A GEL formulation comprising lutetium borate compound with cell-regenerative / virucidal effect and a production method thereof

Abstract

The invention relates to a gel formulation comprising an amorphous (LuxByOz.nH2O) lutetium borate compound with virucidal effect and having cell-regenerative or wound-healing properties, and to a production method thereof.

Description

[0001] DESCRIPTION

[0002] A GEL FORMULATION COMPRISING LUTETIUM BORATE COMPOUND WITH CELL-REGENERATIVE / VIRUCIDAL EFFECT AND A PRODUCTION METHOD THEREOF

[0003] Technical Field

[0004] The invention relates to a gel formulation comprising an amorphous-structured lutetium borate compound (LuxBy0z.nH20) with virucidal effect and cell-regenerative or woundhealing properties, and to a production method thereof.

[0005] Prior Art

[0006] In the state of the art, there are studies on the synthesis and indications of lutetium borate. However, none of these studies disclose a colloidal lutetium borate synthesis having woundhealing or cell-regenerative effects and, at the same time, virucidal effect, nor a gel formulation of a lutetium borate compound produced as a result of such synthesis.

[0007] In the patent document numbered 2023 / 004909 in the state of the art, the antiviral activity of salt compounds derived from the element lutetium, such as lutetium borate, lutetium chloride and lutetium nitrate, against DNA and RNA viruses in infected cells is described. However, no virucidal effect is mentioned anywhere in said document. Furthermore, said document does not disclose any wound-healing or cell -regenerative effect of the lutetium salt compounds. In addition, said document does not provide any synthesis or formulation details for achieving a wound-healing effect. Moreover, no gel formulation having woundhealing properties is disclosed.

[0008] In another prior-art document, which is also owned by the applicant and published as patent document no. 2020 / 04482, a method for synthesizing amorphous and nano- structured lutetium borate hexahydrate for use in the fields of materials, medicine, textiles and defense, and a method for preparing stable colloidal dispersions of the obtained lutetium borate hexahydrate compounds having a hydrodynamic size of less than 150 nm, are described. Although said document discloses a synthesis method for lutetium borate, it does not provide any formulation details or any details of a synthesis method of such a formulation for achieving a wound-healing or cell-regenerative effect. Furthermore, said document does not disclose any gel formulation having wound-healing properties and exhibiting virucidal effect.

[0009] Therefore, in particular, there is a need to develop formulations comprising a lutetium borate compound having wound-healing or cell-regenerative and, at the same time, virucidal effect.

[0010] Summary of the Invention

[0011] The purpose of the invention is to provide a gel formulation comprising an aqueous, amorphous-structured lutetium borate compound (LuxByOz.nTfcO) with wound-healing or cell-regenerative and, at the same time, virucidal effect, and a synthesis method for forming said formulation.

[0012] It has been determined that, by using the produced stable colloidal lutetium borate compound of the invention in a formulation within a specific concentration range and by applying said formulation to the skin, cellular proliferation in the skin is increased. Accordingly, the invention has revealed that lutetium borate, which is known to kill viruses, interestingly also induces the proliferation of cells in which viral infections are present. Owing to this property, gel formulations comprising stable colloidal solutions of lutetium borate can be used as nextgeneration pharmaceuticals and biocides having cell-regenerative or wound-healing properties in addition to their virucidal effect in the treatment of viral infections of the skin.

[0013] Description of the Figures

[0014] Figure 1. It is a flow diagram of the production method of the gel formulation of the invention comprising a lutetium borate compound.

[0015] Figure 2. It is a graph showing the cytotoxic effect of lutetium borate on E6 Vero cells and its inhibitory effect on Sars-Cov2 virus replication.

[0016] Figure 3. It is a graph showing the cytotoxic effect of lutetium borate on MDCK cells and its inhibitory effect on Influenza A virus replication.

[0017] Figure 4. It is a graph showing the cytotoxic effect of lutetium borate on VERO cells and its inhibitory effect on HSV-1 virus replication.

[0018] Figure 5. is a graph showing the virucidal effect of lutetium borate on Herpes simplex virus type 1. Description of the References in the Figures

[0019] To be able to understand the invention better, meanings of the numbers in the figures are given below:

[0020] 100. Method

[0021] 101. Adding purified water and ultrez 21 to a vessel, allowing the mixture to stand, and subsequently mixing it using a mechanical stirrer

[0022] 102. Dissolving boric acid in water in another vessel

[0023] 103. Adding a 20% NaOH solution to said solution

[0024] 104. Adding hyaluronic acid / sodium hyaluronate thereto and mixing,

[0025] 105. Slowly adding the dissolved hyaluronic acid / sodium hyaluronate solution to the Ultrez 21 mixture in the mechanical stirrer, and mixing

