The method of preparation of the medicinal formulation of clotrimazole with amphiphilic copolymer and its application in anticancer therapy and combined anticancer therapy with nifuratel.

Abstract

The invention solves the issue of the method of manufacturing a medicinal formulations of an amphiphilic non-linear copolymer based on polyethers with a hydrophobic core enriched in alkyl or aromatic groups and hydrophilic coating containing diol groups, with clotrimazole which is hardly soluble in water, and the use of this formulation in anticancer therapy and combined anticancer therapy with nifuratel in the form of an aqueous solution or in the form of a hydrogel, preferably in therapy of cervical cancer.

Description

[0001] The method of preparation of the medicinal formulation of clotrimazole with amphiphilic copolymer and its application in anticancer therapy and combined anticancer therapy with nifuratel.

[0002] The object of the invention is the method of producing the medicinal formulation of clotrimazole with amphiphilic copolymer and its application in the anti-cancer therapy and combined anticancer therapy with nifuratel on the example of cervical cancer.

[0003] Cervical cancer is a malignant epithelial cancer that arises in the cervix [1], It is one of the most common causes of death among women worldwide [2], Chemotherapy is one of the main treatments for cervical cancer. However, the use of a single therapeutic agent is not effective in the treatment of this cancer, so it is often necessary to use combination therapy [3], Various combinations of cisplatin, paclitaxel, bevacizumab, carboplatin, topotecan and gemcitabine are recommended as first line therapies [4,5], These drugs have been approved by the Food and Drug Administration (FDA) for the treatment of cervical cancer [6],

[0004] The drugs used in conventional chemotherapy are not selective for cancer cells, which means that both cancer cells and normal cells are simultaneously killed by anticancer drugs, leading to serious side effects [7,8,9,10],

[0005] Due to the high toxicity of the drugs used in chemotherapy, it is necessary to include supportive treatment, i.e. protective against the side effects of chemotherapy. Unfortunately, many of the drugs approved for adjuvant treatment also cause numerous and serious side effects [8-10] in the case of high-dose chemotherapy, it is necessary to carry out a bone marrow transplant.

[0006] In addition to the lack of selectivity toward cancer cells, the drugs used in chemotherapy result in the development of multidrug resistance (MDR, multidrug resistance), which is one of the main reasons for the failure of systemic cancer therapy [11-16],

[0007] That is why it is so important to invent a therapy that is effective and at the same time selective for cervical cancer cells, and the use of a low therapeutic dose would avoid unwanted side effects.

[0008] Clotrimazole is a hydrophobic drug used in antifungal therapies, the action of which is based on the disorder of the calcium pump, the transport of Ca2+in the cell and the inhibition of the synthesis of ergosterol, which leads to damage to the cell membrane of the fungus

[0017] , Clotrimazole is routinely used to treat skin infections caused by dermatophytes, yeasts, moulds and other fungal strains

[0018] , including vulvovaginitis. Due to the poor water solubility of clotrimazole (0.49 mg / L)

[0019] , clotrimazole has both low bioavailability and therapeutic efficacy and is therefore classified as class II drugs under the Biopharmaceutical Classification System (BCS). In topical therapies, only 0.5% of clotrimazole is absorbed, which reduces its effectiveness

[0020] ,

[0009] The antifungal effects of clotrimazole are demonstrated, for example, by patents US4775678A and EP0007595B1. Patent US4775678A describes how to make an oil-in-water emulsion in the form of a cream or lotion containing various amounts of clotrimazole, petrolatum, cetearyl alcohol, propylene glycol, and ceteth-20, with or without other additional ingredients. Patent EP000759B1 describes the formulation of clotrimazole in the form of a cream, gel based on poly(acrylic acid).

[0010] Clotrimazole, in addition to its antifungal activity, also exhibits anticancer properties [21,24], These studies have confirmed the strong effect of the drug on the viability of mammalian cancer cells by affecting cytoskeleton-related glycolysis and inhibition of cellular glycolysis and ATP production [21,22], and that clotrimazole may be an antagonist of calmodulin (Cam)

[0023] , Marinho-Carvalho et al. demonstrated, for example, that the drug inhibited cell glycolysis by directly inhibiting phosphofructose kinase (PFK) and EC 2.7.1.11, a key regulatory enzyme of the glycolytic pathway and calmodulin-binding protein

[0024] , Compared to normal cells, cancer cells show an increased rate of glycolysis

[0025] , and their mitochondria metabolize (in addition to pyruvate) glutamine, which allows faster synthesis of lipids and amino acids necessary for building membranes and proteins

[0026] , This glycolytic preference for cancer cells is called the Warburg effect

