle, J. M., MacFarlane, D. R. Ionic Liquids—An Overview. Australian Journal Chemistry. 2004, 57(2), 113). Moreover, poly(ionic liquids) exhibit unique properties due to the combination of polymer and ionic liquid properties (Kausar, A. Research Progress in Frontiers of Poly(Ionic Liquid)s: A Review. Polymer-Plastics Technology and Engineering. 2017, 56(17), 18231838; Yuan, J., Markus Antonietti, M. Poly(ionic liquid)s: Polymers expanding classical property profiles. Polymer, 2011, 52(7), 1469-1482). From the point of view of designing drug delivery systems, ionic liquids are beneficial for the development of new therapeutic agents, as well as in organic synthesis as green solvents that facilitate the purification and isolation of pharmaceutical compounds. Both ionic liquids and poly(ionic liquids) can influence the solubility of ionically bound drugs (Lu. B., Bo, Y., Yi, M., Wang, Z., Zhang, J., Zhu, Z., Zhao, Y., Zhang, J. Enhancing the Solubility and Transdermal Delivery of Drugs Using Ionic Liquid-In-Oil Microemulsions. Advanced Functional Materials. 2021, 31(34), 2102794; Ait-Touchente, Z., Zine, N., Jaffrezic-Renault, N., Errachid, A., Lebaz, N., Fessi, H., Elaissari, A. Exploring the Versatility of Microemulsions in Cutaneous Drug Delivery: Opportunities and Challenges. Nanomaterials. 2023, 13, 1688; Liu, C., Chen, B., Shi, W., Huang, W., Qian, H. Ionic Liquids for Enhanced Drug Delivery: Recent Progress and Prevailing Challenges. Molecular Pharmaceuticals. 2022, 19, 4, 1033-1046; Shukla, M.K., Tiwari, H., Verma, R., Dong, W.-L, Azizov, S., Kumar, B., Pandey, S., Kumar, D. Role and Recent Advancements of Ionic Liquids in Drug Delivery Systems. Pharmaceuticals. 2023, 15, 702), serve as a prodrug (Cojocaru, O. A., Bica, K., Gurau, G., Narita, A., McCrary, P. D., Shamshina, J. L., Barber, P. S., Rogers, R. D. Prodrug ionic liquids: functionalizing neutral active pharmaceutical ingredients to take advantage of the ionic liquid form. Medicinal Chemistry Communications. 2013, 4(3), 559; Zhang W, Guo Y, Yang J, Tang G, Zhang J, Cao Y. Prodrug Based on Ionic Liquids for Dual-Triggered Release of Thiabendazole. ACS Omega. 2023, 8(3),

3484-3492; Moshikur, R.M., Chowdhury, M. R., Wakabayashi, R., Tahara, Y., Moniruzzaman, M., Goto, M. Ionic liquids with methotrexate moieties as a potential anticancer prodrug: Synthesis, characterization and solubility evaluation. Journal of Molecular Liquids. 2019, 278, 226-233; Cojocaru, O., Shamshina, J., Rogers, R. Review/Preview: Prodrug Ionic Liquids Combining the Prodrug and Ionic Liquid Strategies to Active Pharmaceutical Ingredients. Chimica Oggi - Chemistry Today, 2013, 31(5)), as well as by appropriately selected polymer chain of poly(ionic liquid) and pharmaceutical counterion, it is possible to precisely tune their properties (Pedro, S., Freire, C., Silvestre, A., Freire, M. The Role of Ionic Liquids in the Pharmaceutical Field: An Overview of Relevant Applications. International Journal Molecular Sciences. 2020, 21(21), 8298; Liu, C., Raza, F., Qian, H., Tian, X. Recent advances in poly(ionic liquiO)s for biomedical application. Biomaterial Science. 2022, 10, 2524-2539). Many poly(ionic liquids) exhibit non-toxic, biocompatible and self-assembling properties, which gives them the potential to serve as carrier matrix in drug delivery systems.

