Use of a lutetium salt compound for antiviral effects

Abstract

The present invention relates to the antiviral efficacy of salt compounds obtained from lutetium elements such as lutetium borate, lutetium chloride, and lutetium nitrate against DNA viruses and RNA viruses in infected cells.

Description

Technical Field

[0001] The present invention relates to salt compounds obtained from lutetium such as lutetium borate, lutetium chloride, and lutetium nitrate, which exhibit antiviral efficacy against adenovirus strains, poliovirus strains, herpes simplex type 1 and type 2 strains, coronavirus strains, and norovirus strains in infected appropriate cell lines (HaCaT, Vero, Raw264.7).

Background Art

[0002] Viruses are one of the major causes of diseases and deaths worldwide and can infect all types of cellular life forms, including both eukaryotes and prokaryotes. Viruses consist of a protein structure called an outer capsid and genetic material in the form of single-stranded or double-stranded DNA or RNA. Some viruses also have another structure called an envelope, composed of lipoprotein and having antigenic properties, in addition to the capsid. Viruses, which are also defined as intracellular parasites, can use evolved host cells (bacterial, plant, or animal cells) to produce their viral proteins and genetic material.

[0003] Various drugs have been developed to inactivate viruses or prevent virus replication. For an antiviral agent to be effective, it must directly affect the virus or its replication without affecting the host cell. Antiviral drugs widely used in the prior art have been examined under four main headings. These are drugs effective against influenza virus, drugs effective against herpes virus, anti-HIV drugs, and immunomodulators used in antiviral therapy. Although these drugs are already in use, they are accompanied by many serious side effects such as fatigue, vomiting, stomach upset, diarrhea, dizziness, toxic epidermal necrolysis, peripheral neuropathy, oral ulcers, esophageal ulcers, hepatomegaly, and an increase in diabetes [1]. Furthermore, although preventive measures with vaccines are taken against some viral diseases that cause death such as smallpox virus, there are no existing preventive or treatment methods for persistent or emerging viruses. Therefore, there is a need for new antiviral agents that can be used to overcome the side effects of existing antiviral agents, for combination therapy in patients who develop or experience immunodeficiency, and in cases of antiviral drug resistance.

[0004] The lanthanides are composed of 15 elements known as rare earth elements. In the 1960s, they were discovered to exhibit pharmacological properties such as anticoagulation, anti - inflammation, antibacterial, anti - allergy, and anti - cancer. Due to these properties, compounds containing lanthanide(III) ions (rare earth metal ions) have been of interest since the 19th century [2]. Lanthanide(III) ion compounds play a very important role in medicine, especially in cancer diagnosis and treatment, and these compounds were also used as antibacterial agents for the treatment of tuberculosis in the early 20th century [3]. Lutetium is the last and smallest member of the lanthanide family. Lutetium, which is a silver - white metal as a pure element, has reactivity similar to calcium and magnesium. Lutetium and its compounds are involved in several applications in the petrochemical industry, for example, the preparation of catalysts for decomposing hydrocarbons or the use as effective and reusable catalysts for organic synthesis. In the field of healthcare, lutetium - 177, a radioactive form of lutetium, is used to treat gastrointestinal cancers including those of the stomach, pancreas, and intestine. However, there is no application in the art for the use of lutetium for antiviral purposes.

[0005] The European patent document of European Patent No. 2546839B1, which is a published patent application known in the state of the art, discloses a carrier - free lutetium - 177 compound suitable for use in the medical field and a method for manufacturing the compound. The antiviral effect of lutetium is not disclosed in this document.

[0006] Due to the above - mentioned drawbacks and problems, there is a need in the art for novel compounds exhibiting antiviral effects.

Prior Art Documents

Patent Documents

[0007]

Patent Document 1

Non - Patent Documents

[0008]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Summary of the Invention

Problems to be Solved by the Invention

[0009] The main object of the present invention is to enable the use of a lutetium salt compound in a broad-spectrum antiviral drug formulation in which an antiviral effect against both DNA viruses and RNA viruses is detected within the scope of the present invention. These compounds in which antiviral properties are detected have the potential to be used in the treatment / prevention of diseases in humans, animals, and plants.