[0026] 106. Adding glycerin thereto and mixing

[0027] 107. Adding phenoxyethanol thereto and mixing for a predetermined period of time at a predetermined stirring speed

[0028] 108. Adding a lutetium borate solution thereto, mixing for a predetermined period of time, and obtaining a colloidal lutetium borate solution

[0029] Detailed Description of the Invention

[0030] The invention relates to a gel formulation comprising an aqueous, amorphous-structured lutetium borate compound (LuxByOz.nJUO) with wound-healing or cell-regenerative and virucidal effect, and to a production method (100) of said formulation. For healing wounds on the skin or for providing cell-regenerative and virucidal effect, the gel formulation of the invention comprising a stable colloidal lutetium borate solution comprises predetermined amounts of water, a colloidal lutetium borate solution, boric acid, glycerin, sodium hydroxide, hyaluronic acid / sodium hyaluronate or Ultrez-21 or phenoxyethanol.

[0031] In the formulation, a colloidal lutetium borate solution is added in order to increase the efficacy of the formulation.

[0032] For exhibiting virucidal effect and for healing wounds on the skin, the formulation of the invention comprises, by weight, 37.0-84.9% water, 10-40% colloidal lutetium borate solution, 1-5% boric acid, 1-5% glycerin, 1-5% sodium hydroxide, 1-5% hyaluronic acid / sodium hyaluronat, or 1-2% Ultrez -21 or 0.1-1% phenoxyethanol. For exhibiting virucidal effect and for healing wounds on the skin, the formulation of the invention comprises, by weight, 67.2% water, 20% colloidal lutetium borate solution, 3% boric acid, 3% glycerin, 2.6% sodium hydroxide, 2% hyaluronic acid / sodium hyaluronate or 1.2% Ultrez-21 or 1% phenoxyethanol.

[0033] The active ingredients in the formulation are boric acid, lutetium borate and hyaluronic acid / sodium hyaluronate. Since the product is designed as a gel, a gelling agent is added. Phenoxyethanol is also a preservative. In fact, the formulation itself is inherently antimicrobial due to its constituents. Glycerin is included to provide a moisturizing effect in the medium. By adding a previously unstudied colloidal lutetium borate solution to the formulation, the wound-healing effect is enhanced and, additionally, virucidal effect is imparted.

[0034] The components and proportions of the formulation are given in Table 1.

[0035] Table-1: Composition and proportions of the gel formulation of the invention

[0036] For each formulation, there are specific component values at which the formulation can be stable. Since the best composition providing both virucidal and cell-regenerative effects as well as formulation stability is achieved at these ratios, said ratios have been selected for the formulation of the invention and for the production process of said formulation.

[0037] The production method (100) of a gel formulation of the invention comprising a stable colloidal lutetium borate solution for healing wounds on the skin or for providing cell- regenerative and, at the same time, virucidal effects comprises the following steps: Adding a predetermined amount of purified water and, preferably, Ultrez-21 into a vessel, allowing the mixture to stand for a predetermined period of time, and subsequently mixing it in a mechanical stirrer (101),

[0038] Dissolving a predetermined amount of boric acid in a predetermined amount of water in another vessel (102),

[0039] Adding a predetermined amount of a 20% NaOH solution to said solution (103), Adding a predetermined amount of hyaluronic acid / sodium hyaluronate thereto and mixing for a predetermined period of time (104),

[0040] Slowly adding the dissolved hyaluronic acid / sodium hyaluronate solution to the mixture obtained in step (101) in the mechanical stirrer, and mixing for a predetermined period of time at a predetermined stirring speed (105),

[0041] Adding a predetermined amount of glycerin thereto and mixing for a predetermined period of time at a predetermined stirring speed (106),

[0042] Preferably adding a predetermined amount of phenoxyethanol thereto and mixing for a predetermined period of time at a predetermined stirring speed (107),

[0043] Adding a predetermined amount of a colloidal lutetium borate solution thereto and mixing for a predetermined period of time, thereby obtaining a cell-regenerative or wound-healing gel formulation

[0044] The vessel referred to in step (101) is a 500 mL beaker. In the same step, Ultrez-21 is added in an amount of 1-2% by weight, and the amount of water added is 37.0-84.9% by weight. In a preferred embodiment of the invention, the amount of water is 67.2% by weight and the amount of Ultrez-21 is 1.2% by weight. In step (101), the standing time of the purified water and Ultrez-21 is from 1 to 5 hours, and the mixing time in the mechanical stirrer is from 1 to 5 hours. In the preferred embodiment of the invention, said times are 1 hour.