One of the main problems during therapy is suppressing drug resistance and improving treatment efficiency. Therefore, dual-drug systems have become desirable for combination therapy involving different pharmaceuticals with synergistic effects (Tu, Y., Zheng, R., Yu, F., Xiao, X., Jiang, M., Yuan, Y. Dual drug delivery system with flexible and controllable drug ratios for synergistic chemotherapy. Science China Chemistry. 2021, 64(6), 1020-1030; Xiao, Y., Gao, Y., Li, F., Deng, Z. Combinational dual drug delivery system to enhance the care and treatment of gastric cancer patients. Drug Delivery. 2020, 27(1), 1491-1500; Wei, L., Cai, C., Lin, J., Chen, T. Dual-drug delivery system based on hydrogel/micelle composites. Biomaterials. 2009, 30(13), 2606-2613). There are various approaches to obtaining two-drug systems, but those based on poly(ionic liquids) are not well known at present. There are only a few examples in the literature of using ionic liquid-based polymers for the simultaneous release of different drugs. For example, polymeric nanocarriers based on branched chitosan functionalized with an ionic liquid were successfully obtained for the simultaneous delivery of doxorubicin and methotrexate (Rahimi, M., Shafiei-Irannejad, V., D. Sofa, K., Salehi, R. Multi-branched ionic liquid-chitosan as a smart and biocompatible nano-vehicle for combination chemotherapy with stealth and targeted properties. Carbohydrate Polymers. 2018, 196, 299-312). In turn, carriers based on choline ionic liquid polymers, capable of ionic binding of sulfacetamide or salicylate, were tested for their co-transport with non-ionic drugs such as quercetin, indomethacin or erythromycin (Bielas, R., Siewniak. A., Skonieczna, M., Adamiec, M., Mielańczyk, Ł., Neugebauer. D. Choline based polymethacrylate matrix with pharmaceutical compounds as co-delivery system for antibacterial and anti-inflammatory combined therapy. Journal of Molecular Liquids. 2019, 285, 114-122). Similarly, dual systems were also prepared with the participation of poly(ionic liquid)-based graft copolymers, which delivered fusidan and rifampicin as a drug pair with synergistic action (Niesyto, K., Mazur, A., Neugebauer, D. Dual-Drug Delivery via the Self-Assembled Conjugates of Choline-Functionalized Graft Copolymers. Materials. 2022, 15, 4457).

To date, there are no reports on the preparation of micellar conjugates based on linear copolymers for the simultaneous effective transport of ionic p-aminosalicylate and nonionic isoniazid, which justifies the ongoing research into drug co-delivery systems. Compared to conventional drug systems, amphiphilic linear copolymers have the ability to form stable micelles, which leads to prolonged and controlled release. This release control allows for maintaining a constant concentration and avoiding exceeding the toxicity threshold. Furthermore, the presence of [2-(methacryloyloxy)ethyl]trimethylammonium cations, which constitute the ionic liquid units incorporated into the polymer chain, provides additional biological activity and low carrier toxicity. Such systems for the transport of p-aminosalicylate and isoniazid, substances used in the treatment of tuberculosis, may be useful in combination therapy to prevent the development of drug resistance.

Isoniazid, a derivative of isonicotinic acid, exhibits bactericidal activity against the rapidly reproducing Mycobacterium tuberculosis group and bacteriostatic activity against inactive forms, which is based on inhibiting the synthesis of mycolic acids, which are components of the mycobacterial cell walls. The use of isoniazid in monotherapy leads to increased drug resistance, therefore it is used in combination with p-aminosalicylic acid. The antituberculosis activity of p-aminosalicylic acid as a chemotherapeutic agent is based on its bacteriostatic activity against mycobacteria. This substance prolongs the half-life of isoniazid and increases its plasma concentration, as well as preventing drug resistance of the Mycobacterium tuberculosis strain to isoniazid. Currently, in the treatment of diseases caused by Mycobacterium tuberculosis,

PL 249145 Β1 commercially available ready-made preparations based on isoniazid (Nidrazid) and p-aminosalicylic acid (Granupas). The preparation containing both isoniazid and p-aminosalicylate is not commercially available.

The essence of the invention is a method for obtaining micellar ionic conjugates based on linear copolymers, characterized in that the conjugate with p-aminosalicylate in an amount of 0.01 g to 1 g is dissolved in a 100 to 120-fold excess of a polar solvent in relation to the volume of the conjugate, then isoniazid is added in a 1:1 weight ratio, then deionized water is added dropwise, a two-fold excess in relation to the volume of the solvent used, mixed for 12-48 h, preferably for 24 h at room temperature, the solvent is evaporated and lyophilized. A linear copolymer of the general formula P(MMA-co-TMAMA/PAS) is used as the conjugate, the chain of which contains units of methyl methacrylate and [2-(methacryloyloxy)ethyl]trimethylammonium ionic liquid with a p-aminosalicylate counterion (in the ratio of [2-(methacryloyloxy)ethyl]trimethylammonium ionic liquid with a p-aminosalicylate counterion to methyl methacrylate of 25% to 75%, 50% to 50% or 75% to 25%, respectively). Methanol is preferably used as the polar solvent. Ionic conjugates based on linear copolymers obtained by the above-described method are also of interest for use as a drug in combination therapy, treating diseases caused by Mycobacterium tuberculosis, requiring multidrug therapy.