Means for Solving the Problems

[0010] The present invention relates to the antiviral efficacy of salt compounds obtained from lutetium elements such as lutetium borate, lutetium chloride, and lutetium nitrate against DNA viruses and RNA viruses in infected cells.

Brief Description of the Drawings

[0011] The "use of a lutetium salt compound for an antiviral effect" realized to achieve the object of the present invention is shown in the accompanying drawings.

[0012]

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Mode for Carrying Out the Invention

[0013] The present invention relates to a lutetium salt compound that exhibits antiviral efficacy against adenovirus strains, poliovirus strains, herpes simplex type 1 and type 2 strains, coronavirus strains, and norovirus strains in immortalized human keratinocyte cells (keratinocytes) (HaCaT) infected with the virus. The lutetium salt compound used for the treatment of the diseases mentioned in the present invention is preferably lutetium borate, lutetium chloride, and lutetium nitrate. In the present invention, other water-soluble and water-insoluble lutetium salt compounds formed together with the lutetium element, which are used to bring about an antiviral effect against DNA virus strains and RNA virus strains, may be nitrates, sulfates, acetates, oxides, hydroxides, fluorides, carbonates, phosphates, oxalates, etc. Any one or combination of the above lutetium salt compounds can be used in any products and / or formulations (creams, sprays, lotions, gels, capsules, tablets, toothpaste, oral rinses (mouthwashes), masks, disinfectants, preservatives, powders, polishes, glazes, jellies, foaming pomades) manufactured for medical, medical device, biocidal, and cosmetic purposes.

[0014] To prove the effectiveness of the lutetium salt, the processes of synthesis of the lutetium salt compound, cytotoxicity assay, determination of virus titer, antiviral efficacy analysis, quantitative real-time PCR analysis, and calculation of the infectious titer by TCID50 were carried out in the studies conducted within the scope of the present invention. The above processes are described in detail below.

[0015] Synthesis of lutetium borate: Lutetium borate (LuBO3) produced in a laboratory environment was prepared in an amount of 750 ppm using deionized water.

[0016] Lutetium nitrate solution: Lutetium nitrate hydrate ((Lu(NO3)3xH2O, Cas number 100641-16-5, Sigma) was prepared in an amount of 750 ppm using deionized water.

[0017] Lutetium chloride solution: Lutetium chloride (LuCl3, CAS number 10099 - 66 - 8, Sigma) was prepared in an amount of 750 ppm using deionized water.

[0018] Cytotoxicity assay: The effect of the prepared compound on cell viability was measured by a substance called MTS based on mitochondrial dehydrogenase enzyme activity. In this method, HaCaT (immortalized human keratinocyte cells) were prepared in medium and seeded at 5000 cells / well in a 96 - well culture plate. After the incubation period (24 hours), the medium on the cells was removed, and the above - mentioned compound at a specific concentration was diluted with medium and given to the cells. The response of the cells to the toxicity (cytotoxicity) of the molecule was detected by measuring cell viability after 72 hours. After the incubation period ended, the MTS substance added to the cells together with the medium caused the formation of colored formazan crystals as an indicator of cell viability. This color change was evaluated by measuring the absorbance with an ELISA plate reader. The obtained results were analyzed. The results of the analysis are shown in the graph of Figure 1.

[0019] Determination of virus titer: Virus titer constitutes the most important step in any virological study that requires the use of a specific amount of virus, especially in studies to demonstrate the effectiveness of potential antiviral agents. Since the classical method is quantitative and prone to subjective errors, the colorimetric MTS method was used to determine the virus titer. For this analysis, HaCat cells were seeded at 3×10 4Cells were seeded and incubated at 37°C for 24 hours. The next day, the virus from the virus stock was prepared on ice at a logarithmic scale (Log2) of 2-1 to 2-7. The medium on the monolayer cells was discarded, and the cells were washed three times with PBS. 50 μl of the prepared virus dilution was collected and added to each well in six replicates (repetitions), and the cells were infected by incubating for 2 hours with stirring every 20 minutes. Subsequently, the virus on the cells was removed, and the cells were washed three times with PBS. Virus medium was added to the cells and incubated at 37°C for 72 hours in a CO2 environment. The DMSO (20%) for the positive control and the infected medium for the negative control were aspirated, 200 μl of virus medium containing 10% MTS was added to each well, and incubated for 3 hours. The resulting color change was evaluated based on absorbance measurement with an ELISA plate reader at 490 nm (Figures 2 and 3).