[0045] In step (102), the amount of water is 37.0-84.9% by weight. In addition, the amount of boric acid is 1-5% by weight. In the preferred embodiment of the invention, the amount of water is 67.2% by weight and the amount of boric acid is 3% by weight.

[0046] In step (103), the amount of the 20% NaOH solution is 1-5% by weight. In the preferred embodiment of the invention, the amount of the NaOH solution is 2.6% by weight.

[0047] In step (104), the amount of hyaluronic acid / sodium hyaluronate is 1-5% by weight. In the preferred embodiment of the invention, the amount of hyaluronic acid / sodium hyaluronate is 2% by weight. In the same step, the mixing time is from 1 to 5 hours. In the preferred embodiment of the invention, said time is 1 hour.

[0048] In step (105), the mixing time is from 1 to 5 hours and the stirring speed is in the range of 100 rpm to 2000 rpm. In the preferred embodiment of the invention, said time is 3 hours. In the preferred embodiment of the invention, said stirring speed is 700 rpm. In step (106), the amount of glycerin is 1-5% by weight. In the preferred embodiment of the invention, the amount of glycerin is 3% by weight.

[0049] In step (107), the amount of phenoxyethanol is 0.1-1% by weight. In the preferred embodiment of the invention, the amount of phenoxyethanol is 1% by weight. In the same step, the mixing time is from 1 to 5 hours and the stirring speed is in the range of 100 rpm to 2000 rpm. In the preferred embodiment of the invention, said time is half an hour. In the preferred embodiment of the invention, said stirring speed is 700 rpm.

[0050] In step (108), the amount of the lutetium borate solution is 10-40% by weight. In the preferred embodiment of the invention, the amount of the lutetium borate solution is 20% by weight. In the same step, the mixing time is from 1 to 5 hours. In the preferred embodiment of the invention, said time is 1 hour.

[0051] Depending on the derivative of the formulation to be prepared, new active ingredients may be removed or added while keeping the main active ingredients unchanged. However, the order of addition is important during preparation of the formulation. In particular, the addition of the colloidal nanoparticles must be carried out as the final step, since said solution is extremely sensitive and may have a potential to interact with other chemicals. Therefore, in order to prevent interaction with other chemicals and to protect the colloidal lutetium nanoparticles, the colloidal lutetium nanoparticles should be added as the final component.

[0052] In the method of the invention, the optimum stirring speed is 700 rpm. This is because, at stirring speeds lower than this value, the mechanical stirrer cannot rotate due to the weight of the gel. Sodium hydroxide is added to the formulation in order to adjust the pH. If the amount of NaOH is lower or higher than 2.6% by weight, problems may occur in terms of formulation stability and gel quality. This value is the optimum value. On the other hand, it is essential that the obtained product is compatible both with the pH of the cells and the skin and with the pH range at which the gelling agent operates. The reason for selecting 20% by weight as the optimum amount of lutetium borate is to obtain the maximum effect at an optimum ratio while maintaining formulation stability. For example, if the amount of glycerin is higher than 5% by weight, a sticky feeling is formed in the formulation. The optimum consistency is obtained when the amount of glycerin is 3% by weight. The selected ratios have been determined by taking maximum efficiency and formulation stability into account. In other words, the optimum formulation has been obtained through numerous trials and investigations until the formulation reached its final form.

[0053] Cytotoxicity, virucidal testing and antiviral activity testing were performed for a stable, colloidal lutetium borate solution prepared at a specific concentration and claimed to exhibit virucidal effect. The purpose of repeating the antiviral activity tests was to understand the actual reason underlying the activity initially described as antiviral. This is because the test results demonstrated that the colloidal lutetium borate solution is, in fact, virucidal rather than antiviral. The test findings showed that lutetium borate does not exert its effect by interfering with the viral life cycle, such as viral attachment to the host cell, entry into the cell, replication within the cell, and release from the cell, but instead provides inhibition through a virucidal mechanism, namely by directly killing the virus upon contact. An antiviral agent is a substance that causes inhibition by affecting, in some manner, the cycle of viral attachment to the host cell, entry into the cell, replication within the cell, and release from the cell. In the present case, the antiviral activity of the substance is achieved only by killing the virus upon direct contact. Otherwise, it does not have any effect on intracellular processes. A virucidal agent, in contrast to an antiviral agent, is a substance that kills viruses upon contact with the virus. These two concepts are different from each other.