[0027] ,

[0011] Clotrimazole acts directly on glycolytic enzymes and thus fits into the Warburg effect

[0028] , which was shown by the results of tests on cancer cells and its small effect on normal cells. Unfortunately, clotrimazole has not proven itself in chemotherapy due to its low solubility in hydrophilic media

[0029] , Cancer treatments require new chemotherapy strategies. For example, cervical cancer, one of the most aggressive forms of cancer in women and the fourth most common cancer in the world, is resistant to conventional chemotherapy

[0030] , Effective therapy, especially in the early stages of the disease, can determine the effectiveness of treatment. Due to the beneficial effect of Warburg observed for clotrimazole, increasing its solubility in the aquatic environment may be of key importance for increasing the potential of the drug in anticancer therapies. The purpose of the invention is to overcome the inconveniences indicated by the state of the art. This goal was achieved by developing a formulation solution, a method of its production and a product obtained in this way, allowing to obtain a therapeutic formulation of clotrimazole with high selectivity for cancer cells on the example of HeLa cervical cancer cells, increased solubility and bioavailability in water conditions. The terms used in this description and in the patent claims, if any, have the following meanings.

[0012] ‘Medicinal formulation’ or ‘formulation’ refers to a preparation which is in such form that the biological activity of the active ingredient contained therein is effective and which does not contain additional components that are unacceptably toxic to the individual to whom the formulation will be administered. The term “therapy” or “treatment” refers to the use of a drug containing new compounds according to the invention of formula (I), to treat a disease or medical condition and includes the prevention of an illness or condition, the elimination of a disease or condition, and / or the alleviation of one or more symptoms of a disease or condition. PBGE-PGGE refers to a copolymer with a hydrophobic poly(benzyl-glycidyl ether) core and a hydrophilic coating of poly(glyceryl -glyceryl ether)

[0013] PEBut-PEO-HbPGL refers to poly(l,2-epoxybutane)-poly(ethylene oxide)-hyperbranched polyglycidol

[0014] NIF stands for nifuratel

[0015] CLOT is clotrimazole

[0016] MEDICINE means clotrimazole, nifuratel

[0017] To this end, we demonstrated that the encapsulation of clotrimazole in the structure of nonlinear amphiphilic copolymers with a hydrophobic core made of poly(1.2-epoxybutane) or poly(benzyl-glycidyl ether) and a hydrophilic coating made of poly(glyceryl -glyceryl ether) or polyglycidol results in an increase in bioavailability of the drug, which is seen as its increased activity against cancer cells at a reduced dose. Our previous efforts to improve the bioavailability of clotrimazole in water have shown that encapsulation of the drug with certain amphiphilic copolymers-based polymer carriers resulted in a reduction in its selectivity, i.e. a Warburg effect disorder typical of non-encapsulated clotrimazole

[0031] , Therefore, a treatment aimed at improving the bioavailability of clotrimazole while maintaining its selectivity is essential.

[0018] According to the invention, copolymers with a nonlinear topology on the example of an amphiphilic copolymer with a star topology (three-arm copolymer) with a poly(benzyl- glycidyl ether) or poly(l,2-epoxybutane) core and a poly(glyceryl-glyceryl ether) coating, and a copolymer with a specified three-arm hydrophobic core and a coating composed of hyperbranched polycidol, the hydrophilic part is sufficiently separated from the hydrophobic core by the use of a linker extending the hydrophobic segments made of ethylene oxide polymer with DPn= 30 ± 5 are an appropriate carrier of clotrimazole in the aquatic environment leading to the preparation of a formulation with selective anti-cancer effect on the example of treatment cervical cancer.

[0019] The essence of the invention is the method of manufacturing the medicinal formulation of clotrimazole (a drug that is hard to dissolve in water) and non-linear amphiphilic copolymers with a hydrophobic core made of poly(benzyl-glycidyl ether) or poly(l,2-epoxybutane) and hydrophilic coating with diol groups of poly(glyceryl -glyceryl ether) or hyperbranched polyglycidol and their use to manufacture a drug with increased bioavailability in water conditions, preserving the original action of clotrimazole, consisting in selective activity against cancer cells, such as cervical cancer and the formulation of clotrimazole-nonlinear copolymer in combination anticancer therapy with other biological active substances on the example of nifuratel.

[0020] The way is that in the case of formulation in the form of an aqueous solution, clotrimazole dissolves in methanol, the solutions are mixed together, and left at 50°C to evaporate methanol, after which the mixture is suspended in deionized water, filtered and freeze-dried. In the case of a hydrogel formulation, aqueous solutions of a polyether-based copolymer are mixed together with a hydrophobic core made of poly(benzyl-glycidyl ether) or polyepoxybutane and a hydrophilic coating with poly(glyceryl -glyceryl ether) diols or hyperbranched polyglycidol with encapsulated clotrimazole and boric acid-enriched acrylamide copolymer. As boric acid, 2-acrylamidophenylboric acid is preferably used.