[0020] Anti-viral efficacy analysis: HaCaT cells were added to a 48-well culture plate at 2×10 5 cells per well and incubated at 37°C for 24 hours. At this stage, the medium on the monolayer cells was discarded, and the cells were washed three times with PBS. 100 μl of the virus for which the TCID50 value was determined was placed on the cells. The cells were incubated for 2 hours with stirring every 20 minutes to infect the cells. Subsequently, the virus on the cells was removed, and the cells were washed three times with PBS. The concentration of the substance for which the non-toxic dose was determined was prepared in virus medium, added to the cells, and incubated at 37°C for 72 hours in a CO2 environment.

[0021] Quantitative real-time PCR analysis: The virus medium was taken from the incubated plate into an Eppendorf tube, centrifuged, and the supernatant was transferred to a new Eppendorf tube. Viral DNA was isolated from the collected supernatant according to the kit protocol (Viral nucleic acid kit, Roche). Subsequently, PCR analysis was performed according to the protocol of the quantitative PCR kit (HSV1, HSV2, Argene, Biomerieux) (Figures 4, 5 and 6).

[0022] Calculation of infectious titer by TCID50: Cells were removed from the flask, transferred to a 15 ml Falcon tube, and centrifuged at 500 × g for 5 minutes. The medium on the cells precipitated at the bottom of the Falcon tube was discarded, and 1 ml of fresh medium was added with a pipette and dissolved. Subsequently, the cells were seeded into a 96-well plate to form a monolayer within 24 ± 2 hours and incubated at 37 °C in a 5% CO2 incubator. When it was observed under an inverted microscope that the cells were in a monolayer, the cells were treated. The Vero cell line was used for adenovirus and poliovirus, and the Raw cell line was used for murine norovirus. Substances for which non-toxic doses were determined were prepared using virus medium according to the volume to be used. 225 μl of virus medium was added to a new 96-well plate, and 25 μl of virus was added to the first 6 wells of the 96-well plate, and serial dilutions were performed on a logarithmic scale (Log10). The medium of the prepared monolayer cells was discarded. They were washed twice with virus medium. The serial dilutions prepared in the new 96-well plate were transferred to the cells, and the medium volume was adjusted to 200 μl with the prepared non-toxic dose of the substance. The plate was incubated at 37 °C in a 5% CO2 incubator for 72 hours. At the end of the incubation period, the cytopathic effect (CPE) of the virus suspension on the cells was evaluated under an inverted microscope. The results obtained were evaluated by performing TCID50 calculation by the Spearman-Karber method according to the following formula. M = Xk + d[0.5 - (1 / n)(r)] (Equation 1) In Equation 1, Xk is the highest dilution dose, d is the difference between dilutions, n is the number of wells per dilution, and r is the total of (-) responses. Mv = lg(Va / Vc) = lg(Va) - lg(Vc) (Equation 2) In Equation 2, Mv is the antiviral activity value, lg(Va) is the logarithmic mean of two biological replicates for the control test, and lg(Ve) is the logarithmic mean of two biological replicates for the experimental group.

[0023] The antiviral potencies of lutetium nitrate and lutetium chloride compounds against different types of viruses are shown in the following table.

[0024]

Table 1

[0025] The results of the studies conducted using LuBO3, LuNO3, and LuCl3 demonstrate that the antiviral efficacy is related to the lutetium element. Therefore, it is evaluated that other lutetium salt compounds and / or products / formulations containing these salt compounds may have a similar antiviral efficacy. In the analysis of these three compounds, the effective concentration was detected to be in the range of 100 ppm to 1000 ppm. Therefore, it is evaluated that the antiviral activity values related to lutetium compounds may also apply to other compounds / products / formulations containing the lutetium element.