[0054] The methods used for performing these tests and the findings and results obtained from the tests are as follows:

[0055] EXPERIMENTAL STUDIES AND ANALYSIS:

[0056] 1- Antiviral Test and Cytotoxicity Test:

[0057] Cell line: In the study of the invention, Vero E6 cell lines were used for Sars-Cov-2, MDCK cells were used for Influenza A virus, and VERO cell lines were used for Herpes simplex type 1 (HSV-19). The cells were cultured in T75 cell culture flasks at 37 °C and 5% CO2 using DMEM (Lonza, USA) medium comprising 10% fetal bovine serum (FBS). From cell aliquots stored at -80 °C, one vial was thawed and cultured in a T25 flask in DMEM comprising 10% FBS, and when reached 80-90% confluence, they were expanded in T75 flasks and used for cytotoxicity and antiviral tests.

[0058] Virus(es): In the study, a human Sars-Cov-2 clinical isolate (the complete genome sequence registered in the NCBI GenBank NIH Genetic Sequence Database (GenBank MT955161.1)), Influenza A / PR / 8, and HSV-1 (F strain) were used.

[0059] Preparation of the Test Substance: The test substance, which is ready-to-use and has a water-soluble composition, was used directly from the bottle (after thorough mixing).

[0060] Cytotoxicity Test: The test was performed using 96-well cell culture plates.

[0061] For this purpose, the cells were first prepared as a suspension of 2 * 105 / mL in their own culture medium, and 100 pL / well was dispensed into a 96-well cell culture plate. The plates were incubated overnight at 37 °C in a humidified atmosphere containing 5% CO2. On the following day, after the contents of the plates were discarded and the wells were washed with PBS, the test substance was added to the cells after being serially diluted five-fold (in the range of 20-0,07%) in DMEM medium comprising 2% FBS and penicillin / streptomycin / fungizone. Three wells were used for each concentration. Wells containing no test substance were used as negative controls. The plates were incubated at 37 °C and 5% CO2 for 72 hours. After 72 hours, the plate contents were removed and the wells were washed with PBS. Subsequently, MTT dye (3-[4,5-dimethylthioazol-2,5-diphenyltetrazolium bromide) prepared in DMEM was added to all wells, and the plates were incubated at 37 °C and 5% CO2 for 2 hours. At the end of the period, the contents were discarded and DMSO (0,1 mL / well) was added to the wells to dissolve the intracellular formazan crystals. The absorbance of the resulting dark purple / violet color was measured at a wavelength with an OD of 570 nm.

[0062] Antiviral test:

[0063] The cells prepared in 96-well plates as described above were incubated overnight at 37 °C in a humidified atmosphere containing 5% CO2. On the following day, after the plate contents were discarded and the wells were washed with PBS, it was subjected to the procedures described below for Entry, Full-Time and Post-infection antiviral activity tests. a. Entry Test: The Entry test was performed in order to evaluate the protective / preconditioning effect of the test substance against viral infection by exposing the cells to the test substance for a short period of time and assessing its effect on the cells or on their metabolism. For this purpose, after the plate contents were discarded and the wells were washed with PBS, the test substance, which had been serially diluted two-fold (in the range of 20-0,07%) in DMEM medium comprising 2% FBS and PSF, was added to the cells. Three wells were used for each concentration. Wells not comprising the test substance were used as controls. After incubation for 1 hour at 37 °C in a 5% CO2 incubator, the plate contents were discarded and the wells were washed with PBS. Subsequently, a virus suspension comprising 100 TCID50 was added onto the cells that had been exposed to the test substance dilutions for 1 hour, and incubated for 1 hour at 37 °C in a 5% CO2 incubator. Wells that were not exposed to the test substance but were inoculated with virus and wells comprising only DMEM-2 were used as the Virus Control (VC) and Cell Control (CC), respectively. After a 1-hour virus infection period, the plate contents were discarded, DMEM-2 was added, and it was incubated for 72 hours at 37 °C in a 5% CO2 incubator. b. Full-Time Test: The Full-Time test is performed by exposing the cells to the test substance both before and after infection with the virus. Briefly, as in the Entry test, the cells are first exposed to the test substance, then infected with the virus, and after infection, the cells are exposed again to the test substance. In this manner, it can be determined whether the test substance has an inhibitory effect on viral entry into the cells, on viral replication after entry.