[0021] The application consists in the use of this formulation in anticancer therapy and combined anticancer therapy with nifuratel in the form of an aqueous solution or in the form of a hydrogel on the example of cervical cancer.

[0022] The formulation produced by the method of invention may be in the form of powder, granules, concentrate for the preparation of an aqueous suspension intended for intravenous injection or for use on diseased tissue or for administration in hydrogel form.

[0023] The invention provides:

[0024] - solubility of clotrimazole, a drug that is difficult to dissolve in water,

[0025] - preserving the selective action of the drug against cancer cells and - maintaining high anticancer effectiveness while lowering the dose, which in effect will avoid systemic side effects accompanying the currently used anticancer therapies, as well as prevent the occurrence of phenomena and multidrug resistance, which is one of the main reasons for the failure of systemic anticancer therapy.

[0026] The creators of this invention conducted comprehensive research aimed at determining the structure of copolymers providing not only increased solubility of clotrimazole in the polymer structure, and thus increased bioavailability of the drug, but also found that the two- component drug formulation consisting of copolymer and clotrimazole is characterized by excellent performance in this respect. The creators of the described invention conducted research aimed at determining the exhaustive characterization of copolymers using ID and 2D nuclear magnetic resonance spectroscopy and copolymer-drug formulation using ID nuclear magnetic resonance spectroscopy. It was found that in the obtained formulation the drug is bound to the copolymer on the principle of non-specific effects. The encapsulation of the drug in the structure of the copolymer was confirmed using proton nuclear resonance and dynamic light scattering. In addition, in vitro studies, in addition to increasing the bioavailability of the drug, confirmed the maintenance of its selectivity. Selective action of copolymer-drug formulation against cancer cells was found on the example of cervical cancer cells HeLa (CRM-CCL-2 - ATCC). Therefore, the formulation of a nonlinear amphiphilic copolymer with clotrimazole is expected to be an excellent drug for the treatment of cancer on the example of cervical cancer. In addition, the hydrogel platform produced on the basis of copolymer-drug also has a selective anti-cancer effect on cancer cells, as shown on the example of cervical cancer cells HeLa (CRM-CCL-2 - ATCC).

[0027] Both the aqueous suspension of the copolymer with the drug as well as the hydrogel exert the expected effect when administered to cervical cancer cells. From the obtained values of IC50it is clear that the drug encapsulated in the structure of a copolymer works more effectively compared to a free drug, while, importantly, despite the encapsulation, the drug retains its selectivity toward cancer cells with a simultaneous reduced toxicity toward normal cells (i.e. non-cancerous HMEC-1 - CRL-3243 - ATCC).

[0028] The invention is illustrated in Figure 1, in examples of execution 1-9, in Tables 1-6 and in photographs 1-2, in which Fig. 1 shows the action of pure PBGE-PGGE l hydrogel and the formulation of PBGE-PGGE l hydrogel with clotrimazole; Fig. 2 compares the formulation activity of PBGE-PGGE l hydrogel with clotrimazole, nifuratel and clotrimazole and nifuratel. Structures of star copolymers with a distinguished core of poly(benzyl-glycidyl ether) and a coating of poly(glyceryl -glyceryl ether), on the example of a three-arm copolymer and a hydrophobic copolymer of three-arm poly(epoxybutane) prolonged 30 with a polymer linker 5 of poly(ethylene oxide) with a covalent hydrophilic coating composed of hyperbranched polyglycidol, the copolymer-clotrimazole formulation used is shown in Diagram 1.

[0029] Diagram 1

[0030] The description of the synthesis of copolymers has been carried out according to the procedure described in references

[0032] and

[0031] , The characteristics of the manufactured three-arm copolymers PBGE-PGGE and PEbut-PEO-HbPGL are given in Table 1.

[0031] The formulation of an amphiphilic copolymer with clotrimazole and with another active substance on the example of nifuratel is shown in example ZE 1 and example 2, respectively.

[0032] Table 1.

[0033] * Recalculated based onTH NMR Example 1. Dissolution of clotrimazole in the structure of a non-linear amphiphilic copolymer.