The advantage of the invention is the possibility of co-releasing therapeutic substances in physiological solutions. Such systems can be used in the treatment of diseases caused by Mycobacterium tuberculosis, which require multidrug therapy. The uniqueness of the prepared dual-active systems lies in the use of conjugates based on amphiphilic linear copolymers containing p-aminosalicylate, into which isoniazid is incorporated via encapsulation.

Table 1. Characteristics of the used conjugates with p-aminosalicylate based on a linear copolymer.

Conjugate Degree of chain polymerization Ionic fraction (mol.%) Mn of copolymer (g/mol) p-Aminosalicylate content (mol.%) TIMAL MΜΛ L1_PAS“ 68 204 25 42,500 24 L2PAS 139 51 74 50,300 42 L3PAS ^- 261 18 93 86,500 47

The subject of the invention (Scheme 1) is presented in the following embodiment examples.

Example 1. Preparation of micellar ionic conjugates for co-delivery of p-aminosalicylate and isoniazid L1_PAS7IZO.

LIPAS ^- p-aminosalicylate anion conjugate (0.02 g) was dissolved in a 100-fold excess (w/v) of methanol (2 ml), and an equilibrium amount of 0.02 g of isoniazid was added. A two-fold excess of water (4 ml) was added dropwise to the mixture and stirred for 24 hours at room temperature. The methanol was then evaporated, the aqueous fraction was collected, and the product was lyophilized to obtain a solid product. The content of encapsulated drug in the obtained 1_PAS7IZO system was calculated based on data obtained using UV-Vis spectroscopy (Evolution 300 UV-Vis spectrometer, Thermo Fisher Scientific, Waltham, MA, USA), as a percentage of the drug mass to the total mass of the polymer and drug. The isoniazid content was 47%. In vitro drug release studies for a micellar system transporting isoniazid based on the p-aminosalicylate conjugate L1_PAS7IZO.

Isoniazid transport micellar systems based on L1_PAS7IZO p-aminosalicylate conjugates were dissolved in phosphate-buffered saline (PBS, pH = 7.4) to create a 1 mg/ml solution. A 1 ml solution of the polymer system was placed in a tightly sealed dialysis membrane (MWCO = 3.5 kDa) and then transferred to a vial filled with 45 mL of phosphate-buffered saline. The release process was carried out at 37°C with constant stirring. During the study, 0.5 mL samples were collected at 15-30 minute intervals, and the absorbance of the released drugs was measured using UV-Vis. After 50 hours, 79% (4.2 pg/ml) of p-aminosalicylate and 31% (3.3 pg/ml) of isoniazid were released.

Example 2. Preparation of micellar ionic conjugates for co-delivery of p-aminosalicylate and isoniazid L2_PAS ^- /IZO.

The polymer conjugate with p-aminosalicylate anions L2_PAS ^- in an amount of 0.02 g was dissolved in a 100-fold excess (w/v) of methanol (2 ml), and an equilibrium amount of 0.02 g of isoniazid was added. A two-fold excess of water relative to the solvent (4 ml) was added dropwise to the mixture and stirred for 24 hours at room temperature. The methanol was then evaporated, the aqueous fraction was collected, and the product was lyophilized to obtain a solid product. The content of encapsulated drug in the obtained 2_PAS'/IZO system was calculated based on data obtained by UV-Vis spectroscopy (UV-Vis spectrometer, Evolution 300, Thermo Fisher Scientific, Waltham, MA, USA), as a percentage ratio of the drug mass to the total mass of the polymer and drug. The isoniazid content was 33%.

In vitro drug release studies for a micellar system transporting isoniazid based on the p-aminosalicylate conjugate L2_PAS'/IZO.

Micellar transport systems transporting isoniazid based on L2_PAS'/IZO p-aminosalicylate conjugates were dissolved in phosphate-buffered saline (PBS, pH = 7.4) to create a 1 mg/ml solution. A 1 ml solution of the polymer system was placed in a tightly sealed dialysis membrane (MWCO = 3.5 kDa) and then transferred to a vial filled with 45 mL of phosphate-buffered saline. The release process was carried out at 37°C with constant stirring. During the study, 0.5 mL samples were collected at 15-30 minute intervals, and the absorbance of the released drugs was measured using UV-Vis. After 50 hours, 48% (4.5 μg/ml) of p-aminosalicylate and 48% (3.5 μg/ml) of isoniazid were released.

Example 3. Preparation of micellar ionic conjugates for co-delivery of p-aminosalicylate and isoniazid L3_PAS ^- /IZO.