[0064] For this purpose, after the plate contents were discarded and the wells were washed with PBS, the test substance, which had been serially diluted two-fold (in the range of 20-0,07%) in DMEM medium comprising 2% FBS and PSF, was first added onto the cells. Three wells were used for each concentration. Wells not comprising the test substance were used as controls. After incubation for 1 hour at 37 °C in a 5% CO2 incubator, the plate contents were discarded and they were washed with PBS. A virus suspension comprising 100 TCID50 was added onto the cells that had been exposed to the test substance dilutions for 1 hour, and incubated for 1 hour at 37 °C in a 5% CO2 incubator. Thereafter, the inoculum was removed and the test substance, which had been serially diluted two-fold (in the range of 20-0,07%) in DMEM medium comprising 2% FBS and PSF, was added onto the cells. The plates were incubated for 72 hours at 37 °C in a 5% CO2 incubator. Wells that were not exposed to the test substance but were inoculated with virus and wells comprising only DMEM-2 were used as the Virus Control (VC) and Cell Control (CC), respectively. c. Post-infection test: It is performed by exposing the cells to the test substance after infection with the virus. Briefly, the cells are first infected with the virus and then exposed to the test substance. In this manner, it can be determined whether the test substance has an inhibitory effect on viral replication after entry into the cells. For this purpose, after the plate contents were discarded and they were washed with PBS, a virus suspension comprising 100 TCID50 was added onto the cells and incubated for 1 hour at 37 °C in a 5% CO2 incubator. Thereafter, the virus inoculum was removed and the test substance, which had been serially diluted two-fold (in the range of 20-0,07%) in DMEM medium comprising 2% FBS and PSF, was added onto the cells. Three wells were used for each concentration. Wells not comprising the test substance were used as controls. The plates were incubated for 72 hours at 37 °C in a 5% CO2 incubator. Wells that were not exposed to the test substance but were inoculated with virus and wells comprising only DMEM-2 were used as the Virus Control (VC) and Cell Control (CC), respectively.

[0065] Data Analysis:

[0066] Calculation of Cytotoxicity: The toxic effect of the test substance on the cells at each concentration (% cytotoxicity) is calculated using the following formula: [Test OD / Cell Control OD]*100.

[0067] Calculation of Inhibition: The inhibitory effect (% inhibition) of the test substance at each concentration in preventing the cytopathic effect (cpe) in infected cells is calculated according to the following formula: [(Test OD - Virus Control OD / Cell Control OD - Virus Control OD) x 100],

[0068] Acceptance Criteria of the Study:

[0069] The Cell Control OD must be > 0,5 (non-infected)

[0070] The Virus Control OD must be < 0,7 (infected)

[0071] The findings and results of the antiviral and cytotoxicity tests are presented in the figures. In Figure 1, when the toxic effect of lutetium borate on E6 VERO cells is evaluated, it is observed that, at a high concentration (20%), lutetium borate increases cell proliferation, i.e. cell regeneration, rather than exerting a toxic effect on the cells, and that this effect is lower or absent at lower concentrations. On the other hand, the antiviral effect of lutetium borate against Sars-Cov2 varies depending on the application mode in the Entry, Full-Time (FT) and Post-Infection (PE) tests and, partial inhibition is observed only at high concentrations (20%-10%) in the Entry and Post-infection tests, whereas this effect is relatively higher in the Full-Time test. The graphs shown correspond to the entry, FT and PE antiviral tests. The cytotoxicity line starting from -15 indicates cell regeneration. The value of -15 means that it does not kill the virus and that, instead, it results in an increase of 15 cells.

[0072] In Figure 2, when the toxic effect of lutetium borate on MDCK cells is evaluated, it is observed that, at a high concentration (20%), lutetium borate increases cell proliferation, i.e. cell regeneration, rather than exerting a toxic effect on the cells, and that this effect is absent at lower concentrations. On the other hand, the antiviral effect of lutetium borate against Influenza A virus shows only minor differences compared to the Full-Time and Postinfection application modes, and an inhibition ranging between 100% and 50% is observed within the range of 20%-5%.

[0073] In Figure 3, when the toxic effect of lutetium borate on VERO cells is evaluated, it is observed that, at a high concentration (20%), lutetium borate increases cell proliferation, i.e. cell regeneration, rather than exerting a toxic effect on the cells, and that this effect is absent at lower concentrations. On the other hand, the antiviral effect of lutetium borate against HSV-1 virus varies depending on the Entry, Full-Time and Post-infection application modes, while in the Entry test a minimal effect is observed, an inhibition between 100% and 50% is observed within the range of 20%-2.5%.

[0074] The cytotoxic effect of lutetium borate on three different cells is observed at concentrations > 20%, and interestingly, as the dose decreases, both the cell regeneration effect and the virucidal effect also decrease. At a high concentration, namely at 20%, both the virucidal effect and the cell regeneration activity are observed at a high level.

[0075] When the antiviral activity of lutetium borate against the three different viruses tested is evaluated, based on the Entry test results, it can be stated that lutetium borate does not exhibit an effect such as blocking the receptors on the cell that could be used by the virus. In contrast, when the cells are exposed to lutetium borate prior to and during infection, lutetium borate exhibits antiviral activity against Influenza A and HSV-1 viruses within the concentration range of 20%-2.5%, and against Sars-CoV2 only when lutetium borate is present on the infected cells during the incubation period.