[0034] A base solution of 5 mg / ml of clotrimazole in methanol has been prepared. 100 mg of amphiphilic copolymer as shown in Table 1 were dissolved in 5 ml of methanol. A solution of clotrimazole in a volume range of 2.7 to 6.9 ml has been added to the copolymer solution. The copolymer solution with the drug was mixed for 30 minutes. The methanol was then allowed to evaporate completely at 50°C. The polymer-drug mixture was suspended in two portions of 15 ml deionized water and filtered through a 0.45 pm PTFE (PureLand) hydrophobic syringe filter. The aqueous solution was lyophilized and the amount of encapsulated drug in the copolymer structure of u was determined by 1 H NMR spectroscopy in DMSO using an internal standard (dimethylformamide). The encapsulation capacity of the drug in the structure of non-linear amphiphilic copolymers is given in Table 2.

[0035] Example 2. Dissolution of nifuratel in the structure of a non-linear amphiphilic copolymer.

[0036] A 0.40 mg / ml methanol stock solution of nifuratel has been prepared. 200 mg of polyether- based amphiphilic copolymer was dissolved in 10 ml of methanol. A 26 ml nifuratel solution was added to the copolymer solution. The copolymer solution with the drug was mixed for 30 minutes. The methanol was then allowed to evaporate completely at 50°C. The polymer-drug mixture was suspended in two portions of 15 ml deionized water and filtered through a 0.45 pm PTFE (PureLand) hydrophobic syringe filter. The aqueous solution was lyophilized and the amount of encapsulated drug in the structure of the copolymer was determined by NMR spectroscopy in DMSO using an internal standard (dimethylformamide). The encapsulation capacity of the drug in the structure of a nonlinear amphiphilic copolymer is given in Table 2.

[0037] Table 2.

[0038] Example 3. Formation of a hydrogel based on the copolymer-drug formulation with a total polymer weight fraction equal to 17.25 t% and a concentration of clotrimazole 2.64 10'5mol / g hydrogel.

[0039] The formulation of clotrimazole with the copolymer PBGE-PGGE l (0.0161 g) with a loading capacity of 33.97 mg CLOT / g gel was dissolved in 181 pl of deionized water with pure PBGE-PGGE l (0.0125 g). In the second vessel, the poly(acrylamide-2- acryl amidophenylboric acid) copolymer with a molar ratio of acrylamide to 2- acryl amidophenylboric acid was 90 / 10 (Mw=65600, Mw / Mn=1.55) in an amount of 0,042 g was dissolved in 140 pl of deionized water. Then, the contents of the two vessels were mixed, resulting in the formation of a hydrogel. The concentration of clotrimazole gel was 2.64 10'5mol / g hydrogel.

[0040] Example 4. Formation of a hydrogel based on the copolymer-drug formulation with a total polymer weight fraction equal to 17.25 wt% and a concentration of nifuratel 1.44 10'5moles of the drug / g hydrogel.

[0041] The formulation of nifuratel with the copolymer PBGE-PGGE l (0.0141 g) with a loading capacity of 35.04 mg NIF / g gel was dissolved in 181 pl of deionized water with pure PBGE- PGGE l (0.0125 g). In the second vessel, the poly(acrylamide-2-acrylamidophenylboric acid) copolymer with a molar ratio of acrylamide to 2 -acryl amidophenylboric acid was 90 / 10 (Mw=65600, Mw / Mn=1.55) in an amount of 0,042 g was dissolved in 140 pl of deionized water. Then, the contents of the two vessels were mixed, resulting in the formation of a hydrogel. The concentration of nifuratel was 1.44 10'5mol / g hydrogel.

[0042] Example 5. Formation of hydrogel based on the formulation of the copolymer PBGE- PGGE l-LEK with a total polymer weight fraction equal to 17.25 tt% and a concentration of clotrimazole 2.88 10'5mol / g hydrogel and a concentration of nifuratel 1.54 10'5mol / g hydrogel.

[0043] The formulation of clotrimazole with the copolymer PBGE-PGGE l 90 / 10 (0.0161 g) with a loading capacity of 33.97 mg CLOT / g gel, with the formulation of nifuratel with the copolymer PBGE-PGGE_1 (0.0141 g) with a loading capacity of 35.04 mg NIF / g gel, was dissolved in 181 pl of deionized water. In the second vessel, 0.042 g of poly(acrylamide-2- acrylamidophenylboronic acid) copolymer with a molar ratio of acrylamide to 2- acrylamidophenylboronic acid of 90 / 10 (Mw= 65600, Mw / Mn= 1.55) was dissolved in 140 pl of deionized water. Then, the contents of the two vessels were mixed, resulting in the formation of a hydrogel. The concentration of clotrimazole in the gel was 2.64 10'5mol / g hydrogel and the concentration of nifuratel is 1.44 10'5mol / g hydrogel.