[0026] Synthesis of Lutetium Borate A solution containing sodium hydroxide and boric acid (preferably in a ratio of 1:2) is prepared preferably in 50 mL of deionized water. A lutetium nitrate solution is prepared in polyvinyl alcohol having a total volume of preferably 50 mL. The prepared lutetium nitrate solution is added to the sodium hydroxide and boric acid solution at a constant rate, and the mixture is stirred at 2000 rpm for the first 5 minutes and then at 1000 rpm for 25 minutes. The resulting lutetium borate solution can be used after property evaluation.

[0027] Lutetium Nitrate Solution Lutetium nitrate is prepared using deionized water to preferably an amount of 750 ppm.

[0028] Lutetium Chloride Solution Lutetium chloride is prepared using deionized water to preferably an amount of 750 ppm.

[0029] The lutetium salt compounds and other lutetium salts studied within the scope of the present invention can be prepared and used at appropriate concentrations in different solvents by a similar method.

[0030] In an embodiment of the present invention, solvents such as PVA and glucopan are used to better disperse the compounds formed during the preparation of lutetium borate. Within the scope of the present invention, PVA and glucopan are particularly preferred. This is because LuBO3 does not show a toxic effect on cells while its salts are suspended.

[0031] References [1]. Ekmekyapar, Muhammed, and Sukru Gurbuz. "Antiviral Drugs and Their Toxicities". Eurasian Joumal of Toxicology 1.3 (2018): 77 - 84. [2]. Q.-L. Guan, Y.-H. Xing, J. Liu, W.-J. Wei, X. Wang, F.-Y. Bai, Application of multiple parallel perfused microbiorectors: synyhesis, characterization and cytotoxicity testing ofthe novel rare earth complexes with indole acid as a ligand, J. Inorg. Biochem. 128 (2013) 57 - 67. [3]. S. Ban, S. Suzuki, K. Kubota, S. Ohshima, H. Satoh, H. Imada, Y. Ueda, Gastric mucosal status susceptible to lanthanum deposition in patients treated with dialysis and lanthanum carbonate, Ann. Diagn. Pathol. 26 (2017) 6 - 9.

Claims

1. A lutetium salt compound used to provide an antiviral effect against DNA viruses and RNA viruses (adenovirus, poliovirus, herpes simplex type 1 and herpes simplex type 2, coronavirus and norovirus) in infected cells.

2. The lutetium salt compound according to claim 1, wherein the lutetium salt compound used to provide an antiviral effect against DNA viruses and RNA viruses in appropriate infected cell lines (HaCaT, Vero, Raw264.7) is lutetium borate.

3. The lutetium salt compound according to claim 1, wherein the lutetium salt compound used to provide an antiviral effect against DNA viruses and RNA viruses in infected cells is lutetium chloride.

4. The lutetium salt compound according to claim 1, wherein the lutetium salt compound used to provide an antiviral effect against DNA viruses and RNA viruses in infected cells is lutetium nitrate.

5. Other water-soluble and water-insoluble lutetium salt compounds formed with lutetium element, which are used to provide an antiviral effect against DNA virus strains and RNA virus strains, are nitrates, sulfates, acetates, oxides, hydroxides, fluorides, carbonates, phosphates, oxalates, etc. The lutetium salt compound according to claim 1.

6. Any type of product and / or formulation manufactured for pharmaceutical, medical device, biocidal and cosmetic purposes to provide an antiviral effect against DNA virus strains and RNA virus strains, and containing any one or a combination of the lutetium salt compounds according to any one of claims 1 to 5. A product and / or formulation characterized by this.

7. The formulation according to claim 6, which is in the form of a cream, spray, lotion, gel, capsule, tablet, toothpaste, mouthwash, mask, disinfectant, preservative, powder, polish, brightener, jelly, or foaming pomade.

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