[0076] It should be emphasized that, in light of these findings, lutetium borate is considered to exhibit inhibition via a virucidal mechanism, rather than exerting an effect on the viral life cycle, namely viral attachment (adsorption) to the cell, entry, intracellular replication, and release from the cell. The results indicate that the virus inhibition, i.e. the reduction in viral load, is achieved not through an antiviral effect but through a virucidal effect. The concepts of antiviral and virucidal are quite different and should not be confused with each other. In fact, the present invention provides a wound-healing gel formulation in which lutetium borate kills, i.e. inhibits, viruses upon direct contact with the virus. By way of clarification, for example, alcohol is virucidal, meaning that it kills viruses, whereas the active substances in medicines are antiviral. The alcohol applied to the hands during the coronavirus pandemic kills the virus, while orally administered drugs inhibit viral processes within the cell or interfere with intracellular viral mechanisms. Since lutetium borate is nanosized, it can readily penetrate through the skin and be absorbed by skin cells. For this reason, it causes viral death.

[0077] 2- Virucidal Effect Analysis:

[0078] • Cell Line: The VERO cell line was used for Herpes Simplex Type 1 (HSV-1). The cells were cultured in T75 cell culture flasks at 37 °C and 5% CO2 using DMEM medium comprising 10% fetal bovine serum (FBS). From cell aliquots stored at -80 °C, one vial was thawed and cultured in a T25 flask in DMEM comprising 10% FBS, and when reached 80-90% confluence, they were expanded in T75 flasks and used in the virucidal test.

[0079] • Virus(es): Human HSV-1 (wal strain) was used in the study.

[0080] • Preparation of the Test Substance: Based on the manufacturer’s declaration that the product has a ready-to-use, water-soluble composition, the test substance serially diluted with distilled water to the required percentages (by being thoroughly mixed) in the bottle was directly used.

[0081] Virucidal Test:

[0082] • One part BSA, one part VIRUS and eight parts TEST SUBSTANCE were placed into a tube and vortexed. Samples taken at defined time intervals were neutralized with DMEM comprising 10% FBS and kept on ice until the remaining samples were collected.

[0083] • Ten-fold serial dilutions of the samples taken at time Txwere prepared in DMEM comprising 10% FBS (D-10).

[0084] • Cells susceptible to the virus and grown in T75 flasks were trypsinized, and its suspension at a density of 200 000 cells / mL was prepared in D-10. Subsequently, it was seeded into 96-well plates at 0,1 mL / well.

[0085] • Onto the cells prepared in 96-well plates as described above, ten-fold dilutions prepared from the samples taken after each tx were added (with eight wells used for each dilution).

[0086] • As the Virus Control (VC), samples taken at time tx from a mixture prepared under the same conditions, but using distilled water instead of the test substance, were used. As the Cell Control (CC), a sample comprising neither virus nor test substance was used.

[0087] • The plates were incubated for 4 days at 37 °C, 5% CO2 and under humidified conditions, and were evaluated in terms of cytopathic effect (cpe) using an inverted microscope.

[0088] Data Analysis

[0089] • Calculation of Virucidal Effect: The cpe scores determined for the test substance and the virus control were calculated using the Spearman-Karber formula and expressed as TCID50 / mL. The virucidal effect (%) of the test substance (R: reduction) was determined by subtracting the TCID50 value obtained for the test substance from the TCID50 / mL value obtained for the virus control.

[0090] • Study Acceptance Criteria

[0091] • Cell Control Log TCID50 / mL must be < 0.5

[0092] • Virus Control Log TCID50 / mL must be > 8

[0093] The findings and results of the virucidal activity test are presented in Figure 4.

[0094] In Figure 4, when the virucidal (virus killing / inactivation) activity of lutetium borate against human Herpes Simplex Virus Type 1 is evaluated, it is observed that according to the concentration and exposure time, virus inactivation is achieved at a level of 76% within 5 minutes when lutetium borate is used at a concentration of 20%, and that this activity reaches 90% and 92% within 5 minutes when concentrations of 40% and 80% are used, respectively. When the exposure time is taken into consideration, it is observed that lutetium borate provides its maximum virucidal activity within 5 to 20 minutes, and that extending the exposure time to 40 or 60 minutes does not further increase the activity.

[0095] In light of the obtained results, it has been determined that the concentration of the stable colloidal lutetium borate solution to be used for viral skin infections should be 20%.