[0044] Example 6. Action of an aqueous solution of the medicinal formulation of clotrimazole and a nonlinear amphiphilic copolymer with a distinguished poly(benzyl-glycidyl ether) core and a poly(glyceryl-glyceryl ether) coating (Example 1) on the example of a three-arm copolymer, consisting in a selective increase in cervical cancer cell mortality compared to clotrimazole itself.

[0045] Cervical cancer cells HeLa (CRM-CCL-2 - ATCC) were grown and screened in culture bottles (Nunclon™ Delta Surface for Thermo Scientific Nunc adhesive cell culture) in DMEM medium (11966025 by Gibco Thermo Scientific) supplemented with 10% fetal bovine serum (F9665 FBS Sigma-Aldrich). HMEC-1 non-cancer cells (CRL-3243 - ATCC) were bred and sieved in culture bottles (Nunclon™ Delta Surface for Thermo Scientific Nunc adhesive cell culture) in MCDB 131 medium (10372019 by Gibco Thermo Scientific) supplemented with 10% fetal bovine serum (F9665 FBS Sigma-Aldrich), hydrocortisone (1 pg. Ml-1), L-glutamine (10 mm) and epithelial growth factor (10 ng. ml-1). The cells were seeded into 96-hole transparent plates with a density of 3 x 104cells per hole. After 24 hours of incubation in a suitable medium, clotrimazole and corresponding formulations of this drug were added to the cells in copolymers, prepared in a PBS buffer solution, in the concentration range from 1pm to 500pm. The cells were then incubated in 24 hours and 48 hours, respectively.

[0046] MTT [3-(4.5-dimethylthiazol-2-yl)-2.5-diphenyltetrazolium bromide] was added to the treated cells and incubated for 3 hours. The procedure was followed by the so-called ‘MTT method’, which uses colorimetry based on the reduction reaction of yellow MTT to blue formazate, resulting in a cell viability level.

[0047] The effect of free and encapsulated clotrimazole in the structure of stellar poly(benzyl- glycidyl ether)-poly(glyceryl -glyceryl ether) copolymers on the viability of cervical cancer cells (HeLa) and non-cancerous microvascular endothelial cells (HMEC-1) based on comparison of IC 50 values is shown in Table 3. Table 3.

[0048] Drug concentration HeLa HMEC-1

[0049] [pM] 24h 48h 24h 48h

[0050] Clotrimazol 127.42±7.11 97.33±6.46 >500 >500

[0051] PBGE-PGGE 1 CLOT B 32.74±1.15 38.40±3.13 228.10±4.75 49.95±2.82

[0052] PBGE-PGGE 3 CLOT A 49.31±4.53 30.42±6.02 146.74±4.26 >500

[0053] PBGE-PGGE_3_CLOT_B 41.14±7.17 39.85±6.55 219.02±5.20 199.71±4.55

[0054] Clotrimazole dissolved in the structure of non-linear amphiphilic copolymers is soluble in water, it acts more effectively on cervical cancer cells HeLa than a free drug and at the same time shows reduced toxicity to non-cancerous cells HMEC-1 - selective action on cancer cells.

[0055] Example 7. The effect of an aqueous solution of the medicinal formulation of clotrimazolecopolymer P(BGE-GGE)_1 in combination anticancer therapy with nifuratel (examples 2 and 3) in which cervical cancer cell mortality was selectively increased compared to clotrimazole alone and encapsulated with nifuratel alone.

[0056] Cervical cancer cells HeLa (CRM-CCL-2 - ATCC) were grown and screened in culture bottles (Nunclon™ Delta Surface for Thermo Scientific Nunc adhesive cell culture) in DMEM medium (11966025 by Gibco Thermo Scientific) supplemented with 10% fetal bovine serum (F9665 FBS Sigma-Aldrich). HMEC-1 non-cancer cells (CRL-3243 - ATCC) were bred and sieved in culture bottles (Nunclon™ Delta Surface for Thermo Scientific Nunc adhesive cell culture) in MCDB 131 medium (10372019 by Gibco Thermo Scientific) supplemented with 10% fetal bovine serum (F9665 FBS Sigma-Aldrich), hydrocortisone (1 pg. Ml-1), L-glutamine (10 mm) and epithelial growth factor (10 ng. ml-1). The cells were seeded into 96-hole transparent plates with a density of 3 x 104cells per hole. After 24 hours of incubation in a suitable medium, clotrimazole and the corresponding formulation of this drug in a copolymer, in and without the presence of another biologically active substance nifuratel, prepared in PBS buffer solution, in the concentration range from 1pm to 500pm, were added to the cells. The cells were then incubated in 24 hours and 48 hours, respectively. MTT [3-(4.5-dimethylthiazol-2-yl)-2.5-diphenyltetrazolium bromide] was added to the treated cells and incubated for 3 hours. The procedure was followed by the so-called “MTT method”, which uses colorimetry based on the reduction reaction of yellow MTT to blue formazate, resulting in a cell viability level