The polymer conjugate with p-aminosalicylate anions and L3_PAS ^- in an amount of 0.02 g was dissolved in a 100-fold excess (w/v) of methanol (2 ml), and an equilibrium amount of 0.02 g of isoniazid was added. A two-fold excess of water relative to the solvent (4 ml) was added dropwise to the mixture and stirred for 24 hours at room temperature. The methanol was then evaporated, the aqueous fraction was collected, and the product was lyophilized to obtain a solid product. The content of encapsulated drug in the obtained 3_PAS'/IZO system was calculated based on data obtained using UV-Vis spectroscopy (UV-Vis spectrometer, Evolution 300, Thermo Fisher Scientific, Waltham, MA, USA), as a percentage ratio of the drug mass to the total mass of the polymer and drug. The isoniazid content was 30%.

In vitro drug release studies of a micellar system transporting isoniazid based on the L3_PAS7IZO p-aminosalicylate conjugate. Micellar systems transporting isoniazid based on the L3_PAS7IZO p-aminosalicylate conjugates were dissolved in phosphate-buffered saline (PBS, pH = 7.4) to form a 1 mg/ml solution. A 1 ml solution of the polymer system was placed in a tightly sealed dialysis membrane (MWCO = 3.5 kDa) and then transferred to a vial filled with 45 mL of phosphate-buffered saline. The release process was carried out at 37°C with constant stirring. During the study, 0.5 mL samples were collected at 15-30 minute intervals, and the absorbance of the released drugs was measured using the UV-Vis method. After 50 hours, 50% (5.2 μg/ml) of p-aminosalicylate and 49% (3.2 μg/ml) of isoniazid were released.

All systems are based on linear copolymers, which are classified as poly(ionic liquids). The studied polymers, with well-defined composition, molecular weight, and low dispersity (Mw/Mn < 1.6), were synthesized using controlled atom transfer radical polymerization. The copolymer structures consist of methyl methacrylate units and an ionic liquid, i.e., 2-trimethylammonioethyl methacrylate with a p-aminosalicylate counterion (TMAMA/PAS ^-- ). Conjugates based on linear copolymers of the general formula P(MMA-co-TMAMA/PAS-) (L1_PAS'L3_PAS ^- ) (Table 1) are characterized by an appropriate content of trimethylammonium ionic groups (25-93 mol. %) and drug content, i.e., p-aminosalicylate anions) (24-47%). Linear copolymers of the general formula P(MMA-co-TMAMA) were obtained by a one-step controlled atom transfer radical polymerization (ATRP) reaction involving a monofunctional initiator, i.e., ethyl α-bromoisobutyrate. Both the initial composition of the monomer mixture and their conversion enabled control of the chain length, degree of polymerization, and ionic content, which influenced the molecular weights and dispersity. (Keihankhadiv, S.: Neugebauer, D. Synthesis and Characterization of Linear Copolymers Based on Pharmaceutically Functionalized Monomeric Choline Ionic Liquid for Delivery of p-Aminosalicylale. Pharmaceutics 2023, 15, 860).

The dual bioactive micellar systems of PAS7IZO ionic conjugates, shown in Scheme 1, contain ionically bound p-aminosalicylate and encapsulated isoniazid (2), which physically interacts with the polymer matrix. The studied systems demonstrate the ability to control and gradually release the transported drugs.

Claims (4)

1. A method for obtaining micellar ionic conjugates based on linear copolymers, characterized in that the conjugate with p-aminosalicylate in an amount of 0.01 g to 1 g is dissolved in a 100 to 120-fold excess of a polar solvent in relation to the volume of the conjugate, after which isoniazid is added in a weight ratio of 1:1, then deionized water is added dropwise, a two-fold excess in relation to the volume of the solvent used, the mixture is stirred for 12-48 h, preferably for 24 h at room temperature, the solvent is evaporated and lyophilized, 2. A method for obtaining micellar ionic conjugates according to claim 1, characterized in that a linear copolymer of the general formula P(MMA-co-TMAMA/PAS ^- ) is used as the conjugate, the chain of which contains units of methyl methacrylate and [2-(methacryloyloxy)ethyl]trimethylammonium ionic liquid with a p-aminosalicylate counterion (in the ratio of [2-(methacryloyloxy)ethyl]trimethylammonium ionic liquid with a p-aminosalicylate counterion to methyl methacrylate of 25% to 75%, 50% to 50% or 75% to 25%, respectively). 3. A method for obtaining micellar ionic conjugates according to claim 1, characterized in that methanol is preferably used as the polar solvent. 4. Ionic conjugates based on linear copolymers obtained by the method according to claims 1-3 for use as a drug in combination therapy for the treatment of diseases caused by Mycobacterium tuberculosis, requiring multidrug therapy.