[0096] Herpes Simplex Type 1 Skin Infection and Zosteriform Spread Inhibition Test of the gel formulation of the invention:

[0097] Cell Line: The VERO cell line was used for Herpes Simplex Type 1 (HSV-1). The cells were cultured in T75 cell culture flasks at 37 °C and 5% CO2 using DMEM medium comprising 10% fetal bovine serum (FBS). From cell aliquots stored at -80 °C, one vial was thawed and cultured in a T25 flask in DMEM comprising 10% FBS, and when reached 80- 90% confluence, they were expanded in T75 flasks and used.

[0098] Virus: Human HSV-1 (wal strain) was used in the study. VERO cell-comprising flasks were infected with the virus, and after >90% cytopathic effect was observed in the cells, the infected cells were harvested. Following centrifugation, the virus-comprising supernatant was vialed and stored at -80 °C in a deep freezer. The virus titer was performed by inoculating log dilutions of the virus onto VERO cells grown in 96-well plates, and was found to be TCID50 / mL 1x108.

[0099] HSV-1 Skin Infection and Zosteriform Spread

[0100] • Cutaneous HSV-1 infection experiments were performed using a zosteriform model established by epidermal scarification. Unless otherwise stated, the abdomen flank and dorsal skin of 5-6-week-old female BALB / cJ mice was shaved and the remaining hair was removed by depilation using a depilatory cream to obtain a denuded skin area. After 24 hours, while the mice were under mild ether anesthesia, 10 micro litres of virus (1.0 x 105 virus) was applied onto the denuded area, and the site was scarified using the tip of a 26-gauge needle.

[0101] • Four hours after the virus was applied to the denuded area by scarification, the mice were randomly assigned to a TEST group (n:8) and a CONTROL group (n:8). On the same day, the test and control gels were applied once, and for the following 7 days, the applications were performed twice daily in mornings and evenings to a 4 cm2area including the virus-inoculated site, using a cotton swab. The mice were monitored for 7 days, and the severity of infection was evaluated according to the scoring method shown below.

[0102] Table-1: Scoring table.

[0103] Data Analysis:

[0104] • Calculation of Efficacy: The infectivity scores were calculated using the Student’s independent t-test (two-tailed).

[0105] • Study Acceptance Criteria

[0106] • The mean score of the control group > 5

[0107] Findings and Results of the Herpes Simplex Type 1 Skin Infection and Zosteriform Spread Inhibition Test:

[0108] Table 2. Preventive efficacy of lutetium borate against death associated with cutaneous Herpes Simplex Virus Type 1 infection and zosteriform spread

[0109] In a mouse model of cutaneous infection and infection-associated zosteriform spread established using human Herpes Simplex Virus Type-1, when the infected mice were treated with a lutetium borate-comprising gel (test) and an active-ingredient-free gel (control) once on the same day and subsequently twice daily for 7 consecutive days, it was observed that the lutetium borate-comprising gel statistically significantly inhibited zosteriform spread compared to the control (p = 0,28). On the other hand, with respect to mortality associated with the severity of infection, the control gel provided a survival benefit of 12.5%, whereas the lutetium borate-comprising gel achieved a survival rate of 50%.

[0110] As a result of the conducted tests, the cytotoxicity test results of the parent lutetium borate molecule have demonstrated that this material is not only antiviral but is in fact virucidal, and at the same time exhibits a cell-regenerative effect. Accordingly, a gel formulation having a wound-healing effect has been developed. The wound-healing effect of said gel formulation has also been proved by in vivo experiments.

[0111] Industrial Applicability of the Invention

[0112] The invention relates to a gel formulation comprising an amorphous (LuxByOz.nTfcO) lutetium borate compound with virucidal effect and having cell-regenerative or woundhealing properties, and to a production method thereof, and is industrially applicable and suitable for use in industry.

[0113] The invention is not limited to the descriptions above, a skilled person in the art can perform different embodiments of the invention easily. These should be interpreted within the protection scope of the invention claimed with the claims.

Claims

CLAIMS1. For exhibiting a virucidal effect and for healing wounds on the skin; a gel formulation comprising a stable colloidal lutetium borate solution, characterized by comprising predetermined amounts of water, boric acid, sodium hydroxide, hyaluronic acid / sodium hyaluronate, a colloidal lutetium borate solution and glycerin or comprising Ultrez-21 or phenoxyethanol.

2. The formulation according to claim 1, characterized by comprising by weight 37.0- 84.9% water, 10-40% colloidal lutetium borate solution, 1-5% boric acid, 1-5% glycerin, 1-5% sodium hydroxide, 1-5% hyaluronic acid / sodium hyaluronate, 1-2% ultrez-21 and 0.1-1% phenoxyethanol.

3. The gel formulation comprising the stable colloidal lutetium borate solution according to claim 2, characterized by comprising by weight 67.2% water, 20% colloidal lutetium borate solution, 3% boric acid, 3% glycerin, 2.6% sodium hydroxide, 2% hyaluronic acid / sodium hyaluronate, 1.2% ultrez-21 and 1% phenoxyethanol.