[0021] ,

[0057] Comparing the IC 50 values listed in Table 4, it is clear that drugs encapsulated in a copolymer structure work more effectively in cervical cancer (HeLa), while their toxicity to non-cancerous cells (HMEC-1) is less, which means that the use of the copolymer system retains the selectivity characteristic of encapsulated clotrimazole (described in Example 6). Simultaneous administration of clotrimazole and the biologically active substance nifuratel, encapsulated in the structure of PBGE-PGGE l copolymers, shows significant synergy of action of these substances (Table 4). We talk about significant synergy when the combined effect of two compounds is clearly greater than the sum of the effects of their independent action. Based on the results obtained by the so-called “MTT method”, the interaction coefficient of CDI compounds (or CDI) was determined, co-efficient drug interaction) defined as the quotient of the absorbance ratio of cells treated with a combination of two compounds for control and the absorbance ratio of cells treated with a single compound for control. A CDI value < 0.7 indicates significant synergy

[0022] ,

[0058] Table 4.

[0059] Drug concentration HeLa HMEC-1

[0060] [pM] 24h 48h 24h 48h

[0061] Clotrimazole 127.42±7.11 97.33±6.46 >500 >500

[0062] PBGE-PGGE 1 CLOT C >500 286.89±3.27 389.22±4.75 166.07±2.28

[0063] Nifuratel 339.41±6.62 103.09±6.84 315.47±5.97 116.11±5.45

[0064] PBGE-PGGE 1 NIF 6.91±5.71 20.34±5.24 >500 118.99±2.90

[0065] PBGE-PGGE 1 CLOT C / c c co_1 o c 1

[0066] _ _ _ ,.,r65.27±5.53 45.58±3.77 >500 123.85±1.63

[0067] PBGE-PGGE 1 NIF

[0068] Example 8. Action of hydrogel based on the formulation of clotrimazole and amphiphilic star copolymer with a distinguished poly(benzyl-glycidyl ether) core and a poly(glyceryl-glyceryl ether) coating (Example 4) on the example of a three-spoke copolymer, consisting in a selective increase in the mortality of cervical cancer cells. Cervical cancer cells HeLa (CRM-CCL-2 - ATCC) were grown and screened in culture bottles (Nunclon™ Delta Surface for Thermo Scientific Nunc adhesive cell culture) in DMEM medium (11966025 by Gibco Thermo Scientific) supplemented with 10% fetal bovine serum (F9665 FBS Sigma-Aldrich). HMEC-1 non-cancer cells (CRL-3243 - ATCC) were bred and sieved in culture bottles (Nunclon™ Delta Surface for Thermo Scientific Nunc adhesive cell culture) in MCDB 131 medium (10372019 by Gibco Thermo Scientific) supplemented with 10% fetal bovine serum (F9665 FBS Sigma-Aldrich), hydrocortisone (1 pg. Ml-1), L-glutamine (10 mm) and epithelial growth factor (10 ng. ml-1). The cells were seeded into 12-hole transparent plates, with an ibidi separator (Cat.No:81176) placed, in a density of 3 x 105cells per well. After 24 hours of incubation in a suitable medium, a suitable formulation of clotrimazole in a copolymer, prepared in a PBS buffer solution, was added to the separator chambers. The cells were then incubated within 24 hours. The effects of the formulation were monitored under the Nikon ECLIPSE E200 microscope (Fig.1).

[0069] As you can see in the attached photos, the control cells of both lines divided freely, covering the free (after removing the separator) surface on the plate. The pure gel did not affect the rate of cell division or cell morphology. The use of clotrimazole gel, after 24 hours of incubation, led to the death of HeLa tumor cells (dead cells can be seen as spherical shapes detached from the substrate) and slowed the division of HMEC-1 non-cancer cells causing the death of a smaller pool than cancer cells, which confirms the selective action of the drug encapsulated in hydrogel. When comparing the results obtained with the IC 50 values in Table 4, an increase in the toxic efficacy of the hydrogel drug can be seen compared with the aqueous solution of this formulation against Hela tumor cells.

[0070] Using ImageJ

[0033] , the cell area was evaluated and the cell growth or loss was determined due to clotrimazole gel. When comparing the surfaces occupied by the cells listed in Table 5, after 24 hours of culture (1), an increase in the number of control cells, not treated, in the HeLa tumor cell line by 26.70 %, in the HMEC1 non-cancerous cell line by 64.37 %, (2) the HeLa- PBGE-PGGE l pure gel cell count increased by 26.10 %, HMEC1 line by 45.53 %, and (3) the HeLa-PBGE-PGGE l hydrogel-enriched clotrimazole cell count decreased by 50.29 %, HMEC1 line by 3.31 %, which indicates (i) the lack of effect of pure gel on cell growth and (ii) the selective action of gel formulation enriched with clotrimazole. Table 5. % of area

[0071] Surface area . . . occupied by the Total area occupie y cellscellsinrelations to the total area

[0072] HeLa control t = 0 h 44039.00±22.16 83666.00±17.10 52.64±0.03

[0073] HeLa control t = 24 h 57844.00±25.19 86733.00±2.17 66.69±0.03

[0074] Hmecl control t = 0 h 44893.00±18.78 85408.00±14.20 52.56±0.02

[0075] Hmecl control t = 24 h 73163.00±7.53 84680.00±7.26 86.40±0.01

[0076] HeLa pure gel t = 0 h 49752.00±25.19 86216.00±27.57 57.71±0.03

[0077] HeLa pure gel t = 24 h 63022.00±26.75 86610.00±25.85 72.77±0.03

[0078] Hmecl pure gel t = 0 h 47970.00±19.92 86217.00±15.38 55.64±0.02

[0079] Hmecl pure gel t = 24 h 69272.00±7.50 85552.00±7.37 80.97±0.01

[0080] HeLa gel with clotrimazole t = 0 h 35453.00±23.81 86534.00±17.48 40.97±0.03

[0081] HeLa gel with clotrimazole t = 24 h 17544.00±24.37 86141.00±19.41 20.37±0.03

[0082] Hmecl gel with clotrimazole t = 0 h 42344.00±15.36 86139.00±13.79 49.16±0.02

[0083] Example 9. Action of a hydrogel based on the formulation of a PBGE-PGGE-drug copolymer with different polymer weight fraction and concentration of clotrimazole and nifuratel (example 5) on the example of a PBGE-PGGE copolymer, consisting in a selective increase in cervical cancer cell mortality.

[0084] Cervical cancer cells HeLa (CRM-CCL-2 - ATCC) were grown and screened in culture bottles (Nunclon™ Delta Surface for Thermo Scientific Nunc adhesive cell culture) in DMEM medium (11966025 by Gibco Thermo Scientific) supplemented with 10% fetal bovine serum (F9665 FBS Sigma-Aldrich). HMEC-1 non-cancer cells (CRL-3243 - ATCC) were bred and sieved in culture bottles (Nunclon™ Delta Surface for Thermo Scientific Nunc adhesive cell culture) in MCDB 131 medium (10372019 by Gibco Thermo Scientific) supplemented with 10% fetal bovine serum (F9665 FBS Sigma-Aldrich), hydrocortisone (1 pg. Ml-1), L-glutamine (10 mm) and epithelial growth factor (10 ng. ml-1). The cells were seeded into 12-hole transparent plates, with an ibidi separator (Cat.No:81176) placed, in a density of 3 x 105cells per well. After 24 hours of incubation in a suitable medium, a suitable formulation of clotrimazole and nifuratel in the copolymer, prepared in PBS buffer solution, was added to the separator chambers. The cells were then incubated within 24 hours. The effects of the formulation were monitored under the Nikon Eclipse E200 microscope (fig. 2).

[0085] As you can see in the attached photos, the control cells of both lines divided freely, covering the free (after removing the separator) surface on the plate. The pure gel did not affect the rate of cell division or cell morphology. The use of clotrimazole or nifuratel gel, after 24 hours of incubation, led to the death of most HeLa cancer cells (dead cells can be seen as spherical forms detached from the substrate) and slowed the division of HMEC-1 non-cancer cells causing the death of a smaller pool of cells than cancer cells, what confirms the selective effect of the drug encapsulated in a hydrogel. Combination therapy with clotrimazole and nifuratel resulted in the death of all HeLa tumor cells and only parts of HMEC-1 non-cancer cells within 24 hours of incubation. Thus, the gel containing both drugs was more cytotoxic to non-cancer cells than non-cancer cells, which confirms its selective effect. In addition, it can be concluded that the gel containing both drugs worked more effectively than gels containing single drugs, and comparing the IC50values for aqueous formulations (Table 4, Example 7) also more effectively than its corresponding aqueous formulation.