4. For healing wounds on the skin or for providing a cell-regenerative and at the same time virucidal effect; a production method (100) of a gel formulation comprising a stable colloidal lutetium borate solution, characterized by comprising the following steps:Adding a predetermined amount of purified water and, preferably, ultrez 21 into a vessel, allowing the mixture to stand for a predetermined period of time, and subsequently mixing it in a mechanical stirrer (101),Dissolving a predetermined amount of boric acid in a predetermined amount of water in another vessel (102),Adding a predetermined amount of a 20% NaOH solution to said solution (103), Adding a predetermined amount of hyaluronic acid / sodium hyaluronate thereto and mixing for a predetermined period of time (104),Slowly adding the dissolved hyaluronic acid / sodium hyaluronate solution to the mixture obtained in step (101) in the mechanical stirrer, and mixing for a predetermined period of time at a predetermined stirring speed (105),Adding a predetermined amount of glycerin thereto and mixing for a predetermined period of time at a predetermined stirring speed (106),Preferably adding a predetermined amount of phenoxyethanol thereto and mixing for a predetermined period of time at a predetermined stirring speed (107),Adding a predetermined amount of a lutetium borate solution thereto and mixing for a predetermined period of time, thereby obtaining the colloidal lutetium borate solution (108).

5. The method (100) according to claim 4, characterized in that, in step 101, the amount of Ultrez 21 is 1-2% by weight and the amount of the added water is 37.0-84.9% by weight, and that the standing time of the purified water and Ultrez 21 is from 1 to 5 hours, and the mixing time in the mechanical mixer is from 1 to 5 hours.

6. The method (100) according to claim 5, characterized in that, in step 101, the amount of Ultrez 21 is 1.2% by weight and the amount of the added water is 67.2% by weight, and that the standing time of the purified water and Ultrez 21 is 1 hour, and the mixing time in the mechanical mixer is 1 hour.

7. The method (100) according to claim 6, characterized in that, in step 102, the amount of water is 37.0-84.9% by weight and the amount of boric acid is 1-5% by weight.

8. The method (100) according to claim 7, characterized in that, in step 102, the amount of water is 67.2% by weight and the amount of boric acid is 3% by weight.

9. The method (100) according to claim 8, characterized in that, in step 103, the amount of 20% NaOH is 1-5% by weight.

10. The method (100) according to claim 9, characterized in that, in step 103, the amount of 20% NaOH is 2.6% by weight.

11. The method (100) according to claim 10, characterized in that, in step 104, the amount of hyaluronic acid / sodium hyaluronate is 1-5% by weight, and the mixing time in the same step is from 1 to 5 hours.

12. The method (100) according to claim 11, characterized in that, in step 104, the amount of hyaluronic acid / sodium hyaluronate is 2% by weight, and the mixing time in the same step is 1 hour.

13. The method (100) according to claim 12, characterized in that, in step 105, the mixing time is from 1 to 5 hours, and the stirring speed is from 100 rpm to 2000 rpm.

14. The method (100) according to claim 13, characterized in that, in step 105, the mixing time is 3 hours, and the stirring speed is 700 rpm.

15. The method (100) according to claim 14, characterized in that, in step 106, the amount of glycerin is 1-5% by weight, and the mixing time in the same step is from 1 to 5 hours and the stirring speed is from 100 rpm to 2000 rpm.

16. The method (100) according to claim 15, characterized in that, in step 106, the amount of glycerin is 3% by weight, and the mixing time in the same step is 3 hours and the stirring speed is 700 rpm.

17. The method (100) according to claim 16, characterized in that, in step 107, the amount of phenoxyethanol is 0.1-1%, and the mixing time in the same step is from 1 to 5 hours and the stirring speed is from 100 rpm to 2000 rpm.

18. The method (100) according to claim 17, characterized in that, in step 107, the amount of phenoxyethanol is 0.1-1%, and the mixing time in the same step is from 1 to 5 hours and the stirring speed is from 100 rpm to 2000 rpm.

19. The method (100) according to claim 18, characterized in that, in step 107, the amount of phenoxyethanol is 1%, and the mixing time in the same step is half an hour and the stirring speed is 700 rpm.

20. The method (100) according to claim 19, characterized in that, in step 108, the amount of the lutetium borate solution is 10-40% by weight, and the mixing time in the same step is from 1 to 5 hours.

21. The method (100) according to claim 20, characterized in that, in step 108, the amount of the lutetium borate solution is 20% by weight, and the mixing time in the same step is 1 hour.

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