[0086] ImageJ33evaluated the area of cells and determined the cell growth or loss caused by clotrimazole, nifuratel, and both. Comparing the results from Table 6, after 24 hours of culture (1), an increase in the number of control cells, untreated, in the HeLa tumour cell line by 26.70 %, in the HMEC1 non-tumour cell line by 64.37 %, (2) a 50.29 % decrease in the number of cells treated with PBGE-PGGE l hydrogel-enriched HeLa clotrimazole, in HMEC1 line by 3.31 %, (3) a decrease in the number of cells treated with PBGE-PGGE l Nifuratel-enriched HeLa by 52.10 %, a 73.30 % increase in HMEC1, (2) a decrease in the number of cells treated with PBGE-PGGE l hydrogel enriched with both HeLa drugs by 99.43 %, an increase in HMEC1 by 9.36 %, indicating (i) a selective effect of the gel formulation enriched with clotrimazole, nifuratel and other medication and (ii) increase the effectiveness of the gel enriched with both medications compared to gels containing single medications. Table 6.

[0087] % of the area area occupied occupied by the by cells cells in relation to the total area

[0088] HeLa control t = 0 h 44039.00±22.16 83666.00±17.10 52.64±0.03

[0089] HeLa control t = 24 h 57844.00±25.19 86733.00±2.17 66.69±0.03

[0090] Hmecl control t = 0 h 44893.00±18.78 85408.00±14.20 52.56±0.02

[0091] Hmecl control t = 24 h 73163.00±7.53 84680.00±7.26 86.40±0.01

[0092] HeLa gel with clotrimazole t = 0 h 35453.00±23.81 86534.00±17.48 40.97±0.03

[0093] HeLa gel with clotrimazole t = 24 h 17544.00±24.37 86141.00±19.41 20.37±0.03

[0094] Hmecl gel with clotrimazole t = 0 h 42344.00±15.36 86139.00±13.79 49.16±0.02

[0095] Hmecl gel with clotrimazole t - 24 40802.00±8.52 85848.00±8.45 47.53±0.01 h

[0096] HeLa gel with nifuratel t = 0 h 45241.00±22.86 85705.00±20.52 52.79±0.03

[0097] HeLa gel with nifuratel t = 24 h 21748.00±31.15 86013.00±27.36 25.28±0.04

[0098] Hmecl gel with nifuratel t = O h 42380.00±20.21 85955.00±17.52 49.30±0.02

[0099] Hmecl gel with nifuratel t = 24 h 73103.00±16.26 85556.00±18.61 85.44±0.02

[0100] HeLa gel with clotrimazole and 43878.00±23.02 87320.00±21.49 50.25±0.03 mturatel t = 0 h

[0101] HeLa gel with clotrimazole and 244.00±20.83 85904.00±34.96 0.28±0.02 nifuratel t = 24 h

[0102] Hmecl gel with clotrimazole and 39848.00±20.48 86142.00±15.86 46.26±0.02 mturatel t = 0 h

[0103] Hmecl gel with clotrimazole and 43132.00±12.47 85264.00±12.12 50.59±0.01 nifuratel t = 24 h

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Claims

Claims1. A method of manufacturing the medicinal formulation, characterized in that a polyether- based copolymer with a hydrophobic core composed of poly(benzyl-glycidyl ether) or poly(1.2-epoxybutane) and a hydrophilic coating of a group of diol of poly(glyceryl- glyceryl ether) or hyperbranched polyglycidol, and clotrimazole are dissolved in methanol, the solutions are mixed together, and left at temperature of 50°C for methanol to evaporate, after which the mixture is suspended in deionized water, filtered and freeze- dried.

2. A method of manufacturing the medicinal formulation of clotrimazole with an amphiphilic copolymer in the form of a hydrogel, characterized in that the aqueous solutions of a polyether-based copolymer are mixed together with a hydrophobic core composed of poly(benzyl-glycidyl ether) or polyepoxybutane and a hydrophilic coating with a diol groups of poly(glyceryl ether) polyether or a hyperbranched polyglycidol with encapsulated clotrimazole and boric acid-enriched acrylamide copolymer.

3. The method according to claim 2, characterized in that a 2-acrylamidophenylboric acid is used as boric acid.

4. Medicinal formulation of an amphiphilic copolymer with clotrimazole in the form of an aqueous solution for use in anti-cancer therapy, preferably in the treatment of cervical cancer.

5. Medicinal formulation of an amphiphilic copolymer with clotrimazole in the form of hydrogel for use in anti-cancer therapy, preferably in the treatment of cervical cancer.

6. Medicinal formulation of an amphiphilic copolymer with clotrimazole and nifuratel in the form of hydrogel for use in combination anti-cancer therapy, preferably in the treatment of cervical cancer.

Images